3M Environmental Monitoring Handbook

Environmental
Monitoring
Handbook
for the Food and
Beverage Industries
1st Edition
The 3M Environmental Monitoring Handbook is intended to provide general guidance only. The technical
information, recommendations and other statements contained in this document are based on experience
and information that 3M believes to be reliable, but the accuracy or completeness of such information is not
guaranteed. Such information is intended for persons with knowledge and technical skills sufficient to assess
and apply their own informed judgement to the information, taking into consideration the nature of their
business, existing policies and particular laws and regulations that might apply.
Environmental Monitoring Handbook - Table of Contents
TABLE OF CONTENTS
Environmental Monitoring
Handbook for the Food and
Beverage Industries
Key Terms and Definitions iii
CHAPTER 1
The Importance of Environmental Sampling 1

in Food Safety and Quality Programs
Martin Wiedmann | Cornell University Department of Food Science
Alexandra Belias | Cornell University Department of Food Science
Genevieve Sullivan | Cornell University Department of Food Science
John David | 3M Food Safety
CHAPTER 2
ATP and Protein-based Hygiene Monitoring 11

Louise Roberts | Alimenti Food Sciences Ltd
Gareth Lang | 3M Food Safety
Burcu Yordem | 3M Food Safety
CHAPTER 3
Environmental Monitoring for Indicator Organisms 29

Kelly Stevens | General Mills
Jean-Francois David | 3M Food Safety
Cari Lingle | 3M Food Safety
CHAPTER 4
Environmental Monitoring for Pathogens 41

Christian Blyth | 3M Food Safety
i
Environmental Monitoring Handbook - Table of Contents
TABLE OF CONTENTS (Cont.)
CHAPTER 5
Environmental Monitoring for Spoilage Organisms 59

Randy Worobo | Cornell University Department of Food Science
Abigail Snyder | The Ohio State University Department of Food Science and Technology
CHAPTER 6
Environmental Monitoring for Allergens 73

Thomas Grace | Bia Diagnostics
Ken Davenport | 3M Food Safety
Gabriela Lopez Velasco | 3M Food Safety
CHAPTER 7
Driving Meaningful Change in Your Organization 85

Through Culture and Environmental Monitoring
John Butts | Food Safety By Design
Lone Jespersen | Cultivate
Michele Fontanot | 3M Food Safety
CHAPTER 8
Environmental Sampling Guidance 95

Scott Egan | 3M Food Safety
About the Contributors 107
ii
Environmental Monitoring Handbook - Terms and Definitions
Key Terms and Definitions

Term Definition
Adenosine
Energy molecule present in every cell, alive or dead.
triphosphate (ATP)
Increased frequency and/or scope of sampling in response to a positive sample result. May
Aggressive sampling1
also include addition of post-rinse sampling and other advanced sampling approaches.
Thin, slimy film of densely-packed bacteria that adheres to a surface. Biofilms may form
on rough or scratched surfaces and in hard-to-reach areas, making them difficult to
Biofilm
eliminate. Biofilms can represent a persistent harborage for microorganisms and a source of
contamination of food products, as they can contain spoilage organisms or pathogens.
Microbiota The population of microorganisms found in a specific environment.
Clean out-of-place Method of cleaning equipment items by removing them from their operational area and taking
(COP) them to a designated station for disassembly and cleaning.
Method of cleaning interior surfaces of process equipment, pipes, vessels, filter and
Clean-in-place (CIP)
associated fittings without disassembly.
An action to eliminate a detected nonconformity. These can be immediate activities to

identify and correct a problem that occurred during the production of food, such as re-
Correction2,3 cleaning and sanitizing a line before start-up of production when food residue remains after
cleaning. This should not be confused with corrective action, as it may not address the cause
of the problem.
An action to eliminate the cause of a detected nonconformity or other undesirable situation,

Corrective action2,3 to prevent recurrence. This should not be confused with correction, which may not address
the cause, or preventive action, which is taken to prevent occurrence of a potential problem.
A quality management concept found within GMP, HACCP and ISO standards that aims to
Corrective and rectify a task, process, product or behavior that has resulted in errors or deviations from
preventive action the intended plan. CAPA is split between two distinct functions – corrective actions and
(CAPA) 4 preventive actions – to systematically investigate the cause of the identified problems and
prevent their recurrence or occurrence, respectively.
Critical control point A point, step, or procedure in a food process at which control can be applied and is essential
(CCP) 2,5 to prevent or eliminate a food safety hazard or reduce the hazard to an acceptable level.
A maximum and/or minimum value to which a biological, chemical or physical parameter must
Critical limit5 be controlled at a CCP to prevent, eliminate or reduce to an acceptable level the occurrence
of a food-safety hazard.
iii
Term Definition
Defined program for monitoring the environment of a food manufacturing facility to prevent
cross contamination of the finished product from the environment. The term EMP typically
is used to describe a program that verifies cleaning, sanitation, and other environmental
pathogen control programs, and an EMP typically include sampling sites, frequency, testing
Environmental
methodology, acceptable criteria and corrective actions. More broadly, environmental
monitoring program
monitoring programs often encompass a range of tests – from ATP and indicator organisms
(EMP)
to pathogens, spoilage organisms, and allergens – and may serve to perform either validation
or verification of specific prerequisite programs (e.g., sanitation and sanitary equipment
design) or may be more generally seen as a strategy to monitor the environment for unhygienic
conditions that may lead to food safety and/or quality issues.
Environmental sampling programs use a zone classification to identify the risk level of areas
or sites where product may be exposed to post-lethality environmental contamination. In
most countries and regions, sampling sites in processing facilities are assigned to one of four
zones: (i) Zone 1 is the highest-risk area consisting of exposed food contact surfaces; (ii) Zone
Environmental
2 contains non-food contact surfaces in close proximity to food and food contact surfaces,
monitoring sampling
(iii) Zone 3 contains more remote non-food contact surfaces located in or near the processing
zones1,6,7,8,9
area; (iv) Zone 4 includes non-food contact surfaces outside of the processing areas. In some
countries, sampling sites may be classified into three zones, typically combining Zones 2 and
3 into one zone.
The (often-unsuccessful) approach of repeatedly attempting the same solution on a recurring

Firefighting
problem in effort to obtain microbiological control.
Investigative sampling that follows a positive sample from a product, contact surface or other
For-cause1
verification site.
The conditions and practices for processing safe food under sanitary conditions, including
Good manufacturing personnel, plant and grounds, sanitary operations, sanitary facilities and controls, equipment
practices (GMP) and utensils, processes and controls, warehousing and distribution, and defect
action levels considerations.
Location that supports microbiological growth and is protected from the sanitation process;
Growth niche1
characterized by high microbial counts after cleaning and sanitation.
Harborage site1 Growth niche that contains the pathogen or its indicator.
Any biological, chemical (including radiological), or physical agent that has the potential to
Hazard2,5
cause illness or injury. Hazards may be introduced to or naturally present in the food.
iv
Term Definition
Hazard analysis and A preventive food safety strategy that is a systematic approach to the identification and
critical control points assessment of the risk of hazards from a particular food or food production process or practice
(HACCP) 5 and the control of those hazards that are reasonably likely to occur.
Methods, processes, preservations and technologies used in combination to ensure that

Hurdle
pathogens in food products are appropriately eliminated or controlled.
Division of a food manufacturing facility into different areas to avoid food contamination risks.
Areas are designated based on risk and can include non-production areas (e.g., offices), basic
GMP areas (e.g., raw material storage), and the primary pathogen control area (PPCA) where
Hygienic zoning
processed RTE product is exposed to the environment prior to packaging. Hygienic zones
should not be confused with environmental monitoring sampling zones, which are used to
designate target areas for environmental sampling (i.e., Zones 1-4).
An organism or group of organisms whose presence relates to the possible occurrence of

Index organism
ecologically similar pathogen(s) (e.g., Listeria spp.).
An organism or group of organisms whose presence reflects the general microbiological

Indicator organism
condition of the food or environment (e.g., coliforms, Enterobacteriaceae).
Procedure capable of eliminating the pathogen from the affected area (e.g., heat treatment,
Intervention1
complete disassembly followed by cleaning and sanitation).
Documented regulatory compliance program designed to meet the regulatory needs of the
establishment. The Listeria intervention and control program clearly defines (i) actions taken
Listeria intervention
to verify the effectiveness of the establishment’s control of the environment and (ii) actions
and control program1
taken when a sample from product, contact surface or verification site is positive for Listeria
monocytogenes or Listeria spp.
A defined program for monitoring the environment of a food manufacturing facility for
Pathogen
pathogenic microorganisms. The goal of a PEM program is to find and eliminate pathogen
environmental
contamination in the processing environment. They are typically used to (1) verify an overall
monitoring (PEM)
food safety system (or specific components of a food safety system) and to (2) provide early
program
indication of potential food safety hazards.
v
Term Definition
Periodic deep
Disassembly of equipment or other components of a processing plant beyond the normal
cleaning and
level, followed by cleaning and sanitization.
sanitation1
Samples taken after production, disassembly and the initial rinse but before the application
of soap or sanitizer. Typical sites are below the product line and in areas that tend to collect
spatter from the rinsing process (e.g., machine sides, legs, support structure, floor wall
Post-rinse sampling1 juncture). Post-rinse samples are good broad indicators of the presence of the organism in
the post-lethality exposed product area. Detection of the organism does not mean there is a
harborage site within the scope of the sampled area. Positive post-rinse samples will typically
trigger aggressive sampling.
Preoperative Samples taken after sanitation but before starting production, typically during or after
sampling assembly and setup.
An action to eliminate the cause of a potential non-conformity or other undesirable situation

Preventive action3
to prevent occurrence.
Proactive control measures designed and undertaken to reduce or eliminate food safety
hazards. These include risk-based, reasonably appropriate procedures, practices, and
Preventive control processes that a person knowledgeable about the safe manufacturing, processing, packing,
(PC) 2 or holding of food would employ to significantly minimize or prevent the hazards identified
under the hazard analysis that are consistent with the current scientific understanding of safe
food manufacturing, processing, packaging, or holding at the time of the analysis.
A designated hygiene zone. The PPCA is an area where product is exposed to the
Primary pathogen
environment post-lethal processing. Also known as the ready-to-eat (RTE) area, high-risk area
control area (PPCA)
or high-hygiene area.
Qualitative test A test that determines presence or absence of an analyte(s) in a sample.
Quantitative test A test that measures the level or concentration of an analyte(s) in a sample.
The reading of the amount of light as determined by an individual ATP-based hygiene

Relative light unit
monitoring system. ATP system manufacturers may have different values for 1 light unit and all
(RLU)
measurements are made relative to that value.
vi
Term Definition
Overall process used to manage environmental control; includes both food safety
components and non-regulatory quality components. Regulatory components include
Sanitation process HACCP, SSOPs, prerequisite programs and pathogen control program. ‘‘For-cause’’
control program1 investigative sampling is part of the pathogen control program. ‘‘Not-for-cause’’ sampling
is a part of the sanitation process control program but is not necessarily a component of the
regulatory compliance program.
Written procedures that a food manufacturing facility develops and implements to ensure
Sanitation standard
sanitary conditions and prevent direct contamination or adulteration of food product.
operating procedures
These include written steps for cleaning and sanitation, and are considered as one of the
(SSOPs)
prerequisite programs of HACCP.
A multi-faceted systematic approach to finding sites of persistent strains (niches) in food

processing plants, with the goal of either eradicating or mitigating effects of these strains.
This process has been used effectively to address persistent Listeria monocytogenes
contamination in food processing plants. The continued use of this science-based strategy
can not only control environmental pathogens, but it can also be deployed for controlling
microbial spoilage in ready-to-eat (RTE) foods.
The “seek and destroy” process can help to:

“Seek and destroy”
• Finds pathogenic growth niches
process1
• Finds potential growth niches requiring monitoring and control
• Defines normal level of disassembly
• Defines periodic deep level of disassembly
• Defines frequency of periodic deep level of disassembly
• Qualify a new piece of equipment (e.g., run for 90 days then conduct seek and destroy
investigation)
• Validate effectiveness of equipment cleaning protocol
• Validate effectiveness of intervention applied to a piece of equipment (e.g., heat
treat¬ment or other method)
Time-Action- An approach to evaluate a root-cause failure of a cleaning process by examining the time,
Concentration- mechanical action, concentration of chemicals and/or the temperature of the intervention
Temperature (TACT) process.
vii
Term Definition
Path of travel an organism takes to move from transfer point to transfer point (e.g., the
pathway between the harborage site and a contact surface or product); this typically
Transfer pathway 1
reflects transfer of a pathogen by objects or people. Water, employees, equipment, product,
materials and aerosols are common transfer vectors.
Surfaces that are exposed to cleaning and sanitation and can serve as points of contact
facilitating the transfer of an organism from one surface to another, e.g. gloved hands.
Transfer point 1
Transfer points should not be growth niches when effective cleaning and sanitizing
procedures are used.
Providing scientific evidence that a strategy controls a given hazard. Environmental

monitoring is a key strategy that can be used to validate cleaning and sanitation procedures.
This typically involves testing of equipment, using a “seek and destroy” approach after
Validation 5
cleaning and sanitation have been performed, including complete disassembly of the
equipment and collection of samples on the disassembled equipment to validate that the
procedures used completely clean and sanitize a piece of equipment.
Additional investigative swabbing conducted in all directions, including up and down where
Vector swabbing
possible, from the site of an initial positive detection.
Routine program to verify the consistent application of the sanitation process control
Verification
program; includes sampling of Zone 1, 2, and 3 environmental sites in the ready-to-eat (RTE)
monitoring
area. This program is used for regulatory compliance and is a part of an establishment’s
program1
HACCP or SSOP program.
Testing of Zone 1 (food contact surface) sites is typically the primary verification measure for
Verification sites,
the consistent application of the environmental pathogen control program to prevent product
contact surface
contamination. In high-risk product manufacturing, these sites should be evaluated weekly;
(Zone 1)1
lower risk lines may be evaluated less frequently as long as the process is under control.
Locations sampled during operations to detect the presence of the organism in the normal
operating environment. Verification sites are surfaces that are exposed during the normal
Verification sites
operating conditions and are likely to serve as transfer points (i.e., they are located in transfer
(Zones 2 and 3)1
pathways). Monitoring of verification sites detects the organism as it is being moved from its
harborage location to a contact surface or the product.
viii
Term Definition
Direct food contact surfaces post lethal processing, e.g., slicers, peelers, fillers, hoppers,
Zone 1 1,6,7,8,9
screens, conveyor belts, air blowers, employee hands, knives, racks, work tables.
Non-food contact surfaces in close proximity to food and food contact surfaces, e.g.,
Zone 2 1,6,7,8,9 processing equipment exterior and framework, refrigeration units, equipment control panels,
switches.
More remote non-food contact surfaces located in or near the processing area, e.g., forklifts,
Zone 3 1,6,7,8,9
hand trucks, carts, wheels, air return covers, hoses, walls, floors, drains.
Non-food contact surfaces outside of the processing areas, e.g., locker rooms, cafeterias,
Zone 4 1,6,7,8,9
entry/access ways, loading bays, finished product storage areas, maintenance areas.
ix
References:
1. Malley, T.J., Butts, J., Wiedmann, M. 2015. Seek and destroy process: Listeria
monocytogenes process controls in the ready-to-eat meat and poultry industry. J. Food Prot.
78 (2): 436-445. http://dx.doi.org/10.4315/0362-028X.JFP-13-507
2. United States Food and Drug Administration. 2015. Current Good Manufacturing Practice,
Hazard Analysis, and Risk-Based Preventive Controls for Human Food; Final Rule. Verification
of implementation and effectiveness. https://www.fda.gov/Food/GuidanceRegulation/FSMA/
ucm334115.htm
3. International Organization for Standardization. 2015. ISO 900:2015. Quality management

systems – Fundamentals and vocabulary.
4. United States Food and Drug Administration. 2018. Quality System Regulation. Subpart J –
Corrective and Preventive Action. 21 CFR §820.100. https://www.ecfr.gov
5. Codex Alimentarius Commission. 2003. General Principles of Food Hygiene. CAC/RCP

1-1969. http://www.fao.org/fao-who-codexalimentarius/codex-texts/all-standards/en/
6. Dairy Food Safety Victoria. 2016. Dairy Pathogen Manual. http://www.dairysafe.vic.gov.au/

publications-media/regulations-and-resources/guidelines
7. United States Food and Drug Administration. 2017. Control of Listeria monocytogenes
in Ready-To-Eat Foods: Guidance for Industry; Draft Guidance. https://www.fda.gov/
RegulatoryInformation/Guidances/ucm073110.htm
8. United Fresh Produce Association. 2013. Guidance on Environmental Monitoring and

Control of Listeria for the Fresh Produce Industry. http://www2.unitedfresh.org/forms/store/
ProductFormPublic/guidance-on-environmental-monitoring-and-control-of-listeria-for-the-
fresh-produce-industry
9. Simmons, C.K., Wiedmann, M. 2018. Identification and classification of sampling sites for
pathogen environmental monitoring programs for Listeria monocytogenes: Results from an
expert elicitation. Food Microbiol. 75: 2-17. https://doi.org/10.1016/j.fm.2017.07.005
x
Environmental Monitoring Handbook - Importance
CHAPTER 1
The Importance of Environmental

Sampling in Food Safety and Quality
Programs
By
John David | 3M Food Safety
1.1. Increasing recognition of the food processing environment 2

as a contamination source
1.2. Importance of identifying specific purposes and goals for 5

environmental monitoring programs
1.3. Target analytes for environmental monitoring programs 7
1.4. Importance of coordination and integration of 7

1.5. The business needs for environmental monitoring 8

programs
1
1.1. Increasing recognition of the

food processing environment as a
contamination source
There is increasing recognition that food continuously monitored (“verification”).
processing facilities’ environments, as well The quintessential example for a CCP
as other built environments used in food would be a heat treatment meeting a
production and distribution (e.g., retail food certain minimum temperature and time
handling spaces, restaurants or packing requirement such as pasteurization of milk.
houses for produce) can be important
sources of biological agents, chemical However, HACCP as well as quality
compounds and physical hazards that may management systems that utilize similar
negatively affect food safety and quality. concepts require so-called “prerequisite
programs” to be in place to ensure that
Classical food safety and quality HACCP-based food safety programs and
systems strongly relied on the concept similar food quality programs effectively
of Hazard Analysis and Critical Control work.
Points (HACCP) to ensure food safety
and food quality, with an emphasis on Examples of classical prerequisite
identifying a specific targeted critical programs include pest control, sanitation
control point (CCP) for each hazard and sanitation standard operating
identified as reasonably likely to occur. procedures (SSOPs), personal hygiene and
The specific parameters that would allow Good Manufacturing Practices (GMPs)
for effective control of the target hazard (Figure 1).
at the CCP would have to be established
(“validation”) and would then have to be
Figure 1. HACCP and selected prerequisite programs that can be validated and verified by
environmental monitoring
Safety of water,
steam, ice
Facilities and Prevention

Cleaning and of cross
environment (sanitary sanitation
equipment and design) contamination
(SSOPs)
Condition and
Allergen GMPs cleanliness of food
management contact surface
2
Despite the value of HACCP-based food

safety systems and similarly structured food
quality systems, it has become clear that
a large number of food safety and quality Examples of Listeria
issues experienced around the world are due monocytogenes and
to failures and problems with prerequisite
Salmonella persistence events
programs.
responsible for outbreaks
This includes the lack of validation and
verification of the prerequisite programs, In the United States, the Centers for
particularly sanitation (including sanitary Disease Control and Prevention (CDC)
equipment and facility design), and GMPs and state-level health departments
(including hygienic zoning). continually monitor the number of cases
of foodborne illness. When there is a
Examples of food safety and quality spike in the number of cases caused
issues caused by failures with prerequisite by a given pathogen, this may be an
programs include listeriosis outbreaks indication that an outbreak is occurring.
linked to ready-to-eat (RTE) foods
where contamination could be traced For example, in October of 1998, there
back to locations in the processing plant was a spike in the number of listeriosis
environment. This occurs in growth niches cases in New York, indicating a potential
where Listeria monocytogenes could survive outbreak. In response, the Listeria
over time and contaminate finished product. monocytogenes isolates collected from
Similar issues have also been observed for these clinical cases, as well as cases
Salmonella. in other states, were characterized
by subtyping to determine if their
Microbial spoilage issues in RTE food and “fingerprints” matched. A single subtype
beverages can also often be traced back to was common among several cases from
sources in processing plant environments that October, as well as some isolates
that were not effectively controlled through from previous months initially deemed
sanitation and GMPs. Examples of spoilage to be sporadic cases. Interviews with
organisms typically traced back to sources the patients were then conducted to
in processing plant environments include determine if there were any common
Pseudomonas spp., lactic acid bacteria, as foods consumed among them.
well as yeast and mold.
The results showed that 89 percent
Similarly, allergen contamination issues and of the patients infected with the
recalls can sometimes be traced to failures in outbreak strain had consumed cooked
prerequisite programs. frankfurters, and only 32 percent of
participants not infected with the
outbreak strain had consumed cooked
frankfurters. Of those patients infected
with the outbreak strain, 78 percent
reported eating a single brand of
frankfurters.1
(cont.)
3
With the processing facilities’ association

to sources of food safety and quality
From there, finished product Listeria issues becoming increasingly recognized,
monocytogenes testing was performed the food industry and its regulators are
on the frankfurters of the identified heightening their emphasis of environmental
brand. Subtypes of isolates from monitoring programs, which may target
the finished product matched those the actual analyte of concern (e.g.,
isolated from clinical cases, implicating pathogens, allergens, spoilage organisms) or
this company in the outbreak. indicators. Indicators include any organism
or compound where presence (or their
By the end of the outbreak, there detection above a certain threshold) may
were 108 cases of listeriosis and 14 provide evidence of conditions that are
associated deaths. Even though the unhygienic or otherwise increase the risk of
company had an appropriate HACCP food safety or spoilage issues. Conceptually,
plan, they were still producing unsafe environmental monitoring may serve as
product. It was later determined either validation or verification of specific
that the Listeria monocytogenes prerequisite programs (e.g., sanitation and
contamination originated from the sanitary equipment design) or may be more
processing plant environment. This generally seen as a strategy to monitor the
case illustrates the need for effective environment for unhygienic conditions.
(including appropriate corrective and The increasing importance of environmental
preventive actions) even in facilities monitoring programs is particularly well-
that have HACCP plans. illustrated by recent changes to regulatory
approaches to food safety. The U.S. Food
Similarly, a Salmonella Agona outbreak and Drug Administration (FDA) Food Safety
was traced back to toasted oat cereal Modernization Act (FSMA) and similar
in 1998, which caused 209 cases of regulations in other countries have elevated
salmonellosis.2 The Salmonella was the importance of prerequisite programs.
determined to be coming from the For example, in the FSMA Current Good
processing plant environment. Then, 10 Manufacturing Practice, Hazard Analysis,
years later in 2008, another Salmonella and Risk-Based Preventive Controls for
Agona outbreak was traced back to Human Food Rule (PC Rule), many of the
puffed rice cereal, which caused 28 specified “preventive controls” represent
salmonellosis cases. It was determined programs that would have previously
that the strains implicated in both been classified as prerequisite programs.
outbreaks were of the same subtype, However, FSMA preventive controls
indicating that the Salmonella had include a requirement for verification of the
survived in the plant for a decade. preventive controls, which was not in place
This case illustrates that effective for prerequisite programs.
are not only necessary for Listeria Additionally, the FSMA PC Rule includes
monocytogenes, but are also essential a specific recognition of environmental
for Salmonella, particularly in facilities monitoring as a key verification strategy for
that produce low-water activity certain non-process preventive controls
RTE products. such as sanitation: “Environmental
monitoring, for an environmental pathogen
or for an appropriate indicator organism, if
4
contamination of a ready-to-eat food with This provision demonstrates the growing

an environmental pathogen is a hazard consensus on the importance of
requiring a preventive control, by collecting environmental monitoring programs as an
and testing environmental samples.”3 essential part of food safety and quality
systems.
1.2. Importance of identifying specific

purposes and goals for environmental
monitoring programs
Environmental monitoring programs and purpose-driven environmental monitoring
environmental sampling activities can serve programs could, for example, start with
multiple and sometimes complementary an identification of food safety and quality
purposes. In practice, environmental related “hazards.” A food manufacturer
monitoring programs often encompass a might then determine which specific
range of tests – from ATP and indicator hazards could potentially be transmitted
organisms to pathogens, spoilage through the processing plant environment,
organisms, and allergens – conducted on with recognition given to the fact that the
a variety of samples collected throughout processing plant could be a source or a
a facility at various time points and with vehicle for cross contamination, or both.
varying frequencies. Often, these programs Control strategies (e.g., sanitation, GMPs,
have been used for years and modified sanitary equipment design) would then
over time to address specific customer be prescribed to control each hazard;
and regulatory requirements or specific these would represent the equivalent
issues or concerns. This can lead to of “non-process preventive controls.”
programs that represent an uncoordinated, Subsequently, a facility could identify
non-unified approach that may not use environmental monitoring activities needed
resources effectively, particularly if new to validate that a given non-process
requirements for environmental monitoring preventive control addresses the target
are frequently added. Hence, it is often hazard (which often would be non-trivial).
essential for the food industry and specific It would then verify the effectiveness of the
processing plants to more specifically validated non-process preventive control
define the purpose of current and planned and ensure it is consistently implemented
environmental monitoring programs. (Figure 2).
While there does not appear to be a Importantly, verification may include

universally recognized framework for this, measurements and records other than
there are some potential approaches that classical environmental monitoring tests.
would seem logical and consistent with For example, ATP testing (which can
other aspects of food safety and quality be used to verify cleaning), combined
management, such as HACCP. with records on sanitizer concentration
measurements and check sheets
A HACCP-informed approach to develop that document the length of sanitizer
5
application could be sufficient to verify preventive control), with subsequent

sanitation. Furthermore, corrective actions identification of environmental monitoring
should be developed in the case that the activities needed to validate and verify
verification critical limits are not met. each control. These approaches may
also facilitate realignment of existing
Environmental monitoring programs could environmental monitoring activities,
be developed for specific purposes and including elimination or revision of specific
implemented by identifying key preventive tests that no longer have clearly defined
controls (without necessarily assigning goals and purposes.
specific hazards to be controlled by each
Figure 2. A HACCP-informed approach to environmental monitoring
Identify food safety and quality hazards that can be introduced into the product from
Hazard
Analysis the processing plant environment
Identify non-process preventive control strategies needed to control each hazard

Examples of control strategies include sanitation, GMPs and sanitary equipment design
Identify
CCPs
Determine the limit required to control the hazard

Validate that the critical limit is effective in controlling the hazard
For example, determine equipment disassembly needed as well as the time,
Establish temperature and concentration of a sanitation process/SSOP and validate the process
Critical Limits using intensive environmental monitoring of the target area or target equipment
Develop an enviromental monitoring program (with critical limits) and set a

frequency (e.g., weekly, at least 4 hours after processing starts); possible critical
Monitoring limits may include ATP levels and EB counts below a certain threshold or
"negative for Listeria spp.”
Determine how to respond if a critical limit is exceeded (for example, re-cleaning

Develop and re-sanitation if ATP is above threshold)
Corrective
Actions
Use environmental monitoring to verify that the non-process preventive controls

put in place are working
Verification
Maintain records on the validation, monitoring, verification and corrective actions

Record of non-process preventive controls
Keeping
6
1.3. Target analytes for environmental

monitoring programs
When it comes to designing and protein), allergens, indicator organisms,
implementing environmental monitoring pathogens and spoilage organisms.
programs, it is essential to identify the
proper chemical and biological target An understanding of these target analytes
analytes for testing different samples as well as the sensitivity and specificity
and achieving different goals (such as of the tests used is essential in the design
verification and validation). Typical target and implementation of appropriate
analytes used in environmental monitoring environmental monitoring programs, and
programs include compounds that can more detail on the different target analytes
assess cleaning efficacy (e.g., ATP or is provided in subsequent chapters.
1.4. Importance of coordination and integration

of environmental monitoring programs
Coordinating and integrating different consistent designation of sampling sites

aspects of an environmental monitoring (some facilities may have thousands of
program can increase the program’s sampling sites, all with a unique identifier),
effectiveness and efficiency. For coordinated and integrated analysis
example, in some facilities, ATP testing, of different environmental monitoring
environmental allergen tests and data, regular in-person review of all
environmental microbiological tests may environmental monitoring data (typically
not always be coordinated and data may at least every six to 12 months) as well
not be analyzed together, despite the fact as other approaches to coordinate and
all of them typically help validate or verify integrate different environmental sampling
sanitation practices. Coordinated analyses programs.
of the different tests thus may allow for
rapid and sensitive detection of sanitation Additional strategies and activities that
issues. facilitate coordination and integration
of environmental monitoring programs
For example, coordinated environmental include use of floor plans and trending
sampling programs should include charts that allow for integrated temporal
record-keeping and data analyses of all and spatial analysis of different
environmental monitoring data (ATP, environmental monitoring data as well as
indicator organisms, allergen monitoring SOPs for sample collection and follow-up
and pathogen monitoring) and should on out-of-spec results.
include a standardized sampling site list
that encompasses all sites tested. Best
practices for environmental monitoring
programs may include (but are not
limited to) electronic record-keeping,
7
1.5. The business

Control of environmental needs for
sources of microbial
contaminants is important for environmental
proactively addressing food monitoring programs
spoilage issues
While the primary goal for environmental
Using social media, a single consumer
monitoring programs typically is to
was capable of reaching close to a half
control and reduce food safety hazards
million people with her dissatisfaction
(e.g., allergens, microbial pathogens),
with the premature spoilage of a juice
environmental monitoring programs also
pouch. In this case, the effects were
play an important role for protecting
so powerful that the company was
businesses from potentially expensive
forced to conduct a costly redesign of
recalls. For example, recalls of RTE food
their package so that consumers could
products due to contamination with
see the juice in the pouch to ensure it
pathogens such as Listeria monocytogenes
had not spoiled. Since the processing
and Salmonella can often be attributed to
plant environment is the likely source
environmental sources.
for a number of spoilage organisms,
Effective environmental monitoring
can play a key role in not only
programs, particularly those linked to
improving the safety of food products,
specific goals such as sanitation validation
but also helping with identifying and
and verification, can significantly reduce
eliminating or managing niches of
the risk of these recalls. For example, good
spoilage-causing organisms.
environmental monitoring data are often
essential to allow companies to limit recalls
With social media allowing consumers
to a single lot, production day or production
to easily communicate spoilage
week. This is due to the fact that without
issues to large audiences, proactive
appropriate validation and verification data,
approaches to preventing even
it is challenging to sufficiently prove that
rare spoilage issues are becoming
finished product contamination on a given
increasingly more important. Well-
day could not have been transferred to
designed environmental programs thus
subsequent lots.
provide a number of benefits for food
companies and a larger return-on-
In addition to food safety hazards, spoilage
investment than many may realize.
issues (including problems caused by
organisms introduced from the environment
in processing plants) represent an
increasing business risk for food companies.
Consumers often use social media platforms
to communicate food spoilage issues and
pressure companies into action (Sidebar).
Reduced risks of spoilage issues and

associated recalls due to effective
8
thus represent another benefit to food However, food companies have also seen
companies. that effective environmental monitoring
programs can facilitate extended run-times,
Despite the fact it’s widely known thereby improving production efficiency.
that recalls are extremely costly for For example, environmental monitoring
companies, quantification of the benefits of may identify difficult-to-clean areas that
environmental monitoring programs is still can be eliminated through equipment
often considered challenging. While recalls redesign, which will subsequently allow for
tend to occur rarely, improved foodborne longer production runs.
disease surveillance systems place
companies at an increased risk of being
identified as the source of an outbreak.
Learn more about Connect with a

environmental monitoring 3M Food Safety expert
www.3M.com/EnvironmentalMonitoring www.3M.com/ConnectEnvironmentalMonitoring
9
References:
1. Mead, P. S., Dunne, E. F., Graves, L., Wiedmann, M., Patrick, M., Hunter, S., Salehi, E.,
Mostashari, F., Craig, A., Mshar, P., Bannerman, T., Sauders, B. D., Hayes, P., DeWitt, W.,
Sparling, P., Griffin, P., Morse, D., Slutsker, L., Swaminathan, B. 2006. Nationwide outbreak
of listeriosis due to contaminated meat. Epidemiology and Infection. 134: 744-751. http://doi.
org/10.1017/S0950268805005376
2. United States Centers for Disease Control. 2008. Multistate Outbreak of Salmonella Agona
Infections Linked to Rice and Wheat Puff Cereal (FINAL UPDATE).
https://www.cdc.gov/salmonella/2008/rice-wheat-puff-cereal-5-13-2008.html
3. United States Food and Drug Administration. 2015. Current Good Manufacturing
Practice, Hazard Analysis, and Risk-Based Preventive Controls for Human Food; Final Rule.
Verification of implementation and effectiveness. § 117.165. https://www.fda.gov/Food/
GuidanceRegulation/FSMA/ucm334115.htm
10
Environmental Monitoring Handbook - Hygiene Monitoring
CHAPTER 2
ATP and Protein-based

Hygiene Monitoring
By
Louise Roberts | Alimenti Food Sciences Ltd
Gareth Lang | 3M Food Safety
2.1. Purpose of ATP or protein-based hygiene monitoring 12
2.2. Principle of the methods 12
2.2.1. Principle of ATP testing 12
2.2.2. Principle of protein testing 14
2.3. ATP vs. microbiological results 14
2.4. Development of an ATP or protein sampling program 15
2.4.1. Selection of sampling sites 15
2.4.2. Sampling frequency and number of samples 18
2.4.3. Determination of cut-off levels for ATP 19
2.5. Corrective actions based on ATP or protein sampling results 21
2.6. Data trending and analysis 22
2.7. Other consideratons 25
11
2.1. Purpose of ATP or protein-based

hygiene monitoring
ATP and protein-based hygiene monitoring Organic matter on a surface can act as a
technologies are rapid, simple-to-use food source for microorganisms. Removing
methods for determining the hygienic this organic matter reduces the opportunity
status of surfaces such as those found in for bacteria and mold to multiply or grow,
food processing facilities. Everyday, the thus reducing the microbial risk within
high-risk decision to start food production the processing environment. Removal
needs to be made. These tests can provide of organic matter can also enhance the
a measurable and objective assessment of efficacy of sanitizers, further improving the
the cleanliness of equipment and surfaces overall sanitary status of the facility and
prior to food processing or preparation. reducing risk.
2.2. Principle of the methods

2.2.1. Principle of ATP testing
ATP (adenosine triphosphate) is present in present in residues from organic sources
every cell. It is the energy molecule for the such as:
cell and is broken down to ADP (adenosine
diphosphate), releasing energy for the cell • Food debris remaining on a surface
to utilize. after cleaning.
• Biofilms produced by bacteria.
As well as being present in living cells, it is • Surfaces touched by operators.
Figure 1. How ATP indicates direct and indirect risks
• Where the cleaning regime is inadequate or fails, residue from organic sources may
remain on the surface.
• Where this occurs, both direct and indirect food contamination risks exist.
Microorganisms Organic residues
DIRECT RISK INDIRECT RISK

Food poisoning, spoilage Encourage
and shelf-life reduction microorganism growth
12
The amount of ATP present in a cell Luciferase to produce light, the same
will vary depending on a number of chemical reaction used by fireflies.1
factors, including whether it is bacterial
(prokaryotic) or somatic (eukaryotic). It is In the bioluminescence reaction, luciferase
much easier to detect ATP from food cells utilizes ATP to catalyze the oxidation
than microbial cells, as the amount of ATP of luciferin to oxyluciferin, yielding
in a eukaryotic cell can be 107 times more light (Figure 3). The light produced is
than a prokaryotic cell (Figure 2). proportional to the amount of ATP present.
By measuring the light produced, a
ATP hygiene monitoring utilizes the energy correlation can be formed with the amount
present in the ATP molecule along with of ATP present and therefore the amount of
an enzyme complex known as Luciferin- ATP-containing organic matter present.
Figure 2. ATP content in different cell types
The amount of ATP in a cell will vary. This is mostly related to the size of the cell.
Food (eukaryotic) Microbial (prokaryotic)

cells may contain cell may contain
10 ng ATP 1 fg ATP
1 ng = 10–9 g 1 fg = 10–15 g
Figure 3. Measuring ATP with bioluminescence
D -luciferin oxyluciferin
HO S S O S S
N N OH N N
O
O
+ATP + O2 Firefly luciferase +AMP +PP1 + CO2 + Light
+ Mg2+
13
2.2.2. Principle of protein testing

Protein testing is a qualitative or semi- (BCA) can then form a complex with the
quantitative color-based test for the Cu+ ions, resulting in a color change.2
presence of protein residue and therefore
cleanliness. Results from protein-based tests are
generally available within several minutes
The depth of color produced indicates the (compared to seconds for ATP-based
level of protein present. However, as with tests) and are less sensitive than ATP
ATP testing, the technology cannot indicate technology. The results are also generally
if the source of the protein is microbial or only qualitative or semi-quantitative,
otherwise. limiting their usefulness in data analysis
and trending. A significant benefit is that
Protein-based tests generally utilize the protein tests can often be performed with
well-understood copper-based Biuret no specialized equipment. They are also
reaction (Figure 4). In this reaction, cupric often temperature-stable, making them
ions (Cu2+) form a complex with the peptide particularly useful to facilities with limited
bonds of protein, reducing the cupric ions resources, such as auditors and food
to cuprous icons (Cu+). Bicinchoninic acid service outlets.
2.3. ATP vs. microbiological results

Microorganisms Organic residues
While ATP and protein testing are well- role of ATP or protein-based tests is to
established methods for measuring assess the levels of cleanliness, which
hygiene, it is important to note that then relates to increased risk of microbial
the technologies cannot be used as a contamination. An effective environmental
replacementDIRECT
for traditional
RISKmicrobiology. monitoring
INDIRECTprogram will make use of a
RISK
The amount ofpoisoning,
Food ATP or protein
spoilagein a single combination of these technologies in a
Encourage
microbial cell is far below
and shelf-life detectable
reduction methodically planned
microorganism and well-justified
growth
levels using ATP or protein-based tests. manner. In addition, hygiene monitoring
Therefore, these technologies cannot results are immediately actionable,
be used to quantify microbes or directly allowing any corrections to be taken
correlate with microbiology results. The without delay.
Figure 4. The Biuret reaction utilized in protein tests
OH1 2 BCA
PROTEIN
BCA = Cu+ Purple
(Cysteine, cystine
tyrosine and tryptophan Cu2+ Cu+ Complex Abs=562nm
amino acid residues)
14
2.4. Development of an ATP or protein

sampling program
Development of an environmental hygiene chemicals or processes are introduced,
monitoring program will typically involve new equipment is used or new products are
three steps. manufactured.
First is the initial program to validate the The ongoing verification program is
cleaning regime. This is followed by a then generally conducted at a reduced
program for routine verification of the frequency using fewer test points.
regime and finally ongoing review and However, the data generated during
adjustment of the program. this time should be routinely reviewed
and analyzed to determine if there are
The initial validation program will typically any trends or areas of concern, and also
involve a much higher testing frequency to confirm that pass/fail levels and the
and more test points, and the data program itself is adequate and adjusted as
gathered during this program can be used necessary.
to establish baseline levels. Revalidation
should take place whenever changes Specific aspects of a sampling program will
are made, such as when new cleaning be discussed in other sections.
2.4.1. Selection of sampling sites
Sampling site selection should begin with noted that the ATP test points may differ
a mapping exercise to give an overview from microbiological sampling sites.
of the complete facility and production
process. This will involve a division of the Some of the main things to be considered
facility into several areas (zones) based on by the team are:
the microbial risk to the product (Figure
5).3, 4, 5, 6 1 - Stage of processing. In any
manufacturing process using a step to
Once the overall environment has been reduce microbial risk, all processing
mapped, a process can be undertaken to environments occurring after that step
determine the most appropriate test points, can be considered higher risk due
keeping in mind that the aim is to assess to the potential for post-processing
cleanliness and control the risk posed by contamination. Any processing
having an unclean surface. environment situated prior to the microbial
reduction step can be considered a lower
This process is best undertaken as a team risk area since it is preceding the hazard
approach with input from cleaning crew control point. Microbial reduction steps
and quality, combining an understanding can take many forms, from pasteurization
of the purpose of the ATP test and a risk- to peeling of fruit.
based approach to sampling. It should be
15
It should be noted that the lower risk 3 - Ease of cleaning and condition of
rating assigned to areas prior to microbial the surface to be tested. While sanitary
reduction must be viewed in context of the design and good maintenance should be
validated microbial reduction step. If these fundamental in any facility, circumstances
areas were to be insufficiently cleaned, may arise where these aspects are less
it may lead to cumulative microbial than optimal. To address this risk the
contamination, rendering the later level of difficulty in cleaning, a surface
processing steps insufficient. must be considered to assess if the
surface condition or material can reduce
2 - Proximity to the food and potential for the effectiveness of the cleaning. The
cross-contamination. Generally, a surface level of risk associated with the surface
that has direct contact with a product may increase where cleaning is difficult.
that will not be processed further to Examples include older equipment, porous
eliminate microbial risk is a high-risk point. surfaces, scratched or marked surfaces and
In contrast, a surface that does not have poor accessibility.
contact with the product and/or where
the product will be processed further to A simple and convenient way to conduct
eliminate microbial risk is a lower risk point. the risk analysis (Figure 6) and understand
the potential risk to be mitigated through
In addition to direct contact surfaces, the the use of hygiene monitoring can be
potential for cross contamination should summarized as follows:
also be considered including:
Risk Analysis:
• Proximity of the surface to the product,
e.g., whether the equipment is above • How significant is the hazard? = How
the product and whether there is risk of close is the surface to the food?
contamination such as water droplets
in a humid environment. • What is the probability the hazard
will occur? = How hard is it to clean the
• Control panels, utensils or tools surface?
and whether there is a risk of cross
contamination by operators.
16
Figure 5. Environmental monitoring sampling zones
ZONE 1
Product Contact Surfaces
(Slicers, peelers, fillers, hoppers, screens, conveyor belts,
air blowers, employee hands, knives, racks, work tables)
ZONE 2
Non-Food Contact Surfaces in Close Proximity to
Food and Food Contact Surfaces
(Processing equipment exterior and framework,
refrigeration units, equipment control panels, switches)
ZONE 3
More Remote Non-Food Contact Surfaces Located
In or Near the Processing Areas
(Forklifts, hand trucks, carts, wheels, air return covers,
hoses, walls, floors, drains)
ZONE 4
Not typically Non-Food Contact Surfaces Outside of the Processing Areas
tested for ATP (Locker rooms, cafeterias, entry/access ways, loading
bays, finished product storage areas, maintenance areas)
Figure 6. Identification of high-risk sampling sites
| General
NE 4 Are
ZO a
Close Prox
3 | im
NE ity
ZO
| IndirectC
E 2 o n
N
ta
ZO
ct
PROBABILITY
(Difficulty of Cleaning)
ZONE 1
Direct Contact
Low Medium High
High
(Proximity to Food)
(generally Zone 1)
HAZARD
Medium
(generally Zone 2 and 3)
Low
(generally Zone 4)
Color = Risk Level
17
Based on these principles, the use of such as catering kitchens, the ability to
hygiene monitoring technologies such as conduct microbiological testing may be
ATP and protein-based swabs is typically limited. In these cases, ATP testing may be
directed towards Zone 1 (product or increasingly used in Zone 2, indirect food
packaging contact) test points. In a facility contact surfaces which represent a risk for
that is “under control,” Zone 1 areas will cross contamination.
be free of pathogens and have low levels
of indicator organisms (both discussed This same approach can be used for any
in other chapters). With the reduced facility, although in cases like facilities
likelihood of direct risks at these points, the utilizing a CIP cleaning system, the ability
primary focus should be to control indirect to access higher risk surfaces may be
risks such as unclean surfaces that can lead limited. In these cases, ATP testing of the
to the development of direct risks or impact final rinse water can be used to indicate the
product quality. level of cleanliness achieved.
In larger food production facilities, the Additional test points may also be
equipment is likely to be more complex included as a result of corrective and
and involve both manual and clean-in- preventive action (CAPA) activities or
place (CIP) systems. In such facilities, during any validation activities following a
a comprehensive program involving process change, such as construction or
indicator and pathogen testing should modification of existing equipment.
also be established. In smaller facilities
2.4.2. Sampling frequency and number of test points sampled

Once sampling sites have been identified, Highly manual production processes may
a combination of the testing aims (cleaning warrant more Zone 2 test points to be
validation or ongoing verification) and included in the sampling plan, as hands-on
outcomes of the previously conducted operation means an increased risk of cross
risk rating exercise should be used to contamination by production staff.
determine the frequency and number of
test points to sample. Zone 1 areas should have the highest
sampling frequency and should be
The primary factors determining the conducted daily, ideally during every
number of test points to sample is the cleaning and sanitation process and
physical size of the manufacturing possibly also as part of the production
operation and complexity or number start-up routine. This ensures that
of steps involved in the manufacturing corrective actions can be undertaken
process. For example, where several before the finished product is
manufacturing steps or pieces of compromised. Where there are a high
machinery are involved and considered a number of test points, it may be more
risk, each should be sampled. In cases of economical to randomize or rotate a
complex or large machinery, multiple test portion of testing, but careful consideration
points should be considered. should be given to ensure overall hygiene is
still being achieved.
18
For Zone 2 or lower risk areas, the sampling For any test points where an ATP or
regime may occur at a lower frequency but protein-based test is not performed, for
should still be sufficient enough to ensure example due to rotated sampling, a visual
cleaning and hygiene levels are maintained inspection should still be conducted and
before they can lead to more extensive findings or corrective actions undertaken
issues. Sampling frequencies for Zone 2 recorded. A visual inspection can also be
could include rotating sampling throughout used prior to ATP or protein testing.
a given time frame until all areas are tested,
a periodic (weekly, for example) check
of all test points or a daily randomized Increase in organisms
selection. or organic residues
2.4.3. Determination of cut-off levels for ATP

ATP:
As with any test method, hygiene
Adenosine Triphoshate protein-based systems also in
Increase have an option
monitoring results or levels which aremolecule
The “energy currency” to set a caution range
ATPthat sits between a
levels
outside of acceptable limits
of all and
living require
organisms pass and a fail.
corrective action must be established.
While many other test types have With ATP detection technology, multiple
well-established or regulated
Luciferin /levels at
luciferase methods can be used to determine these
which corrective action must be taken, levels, with some methods increasing the
acceptable hygiene levels are very user- level of complexityIncrease in
and accuracy more than
light (RLU)
specific and should reflect the needs of the others. These can be summarized into three
individual facility or process. Many ATP or main methods (Figure 7).
Figure 7. Common methods of determining ATP test thresholds
COMPLEXITY
1 2 3
Manufacturer Before & After Statistical
Guidance Cleaning Analysis
(No Data Points) (Minimal Data Points) (Many Data Points)
ACCURACY
19
1. Manufacturer Guidance If the aim is to achieve an immediate

The simplest method, and often the improvement in hygiene rather than
first step used to determine cut-off maintain current levels, clean levels
levels is to seek guidance from the may be based on results from deep
manufacturer of the ATP system cleaning rather than routine cleaning.
being used. In this situation, the
guidance should reflect the types of 3. Statistical Analysis
product manufactured and/or the While more complicated, using a
types of equipment or surfaces being statistical analysis will result in the
sampled. Similar guidance may also most meaningful pass/fail values
be sought through industry contacts, being set. Performing a statistical
publications or manufacturers of the analysis will involve the collection
production equipment being used. of a larger number of results (data
points) from cleaned surfaces, with
Regardless of the source of the a minimum of 30 being required for
guidance, the levels should be the analysis to be meaningful. Ideally,
reviewed as soon as data is available the minimum of 30 data points will
to ensure that they are meaningful. At be collected from each test point
a minimum, this should involve testing and analyzed individually, although
both clean and unclean surfaces to it is also possible to group similar
ensure they pass/fail as expected. test points (in terms of surface type,
product and risk, etc.) to obtain the
It must be clearly noted that 30 data points for analysis.
manufacturers of ATP systems use
different measurement scales, so The statistics used can vary, although
pass/fail values cannot be used two common approaches use either
interchangeably between one standard normal distribution or an
another. accepted percentage of pass/fails.
Both are described below. For more
2. Before and After Cleaning detailed guidance and tools to help
A relatively simple and more determine pass/fail levels, the ATP
customized approach, this method system manufacturer should be
can have several variations. At contacted.
a minimum, it will involve taking
measurements over several days of For both types of analysis described
representative test points before here, an initial review should be
and after cleaning. It may also conducted to confirm that the
involve taking several measurements data set is acceptable. This can be
following a deep clean to show what achieved by performing a simple plot
is achievable. of the relative light unit (RLU) values
over time followed by a review to
Once the data has been collected exclude any obvious outliers (high
it should be reviewed to establish RLU values) that may skew the results.
how easily clean and unclean can be This review should be performed
differentiated and pass/fail levels using a scale that takes into account
applied as appropriate. An example the results that would be expected
may be to use a pass level that from an unclean surface. If the results
is twice the average clean value, are erratic, it indicates the cleaning
provided that clean and dirty can still process is highly variable and should
be clearly differentiated. be investigated and stabilized.
20
Once an acceptable data set has been (e.g., 95 percent) is determined to be the
obtained, the pass/fail levels can be pass/fail level.
statistically determined. To use a method
based on standard normal distribution, Once pass and fail levels have been
the mean and standard deviation established, they should be reviewed
must be calculated. The fail level can to ensure they are reflective of actual
then be determined by adding two or cleaning performance. Where ATP testing
three standard deviations to the mean, is used effectively and a CAPA process
corresponding to ~95 percent or ~99 implemented, there will typically be an
percent of results respectively. improvement in hygiene levels and a
subsequent lowering of average ATP
An alternative method utilizes an results within a short space of time.
accepted level of cleaning efficacy that
the company believes they are achieving To take into account the improved
(e.g., 95 percent) or can be viewed as the hygiene levels, the pass and fails levels
percentage improvement in cleaning they should be reviewed as soon as sufficient
would like to achieve (e.g., 5 percent). To additional data is available. Subsequent
use this method, a histogram of the results to then, ongoing periodic reviews should
is generated and the level at which the be completed as part of a continuous
required number of pass/fails is reached improvement approach.
2.5. Corrective actions based on ATP

or protein sampling results
As discussed, one of the key benefits While data trending and analysis is
of these hygiene monitoring methods is conducted in more detail in the following
the speed at which results are available, section, repeated fail or caution results
therefore allowing immediate corrections should be investigated as a priority by
to be taken. those on site who have knowledge of
the process, and appropriate preventive
The corrections to be taken in the event actions should be implemented.
of a failing result should be documented
as part of the quality system and followed Along with speed and sensitivity, a key
up with corrective actions to prevent benefit of ATP hygiene monitoring is the
a recurrence. In the case of hygiene ability to trend and analyze the generated
monitoring, a failed test outcome will data over time. This provides a better
typically result in recleaning and retesting understanding and ultimately control
until a pass is achieved. Sometimes, a of the facility’s hygiene and production
caution range may be implemented in processes.
the system. In such cases, the corrective
action may not warrant immediate action,
but instead a more thorough cleaning
and/or increased scrutiny before the next
production run.
21
2.6. Data trending and analysis

ATP system manufacturers will typically When trending and analyzing data, aspects
provide software for managing data, that should be routinely monitored include
although the data analysis capabilities and cleaning consistency, suitability of pass/
ability to present meaningful data varies fail levels, trends or patterns and areas of
with each (Figure 8). concern.
Figure 8. Data trending software for cleaning consistency
All Results Overview

Test# Test Name Points (#) Points (%) Results
1 1st Test 278 100% 50 35 167 26
2 Retest (1) 50 18% 20 20
3 Final Retest 20 7% 18
Daily Master Sanitation Schedule

Su M Tu W Th F Sa Su M Tu W Th F Sa Su M Tu W
Test Point 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Pastry cutter 89 179 123 123 34 358 68 89 28 262 149 143 148 111 102 58 79 145
Cheese grater 234 65 178 197 345 1 245 78 37 66 231 199 78 81 199 232 456
Depositor 203 143 65 69 23 78 37 198 9 89 25 49 75 81 23 123 48 66
Cheese mixer 56 23 56 45 48 25 45 25 24 156 32 67 37 56 82 14 22 69
Moulder 12 14 51 32 45 101 67 85 15 564 12 54 62 95 32 75 76 11
Dough station 1 45 6 5 8 12 65 45 12 9 5 48 23 2 45 2 15 65 45
Dough toss 1 98 100 101 87 45 65 20 15 89 97 45 85 86 12 45 86
Mixer 1 64 91 200 80 95 91 24 86 32 84 75 1 6 8 45 54 78 52
Mixer 2 98 654 32 48 19 27 83 64 58 98 73 71 81 97 95 48 56 84
Grinder P P P C P P P P P P P P P P F P P P
Depositor 2 15 1 4 3 9 50 7 6 5 90 4 8 3 1 4 8 6 4
Depositor 2 78 82 94 76 80 54 80 90 64 125 36 1 65 75 84 64 21 45
Moulder 100 105 90 89 87 86 8 9 4 890 78 72 91 83 49 76 5 4
Cheese grinder P P P P P P P P P F P P P P P P P P
Moulder 2 P P P P P F P P P C P P P P P P
Moulder 3 1 7 3 5 6 759 2 9 5 8 6 4 5 3 2 9 4 7
Legend: Pass Caution Fail Untested
22
Cleaning consistency can be assessed Trends or patterns can also be identified

through a simple line graph (Figure 9a- using longer term data sets in the form of
b). If the graph shows a high degree of a line graph (Figure 10). These trends can
variability, it indicates that the cleaning reflect improving or declining hygiene
process is not in control. levels at plant-level or test point-level,
and can be used as evidence of improved
Common areas to investigate to hygiene practices or identification of areas
understand the root-cause of poor cleaning needing investigation.
control include staff training, cleaning
methods/tools, variations in the products
produced and sampling technique.
Figure 9a. High degree of variance / inconsistency in results over time. Cleaning not in control.
Food Prep line 1 : high risk : CONVEYOR TWO : UXL 100 (Surface ATP)
1000
800
RLUs
600
Food Prep line 1 : high risk : CONVEYOR TWO : UXL 100 (Surface ATP)
400
1000
200
800
0
RLUs
600
7/26 8/2 8/9 8/16 8/23 8/30
400
200
0
Food Prep line 1 : high risk : HOPPER TWO : UXL 100 (Surface ATP)
Figure 9b.7/26
Low degree 8/2
Food of
Prepvariance
8/9
/ :inconsistency
line 1 : high risk in8/16
CONVEYOR TWO : UXL results
100 (Surfaceover
8/23 8/30
ATP) time. Cleaning in control.
1000
1000
800
800
RLUs
RLUs
600
600 Food Prep line 1 : high risk : HOPPER TWO : UXL 100 (Surface ATP)
400
400
1000
200
200
0
800
RLUs
07/26 8/2 8/9 8/16 8/23 8/30

600
7/26 8/2 8/9 8/16 8/23 8/30
400
200 Food Prep line 1 : high risk : HOPPER TWO : UXL 100 (Surface ATP)
0
1000
800 7/26 8/2 8/9 8/16 8/23 8/30
PIZZA LINE 1 : Dough Mix : DEPOSITOR 1 : UXL 1
RLUs
600 PIZZA LINE 1 : Dough Mix : BLENDER : UXL 100 (Surface ATP)
400
200
300
0
250 10: Cleaning trend identification from long-term ATP data
Figure PIZZA LINE 1 : 8/2
200 7/26 Dough Mix : BLENDER
8/9 : UXL 100 8/23 PIZZA LINE
8/16(Surface ATP) 8/301 : Dough Mix : DEPOSITOR 1 : UXL 10
RLUs
150
100
300
50
250 0 PIZZA LINE 1 : Dough Mix : BLENDER : UXL 100 (Surface ATP) PIZZA LINE 1 : Dough Mix : DEPOSITOR 1 : UXL 100 (Surface ATP)
200
RLUs
150 2017 2018 2017 20

300
100
250
50
200
RLUs
0
150
100 2017 2018
50 2017 201
0
2017 PIZZA LINE 1 : Dough2018
Mix : FILLER2017
HEAD 1 : UXL 100 (Surface ATP)
2018
300
250
PIZZA LINE 1 : Dough Mix : FILLER HEAD 1 : UXL 100 (Surface ATP)
RLUs
200
150 23
300
100
300
250
250
50
ss
200
200
Any adverse trends observed should be 11). Assessing if the levels are appropriate
investigated to understand the root-cause can be handled in several ways, but can
and may include the following observations include evaluating each test point for
and causes: its history of failing. If over an extended
period of
Food Prep line 1 : high risk : CONVEYOR time
TWO or many
: UXL measurements
100 (Surface ATP) the
• Long-term
1000 trends may be associated test point has never failed, it is likely that
with
800 seasonal variation or equipment either the pass/fail levels are too lenient
surfaces becoming worn. In these or the level of risk associated with that
RLUs
600
cases,
400 hygiene could be improved point should be reviewed. Following the
through
200
increased cleaning at review, appropriate pass/fail levels may be
appropriate times of the year or worn set as discussed in the previous section. A
0
equipment could be replaced. system of continuous improvement through
7/26 8/2 8/9 8/16 8/23 8/30
• Regularly occurring patterns may regular lowering of pass/fail levels is also a
be linked to scheduled production common practice.
of different products or changes in
Food Prep line 1 : high risk : HOPPER TWO : UXL 100 (Surface ATP)
cleaning crews. Overall, the use of ATP and protein-
• Step
1000 changes may indicate a change based tests should be seen not only as a
in800
cleaning practices, chemicals or convenient tool for quickly determining the
RLUs
equipment.
600 hygiene levels of a surface before starting
400 production but also as an investment in
Areas200of concern can be identified by data related to the production process.
analyzing0 test points for frequency of Once generated, the data should be
failing results,
7/26 indicating that 8/2the test point 8/9 analyzed8/16
and used as a tool 8/23to manage 8/30
is consistently difficult to clean. This level the site in a more effective manner and
of analysis becomes more complex and demonstrate that hygiene targets are
generally requires the use of a software being achieved. It can lead to an informed,
system capable of performing
PIZZA LINE such
1 : Dough Mix a
: BLENDER : UXL 100 focused approach
(Surface ATP) to managing
PIZZA the hygiene
LINE 1 : Dough Mix : DEPOSITOR
task automatically, although it could also of any given area and can also be used
be done manually if sufficient time was as a training aid or to optimize cleaning
300
dedicated
250 to the analysis. regimes, production run times or use of
200 cleaning chemicals/sanitizers.
RLUs
150
Suitability of pass/fail levels should also
100
be reviewed
50 on an ongoing basis (Figure
0
2017 2018 2017
Figure 11. Adoption of more stringent pass/fail levels for continuous improvement of
hygiene control
300
250
RLUs
200
150
100
50
0
2017 2018
24
2.7. Other considerations

A trial of any rapid hygiene monitoring In addition, ATP testing can be
system is strongly recommended and this complemented by visual inspection and
should mimic a part of the full sampling microbiological testing. Visual inspection
schedule. As previously mentioned, it is can quickly give a big-picture view about
also very important to note that while all the effectiveness of cleaning processes, but
ATP systems give results in “RLU,” the it has limitations because microorganisms
readings from different manufacturers cannot be seen by the naked eye.
are not interchangeable. For instance, a Microbiological testing can enumerate
reading of 10 RLU from one manufacturer organisms that may cause contamination;
may be equivalent to 50 RLU for a different however, it cannot provide immediate
manufacturer, so pass/fail levels must results on the manufacturing floor. A robust
be independently determined for every hygiene monitoring program would utilize
system. all three complementary methods.
CASE STUDY
This case study has been selected as Using the ATP system as an objective
an illustration of how a rapid hygiene measure of cleanliness, the cleaning staff
monitoring system can be used to were then able to improve their cleaning
measure and manage the hygiene of food methods to bring the ATP results back
preparation areas. down to levels known to be achievable
in similar facilities. This phase in the ATP
The manufacturing site system implementation was used to
The manufacturer was operating a stabilize cleaning and show that it was in
medium-sized cook-chill facility, providing control before progressing to further data
prepared raw meats, vegetables and analysis to refine and customize pass/fail
various prepared meals. It had been levels. The use of an objective measure of
purpose-built with fully segregated cleanliness also reinforced the importance
low-risk and high-care areas, as well as a of having correctly implemented cleaning
butchery and vegetable preparation areas. practices and embedded a culture of good
hygiene amongst the staff.
ATP-based rapid hygiene monitoring was
introduced at the site during the pre- The system was initially used solely to
production phase as both a training tool confirm the hygiene of the high-care area,
for staff and to collect baseline data for but once established and fully operational,
determining the pass/fail levels that would use of the system was expanded to monitor
be used during routine production. and help improve hygiene practices in
other areas (e.g., butchery and wash-up),
The belief that visual assessment was although on a less frequent basis in line
sufficient to determine the hygienic status with the lower risk ratings in those areas.
of a surface was quickly dispelled, as the
ATP results showed readings much higher
than should be achievable for the types of
surfaces and processes being used.
25
Over time, the range of products prepared correlated well with the hygiene levels,
at the facility expanded to include more although occasional discrepancies were
ready-to-eat (RTE) foods, which were observed, highlighting that a combination
given a defined, separate area. Analysis of of test methods is required to manage
historical data from the other production the microbial risk in a facility. There were
areas was used to determine that a lower microbiological swab results that fell
pass/fail value could be implemented in outside of acceptable limits from time to
this area, recognizing the higher levels of time, but these were infrequent and quickly
hygiene expected for RTE products. resolved, resulting in a “pass” at the retest.
Ongoing use of the ATP system has allowed Ongoing analysis of the data has enabled
the different areas to be released for the facilities’ hygiene to be managed
production with confidence that high levels in a proactive rather than reactive
of hygiene are being met and maintained. way, enabling a system of continuous
The microbiological test results also improvement.
Learn more about hygiene Connect with a

monitoring 3M Food Safety expert
www.3M.com/ATPMonitoring www.3M.com/Connect/ATPMonitoring
26
References:
1. Chappelle, E.W., Levin, G.V. 1968. Use of the firefly bioluminescence reaction for
rapid detection and counting of bacteria. Biochemical Medicine. 2: 41– 52. https://doi.
org/10.1016/0006-2944(68)90006-9
2. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D.,
Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C. 1985. Measurement of protein using
bicinchoninic acid. Analytical Biochemistry. 150: 76-85. https://doi.org/10.1016/0003-
2697(85)90442-7


27
Environmental Monitoring Handbook - Indicators
CHAPTER 3
for Indicator Organisms
By
Kelly Stevens | General Mills
Jean-Francois David | 3M Food Safety
3.1. Purpose of environmental monitoring for indicator organisms 29
3.2. Indicator organisms and their significance in the food 29

processing environment
3.2.1. Total Plate Count 30
3.2.2. Coliforms 31
3.2.3. Enterobacteriaceae 31
3.3. Development of an indicator sampling program 32
3.3.3. Data trending, analysis and establishing a baseline for 35

indicator organisms
3.3.4. Determination of cut-off levels for indicator organisms 36
3.4. Corrective actions based on indicator organism results 37
3.5. Identifying sources of indicator organisms 37
3.6. Summary 38
28
3.1. Purpose of environmental monitoring for

indicator organisms
The term indicator organism is defined The role of testing for indicator organisms
as an organism or group of organisms is still often misunderstood by food
that reflects the general microbiological microbiologists, quality assurance
condition of a food or the environment.1 The personnel, and others; many incorrectly
presence of indicator organisms does not assume that detection of indicator
provide any information on the potential organisms above a certain level suggests
presence or absence of a specific pathogen the presence of pathogens. In contrast
nor does it provide an assessment on to “indicator organisms,” organisms
potential public health risk. However, data whose presence (or detection above a
from environmental monitoring programs threshold) actually suggest an increased
that incorporate indicator organisms can risk for the presence of an ecologically
be used to: similar pathogen are referred to as
“index organisms.” There is, however,
• Determine the hygienic status of considerable skepticism among many
the processing equipment and whether there are any organisms that could
environment. accurately be considered as true “index
• Understand the microbial ecology of organisms,” with the possible exception of
the processing environment. Listeria spp.
• Validate and/or verify cleaning and
sanitation (typically testing for indicator
organisms would validate or verify
sanitation, while ATP testing (see
Chapter 2) would be used to validate or
verify cleaning).
• Verify process control steps.
• Assess post-processing contamination
risk.
3.2. Indicator organisms and their significance

in the food processing environment
Once thought to be an indication of fecal Microbiologists knew if the manufacturing
contamination or potential pathogenic process was truly under control, the
contamination, indicator organisms were number of indicator organisms would
incorporated into food microbiological also be in control. Indicator organisms
testing during the beginning of the 20th that can be used for environmental
century. Testing for these organisms gave monitoring programs include those found
a broader view of organisms in ingredients, by Total Plate Count, coliforms, and
finished product and the environment Enterobacteriaceae tests.
rather than looking for a specific species.
29
Enterococcus faecalis
Bacillus
E. coli
3.2.1. Total Plate Count

Total Plate Count (TPC), also referred to as TPC has many applications. For instance,
Aerobic Plate Count (APC), Standard Plate the total number of organisms present can
Count (SPC), Total Viable Count (TVC) affect both quality and potential spoilage
or Mesophilic Count (MC), represents risk of a finished product. In its application
one of the most common indicator tests, as an indicator organism, TPC is used to
although methods used throughout the provide an indication of the total microbial
world vary slightly. At the core, these population on a surface or in a sample.2
methods have key commonalities: a non- More specifically, TPC is an extremely
selective nutrient medium incubated under valuable method to validate and verify
aerobic conditions used for enumeration. sanitation procedures. TPC counts above
The purpose of the method is to provide a certain threshold would typically suggest
information on the total population of that sanitation of the specific environment
bacteria present capable of growing in or equipment was ineffective or improperly
the presence of oxygen at mesophilic performed.
temperatures.
30
3.2.2. Coliforms
Coliforms are a diverse group of Gram-
Utilizing coliforms as an negative, non-spore forming rods that are
indicator for environmental defined by their ability to ferment lactose
monitoring to produce acid and/or carbon dioxide
gas. The precise definition varies by
While there is general agreement that internationally accepted standard methods.
coliform detection does not provide Traditionally, testing of coliforms derived
evidence for fecal contamination, from the search for E. coli, and presence of
a number of countries (e.g., Japan) coliforms had long been thought to indicate
and industries (e.g., dairy industry in fecal contamination. However, decades
the United States) have regulations of research regarding this diverse group
on coliforms. For example, in Japan, of bacteria indicates that only a fraction
coliforms are historically well- are fecal in origin, while the majority are
recognized in several regulations for environmental contaminants.3
food industries. Therefore, coliforms are
widely used as indicators in Japan for Coliform testing is used as an indication of
monitoring production environments. improper cleaning, insanitary conditions or
post-process contamination. Importantly,
Environmental monitoring for coliforms however, coliform testing only detects a
is considered valuable since coliform subset of the organisms that may be present
presence in finished products would in a food processing facility. For example,
typically result from environmental members of the genus Pseudomonas, which
sources after the critical control points represent important spoilage organisms for
(CCP), usually the heat treatment step, many foods, are not detected with coliform
except for rare instances where it may tests. For this reason, a coliform test may not
indicate a failure of the CCP. When detect certain problems with a sanitation
coliforms are used in environmental program, which could be detected with
monitoring, high levels of coliforms another test (e.g., TPC). Therefore, coliform
may sometimes even trigger additional tests are best used in conjunction with other
follow-up pathogen testing. Therefore, tests, such as TPC, to validate or verify
despite an increasing preference sanitation procedures and protocols.
for Enterobacteriaceae testing over
coliform testing, coliform testing of
environmental samples may still be 3.2.3. Enterobacteriaceae
common in a number of countries and Enterobacteriaceae represent a diverse
industries. group of Gram-negative bacteria,
which includes all coliform bacteria.
Enterobacteriaceae are non-spore forming,
oxidase-negative rods that ferment glucose
to acid and/or carbon dioxide gas. Although
the Enterobacteriaceae group includes
genera known to be pathogenic, such as
Salmonella, it is considered an indicator
test group and not a method for monitoring
the presence of pathogens. If information
regarding the presence or absence of a
specific pathogen is required, it is advised to
perform a specific test for that organism as
opposed to relying on indicator tests.
31
Enterobacteriaceae testing serves the Similar to coliform testing procedures,

same purpose as coliform testing in that Enterobacteriaceae tests will also not detect
it indicates improper cleaning, insanitary all Gram-negative bacteria, for example,
conditions or post-process contamination. Pseudomonas species.
3.3. Development of an indicator

sampling program
Development of an indicator sampling plan collecting samples or analyzing data from
should be initiated under the direction environmental monitoring programs.
of a person trained and experienced Training should include aseptic technique,
in microbiological indicators, testing properly collecting samples at each
methodology, sampling methodology, location, ensuring the proper location
microbiological results interpretation and is swabbed and understanding safety
with knowledge of the processing system considerations for each location. Collectors
that will be sampled. Sampling sites, should be retrained if there are any
sampling frequency and collection times incidences or indications of any improper
should be determined based on risk and handling or swabbing. Additionally, annual
processing schedules. Once the sampling training should take place to ensure
plan is fully developed, training and that proper technique and sampling is
documentation should be facilitated. maintained year after year. To see how each
operator collects a sample, training and
Sample collectors and data reviewers evaluation of this technique is ideally hands-
should always be trained prior to them on as opposed to classroom training.

The first step when selecting sampling or conditions which could lead to product
sites should be to map the manufacturing contamination. In-process sampling of
process and identify the processing steps Zone 1 sites for indicator organisms can
(e.g., filling, freezing, slicing), functional also be used to define appropriate run
units (e.g., processing lines, which times for different lines and could be used
typically consist of multiple pieces of to provide scientific support for extended
equipment), and equipment, noting the run times.
construction materials used (e.g., stainless
steel, rubber, high-density polyethylene Additionally, indicator testing for Zone 1
[HDPE]). Mapping and sampling sites and 2 sites represents a supplementary
should focus on Zone 1 (product contact method for monitoring the condition of
surfaces) and Zone 2 (surfaces adjacent equipment and prescribing the frequency
to product contact surfaces), as indicator of preventative maintenance or repairs. For
testing in these areas provides the most example, trends towards higher numbers
value in terms of sanitation effectiveness. of indicator organisms in certain sites may
In-process sampling of Zone 1 sites also point towards the need for (more frequent)
provides quantifiable data that can be used replacement of gaskets or other rubber and
to indicate possible loss of process control plastic parts.
32
Incorporation of Zone 3 sites into the sanitize. The sampling plan should include
indicator sampling plan may be valuable a representative site from each processing
during investigations or root-cause step as well as sites that include each of
analysis, as these sites are likely to have the different types of material used in the
fluctuating levels of the different target construction of equipment.
bacteria, which may result in erratic trends.
Once sites are selected, the appropriate
Similar to selection of sites for pathogen tool for sampling each should also be
testing (see Chapter 4), indicator sampling decided. If the site is a small niche or
sites should be selected with the aim of crevice that is difficult to access, a swab
finding potential issues rather than sites may be the best option. For areas that are
that are easy to clean and sanitize or larger, a sponge would be best as it allows
will always meet the acceptable limits. for more effective collection through higher
For example, large, flat stainless-steel mechanical action. On easy-to-clean,
surfaces are typically easier to clean and flat surfaces in which a higher sensitivity
sanitize (and therefore tend to not be the test method is desired (as low counts are
best sampling sites, particularly if these expected), direct contact of a medium to
are the only sites used) while a fabric- the surface may be used (Figure 1).
backed belt is more difficult to clean and
Figure 1. Examples of direct contact and swab sampling using 3MTM PetrifilmTM Plates
33
3.3.2. Sampling frequency and number of samples
Sampling frequency for indicators as Increased sampling should also take place
part of an environmental monitoring following an out-of-specification result,
program (which typically is designed as a particularly for coliforms and
verification activity) should be risk-based Enterobacteriaceae.
and take into consideration the type of
product being produced (ready-to-eat, This section outlines considerations
ready-to-cook or raw; high or low-water for sampling frequency of routine
activity), the level of risk at each process (verification) environmental monitoring
step and other considerations specific to programs that utilize indicator organism
the processing environment such as: tests. However, indicator organism testing
is also an essential tool for validation of
• Processing lethality. sanitation procedures, such as specific
• Sanitation frequency. high-risk pieces of equipment. As detailed
• Facility characteristics. in other chapters, validation of cleaning
• Potential for cross contamination. and sanitation may include multiple testing
methods (e.g., ATP, indicator, and possibly
The frequency of in-process sampling pathogen tests).
is also influenced by the microbial
susceptibility of the product being
produced, microbial load of ingredients
and normal flora of ingredients.
A risk-based sample selection approach

should allow one to test only a portion of
all the available sampling sites, but still be
able to verify control of the environment or
sanitation procedures.
When used for verification of sanitation

efficacy, sampling should take place
after every sanitation cycle and prior to
production startup to allow for trending of
results and early identification of issues.
If the production equipment is complex
or contains difficult-to-access areas, it
may also be helpful to sample while the
equipment is operating but before starting
the processing of food. This may mean
that certain equipment (e.g., conveyer
belts) may need to be run for a certain
time period (e.g., 15 minutes) before
sampling. This will increase the likelihood
that residual microbial populations
that remained after sanitation become
accessible to sampling.
34
3.3.3. Data trending, analysis and establishing a baseline

for indicator organisms
The quantitative results that can be consistent basis throughout the process/
obtained from indicator organism testing facility and can therefore be used to
are particularly useful, as they can be expose results that are out of specification
further analyzed and used to determine in terms of sanitation effectiveness. The
baseline levels. Analysis of the data should baseline can be determined in a number
take place regularly to identify trends and of ways, including collecting samples after
specific issues to allow for appropriate consecutive sanitation cycles from each
corrections and corrective actions. For test point. The results can then be plotted
example, frequent analysis can help in a process control chart to establish the
identify a trend of increasing indicator baseline (Figure 2).
organism numbers, which may then allow
facilities to take action before a failure Importantly, standard operating
point is reached. Longer term analysis can procedures (SOPs) for indicator testing
also foster understanding of seasonality should include specific instructions for
effects and identify opportunities for trending, including the frequency of formal
operational and product improvements. reviews of indicator testing data.
The baseline levels represent what a

sanitation program can deliver on a
Figure 2. Example of coliform counts and baseline-level post-sanitation
Coliform Count
2
1.9
1.8
1.7
1.6
Log CFU/cm2
1.5
1.4
1.3
1.2
1.1
0.9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
35
3.3.4. Determination of cut-off levels for indicator organisms

Acceptable limits of indicator organisms Example guidance from the Almond Board
should be established for each sampling of California (Table 1) would suggest the
location. The limits can be determined following as appropriate and achievable
in several ways, including utilizing the levels for indicator organisms. Presence
baseline levels and leveraging historical or levels of indicator organisms above
data. the acceptable limit demonstrate that
conditions exist that could lead to loss of
Following sanitation, low levels of indicator process control, potentially resulting in
organisms are expected on surfaces. product contamination.
Table 1. Recommended micobiological indicator limits for equipment cleaning before and
after application of sanitizer provided by Almond Board of California.4
Post-Heat
Post-Heat Treatment
Quantitative Target/ Treatment Taken
- Pre-op Taken After
Microbiological Acceptable Before Sanitizer
Sanitizer
Indicator Test Limits (cfu/40 in2 [250
(cfu/40 in2 [250 cm2])
cm2])
Target <100 <10
Aerobic Plate Count
Acceptable <500 <100
Target <10 <10
Coliforms
Acceptable <100 <50
Target <10 <10

Total
Enterobacteriaceae
Acceptable <100 <50
Improvements to sanitation, repair of equipment and changes in processes may allow for a new
baseline and lower acceptable limits to be applied.
36
3.4. Corrective actions based on indicator

organism results
Corrective action documentation should indicate seasonal effects, abnormal
include the actions taken, results of microbial load of an ingredient or
those actions, dates and people involved. equipment in need of repair.
Significant deviations should trigger
re-evaluation of the plan and sample As an example of corrective action
collection retraining. Once the corrective resulting from an identified seasonal
action has been put into place, additional effect, consider a scenario in which counts
sampling at strategic locations in the area at a certain point in the process during
of the failure should transpire to ensure winter may take eight hours of operation
the effectiveness of the corrective action. to become elevated to a point that the
Corrective actions should be reapplied in equipment needs to be cleaned. However,
locations or lines where similar conditions in the summertime it takes four hours to
or risks occur. reach the same level of bioburden at the
same location. The process improvement,
Corrective actions may not always be in this case, may be to increase the
a consequence of a failure; they may frequency of cleaning during the summer
also be in the form of quality or process months on that product line.
improvement. In-process sampling may
3.5. Identifying sources of indicator organisms

As described above, indicator organisms Sporadic increases in indicator organism
are commonly present in the production counts could also stem from equipment
environment, and more broadly in nature. failures or improper cleaning. Equipment
Classical sources of indicator organism failures could include cracks in gaskets
are cross contamination from outside of or fractures in conveyor belts creating a
the production area or in process (potable) growth niche or harborage site (Figure 3).
water. Indicator organisms introduced
to Zone 1 and Zone 2 areas are most Furthermore, if all equipment and
likely to stem from ingredients and raw machinery is not dismantled for cleaning
materials. When assessing sources of on a regular basis, or if difficult-to-clean
elevated indicator organism levels, it also areas exist, biofilms may form and result
is important to take into consideration any in contamination of product. While these
atypical activity that may be occurring issues should typically be identified during
in the facility such as construction or validation of cleaning and sanitation SOPs,
new product runs on an adjacent line. they may sometimes be identified through
New activities, equipment, change in verification sampling that targets indicator
sanitation chemicals or personnel could organisms.
also contribute to increases in indicator
organism counts.
37
Figure 3. Examples of potential growth niches in equipment
Interface
Growth niches in
rubber-plastic interface
of filler cup (cracking)
Underside strip
Milk Filler
Bolts
Hollow roller on a conveyor belt
Poor welds
3.6. Summary
A robust environmental monitoring defined acceptable limits can indicate
program should include testing for insufficient cleaning and sanitation or
indicator organisms, especially post- operating conditions. Use of indicator
sanitation and on Zone 1 and 2 surfaces. testing can act as an early warning system
Indicator organisms TPC, coliforms, to identify and prevent potential product
and Enterobacteriaceae may be used contamination issues. If results exceed
to verify efficacy of sanitation activities the established control limits, facilities are
and that plant operating conditions are expected to take appropriate corrective
under control. The presence of indicator action and to document the actions taken
organisms does not indicate the presence and results obtained.
of a pathogen, but their levels above
Learn more about indicator Connect with a

organism testing 3M Food Safety expert
www.3M.com/MicroIndicators www.3M.com/Connect/MicroIndicators
38
References:
1. Chapin, T.K., Nightingale, K.K., Worobo, R.W., Wiedmann, M., Strawn, L.K. 2014.
Geographical and Meteorological Factors Associated with Isolation of Listeria Species in New
York State Produce Production and Natural Environments. Journal of Food Protection. 77: 1919-
1928. https://doi.org/10.4315/0362-028X.JFP-14-132
2. Downes, F. P., Ito, K., and American Public Health Association. 2001. Compendium of
methods for the microbiological examination of foods (4th ed.). Washington, DC: American
Public Health Association.
3. Martin N.H., Trmčić, A., Hsieh, T., Boor, K.J., Wiedmann, M. 2016. The Evolving Role of
Coliforms As Indicators of Unhygienic Processing Conditions in Dairy Foods. Front. Microbiol.
7:1549. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043024/pdf/fmicb-07-01549.pdf
4. Almond Board of California. Preventing Salmonella Recontamination: Pathogen

Environmental Monitoring Program Guidance Document. http://www.almonds.com/
processors/processing-safe-product/pem
39
Environmental Monitoring Handbook - Pathogens
CHAPTER 4
for Pathogens
By
Christian Blyth | 3M Food Safety
4.1. Purpose of environmental monitoring for pathogens 41
4.2. Pathogens of concern and their significance in the 43

food processing environment
4.2.1. Listeria and Listeria monocytogenes 44
4.2.2. Salmonella 44
4.2.3. Cronobacter 45
4.3. Development of a pathogen sampling program for 46

verification of environmental pathogen control strategies
4.3.1. Zoning and selection of sampling sites 47
4.4. Corrective actions based on pathogen testing results 51
4.5. Identifying sources of pathogens and development of 52

preventive controls
4.6. Advanced sampling approaches to control 53

environmentally transmitted foodborne pathogens
40
4.1. Purpose of environmental monitoring for

pathogens
In general terms, companies perform monitoring approaches, including ATP
environmental monitoring for foodborne testing, to validate cleaning and Total Plate
pathogens in processing and other Count (TPC) methods to validate sanitation.
food handling facilities to identify and Often, use of these tests is supplemented
eliminate environmental pathogen sources, with pathogen testing to identify specific
consequently reducing the risk of food harborage sites that allow for pathogen
contamination and the associated risk growth or survival. The process used to
of recalls, foodborne illness cases and identify specific harborage sites or niches
outbreaks. Pathogen environmental (e.g., as part of validation or similar type
monitoring (PEM) programs thus are efforts) is often referred to as the “seek and
often considered to represent a proactive destroy” technique.2
approach to microbial food safety. They
can identify challenges and pathogen In addition to validation and verification,
sources before they lead to contamination testing of environmental samples for
of finished food products. PEM programs pathogens is used to support root-cause
are particularly important since foodborne analysis efforts and to verify that corrective
pathogen contamination of finished actions taken are effective in addressing
products typically occurs at low frequency, specific pathogen-related problems. These
which makes finished product testing alone activities may be part of “for-cause” and
an ineffective strategy for ensuring food “not-for-cause” investigations.
safety.
While PEM programs are most commonly
More specifically, PEM programs are used in processing facilities that
typically used to (1) verify an overall food manufacture ready-to-eat (RTE) products,
safety system (or specific components of a these programs are also increasingly used
food safety system) and to (2) provide early in produce packing houses, often to help
indication of potential food safety hazards.1 with control of L. monocytogenes. They
However, testing of environmental may also be valuable to verify pathogen
samples for pathogens is usually not an control strategies in other establishments
effective means for validation of food that handle RTE food, such as institutional
safety practices, prerequisite programs and kitchens that serve high-risk populations.
“non-process preventive controls” (e.g.,
sanitation standard operating procedures Importantly, there are a number of
[SSOPs]). This is because the absence of industry and commodity-specific
pathogens may suggest a control strategy guidance documents for establishing and
was effective when really the target implementing PEM programs (in particular
pathogen was simply not present even for Listeria) that should be consulted (Table
before the control strategy (e.g., sanitation) 1). These guidance documents typically
was applied. provide a level of detail that considerably
exceeds what is covered in this handbook.
Validation of sanitation procedures
and other control strategies typically
requires the use of multiple environmental
41
Table 1. Examples of guidance documents for the establishment of pathogen environmental

monitoring programs.
Document Title Organization Target Industry Target Pathogen
Dairy Food Safety Salmonella and

Dairy Pathogen Manual 3 Dairy
Victoria (Australia) L. monocytogenes
Listeria monocytogenes
Grocery
Guidance on Environmental Ready-to-eat
Manufacturers L. monocytogenes
Monitoring and Corrective (RTE) foods
Association
Actions in At-Risk Foods 4
Control of Listeria
monocytogenes in U.S. Food and Drug
RTE L. monocytogenes
Ready-to-Eat Foods: Administration
Guidance for Industry 5
Control of Listeria Innovation Center

monocytogenes: Guidance for for Dairy L. monocytogenes
the U.S. Dairy Industry 6 U.S. Dairy
Guidance on Environmental
Monitoring and Control of United Fresh
Fresh Produce L. monocytogenes
Listeria for the Fresh Produce Produce Association
Industry 7
FSIS Compliance Guideline:

Controlling Listeria U.S. Department
monocytogenes in Post- of Agriculture
RTE L. monocytogenes
lethality Exposed Ready-to- Food Safety and
Eat Meat and Poultry Inspection Service
Products 8
42
4.2. Pathogens of concern and their

significance in the food processing
environment
While a considerable number of pathogens manufacturing of powdered infant formula.
cause foodborne illness, there are only a
few pathogens for which sources in food While not covered in more detail here,
processing and handling environments food processing environments cannot be
have been linked to foodborne illness cases excluded as the source of other foodborne
and outbreaks. Key pathogens targeted in pathogens, including Gram-negative
PEM programs include L. monocytogenes, pathogens in the Enterobacteriaceae
with testing typically targeting all Listeria family (e.g., pathogenic E. coli, such as
spp., rather than the specific pathogenic Enterohemorrhagic E. coli [EHEC]) and
species L. monocytogenes and Salmonella. even Yersinia. Hence, some facilities may
In addition, environmental sources include pathogenic E. coli as a target in
of Cronobacter spp. are a concern in their PEM programs.
Listeria
Cronobacter
Salmonella
43
4.2.1. Listeria and Listeria monocytogenes

Listeria is a bacterial genus that, as of 2016, However, a site that is positive for Listeria
is comprised of 17 species. Nine Listeria species could also be positive for L.
species have been newly described since monocytogenes and follow-up on each
2009.9 Genomic and phenotypic data Listeria positive sample needs to be
clearly define a distinct group of six species conducted “as if the site were positive
(Listeria sensu strictu) that share common for L. monocytogenes.” If this approach is
phenotypic characteristics (e.g., ability followed consistently and appropriately, it
to grow at low temperature); this group provides a more sensitive approach to food
includes the human pathogen Listeria safety and environmental monitoring than
monocytogenes. The other 11 species specific testing for L. monocytogenes.
(Listeria sensu lato) represent three distinct
groups, which have been proposed to There are specific situations, however,
represent three different genera that are where testing of environmental samples
distinct from Listeria sensu strictu.9 Listeria to specifically find L. monocytogenes may
tests virtually always detect all members be appropriate, for example in a for-cause
of Listeria sensu strictu but may not always investigation that is triggered by a finished
detect all members of Listeria sensu lato. product positive for L. monocytogenes. In
It is important to select a method that has this context, it is important to emphasize
been tested for the ability to detect these that finished product testing approaches
species.10 with regard to testing for Listeria spp. or
L. monocytogenes may differ considerably
While L. monocytogenes is the pathogen by country and region. For example, in the
of concern, PEM programs typically test United States, one would virtually always
for Listeria spp., which means a positive test for Listeria monocytogenes and not
test result indicates the presence of a for Listeria spp., as regulatory agencies
Listeria species that may or may not be L. typically expect speciation of Listeria
monocytogenes. This strategy provides for spp., isolated from finished RTE products.
a more sensitive approach to identify (1) In other countries however, screening of
conditions that allow for L. monocytogenes finished products for Listeria spp. may be
presence or introduction and (2) harborage the more common approach.
sites that could support L. monocytogenes.
4.2.2. Salmonella
The genus Salmonella includes two pathogen, there is clear evidence that the
species, Salmonella enterica and environment of processing facilities and
Salmonella bongori. PEM testing in facilities other food associated environments can
where Salmonella has been identified as be an important source of Salmonella,
a hazard reasonably likely to occur from particularly, but not limited to dry
environmental sources will virtually always environments. For example, Salmonella
target Salmonella spp., using tests that has been shown to persist for at least 10
detect both of these species. years in dry food processing facilities.11
Hence, the identification of Salmonella
While Salmonella may classically be harborage sites is important for certain
thought of as a fecally transmitted types of RTE food facilities.
44
Be aware of the square inch 4.2.3. Cronobacter

or cm2 mentality
The genus Cronobacter (formerly
Many training materials and even Enterobacter sakazakii) has been amended
government guidance documents over the last several decades due to the
specify a certain area that should continued genotypic and phenotypic
be sampled when environmental investigation of various strains that
pathogen monitoring is performed. have emerged over time.12,13 Sources
Areas of 12 inches by 12 inches or 30 of Cronobacter have been shown to be
centimeters by 30 centimeters are often primarily plant-based matrices (corn, soy,
mentioned.8 These recommendations wheat, rice, herbs, spices) as well as milk
are problematic, however, as virtually all powder and powdered infant formula.14
potential niches that should be sampled Cronobacter species are opportunistic
as part of an environmental monitoring pathogens, and have been found to be
program are not square or even flat the cause of life-threatening illnesses in
areas. Think about hollow table legs neonates, infants and immunocompromised
or rollers, floor wall junctures or floor older individuals.
cracks.
Contamination of powdered infant formula
Hence, it is important to provide training has been the primary cause for infections
on sampling that emphasizes the need in neonates and infants, resulting in many
to sample potential niches on irregular outbreaks worldwide and associated recalls
areas, rather than only flat surfaces. of powdered infant formula.15 In addition
Sometimes a good sample may be to finished product, Cronobacter has been
600 centimeters (6 meters) of a floor isolated from milk powder and powdered
seam that is 0.5 centimeters wide. Or infant formula plant environments (including
sometimes it may have to be any surface roller dryers, drying towers, and tanker
of a hollow table leg that is accessible to bays) and has been shown to persist in these
sampling. environments for long periods of time due
to its resistance to desiccation and ability
to survive spray drying.14,16,17 Monitoring for
Cronobacter in milk powder and powdered
infant formula plant environments is critical
to prevent contamination of the finished
product. Due to the changes in taxonomical
classification, it is especially important to
select a method that reliably detects all
species of Cronobacter.18
45
4.3. Development of a pathogen sampling

program for verification of environmental
pathogen control strategies
This section will focus on the development uncracked floor section) can have a
of PEM programs that provide verification huge impact on whether pathogens
of safety systems. It will not cover are detected. Hence, it is important to
sampling strategies for validation of food design the sampling plan and the overall
safety programs, prerequisite programs PEM program to avoid intentionally or
and non-process preventive controls unintentionally providing incentives for the
(such as sanitation procedures), as this sample collectors to not collect samples
would typically involve a combination of that would likely yield pathogen positives.
multiple testing approaches, including
ATP, TPC and, potentially, pathogen tests. For example, setting numeric targets or key
Development of a PEM program and performance indicators for the percentage
associated sampling plans involves multiple of positive PEM samples may simply lead
steps. A possible step-wise framework to sample collectors not collecting samples
for this is detailed in Table 2; however that will likely yield positives. The goal of
individual facilities must typically refine and a PEM program is to find and eliminate
expand this framework. pathogen contamination in the processing
environment, and this goal cannot be
Importantly, a key challenge with PEM achieved if there are incentives against
programs is that the specifics of sample collecting a positive sample.
collection, including the pressure applied
to a sponge and the specific locations
tested (e.g., a floor crack vs. an adjacent
46
Table 2. Key steps for development of a PEM program
Steps Comments and Suggestions

Should be a cross-functional team that, at minimum, includes
1 Assemble PEM team representation of quality assurance, microbiologist, sanitation and
plant management functions
Includes floor plans, details on equipment and equipment location,

Assemble documentation
PEM results obtained previously in the same facility, other
2 and information needed for
environmental programs already implemented (e.g., ATP testing),
PEM program development
and validation data for food safety programs (if available)
Identify regulatory and

Should also include identification of industry and regulatory
3 customer requirements for
guidance documents
PEM (if any)
Includes target organisms, testing procedures, sample sites,

Decide on key parameters sampling frequency, number of samples collected per week or
4
of PEM program month, sampling time and day and testing lab (in-house versus
third-party lab)
Includes record-keeping system, SOPs and written guidance for

Develop written follow-up on positive results (corrective actions). All tasks need
5
documentation to be assigned to specific individuals with written record of these
assignments
Includes training SOPs, records of training and results for tests.

6 Train sample collectors Training should be delivered in a form that is easily understandable
by all personnel
Regular review of sampling plans, results and corrective actions

should occur every 6 to 12 months and needs to include the
complete PEM team (Step 1). This may include a regular (e.g.,
yearly) PEM sampling performed by an independent or outside
7 Schedule regular review
group (e.g., consultants or a corporate food safety team), which
may collect a large set of environmental samples to assess
whether the implemented routine sampling plan is effective at
detecting the target pathogens
4.3.1. Zoning and selection of sampling sites

Virtually all PEM programs use the concept (see Figure 1) with Zone 1 representing food
of sampling “zones” when developing contact surfaces (i.e., surfaces directly
a sampling plan. In most countries and contacted by an exposed RTE food) and
regions, sampling sites in processing Zone 4 representing areas outside of the
facilities are assigned to one of four zones RTE area (such as locker rooms, loading
47
docks, etc.).3,5,7,19 In some countries, considered Zone 1, but may be classified

sampling sites may be classified into three into Zone 2 if zone classification is
zones. Under this scheme, Zones 2 and 3 performed during a time of low humidity
of the “four-zone” scheme are typically when no visible condensation is present
combined into one zone. and when the team may not be aware of the
condensation potential. Additionally, while
Assignment of sampling sites to zones is drains are typically classified into Zone 3,
not always straightforward. For example, drains that are located immediately under
surfaces above exposed RTE foods, which food contact surfaces may be considered
show condensation that can drop onto Zone 2 sites.
the exposed food, would typically be
Figure 1. Environmental monitoring sampling zones
ZONE 1
Food Contact Surfaces
(Slicers, peelers, fillers, hoppers, screens, conveyor belts,
air blowers, employee hands, knives, racks, work tables)
ZONE 2
Non-Food Contact Surfaces in Close Proximity to
Food and Food Contact Surfaces
(Processing equipment exterior and framework,
refrigeration units, equipment control panels, switches)
ZONE 3
More Remote Non-Food Contact Surfaces Located
In or Near the Processing Areas
(Forklifts, hand trucks, carts, wheels, air return covers,
hoses, walls, floors, drains)
ZONE 4
Non-Food Contact Surfaces Outside of the Processing Areas
(Locker rooms, cafeterias, entry/access ways, loading
bays, finished product storage areas, maintenance areas)
48
Typically, an initial step in the design of a part of seek and destroy missions triggered
PEM program is to select possible PEM by other events, such as positive samples
sites. The result of this effort is usually a found through verification sampling.
master list of sampling sites with a unique
identifier for each sampling site. Sufficiently Each sample site will be assigned a zone,
detailed descriptions should be included to and zone definitions may differ by country,
ensure subsequent sampling of the same region and even regulatory agency. A
site can be reproducible, and preferably, written definition for what constitutes a
this list would be created and maintained in given zone should be included in each
an appropriate database that is compatible sampling plan. Importantly, while this
with other databases such as laboratory list represents all potential verification
information management systems (LIMS). sampling sites, this does not mean that
Selection of the sampling sites typically samples from all sites will be collected
involves a walk-through by the PEM team during each sample collection.
(Table 2, Step 1), to identify sampling sites,
including hard-to-clean areas, potential For example, it would not be unusual for
niches, harborage sites, high-traffic areas a medium-sized food processing facility
and pathways that may facilitate pathogen to have a master list of 400 to 500 sites,
movement in the facility. but only collect samples from 40 to 50 of
these sites a week. However, it is important
Since disassembling equipment in order that the individuals responsible for sample
to allow sampling of actual harborage collection are given the freedom to also
sites is not feasible for routine verification collect at least some samples that are not
sampling, companies may instead select included in the sample site list, to allow
representative sampling sites that are them to collect samples from high-risk
contiguous or adjacent to areas of potential sites like pooled water, drain back-ups or
harborage. Sampling of actual harborage new cracks in the floor that may become
sites post-disassembly is typically apparent during sample collection.
performed for validation sampling or as

The standard guidance for sampling produces foods that have been linked to
frequency and number of samples suggests listeriosis outbreaks regardless of whether
that both of these need to be determined or not they typically would not allow for
“based on risk.” This definition tends to growth (ice cream is a good example of this
not be very helpful as there are few, if any, latter case).
guidance documents that specify how to
quantitatively assess the risk associated The sampling frequency can be reduced
with environmental pathogens. Generally, to monthly (or less, in rare cases) if there is
facilities where RTE foods are exposed substantial evidence that there is a low risk
to the environment would be considered of Listeria contamination. For example, a
high-risk and would require, at a minimum, very small facility that processes less than
weekly sampling for target pathogens. 3 to 4 days per week may be able to justify
Specifically, Listeria would be a target a lower sampling frequency. Similarly,
pathogen for weekly sampling for any such facilities that only produce RTE foods that
facility that either produces RTE foods that undergo in-package listeriocidal treatment
allow for Listeria growth (e.g., cheese, fluid and that do not allow Listeria growth may
milk, RTE deli meats, RTE seafood) or that be able to justify sampling less than weekly.
49
Salmonella would be a target pathogen Similar to sampling frequency, there are

for weekly sampling for any facility that very few, if any, recommendations for
produces RTE foods that are exposed to the number of samples to be taken as
the processing facility environment and part of PEM programs, other than that
have previously been linked to either sample number determinations should be
salmonellosis cases or outbreaks, or “risk-based.” One of the few documents
contamination events that could be linked that provides guidance on sampling
to sources in the processing facilities. frequency is a United States Department
Facilities that would typically be required of Agriculture Food Safety and Inspection
to execute stringent Salmonella sampling Service (USDA FSIS) Listeria guidance
plans involving weekly sampling include, document that suggests collection of 3 to
but are not limited to, those that produce 5 food contact surface (Zone 1) samples
chocolate, dry cereals, dairy powders, per production line per sampling.8 This
and many other low-moisture RTE food could range from weekly to every 6
products. months for extremely low-risk facilities
(Table 3), but only covers Zone 1 (food
contact surfaces).
Table 3. Description of sampling frequency for the different food processing facility
alternatives classified by USDA FSIS8
Minimum Frequency of Food

HACCP Production
USDA FSIS Description of Contact Surface Testing
Classification Volume/ Day
Alternatives Alternatives (note: 3-5 samples should be
Size (lbs.)
collected per line)
Alternative 1 2 times/ year/ line (every 6

PLT & AMAP n/a n/a
(Alt. 1) months)
Alternative 2,
PLT, only n/a n/a 4 times/ year/ line (quarterly)
Choice 1 (Alt. 2a)
Alternative 2,
AMAP, only n/a n/a 4 times/ year/ line (quarterly)
Choice 2 (Alt. 2b)
Sanitation,
Alternative 3 (Alt.
only (neither
3); non-deli or n/a n/a 1 time/ month/ line (monthly)
PLT nor
non-hotdogs
AMAP)
Very small 1 – 6,000 1 time/ month/ line (monthly)

Sanitation,
Alternative 3 (Alt. only (neither 6,001 – 2 times/ month/ line
Small
3); deli or hotdogs PLT nor 50,000 (every 2 weeks)
AMAP) 50,001 -
Large 4 times/ month/ line (weekly)
>600,000
Figure legend: Post-lethality treatment (PLT): a process used to reduce or eliminate

L. monocytogenes in the product; examples include pasteurization and high-pressure
processing. Antimicrobial agent or process (AMAP): an agent or process used to limit or
suppress the growth of L. monocytogenes in the product.
50
4.4. Corrective
actions based on Air testing for pathogens
pathogen testing A frequent question is whether air
results
should be tested for pathogens. Unlike
for mold spores, there is no evidence
that vegetative bacterial pathogens are
For a routine verification program, which transmitted by air in food processing
is described here, it is essential that a clear facilities. However, aerosols (extremely
written plan and outline exists for corrective fine and small water suspended in
actions. These plans should include details air) can be a very effective vehicle
on: for transmission of pathogens in
processing facilities. Confusion
• Minimum number of vector swabs to about the role of air versus aerosols
be collected after an initial positive, may explain why questions about air
including a protocol to determine the sampling for pathogens are brought up
specific vector swabbing procedures. frequently.
• Deep cleaning procedures to be used in
follow-up to a positive test result. Rather than air testing to identify
• Root-cause analyses procedures to be the role of aerosols in pathogen
used, including details on the team that transmission in a processing facility,
will conduct these analyses. testing the sources and deposition
• Procedures to be used to translate areas of aerosols would be a more
findings into a corrective and preventive appropriate strategy to address this
action (CAPA) plan, including concern. In addition, minimizing
requirements for CAPA close-out. aerosolization (for example by removing
high-pressure hoses in processing
Vector swab sites should be selected to facilities and minimizing water use
represent areas and sites that could be the during processing) is essential to
source of the initial positive findings. This reduce pathogen transmission in
could mean nearby potential harborage processing facilities.
sites, such as floor-wall junctures, drains,
overhead drip pans, or traffic path sites that Another air-associated potential source
intersect with the initial positive site to which of pathogens maybe high-pressure
the organism could have spread. air; the air hoses may be a niche for
pathogens. Hence, testing high-
If the routine (“verification”) environmental pressure air may be advised in facilities,
sampling is used to verify a validated food particularly if high-pressure air is used
safety program, prerequisite program or to clean food contact surfaces.
non-process preventive control (e.g., a set of
SSOPs, sanitation procedures), the written
plan should also include details on the
procedures to be used for revalidation of the
affected non-process preventive controls.
51
4.5. Identifying sources of pathogens and

development of preventive controls
A key part of a PEM program is to identify For example, excessive sanitizer use
harborage sites where actual pathogens (including floor sanitation) performed
survive and grow, often because they are immediately prior to vector swabbing may
protected from sanitizers. The food safety yield negative results, when in reality it
goal is to identify and eliminate growth simply led to a situation where persistent
niches (i.e., areas that support general pathogens, index organisms or harborage
bacterial growth) as well as potential sites were covered up rather than truly
harborage sites during the validation of eliminated. This approach could also
sanitation procedures and before they lead to situations where a pathogen or
become contaminated. Routine verification index organism positive sample may be
PEM programs confirm the effectiveness of misinterpreted as a sporadic positive when
sanitation procedures and other preventive it actually was an indication of pathogen
controls that have been implemented. persistence. Appropriate, well-planned
and executed follow-up to each pathogen
The initial objective of the PEM program is or index organism positive sample is thus
to identify and eliminate harborage within essential for effective PEM programs.
the exposed product area. However, it is
possible that niches may be missed during
the validation process (and subsequent
seek and destroy missions), as they may
sometimes only be identified by ongoing
verification sampling. An example would
include a niche that was not present at the
time of validation sampling but developed
over time. In addition, areas that do not
initially represent potential niches and
harborage sites may become actual niches
and harborage sites because equipment
and equipment parts, such as gaskets,
wear out.
Well-designed and implemented

verification PEM programs can and should
detect these sorts of issues. However,
detection of actual pathogen sources
can sometimes be hindered if follow-up
activities to an initial positive (e.g., vector
swabbing, deep cleaning) are not executed
correctly.
52
4.6. Advanced sampling approaches to control

environmentally transmitted foodborne
pathogens
As detailed throughout this chapter, basic a growth niche to measure the level of
environmental pathogen monitoring control exerted by the sanitation process
programs generate data needed to validate control system. In addition to pathogen
and verify environmental pathogen control detection, indicator site sampling can also
strategies. This includes “for-cause” use TPC, ATP and other analysis methods.
investigative sampling after verification
samples yield positive results. Investigative sampling following pathogen
detection in an indicator site would be
Food processing facilities that have robust considered “not-for-cause,” as this is
validation and verification sampling conducted as part of a sanitation process
strategies often develop and implement control program but is not necessarily a
advanced sampling strategies. These component of a regulatory compliance
sampling methods enable the creation of program. Employing process control
preventive controls and other strategies sampling that utilizes indicator sites
that further improve the ability of these provides facilities not only with an “early
facilities to prevent microbial contamination warning system,” but can also encourage
events from environmental sources. For stringent testing strategies, as positive
example, some RTE meat processors in the results at an indicator site would not
U.S. perform “process control” sampling necessarily indicate a systematic failure of a
in addition to verification and validation food safety system that requires “for-cause”
sampling activities, with validation sampling investigative sampling.
using the seek and destroy approach to find
and eliminate niches and harborage sites
(Figure 2).
Process control sampling utilizes “indicator

sites” (not to be confused with indicator
organisms) for sampling, which are early
warning sites where pathogen detection
does not yet indicate an acute food safety
issue. These include areas of the facility and
equipment with sanitary design concerns
as well as Zone 4 to Zone 3 transfer
pathways where presence or ingression
of a target pathogen can be identified
before it reaches a verification sampling
site. Indicator sites are typically located
near hurdles and barriers to measure the
effectiveness of the obstacle, or at or near
53
Figure 2. Example of the seek and destroy process. 2
Seek and Destroy Process

Normal Cleaning and
Sanitation Process
Are All Components Being

Observe Flood Sanitation Adequately Sanitized
(Chenical or Heat)?
Observe Assembly
Observe Post-Assembly Are GMPs Being

Sanitizer Application Followed?
Observe Normal Setup

and Startup Activities
Inspect and Swab Any
Suspect Areas
Stop Operation Before Product
is Placed on the Line
If APC Growth
is Supported, Then
Suspicious Area is a
Disassemble to Normal Growth Niche
Daily Level
Evidence of
Unacceptable
Organic
Disassemble Any Remaining Buildup?
Machine Components Root-cause
If Positive for Listeria,

Clean and Flood or Heat- Are Cleaning Is Level of
the Suspicious Area
sanitize All Disassembled Methods Disassembly
is a Harborage Site
Line Components Acceptable? Acceptable?

pathogen testing 3M Food Safety expert
www.3M.com/PathogenTesting www.3M.com/Connect/PathogenTesting
54
References:
2. Malley, T.J., Butts, J., Wiedmann, M. 2015. Seek and destroy process: Listeria
monocytogenes process controls in the ready-to-eat meat and poultry industry. J. Food Prot.
78 (2): 436-445. http://dx.doi.org/10.4315/0362-028X.JFP-13-507

4. Grocery Manufacturer’s Association. 2014. Listeria monocytogenes Guidance on

Environmental Monitoring and Corrective Actions in At-Risk Foods. https://www.gmaonline.
org/forms/store/ProductFormPublic/LEMP
6. Innovation Center for U.S. Dairy. 2015. Control of Listeria monocytogenes: Guidance for the
U.S. Dairy Industry. http://www.idfa.org/docs/default-source/resource-library/guidance-for-
the-us-dairy-industry-10-19-15.pdf

8. United States Department of Agriculture Food Safety and Inspection Service. 2014. FSIS
Compliance Guideline: Controlling Listeria monocytogenes in Post-lethality Exposed Ready-
To-Eat Meat and Poultry Products. https://www.fsis.usda.gov/wps/wcm/connect/d3373299-
50e6-47d6-a577-e74a1e549fde/Controlling-Lm-RTE-Guideline.pdf?MOD=AJPERES
9. Orsi, R., Wiedmann, M. 2016. Characteristics and distribution of Listeria spp., including
Listeria species newly described since 2009. Appl. Microbiol. Biotechnol. 100: 5273-5287.
https://doi.org/10.1007/s00253-016-7552-2
10. 3M Food Safety. 2018. 3M™ Molecular Detection Assay 2 – Listeria Performance Summary.
11. United States Centers for Disease Control. 2008. Multistate Outbreak of Salmonella Agona
Infections Linked to Rice and Wheat Puff Cereal (FINAL UPDATE). https://www.cdc.gov/
salmonella/2008/rice-wheat-puff-cereal-5-13-2008.html
55
12. Joseph, S., Cetinkaya, E., Drahovska, H., Arturo Levican, A., Figueras, M.J., Forsythe, S.J.
2012. Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis
sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg
infection, water and food ingredients. Int. J. Syst. Evol. Microbiol. 62: 1277–1283. http://dx.doi.
org/10.1099/ijs.0.032292-0
13. Jackson, E.E., Masood, N., Ibrahim, K., Urvoy, N., Hariri, S., Forsythe, S.J. 2015. Description
of Siccibacter colletis sp. nov., a novel species isolated from plant material, and emended
description of Siccibacter turicensis. Int. J. Syst. Evol. Microbiol. 65: 1335–1341. https://dx.doi.
org/10.1099/ijs.0.000108
14. Forsythe, S.J. 2018. Updates on the Cronobacter Genus. Annu. Rev. Food Sci. Technol.
9:23–44. http://dx.doi.org/10.1146/annurev-food-030117-012246
15. Norberg, S., Stanton, C., Ross, R.P., Hill, C., Fitzgerald, G.F., Cotter, P.D. 2012. Cronobacter
spp. in Powdered Infant Formula. J. Food Prot. 75: 607–620. https://doi.org/10.4315/0362-
028X.JFP-11-285
16. Craven, H.M., McAuley, C.M., Duffy, L.L., Fegan, N. 2010. Distribution, prevalence and
persistence of Cronobacter (Enterobacter sakazakii) in the nonprocessing and processing
environments of five milk powder factories. J. Appl. Microbiol. 109:1044–52. https://doi.
org/10.1111/j.1365-2672.2010.04733.x
17. Osaili T., Forsythe S. 2009. Desiccation resistance and persistence of Cronobacter
species in infant formula. Int. J. Food Microbiol. 136:214–20. https://doi.org/10.1016/j.
ijfoodmicro.2009.08.006
18. 3M Food Safety. 2017. Evaluation of the 3M™ Molecular Detection Assay 2 – Cronobacter
and Three Commercial Methods for the Detection of Cronobacter spp.
56
Environmental Monitoring Handbook - Spoilage Organisms
CHAPTER 5
Environmental Monitoring for

Spoilage Organisms
By
Randy Worobo | Cornell University Department of Food Science
Abigail Snyder | The Ohio State University Department of Extension
5.1. Purpose of environmental monitoring for spoilage 58

organisms
5.2. Spoilage organisms and their significance in the 58

5.2.1. Yeast and molds 59
5.2.2. Total Plate Count 59
5.2.3. Lactic acid bacteria 59
5.3. Development of a spoilage organism sampling 61

program
5.3.3. Data trending and analysis for spoilage 65

organisms
5.3.4. Determination of cut-off levels for spoilage 67

organisms
5.4. Corrective actions based on spoilage organism 67

results
5.5. Identifying sources of spoilage organisms 68
5.6. Additional aspects to consider 68
57
5.1. Purpose of environmental monitoring

for spoilage organisms
Environmental monitoring allows Environmental monitoring is often used as
companies to take a proactive approach a verification activity for sanitation regimes
to microbial spoilage, rather than since the processing environment is one of
retrospectively addressing failures as the main contributors to microbial quality
they arise. This is particularly useful in failures that manufacturers seek to control.
quality management systems, as spoilage Poor environmental sanitation increases the
incidents often arise sporadically and, risk of an unintended microbial spoilage
without consistent baseline measurements, incident.
underlying or chronic issues can go
unnoticed.
5.2. Spoilage organisms and their significance

in the food processing environment
Food processing environments are non- Particular production methods or product
sterile, and the microorganisms which types are associated with certain spoilage
colonize these environments are often organisms. Facilities should consider which
well-adapted to using the manufactured spoilage organisms are most pertinent
food product as a substrate for growth. based on these parameters to determine
This adaption increases the risk of spoilage if the use of a targeted environmental
if cross contamination occurs. monitoring strategy, one which focuses
on a specific type or class of organisms,
Moreover, facility-specific spoilage or a more generalized environmental
microbes are often adapted to withstand monitoring strategy, one which relies on
facility-specific production controls. relevant indicators, is most appropriate.
Heat-tolerant bacteria and fungi are For example, hot-fill facilities would likely
more frequently isolated from products address heat-resistant molds as part of
and facilities with thermal processes. their environmental monitoring program.
Preservative-resistant yeast are more
frequently isolated from facilities The resilience of spoilage organisms
employing those preservatives. Long- to the inactivation process employed,
term use of sanitizers that are not broad- the tolerance of the spoilage organism
spectrum, or poor cleaning practices can to the formulation conditions and the
also result in higher levels of environmental affinity of the spoilage organism for the
spoilage organisms. The power of selective raw ingredients should be evaluated
pressure can lead to environmental in identification of specific spoilage
harborage of troublesome spoilage organisms. Relevant spoilage organisms
microbes. are often grouped by a combination of
taxonomy, function and detection methods.
Microbial spoilage can result in decreased Commonly used groups include yeast and
shelf-life, inferior organoleptic properties, molds, Total Plate Count and lactic acid
and in some instances, product recalls bacteria.
and withdrawals. These outcomes have
significant economic and consumer
perception consequences.
58
5.2.1. Yeast and molds

Yeast and molds are fungi, eukaryotic vectors, or due to insufficient sanitation
spoilage organisms that are highly resistant practices.
to many processing and formulation
controls.1 Yeast and molds reportedly Filamentous fungi (molds) are resilient
persist and propagate even under to low pH, water activity, and some
extremely harsh environmental conditions. are extremely heat-resistant. They are
particularly associated with shelf-stable
Yeast, single-celled eukaryotes that appear or extended shelf-life (ESL) products,
similar to bacteria on a Petri dish or under those foods which have been processed
the microscope, are resilient to low pH and formulated in a way to control other,
and are particularly associated with the faster growing spoilage organisms. Mold
spoilage of high-water activity and/or high- transmission frequently occurs through air
sugar foods such as pasteurized juices, due to the high aerosolization potential of
syrups, fresh-cut fruit and yogurt. Yeast spores, in addition to the other mechanisms
transmission often occurs through food, relevant to all spoilage organisms.
beverage or processing/cleaning water
5.2.2. Total Plate Count

Total Plate Count (TPC), or more products which support the growth of
accurately, total aerobic plate count, only a select few spoilage organisms. TPC
refers to all culturable microorganisms results are usually dominated by bacterial
recovered on rich, complex media under growth, which out-competes slower
aerobic conditions.1 TPC may be used as growing fungi.
an indicator of general sanitation and to
evaluate the total microbial load in the Processing environment designs that are
processing environment. sensitive to environmental contamination
may also be usefully evaluated using TPC.
Detection using this method may be These systems could include filler areas,
specifically relevant to highly perishable cooling water reservoirs and hard-to-clean
products subject to spoilage from a diverse niches in production lines.
array of commensal organisms, rather than
5.2.3. Lactic acid bacteria

Lactic acid bacteria represent a diverse, microbial growth – notably, lactic acid.
functional collection of bacteria that Homofermentative lactic acid bacteria
cause spoilage of fresh meat and meat exclusively produce lactic acid as a
products, ready-to-eat (RTE) products like byproduct of their metabolic activity,
fresh-cut fruit and modified atmosphere whereas heterofermentative lactic acid
packed (MAP) lunch meats, beer and bacteria variably synthesize lactic acid,
wine.1 Spoilage is characterized by acetic acid, carbon dioxide and other
off-flavor metabolites produced during organoleptic metabolites.
59
Lactic acid bacteria are a significant formation and package bloating due
challenge for the meat industry. Meat is to carbon dioxide production among
a high-value, highly perishable product heterofermentative strains. Lactic acid
that is commonly associated with lactic bacteria are ubiquitous; contamination
acid bacteria spoilage. Subsequently, it is comes from the environment and can be
one of the best studied product/spoilage mitigated through strong environmental
relationships and the quality defects are and utensil sanitation practices, along with
well-characterized. Spoilage due to lactic control of the storage conditions during
acid bacteria outgrowth can be recognized shelf-life.
by off-flavors and aromas, slime (dextran)
Figure 1. Chronology of spoilage-induced recall for a hypothetical U.S. food manufacturer
Consumers report
package bloating
due to microbial
spoilage initiated
an investigation.
Initial assessment of the

Total Plate Count and
FDA becomes involved yeast and mold count
and, initially, proposes is inconclusive about
a Class I Recall.* the cause of the spoilage.
Additional testing reveals the spoilage

organism to be lactic acid bacteria, the
same microbes detected in the processing
environment. Consequently, the spoilage issue
is treated as a Class II Recall.*
As a part of long term corrective actions,

the facility improves their environmental
sanitation regime and changes the
container closure to facilitate
decontamination during processing.
*Class I Recall: Dangerous or defective products that predictably could cause serious health
problems or death. Class II Recall: Products that might cause a temporary health problem, or
pose only a slight threat of a serious nature.2
60
5.3. Development of a spoilage organism

sampling program
A prescriptive environmental monitoring Environmental monitoring of surfaces can
program can target problem areas to be accomplished by direct plating and
reduce spoilage in the short term, and indirect plating through the use of sponges.
allows for tracking and trending to control
quality threats in the long term. This Figure 2: Example of air sampling using
aids in root-cause analysis and can help 3MTM PetrifilmTM Plates
distinguish between failures in policy
versus failures in the execution of policies.
Sampling plans should be structured
around several factors:
• Identification of an appropriate
microbial target.
• Selection of sampling sites.
• Determination of the frequency of
sampling.
• Establishment of actionable cut-off
levels and associated corrections.
Sampling programs should be feasible for

the facility, and decisions around these
parameters may necessarily involve several
members of the food quality team.
Facilities should also consider the method Direct contact plating is a rapid, easy-to-
of detection most appropriate for their apply method for detecting low levels of
spoilage organism of concern. Facilities microbes from non-food contact surfaces.
targeting molds which produce spores that However, if sampling of larger surface
are readily aerosolized may consider the areas is required, common indirect contact
use of air sampling methods to monitor methods utilizing swabs or sponges can be
spore load. used.
Microbial air quality can be evaluated Indirect plating also allows for additional
through quantitative air sampling or using sample processing. For example, for
the settle plate method. Location and time selection of heat-resistant spoilage
of sampling should both be considered in organisms, a heat shock can be applied
development of a monitoring plan. Areas to the sample before plating to reduce
of high-air circulation, high-sensitivity background microbiota. Additionally, this
(i.e., exposed product), or high-microbial method allows for plating on multiple
prevalence (e.g., depalletizing area) media if several microbial targets are of
are relevant air sampling locations. interest.
61

Site selection should be based on the goals monitoring day. If particular sites are
and targets of the environmental sampling notoriously problematic or indicative of
plan. Environmental monitoring plans sanitation efficacy, the facility may choose
targeting spoilage organisms can serve to incorporate those more frequently into
as a verification of sanitation procedures the rotation among a randomized subset. It
or a “seek and destroy” technique for is advisable to periodically re-evaluate the
targeting specific spoilage organisms in the master list, and invite alternative opinions
environment. Both goals can be addressed of relevant sampling sites to add to the list.
through the same plan, but the primary goal Moreover, employees should be trained on
may influence aspects of the procedure. where, specifically, to sample sites based
on the description in the sampling plan.
A master list of sampling sites should be Figure 3 illustrates how multiple, highly
developed, and from that collection of relevant sites can be identified on the same
sites, a subset should be tested on each piece of equipment.
Figure 3. Example of multiple sampling sites from one piece of equipment
Joints in conveyor belt links and underneath belt
Inside roller and gaps
Leg harborage areas
62
Verification of sanitation is supported For control of spoilage organisms, facilities

through the selection of a diverse array of may choose to direct sampling activity to
changing sites, along with targeted checks surfaces increasingly distant from food
of difficult-to-clean sites. Seek and destroy production as they contribute to cross-
approaches to eliminate specific spoilage contamination. Zone 2 surfaces such as
organisms from the environment should be overhead pipes directly above food contact
informed by the transmission mechanism surfaces, Zone 3 surfaces such as fan
and probable sources associated with the blades and cooling water reservoirs, and
organism, as described earlier. Zone 4 surfaces such as air intake vents
all represent areas prone to harboring
Generally speaking, swabbing larger areas, problematic spoilage organisms, depending
compared to the investigation of small on the facility.
niches often sampled in Listeria-targeting
programs, has been shown to improve Moreover, Zone 1 surfaces are easily
environmental monitoring programs to included in an environmental monitoring
prevent spoilage. Environmental swabs program which targets spoilage organisms,
may serve a dual purpose since spoilage and the findings may inform sanitation
organisms and pathogens, or their interventions which pertain to safety as
indicators, can be detected from a single well. Table 1 contains a list of common
sample. However, in some instances, problematic areas in processing facilities
sampling site selection may vary between that arise in all four zones.
pathogen and spoilage environmental
monitoring programs based on the zones
which are selected.
Table 1. Example sample sites often associated with spoilage organism harborage
Site Quality threat Zone

Lack of turbulent flow leads to accumulation and growth of
Dead leg 1
spoilage bacteria and yeast.
Complex equipment that may directly contact product

and may also include hollow rollers, rough welds and
Conveyor microcracks. Moreover, employee overspray during sanitation 1+
can contaminate this equipment and contribute to cross
contamination.
Biofilm development contributes to post processing

Cooling water reservoirs 2
contamination of hot-filled or retorted product.
Fan blades
Accumulation of fungal spores and dust particulate leads to
3
circulation through air streams in the production environment.
Air vent
Harborage site, particularly associated with machinery mold,

Cooler seals/gaskets that is difficult to clean without dedicated attention in the 3
master sanitation schedule.
63
Figure 4: Example of spoilage organism sample sites in Zones 1 and 2
In the graphic below, a baked product exits an oven on a conveyor while a topping is added
from a dispenser. Above the line is an overhead pipe on which condensation forms during
production. The arrows in the figure indicate potential sampling sites for spoilage organisms
in this area of production.
Underside of overhead pipe
Dead leg
Dispense head
Conveyor

The number of samples taken on each alternatively be established in relationship
monitoring day should be based on the to the timing of a sanitation event or high-
size and complexity of the facility, in risk processing activity that may require
addition to the practicality of implementing additional monitoring to prevent quality
the program. The frequency of sampling deviations.
should be evaluated in accordance with the
relative risk of a quality failure, should pre- Depending on the facility, sampling
established cut-off levels be exceeded. may need to be adjusted seasonally or
as a result of intermittent events. For
Facilities in which environmental example, the concentration of airborne
monitoring results frequently reveal poor fungal spores increases during the
sanitation or emerging microbial harborage spring and manufacturers sensitive to
sites should increase the frequency of mold spoilage at the fill step may adjust
sampling. This same risk evaluation should accordingly. Alternatively, co-packers
be used in determining how frequently and facilities handling multiple SKUs on
results should be evaluated by a food safety shared processing lines may consider their
and quality team. Sampling frequency may sampling schedule as a part of a mitigation
64
strategy to prevent the introduction of more may be increasingly informative. In

problematic spoilage organisms, or their most cases, sampling frequency should
growth substrates, into sensitive products. occur at least monthly. Both frequency
and the number of sites increase as size of
Generally speaking, one swab per 1,000 the facility, pace of production, age of the
square feet (roughly 100 square meters) facility and equipment, and quality threat
of processing space may be used as a risk aversion increase.
baseline for quality management, although
5.3.3. Data trending and analysis for spoilage organisms
Different visualization methods allow the location or type of sampling site can
food quality team to address different address various issues that can arise in a
questions. It often proves useful to present production facility. Manufacturers should
environmental monitoring data from an take the time to analyze their results in
extended period of time in the form of a order to gain the full benefit of instituting
graph, so that trends and patterns become an environmental monitoring program for
apparent compared to visualization in a spoilage organisms. Figures 5a-c illustrate
spreadsheet or as a collection of sampling how a company may choose to analyze
reports. Sorting data based on date, their environmental monitoring data.
Figure 5a. Example of environmental monitoring data visualization: Total Plate Count
Total Plate Count

1600
Total Plate Count CFU/g
1400
1200
1000
800
600
400
200
0
AY
V
L
T
N
PR
EC
JU
SE
C
FE
O
JA
JU
AU
A
D
M
This chart is an illustration of Total Plate Count results in one location over the course of a year.
During the warmer summer months (June, July, August), counts increase due to the season. A
sharp, significant increase can be seen in late November that is unusual for the season. A root-
cause investigation would need to be conducted to understand the cause for these irregular
results.
Yeast and Mold Counts from Air
1200
ounts CFU/60cm2
1000
65
800
600
Total Plate Count CFU/g
1400
0
AY
V
L
T
N
PR
EC
1200
JU
SE
C
FE
O
JA
JU
AU
A
D
M
N
1000
800
600
400
Figure 5b.
200
Example of environmental monitoring data visualization: Yeast and mold
0
Yeast and Mold Counts from Air
AY
V
L
T
N
PR
EC
JU
SE
C
FE
O
JA
JU
AU
A
D
M
N
1200
Yeast and Mold Counts CFU/60cm2
1000
800
600
400 Yeast and Mold Counts from Air

200
1200
Yeast and Mold Counts CFU/60cm2
0
1000
LINE 1 LINE 2 LINE 3 LINE 4
800
600
Yeast and mold counts from air samples from multiple locations in a facility can be monitored
400
by comparing counts side-by-side using a bar graph. In this example, the counts on Line 2
are higher than the other locations, thus making the food produced on this line at a higher
200
risk for yeast and mold contamination. Steps can be taken to mitigate the risk by determining
Lactic
the source0of the yeast Acid
and Bacteria
mold, putting Counts
equipment from Equipment/Environment
in place to shield the product from
LINE 1
contamination or implementing LINE
a process to2 eliminate the yeast
LINE 3and mold after this
LINE 4
point in the
production.
Drain
Figure 5c. Example of environmental monitoring data visualization: Lactic acid bacteria
Funnel
Seal
Lactic Acid Bacteria Counts from Equipment/Environment
Belt
Drain
Filler Nozzle
Funnel
0 50 100 150 200 250 300 350 400
Seal
Lactic acid bacteria counts CFU/cm2
Belt
Filler Nozzle
0 50 100 150 200 250 300 350 400
Lactic acid bacteria counts CFU/cm2
In this chart, lactic acid bacteria counts have been monitored at various locations. If a finished
product is contaminated, this information may be useful to start the investigation to determine
the root-cause of the failure. In this example, the lactic acid bacteria counts on a seal are higher
than expected and the seal should be checked for cracks or improper cleaning.
66
5.3.4. Determination of cut-off levels for spoilage organisms

Cut-offs are the quantitative standards A facility may choose to stratify their cut-
which delimit acceptable results for the offs, and corrections, based on the type
environmental monitoring program, and of surface from which the sample was
are best established through in-plant taken. For example, a baseline appropriate
experience. In order to make an informed TPC for a drain likely differs from that of
decision about appropriate levels, a a food contact surface. The initiation of a
facility should track the results of their corrective response should be appropriate
environmental monitoring program for 10- for the findings.
20 rounds of monitoring. This data creates
a baseline from which normal variation Environmental monitoring programs often
can be observed, and cut-offs can be become burdensome for companies
extrapolated. when ineffective cut-offs or overzealous
corrective actions are mandated. Since
The baseline method is particularly suited environmental monitoring programs are
for establishing cut-offs for indicator largely preventive instead of reactionary,
organisms and quantitative microbial sustained trends in microbial detection
evaluations. In contrast, environmental may also warrant an investigation. Again,
monitoring programs that target specific slight variations in counts are expected and
spoilage organisms with the goal of guide baseline calculations, but facilities
their total exclusion from the processing may choose to adopt a policy wherein an
environment (e.g., heat-resistant molds) upward trend of 5-10 consecutive sampling
may choose to identify the presence of events may trigger a correction prior to
any detectable target sufficient to initiate a reaching cut-off levels.
correction.
5.4. Corrective actions based on spoilage

organism results
When cut-off levels are exceeded, short- opt to include a re-sampling step in their
term corrections and long-term corrective corrections following this sanitation
actions must be initiated. Immediate procedure to verify the contaminant was
corrections universally include a sanitation removed or reduced to an acceptable
step, one which either targets a particular level. It is advisable to include this site,
location or is a general deep cleaning. additionally, in the next monitoring cycle
to determine if the source or cause of
A procedure detailing the steps and the contamination was removed or if,
focus of the cleaning practices initiated instead, the same location becomes re-
after exceeding the established contaminated.
environmental monitoring cut-offs
should be documented, and employees Long-term solutions and root-cause
responsible for interpretation of the analysis should be based on data from
environmental monitoring results and several observation cycles, and may
initiation of corrections should be trained include retraining employees, evaluating
for these responsibilities. Many facilities cleaners and sanitizers, modifying
67
cleaning and sanitizing procedures or This should be recorded in a policy with

sanitation schedules and considering the environmental monitoring program
pertinent changes in production. These before any breach of established cut-off
corrective actions may be dependent levels occurs. Some facilities also elect
on the risk aversion of the company and to increase the number or frequency of
the probability that a spoilage issue will their sampling following a violation of
result subsequent to the environmental their cut-off levels. This, theoretically,
monitoring observations. could direct targeted sanitation towards
the contamination source through vector
Both the probability and severity of swabbing from ATP tests, but it also
potential product spoilage should be increases the level of control a facility has
used to determine if finished product over maintaining an acceptable sanitary
needs to be reprocessed or destroyed. level in their production environment.
5.5. Identifying sources of spoilage organisms

Identifying areas in the processing facility environment during every production run.
contaminated with spoilage organisms Equipment selection and design may also
is a useful quality management strategy. work against the environmental monitoring
However, spoilage organisms may be system if it allows for cross contamination,
continuously reintroduced into the system or even if it fails to actively exclude
if the point source is not eliminated. contaminants. Isolation of activities, age of
Continually detecting problematic levels of equipment and the building, and the degree
spoilage organisms from the same site may to which processes are enclosed are all
indicate additional underlying issues that factors that impact the environmental
are not being addressed through routine or microbiota and the probability of
specialized sanitation of this site. contamination. Consider long-term
findings from the environmental monitoring
Facilities should consider their risk levels program for spoilage organisms in the
for introduction of spoilage organisms from development of preventive maintenance
various sources. Common sources include and approved supplier programs.
poor-quality raw ingredients, which can
continuously reintroduce microbes into the
5.6. Additional aspects to consider

Environmental monitoring for spoilage practices (GMPs) also serve to control
organisms is diagnostic, and should not spoilage microbiota. However, only GMP
be considered a standalone system for violations which directly contribute to
control. Microbial analysis of cooling changes in surface or air contamination
water, ingredients and pressurized may be detected by the environmental
air may all be important supporting monitoring program and other avenues of
analyses to an environmental monitoring contamination should be considered.
program. Rigorous good manufacturing
68
A concerted effort across a broad-based these programs in order to leverage the

team is the best strategy to minimize the findings.
risk of a spoilage incident. Additionally,
the data from this program may be Employees should also consider the
broadly beneficial as the food safety and impact of GMPs on spoilage microbiota
quality team evaluates various systems. present in the facility. Re-evaluation of
An increase in spoilage potential may the environmental monitoring program
signal systemic problems that preempt itself should be conducted every 1-3 years.
future potential food safety failures as Changes to the processing system or
well. Environmental monitoring systems formulation may not only change the level
which detect spoilage organisms support of spoilage risk, but could also impact the
proactive responses, but companies need type of spoilage organisms relevant for a
to be prepared and have personnel with given manufacturer.
sufficient time to evaluate the results from
Learn more about spoilage Connect with a

organism testing 3M Food Safety expert
www.3M.com/SpoilageDetection www.3M.com/Connect/SpoilageDetection
69
References:
2. United States Food and Drug Administration. 2010. FDA 101: Product Recalls.
https://www.fda.gov/ForConsumers/ConsumerUpdates/ucm049070.htm
70
Environmental Monitoring Handbook - Allergens
CHAPTER 6
for Allergens
By
Thomas Grace | Bia Diagnostics
Ken Davenport | 3M Food Safety
Gabriela Lopez Velasco | 3M Food Safety
6.1. Purpose of environmental monitoring for allergens 72
6.2. Allergens and their significance in the food 73

processing environment
6.3. Specific vs. non-specific allergen testing 74
6.4. Development of an allergen sampling program 76
6.4.3. Determination of cut-off levels for allergens 78
6.5. Corrective actions based on allergen testing results 78
6.6. Identifying sources of allergen contamination 79
6.7. Additional aspects to consider 80
6.8. Summary 81
71
6.1. Purpose of environmental monitoring

for allergens
Food allergens have increasingly become among the leading causes of food recalls in
major concerns for food and beverage the U.S., which significantly impacts food
manufacturers. In 2004, it was estimated manufacturers.4
that approximately 2 percent of adults
and about 5 percent of infants and While having dedicated facilities for
young children in the United States suffer allergen-containing and allergen-free
from food allergies each year (Figure 1).1 manufacturing would be ideal, the reality
Additionally, about 30,000 individuals is that food not intended to contain
require emergency room treatment and particular allergens may be manufactured
150 individuals die each year due to allergic in the same facility and, often, on the same
reactions to food.1 equipment as allergen-containing foods.
Consequently, a robust environmental
The number of people diagnosed with monitoring program should include
food allergies has increased significantly considerations for allergen detection on
over the last several years as well as the manufacturing equipment after cleaning
number of hospital visits. This has a direct and before production of the next
impact on public health expenditures commodity. Also, the presence of allergens
and lost productivity.2,3 At the same time, should be assessed in the environment
allergens that are not declared on food and to prevent cross-contact of food with
beverage labels have consistently been allergens.5
Figure 1. Percentage of U.S. children with food allergies over time
6%
5%
4%
3%
2%
1%
0%
2000-2002 2003-2005 2006-2008 2009-2011
72
6.2. Allergens and their significance in the

The types of foods that can cause allergic example, “seafood” is an all-encompassing
reactions are wide and varied. However, category for Canada. However, it is
the most common sources can be grouped subdivided in the U.S. as “fish” and
into a few categories. These categories are “shellfish;” the latter of which is again
not consistent across regulatory agencies, further divided in the European Union (EU)
which adds complexity to the classification as “crustaceans” and “mollusks.”6 Some
(Table 1). countries go so far as to define specific
species of fish or fish component. In Japan,
In some cases, the specificity of the fish are recommended to be labeled
definitions of a category can determine the specifically as “mackerel,” “salmon,”
number of foods included in the list. For “salmon roe,” etc.7
Table 1. Regulated allergen foods in the United States, Canada, Australia/New Zealand and EU1,6,7
Canada Australia/New Zealand EU

United States “Big 8”
(10 Allergens) (12 Allergens) (14 Allergens)
Milk Milk Milk Milk
Egg Egg Egg Egg
Peanut Peanut Peanut Peanut
Soybeans Soy Soy Soya
Gluten (Including
Gluten (Including Wheat,
Wheat Wheat Wheat, Barley,
Barley, Rye, etc.)
Rye, etc.)
Tree nut Tree nut Tree nut Tree nut
Fish Seafood (fish) Fish
Seafood Crustacean
Crustacean shellfish Shellfish
Mollusks
Mustard Mustard Mustard
Sesame Sesame Sesame
Sulphite* Sulphite* Sulphite*
Lupin Lupin
Celery
*Not an allergen but regulated in a similar way as adverse reactions can occur in some
individuals.
73
The Food Safety Modernization Act foods and foods not intended to contain
(FSMA) requires manufacturers in the allergens, it is essential to take appropriate
U.S. or that export to the U.S. to include actions to ensure that there is no cross-
allergen controls in their food safety plan.8 contact between the foods. In some cases,
Similarly, the various schemes commonly this can be handled by the scheduling
employed for compliance with the Global of manufacturing operations to limit the
Food Safety Initiative (GFSI) also require risk. However, this does not eliminate the
allergen controls to be identified and possible risk of cross contamination alone,
monitored. While not explicitly required even with a robust cleaning program in
in Hazard Analysis and Critical Control place.
Points (HACCP) plans, there is the implicit
expectation that allergens should be Because of this, environmental monitoring
identified as hazards, and that critical is required for both the initial validation of
controls should be in place to prevent the cleaning procedure and the ongoing
inadvertent contamination of products with verification that the cleaning has been
allergens. executed according to written procedures.
In facilities and production lines that

manufacture both allergen-containing
6.3. Specific vs. non-specific allergen testing

Food manufacturers use a variety of There are two general approaches to
approaches and tests as part of a food allergen testing that have been traditionally
safety allergen program (Figure 2).5 Thirty employed for cleaning verification: specific
percent of today’s food and beverage and non-specific allergen tests.
manufacturers report the use of multiple
allergen tests.5
Figure 2. Food manufacturers’ allergen testing by method5
42.6%
35.7% Allergen-Specific Tests
24.3% Protein-Specific Tests
ATP-Based Tests
74
Specific allergen tests use a target there is a high-degree of certainty that

recognition approach to detect proteins the surface or rinse water sample is
within the allergenic food. These tests can contaminated with gluten. Because of
be used to identify and/or quantify the this selectivity, specific allergen tests are
amount of the allergenic food that may be required by GFSI for process validation.
present in a sample. For example, a facility
that makes both peanut butter ice cream If a cleaning process is designed to
and vanilla ice cream needs to ensure that remove milk from processing equipment
the peanut butter ice cream is completely prior to manufacturing soy milk, then a
removed from the manufacturing milk-specific ELISA or LFD is needed to
equipment. They could use an antibody- validate that the process is capable of
based test such as lateral flow device (LFD) removing residual milk. This is typically
or enzyme-linked immunosorbent assay done by testing before and after cleaning
(ELISA) to detect and/or quantify peanut to show, specifically, that milk residues are
proteins using antibodies raised against the effectively removed. LFDs and ELISAs can
purified protein. help define a HACCP system by surveying
the processing equipment and finding the
Additionally, processing can affect the “hot spots.” This can expose which areas
recognition of target proteins by the (e.g., valves and equipment interfaces)
antibodies in the test. For example, heat need future monitoring or need to optimize
treatment of the food (e.g., boiling, baking, clean-in-place cycles.
roasting, etc.) or even the temperatures
used during cleaning (e.g., steam-cleaning) After validation is complete, routine testing
may alter the sensitivity of the test to the following cleaning allows users to verify
allergen residues in the environment. It is that the validated cleaning procedures are
important to ensure that the test selected effectively being carried out. For example,
for environmental monitoring is capable of results that determine that the allergenic
detecting both non-thermally processed residues are at low or undetectable levels
as well as thermally processed allergens. following routine cleaning during a line
Users should take extra caution with foods changeover would serve as a useful
that go through fermentation (e.g., soy verification.
sauce, wheat beer) or enzymatic/chemical
digestion (e.g., hydrolyzed proteins used While most companies know the specific
in some infant formula). Food processes allergen they need to monitor, the
where the proteins may be severely specificity of the ELISAs and LFDs also
fragmented into small peptides may make represent a drawback when dealing
the allergenic foods undetectable by with foods containing multiple allergens.
traditional ELISA or lateral flow tests. For For example, a production line of salad
this reason, it is important that the selected dressing containing egg, milk, gluten and
method used for cleaning verification is fit soy scheduled to next produce a vinaigrette
for purpose and thus capable of detecting lacking all of these allergens would require
the allergens of concern in the users’ verification that these allergens have been
process. removed by using egg, milk, gluten and
soy-specific tests. It is possible that one
The use of an allergen test based on the can choose a single target allergen that
application of specific antibodies has will be representative of all four allergens
an advantage in its high specificity. If an and can indicate that there is no residue of
antibody-based test results in a positive the previous salad dressing present. In this
outcome with a gluten test, for example, instance, one might choose the highest
75
concentration in the matrix, e.g., milk, or surface during cleaning. The rationale
the allergen that is most difficult to remove, is that if proteins have been removed to
e.g., egg. an undetectable level (e.g., less than 3
micrograms per 100 square centimeters),
In these situations, a non-specific allergen then allergenic proteins have also been
test may be an alternative to ELISAs and removed to a very low level. In situations
LFDs. Non-specific allergen tests include with multiple allergens, such as the salad
ATP and protein surface swabs. While ATP dressing example, determining that you
does not directly measure allergens, it have less than 3 micrograms of total
stands to reason that if a surface is cleaned protein directly demonstrates that you have
sufficiently well to remove ATP to a low less than 3 micrograms of protein from any
level then the cleaning has been adequate and all of the allergenic food sources in a
to remove allergens. single test.
That said, it’s known that the solubility of Ultimately, the choice of whether to use an
ATP, a small negatively charged molecule, allergen-specific test or a non-specific test
can be very different from allergenic depends on many factors. Among these
proteins in the food that may be baked onto are the difference in the number and type
the surface. Additionally, some allergenic of allergens in the products produced in
food sources like egg white have low ATP the same area or production line, the time
levels, making ATP a poor surrogate for required for the testing, the necessity of
removal of these allergenic proteins. For quantitative results, the relative technical
this reason, highly sensitive protein swabs aptitude of the technician and the
offer a direct assessment of the success requirements of the customers for whom
in removing allergenic proteins from a the products are being produced.
6.4. Development of an allergen sampling

program
The selection of sampling sites mimics the areas of dust, liquid and other residue
same process as is used for ATP testing and build-up that might lead to cross-contact.
microbial indicator testing. While most of For cleaning verification, a risk-based
the testing focus for allergens should be approach should be used looking at both
on immediate post-cleaning verification the impact on the food should a surface
of Zone 1 and Zone 2 test points prior to be contaminated (the hazard) and the level
release of the line for production, there is of difficulty in getting the surface cleaned
also value in periodic testing of all of the properly (the probability) (Figure 3).
environmental sampling zones to identify
76
Figure 3. Identification of high-risk areas of allergen testing
| General
NE 4 Are
ZO a
Close Prox
3 | im
NE ity
ZO
| Indirect C
E 2 on
N
ta
ZO
ct
PROBABILITY
(Difficulty of Cleaning)
ZONE 1
Direct Contact
Low Medium High
High
(Proximity to Food) (generally Zone 1)
HAZARD
Medium
(generally Zone 2 and 3)
Low
(generally Zone 4)
Color = Risk Level

Those areas with direct food contact (Zone food and beverage manufacturers can
1) and very close indirect contact (Zone ensure that they get the most risk-
2) that are judged to be difficult to clean reduction for the resources spent
should be prioritized for most frequent on testing.
testing. Those areas that are distant from
the food (Zones 3 and 4) or are very easy The number of samples depends on both
to clean (smooth, flat surfaces with easy the complexity of the manufacturing
access) should be prioritized lower. equipment/line and the practical
considerations for testing budget. For
High-risk areas (Red, in Figure 3) should a typical testing line, 5 to 10 test points
be tested every time the line is cleaned or per line should be tested to get enough
perhaps at a high frequency such as once coverage to substantially reduce the risk
per week. Moderate-risk areas (Yellow) of undetected poor cleaning. The exact
could be tested at a lower frequency of number, however, is at the discretion of the
once per week to once per month. Low-risk quality team, and the rationale should be
areas (Green) should be tested at a low planned and documented in the facility’s
frequency of, perhaps, once per month or food safety plan or HACCP plan.
once per quarter. By modifying the testing
frequency based on the risk assessment,
77
6.4.3. Determination of cut-off levels for allergens
The topic of allergen thresholds has been volume units of measure have no meaning.
the focus of debate over the last decade Historically, this likely came about by the
with only pockets of resolution. Gluten use of ELISA methods that give results
thresholds in the finished product seem as ppm to analyze environmental swabs.
to be generally accepted at less than 20 Regardless of the source, it has produced
parts per million (20 ppm, or 20 μg/g).9 additional confusion in the marketplace, as
Certain special interest groups for celiac even some standard-setting bodies have
and gluten-sensitive communities are discussed applying 5 ppm as a threshold
advocating for lower thresholds (5 ppm-10 for environmental samples.
ppm) than what is required by regulations.
Other allergenic foods have less clarity, That being said, the current expert opinion
as a patchwork of thresholds is emerging from the Food Allergy Research and
from Voluntary Incidental Trace Allergen Resource Program (FARRP) is that a pass
Labelling (VITAL) in Australia, EU, Japan result using an ELISA kit should be below
and other national/regional regulations.10 the limit of quantification (LOQ – for most
kits 2.5-5 ppm(μg/g), or possibly equivalent
While there is currently little consensus to 1.25-2.5 μg/100cm2, depending on the
on the thresholds for finished food, protocol for swab extraction) to effectively
there is even less for what is acceptable reduce the risk to the end-consumer.11 This
on equipment and environmental represents a very practical approach to
samples. This is doubly compounded setting environmental thresholds for testing
by concentration units of measure for systems despite the lack of clarity from
food (ppm) being improperly applied regulatory bodies.
to surfaces where weight or per weight
6.5. Corrective actions based on allergen

testing results
The immediate corrective actions to • Moderate-risk (Yellow) samples
be taken when an allergen test from can receive a bit more discretion
environmental monitoring programs is depending on the type of product
above the threshold depends on the risk produced. Ideally, the area should be
level of the sample as determined in Figure re-cleaned prior to production, though
3. increased monitoring and/or deep
cleaning of the area in the future might
• High-risk (Red) samples that are also be an acceptable response.
positive require re-cleaning of the
equipment and re-testing prior to • Low-risk (Green) positives should be
clearing the line for production. scheduled for additional cleaning at a
future date followed by post-cleaning
testing.
78
Longer term corrective actions should • Changing the cleaning process to

include root-cause analysis for determining remove variability or increase the
the source of the allergen contamination effectiveness.
or the cause of failure in the cleaning • Assessing equipment for upgrades or
procedure. Additional long-term corrective replacement.
actions could include: • Upgrading plant design to improve
cleaning.
• Changing the cleaning frequency. • Improving raw material/ingredient
• Revalidating the cleaning procedure. segregation.
6.6. Identifying sources of allergen

contamination
As with any food safety failure, a root- determine why the food residues were
cause analysis to determine the source of not adequately removed. The focus of
allergens or origin of the failure and follow- the root-cause should be on the cleaning
up action to remove them is required to process and on a potential failure in the
ensure the failures do not repeat. There Time, mechanical Action, concentration
may be situations where the source of the of Chemicals, or the Temperature of the
contamination is not known. In these cases, process, commonly referred to as TACT
the tests for specific allergen residues will (Figure 4).
likely be much more valuable in the root-
cause analysis than the use of non-specific Additional considerations could include
tests such as ATP or protein swabs. changes, intentional or unintentional,
to the manufacturing process, such
If the failure occurred on Zone 1 or Zone 2 as excessive cooking that makes the
where the source of allergens is obvious food residues more difficult to remove,
(i.e., they were present in the previous equipment failures that cause spattering or
product run on the equipment), then product accumulation, or changes in the
the core of the root-cause analysis is to raw materials.
Figure 4. TACT approach to evaluate root-cause failure on the cleaning process
TACT – Every day, every time
T A T Time – enough time for effective cleaning
A Action – sufficient mechanical action applied
T C C Chemical – type/concentration
T Temperature – too hot/too cold
79
If the failures occurred in Zone 3 or Zone other causes may result in the migration
4, the focus of the root-cause should be of allergen-containing residues from the
on the source of the allergenic materials manufacturing area to Zones 3 and 4. Air-
and their potential transportation to handling equipment, fans and construction
these zones. People, spatter from the might also cause the inadvertent transport
manufacturing process, fine powder of allergen residues.
drift, traffic patterns for fork-lifts and
6.7. Additional aspects to consider

Selecting the proper allergen detection that the test is fit for purpose and can
test sometimes requires more detailed reliably detect the allergen source present
knowledge about the targets of the test. in the specific food matrix.
For example, many commercial milk assays
target the protein casein, which is about The detection of gluten and wheat also
80 percent of the protein in cow’s milk. has a number of challenges. Gluten is the
This is a good indicator for producers using protein that triggers celiac disease (a non-
products containing whole-milk or cheese allergenic disease) as well as triggering
powders. the symptoms in those people with gluten
sensitivity. Gluten is the main protein found
However, if the milk-containing products in a wide range of grains including wheat,
only contain whey powder, the casein barley and rye and their sub-cultivars.
test will not detect residues from these
products, as the content of casein in whey In contrast to celiac disease, there are
is very low. For companies with whey- or people with a specific allergy to wheat
whey protein isolate-containing products, proteins that include gluten. To complicate
tests that target beta-lactoglobulin (the this matter further, there are some test
major protein in whey) would be required methods which employ gluten antibodies
in order to measure the carryover of whey that are very specific for wheat gluten
protein in their non-milk labeled product. with low affinity for barley gluten, while
Similar concerns exist for food containing others can have greater than a four-fold
yolk or egg white, as most tests for egg stronger reaction to barley gluten than
proteins focus on the ovalbumin from the to wheat gluten. The wheat-specific
egg white, but would be ineffective in gluten antibodies may indicate that there
detecting the presence of egg yolk. is no gluten present when there are
significant amounts contributed from
One of the interesting “quirks” of the barley contamination. In contrast, barley-
allergen groupings in the U.S. and other specific gluten antibodies may indicate
regions is the grouping of certain allergen that there is 40 ppm of gluten when in
sources into large categories, such as the reality the concentration is only 10 ppm of
seafood/fish/shellfish categories. Some barley gluten. Then for this particular case,
antibody sources and, in turn, ELISAs and it would be important to verify that the
LFDs may be specific for certain species selected method can specifically detect
within the category while others may be and quantify rye, barley and wheat.
more broadly applicable to a wide range
of species. It is important to perform a
validation for any test selected to ensure
80
6.8. Summary
• Food allergies have increased over the • Selection of a testing method should be
years, which may have a severe impact supported by a risk-based analysis that
on public health, especially in infants helps determine that the verification
and young children. measurements will support allergen
• Current food demand may require control plans.
sharing of production facilities to • Testing methods for allergen detection
manufacture foods containing allergens are often based on the specific
and foods expected to be specific recognition of a particular protein. It is
allergen free. Thus, robust food safety important to perform a validation for
programs that consider environmental any test selected to ensure that the
monitoring and allergen control are test is fit for purpose and can reliably
essential. detect the allergen source present in
• An effective allergen control program the specific food matrix.
should be able to identify and monitor • Currently, allergen thresholds are a hot
potential areas of cross-contact and topic of debate without clear guidance.
ensure through a comprehensive Based on expert opinion by FARRP, a
validation that the cleaning process in a pass result using an ELISA kit should be
food manufacturing facility is effective below the limit of quantification of the
to minimize contamination with food specific method (2.5 to 5 ppm for most
allergens. commercial kits).
• Verification of allergen control • Environmental monitoring for allergen
measurements can be achieved control should include a sampling plan
through allergen testing. There are two that supports verification of food safety
general approaches that can be used: or HACCP plans. Identification of high-
risk areas (Zones 1 and 2) should be
-- Highly specific allergen testing that prioritized for higher testing frequency.
relies on the recognition of specific
Consideration should also be given to
proteins yielding a qualitative
or quantitative result. These are moderate- and low-risk areas (Zones
recommended for a cleaning process 3 and 4) that may be tested with lower
validation, to test allergen-free frequency.
final product and for environmental • A complete allergen control strategy
monitoring. should consider short- and long-
term corrective actions within the
-- Non-specific allergen testing that environmental monitoring program,
generally detects ATP and proteins as well as root-cause analysis to
whose presence may indicate an determine potential sources of
inadequate cleaning process. These allergens and anything that may cause
are useful when food manufacturing
a failure in their removal during the
includes products containing various
allergens in a single product, or cleaning process or their exclusion in
when it’s necessary to assess overall final product.
cleaning processes.

allergen testing 3M Food Safety expert
www.3M.com/AllergenMonitoring www.3M.com/Connect/AllergenMonitoring
81
References:
1. Food Allergen Labeling and Consumer Protection Act of 2004. Public Law 108-282, Title II.
https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/
Allergens/ucm106187.htm
2. Jackson, K.D., Howie, L.D., Akinbam, L.J. 2013. Trends in Allergic Conditions among
Children: United States, 1997-2011. National Center for Health Statistics Data Brief. http://
www.cdc.gov/nchs/products/databriefs/db121.htm
3. Gupta, R., Holdford, D., Bilaver, L., Dyer, A., Meltzer, D. 2012. The high economic burden of
childhood food allergy in the United States. J. of Allergy and Clin. Immunol. 131: AB223-AB223.
https://doi.org/10.1016/j.jaci.2012.12.1464
4. Food Safety News. 2017. Undeclared allergens a leading cause of food recalls in U.S.
https://www.foodsafetynews.com/2017/04/undeclared-allergens-a-leading-cause-of-food-
recalls-in-u-s/
5. Ferguson, B. 2018. Testing and Sanitation for Allergen Control. Food Safety Magazine.
https://www.foodsafetymagazine.com/magazine-archive1/februarymarch-2018/testing-and-
sanitation-for-allergen-control/
6. Regulation EU No. 1169/2011 of the European Parliament of the council. 2011. Annex II.
7. Food Allergy Research and Resource Program. 2017. International Regulatory Chart. Version
September 21. https://farrp.unl.edu/documents/Regulatory/International%20Allergens%20
9-21-17.pdf
9. Codex Alimentarius. International Food Safety Standards. CODEX STAN 118-1979. Revised
2008. Standard for foods for special dietary use for persons intolerant to gluten.
10. Taylor, S.L., Baumert, J.L., Kruizinga, A.G., et al. 2014. Establishment of reference doses for
residues of allergenic foods: Report of the VITAL expert panel. Food Chem. Toxicol. 63: 9-17.
https://doi.org/10.1016/j.fct.2013.10.032
11. Taylor, S.L. 2016. Validation and Verification of Allergen Control Plans. Food Allergy
Research and Resource Program Effective Food Allergen Management Workshop.
Rosemont, IL.
82
Environmental Monitoring Handbook - Driving Change
CHAPTER 7
Driving Meaningful Change in Your

Organization Through Culture and
By
John Butts | Food Safety By Design
Lone Jespersen | Cultivate
Michele Fontanot | 3M Food Safety
7.1. The path to microbiological process control 84
7.2. Benefits of microbiological process control 88
7.3. Company culture and predictive microbiological 89

process control
7.4. Cultural dimensions, tactics and environmental 89

monitoring target behaviors
83
As described throughout this handbook, manufacturers regard environmental

an environmental monitoring program is monitoring programs as critical and
fundamentally a tool to measure and reflect invest the resources necessary to ensure
control. With renewed industry focus on effective execution. Once implemented,
the programs underpinning Hazard Analysis it is also vital that the programs evolve
and Critical Control Points (HACCP) and with the organization to result in ongoing,
a greater-than-ever understanding of the microbiological process control of facilities
important role environmental monitoring and to foster an effective and positive food
plays in delivering safe products to safety culture within the organization.
consumers, it is imperative that food
7.1. The path to microbiological

process control
How effectively an environmental Step 2. The effectiveness of barriers and
monitoring program is applied largely hurdles to entry and movement within the
defines a food manufacturer’s ability to exposed product area measure the control
attain microbiological process control of its of movement.
environment – and therefore in its finished
product. Step 3. Degree of microbiological
process control is evaluated by plotting
Microbiological process control is a three- collected data (variable and attribute) on
step process: control charts and calculation of statistical
capability indices.
(1) Eliminate the resident organisms of
concern from the processing environment. Environmental monitoring measures the
risk present in the processing environment
(2) Control movement by managing the and also assesses the hurdles established
vectors and pathways. to control entry of pathogens. This requires
process control, or indicator sites as well as
(3) Utilize process control methodology to verification sites to be sampled individually
measure and predict loss of control. and in conjunction with one another. These
results indicate the level of control in the
The concept of complete microbiological facility and help identify when failures
process control uses environmental occur or when interventions or additional
monitoring as a tool to measure the level of actions are required to bring the process
control being achieved. back into required levels of control.
Step 1. Elimination of the resident

organisms of concern is measured by
presence or absence in the verification,
indicator site and investigative sampling
programs. Obtaining negative results
from these sites over a long term is a key
indicator of elimination.
84
Process control (aggressive sampling

looking for positives)
Experience of the U.S. processed
• Indicator sites meat industry during their time of
ŔŔ Facility and equipment sanitary “Enlightenment” and the introduction
design concerns of sanitary design
ŔŔ Zone 4 to Zone 3 transfer pathways
(hurdles) In the late 1980’s and early 1990’s, the
ŔŔ Effectiveness of hygienic zoning United States processed meat industry
ŔŔ Post initial rinse was well aware of the hazards of Listeria
but was unaware of how to control it in
• Verification sites (indicates process the processing environment. Despite
control failure) their best efforts, corrections and
ŔŔ Zone 1 contact surfaces attempts to rid harborage sites or niches
ŔŔ Zone 2 and Zone 3 transfer following a positive environmental
pathways and vectors result were often ineffective. Thorough
cleaning and sanitization didn’t address
Importantly, microbiological process control the root cause and prevent the sites from
measures the conditions for growth, e.g. via routinely being recontaminated, keeping
ATP tests and Total Plate Count (TPC) as the industry at large stuck in a phase
well as transfer of the indicator organism. of awareness for a prolonged period of
The path to microbiological process control time.
is one of increasing maturity which can
typically be broken into five stages1: As it reached an eventual state of
Enlightenment, the industry experienced
Figure 1. Five stages of microbiological still further setbacks. The clean and
process control maturity sanitize approach that was being
employed following a positive result
created a frustrating dynamic of
1 Doubt “firefighting,” or solving the same
problem repeatedly with the same
results (Einstein’s definition of insanity).
2 Awareness
Only with the implementation of true
corrective actions in the form of new
3 Enlightenment sanitary design principles for equipment
were harborage sites and growth niches
reduced or eliminated.
4 Preventive
5 Predictive
85
Doubt • Increased sanitation efforts, costs and

The initial stage in the journey to labor (these remain until preventive
microbiological process control can often and predictive stages are attained).
be described as one of Doubt. In this stage, • Stress caused by the inability to clean
management often sees environmental the uncleanable.
monitoring as an unnecessary cost offering • Greater amounts of retained product,
no real benefit, typically pointing to the fact which brings with it more risk
that they have a HACCP program in place associated with loss of control, the
and believe that their facility is somehow potential of recalls, more involvement
different or better managed than others and efforts from top management,
and therefore environmental monitoring is need for more storage space and, all
not something that should apply to them. in all, unnecessary stress to the entire
system.
Awareness
The next stage after doubt is Awareness. In Preventive
this stage, the food manufacturer becomes The Preventive state exists when a known
aware of the potential of an environmental growth niche or harborage site can be
microbiological hazard, but is not aware of brought back to an acceptable sanitary
the root cause or source of the hazard, thus condition, e.g. harborage-negative
unable to control the hazard. growth conditions equal to or less than
preoperative upper specification limit.
Enlightenment
The stage of Enlightenment is reached The Preventive state is characterized by
when growth niches are eventually cleaning-out-of-place (COP) for all small
identified in a facility. The discovery of parts, maintenance tools and operator
these niches during this stage of maturity tools. All equipment in high-risk areas
is typically the result of investigations have validated interventions. Microbial
following more serious incidents, e.g., movement is minimized by effective good
positives showing up in end products or manufacturing processes (GMPs) and floor
upon discovery and testing of residues sanitizer. Additionally, a physical hygienic
during machinery disassembly. zone separation is accompanied by hurdles
at zone barriers.
The process of moving from Awareness to
Enlightenment is often one of significant Predictive
stress and tension within a facility as they The Predictive stage exists when a growth
begin to comprehend the situation and niche or harborage site can be managed
attempt to deal with the issue through with sampling and analysis of indicator site
corrective actions, almost universally being data. Out-of-control or out-of-specification
a process to clean and sanitize without indicator site results define when to apply
getting to the root cause (Table 1). the chosen intervention to manage the
contaminant.
This state of control can often be
associated with: Unfortunately, today’s technology does
not provide the ability to eliminate through
• Management and employee redesign all sanitary design issues that
frustrations associated with the could result in food safety or product
inability to solve chronic problems quality issues. Those that require control
(“The problems just do not go away.”) may be managed in a Preventive and
• Tension created between Predictive method by the use of indicator
departments. sites.
86
Table 1. Five stages of microbiological process control within plants1
Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Doubt Awareness Enlightenment Preventive Predictive
Early Preventive
phase positive
results are
dominated by
Expanded and indicator sites
regular sampling of such as post rinse. No contact
contact surfaces In final phase surface positives.
Contact and environmental of Preventive, Zone 4 positives
Sampling surface and sites. Intermittent only rare predominate.
Results product positives on contact surface Hurdle transfer
positives contact surfaces. positives. No point sampling
Routine positives product positives. produces rare
on environmental Investigative positives.
sites. facility-based
No testing or positives dominate
only testing the ready-to-eat
as required (RTE) processing
to meet area.
regulatory
requirements.
Unfortunately, Aggressive early
Potential growth
sampling warning sampling in
niches mapped.
is often Recognize place. Intervention
Some scheduled
conducted in a Sample existence of growth practices in
intervention
manner not to product. niches. Sample place with all
practices in
Control find Listeria. Recognition of contact surfaces RTE processing
place. Managing
Methods environmental and some floor equipment. Focus
“critical factors”
nature of and environmental on Zone 4 and
of the sanitation
Listeria. areas. Starting the facilities. Advanced
process. Engaged
redesign phase. phases of both
in equipment and
equipment and
facility redesign.
facility redesign.
Sample product, Sample product,

contact surfaces contact surfaces
Sample Sample product and
Verification and primary and transfer points
product contact surfaces.
transfer vectors in (Zones 1, 2, 3) in the
the RTE area. RTE area.
87
7.2. Benefits of microbiological

process control
Once microbiological control is obtained, • Production downtime caused by
the benefits include: positive results becomes rarer.
• The Predictive phase enables time-
Productivity gains consuming and equipment-stressing
interventions to be applied only when
• Order fulfillment becomes more necessary.
predictable. • Data collection is less expensive and
• Fewer problems are encountered statistical analysis is more easily and
during normal production. reliably applied.
• Plant performance exhibits greater • Insurance costs are reduced.
overall equipment effectiveness (OEE). • Food safety and quality professionals
• Process and product qualifications spend less time managing the
work in a systematic manner and sampling process.
provide data to validate. • Sampling costs are lower while more
• The Predictive phase enables time- sites are sampled for multiple reasons:
consuming and equipment-stressing ŔŔ Firefighting ceases and for-
interventions to be applied only when cause investigational sampling is
necessary. eliminated.
ŔŔ Indicator tests (e.g., TPC) become a
Risk mitigation larger portion of total testing.
• The Predictive phase predominately Continuous improvement

manages growth niches as opposed to
harborage sites. • Understanding of sanitary design
• Organizations gain a higher level of failure leads to improved sanitary
brand protection. design and reduced sanitation costs
• Facility- and equipment-based and labor.
positives are eliminated. • Plants can be more aggressive with
• The focus of control is on Zone 4 and indicator testing.
raw materials. • More consistent and predictable
quality and shelf-life is realized.
Direct cost reduction
• The sustainability and financial costs

associated with products that are
destroyed or diverted are reduced.
• Labor and overhead costs associated
with managing retained product,
and with managing the effects of
in-process testing, verification and
requalification, diminish.
88
7.3. Company culture and predictive

microbiological process control
Connecting microbial process control and an organization’s culture
The relationship between effective organizational, and food safety, culture
environmental monitoring programs and an is critical. It creates “line of sight” to the
organization’s culture is more significant corporate vision, principles and values
than most food safety practitioners and and subsequent team – and individual
business leaders realize. As such, much behaviors.
angst can spread throughout a food
company when positives are detected The Global Food Safety Initiative (GFSI)
through verification activities, especially has defined food safety culture as “A
in cultures at Doubt and Awareness stages company’s shared values, norms, and
(Table 1) where food safety activities beliefs that affect mindsets and behaviors
are largely completed by food safety toward food safety in, across, and
professionals. throughout the company”.2
Food safety in these stages is crisis As one looks at the descriptors for the
management-driven, with leaders stressing Predictive stage, they find a reliance
the importance of “doing things right” on Zone 4 and equipment – and facility
while conducting investigations that fail to design to eradicate and control organisms.
get to the root cause. The development of In other words, a culture that believes
such effect-driven behaviors that wait for a in keeping the organisms as far away
crisis to engage operations professionals is from food products and a mindset that
harmful to consumers, brands and overall investing in re-designing equipment and
company financial performance. infrastructure is an important and ongoing
activity. Organizations are wise to look
The separation of process control and introspectively at some of the cultural
verification enables celebration of process tactics that they can apply to create this
control positives and focus on prevention linkage and move towards a Predictive
rather than control via crisis. Linking stage for microbial process control.
environmental monitoring programs to
7.4. Cultural dimensions, tactics and

environmental monitoring target behaviors
Organizations cannot get to the Predictive It is important to note that no two cultures
stage without understanding the multi- are the same and that, not unlike the
dimensional aspect of a culture of food scientific expertise that many rely on to
safety. Based on the five dimensions to design effective environmental programs,
a culture of food safety3, an integrated experts might have to be engaged to help
set of tactics might help move a culture. build a plan specific to the organization and
Food manufacturers will find “target its needs.
behaviors” that, if tactics are implemented
effectively, should be consistently seen
from employees (Table 2).
89
Table 2. Cultural tactics and target behaviors
Cultural Environmental Monitoring Target

Tactic
Dimension Behaviors
Leaders of all functions actively ask questions

• Integrate environmental
about food safety and environmental
monitoring to company/
monitoring in strategy and budget discussions
Mission and plant/business strategic and
values operational cycle
Leaders of all functions integrate food safety
• Enable all leaders to message
and environmental monitoring messages in
their ongoing communications
All employees are expected to take company

food safety education as part of their role-
• Food safety education for
specific competencies
everyone: “Put a swab in
People
everybody’s hands…”
All environmental monitoring insights – good
• Multidisciplinary team
and bad – are investigated by teams from
multiple functions
Team leaders use indicator sites and positive

consequences (e.g., reward findings), resulting
Adaptability • Carrot vs. stick in problem prevention and continuous
improvement that builds trust in the food
safety process
Leaders design food safety and environmental

monitoring into the company rhythm (i.e.,
board discussions, leadership meetings, plant
• Communication rhythm
huddles and frontline team discussions)
• Insights driven by
Consistency
Environmental monitoring data are integrated
data
into the company business intelligence
solution and insights discussed from board
room to frontline
Technical team members generate ongoing

Risks and • Environmental monitoring
messages and stories for others to use in team
hazards pictures and stories
member onboarding and engagement

environmental monitoring 3M Food Safety expert
www.3M.com/ImprovedMonitoring www.3M.com/Connect/ImprovedMonitoring
90
References:
1. Butts, J. 2011. A Team Approach for Management of the Elements of a Listeria Intervention
and Control Program. Agric. Food Anal. Bacteriol. 1:6-14.
2. Global Food Safety Initiative. 2018. A Culture of Food Safety: A Position Pager from the
Global Food Safety Initiative (GFSI). Version 1.0 – 4/11/18. https://www.mygfsi.com/news-
resources/news/news-blog/1419-a-culture-of-food-safety.html
3. Jespersen, L., Griffiths, M., Wallace, C.A. 2017. Comparative analysis of existing food safety
culture evaluation. Food Control. 79: 371-379. https://doi.org/10.1016/j.foodcont.2017.03.037
91
Environmental Monitoring Handbook - Sampling Guidance
CHAPTER 8
Environmental Sampling
Guidance
By
Scott Egan | 3M Food Safety
8.1. Sampling neutralizers 93
8.2. Selection of sampling device 97
8.3. Sampling methods 98
92
8.1. Sampling neutralizers

Sampling from food processing merely maintain the population at or similar
environments can present several to the level at the time of sampling.
challenges. Trying to obtain meaningful
results that accurately reflect the level of These aspects can often be overlooked not
microbial contamination on a surface is only during initial selection of a neutralizer,
no small chore. One such challenge is the but also when sanitizers, test methods or
presence of sanitizers that may continue sampling regimes change.
to have bactericidal or bacteriostatic
activity after the sampling event. This Most commercially available swabs and
continued activity can reduce the microbial sponges will incorporate a combination
population within the sample prior to of neutralizers as part of standard or
detection or enumeration taking place proprietary formulations. The most
(e.g., during transportation) or inhibit an common neutralizing or sampling liquids
organism’s growth on the culture media and their various levels of effectiveness are
used in the actual testing process. This summarized in this chapter’s table content.
can ultimately result in reduced counts In the case of proprietary formulations,
for quantitative methods or negative the manufacturer should be contacted
results for qualitative methods and thus to obtain information on the components
not truly represent the risks present in the or the sanitizers it has been shown to be
production environment. effective against.
To overcome this challenge, sample Letheen Broth is commonly used

collection devices such as swabs or for environmental sampling in the
sponges should incorporate components food, nutraceutical, cosmetic and
that are effectively able to neutralize pharmaceutical industries.1,2 It has
any sanitizers present. The selection of a neutralizing capability with iodine,
neutralizer (or combination of neutralizers) quaternary ammonium compounds
should be undertaken with knowledge of and chlorine sanitizers. However, it
the types of sanitizers used within a facility, has no ability to neutralize mercurials,
as not all neutralizers/combinations are formaldehyde or glutaraldehyde, so once
equally effective against different types of again the sanitizer being used must be
sanitizers. taken into consideration.
Two other important aspects to consider Additionally, Letheen Broth has some
when selecting neutralizers are: enrichment capabilities, so the surface
compatibility with the test method to be should be resanitized after sample
used, and whether the method is qualitative collection.
or quantitative. If quantification is the
aim, the neutralizers selected should not
support the growth of the organisms, but
93
Table 1. Composition of Letheen Broth
Composition: (typical formula g/L)
Enzymatic Digest of Animal Tissues 10.0 g
Beef Extract 5.0 g
Polysorbate 80 5.0 g
Sodium Chloride 5.0 g
Lecithin 0.7 g
D/E Neutralizing Buffer was developed an indicator dye and has enrichment
by Dey and Engley to neutralize a properties. Its broad neutralizing
broad spectrum of disinfectants and capabilities may be more than required
preservative antimicrobial chemicals. since few food processing plants sanitize
It was designed for testing the with the toxic agents such as mercurials,
efficacy of disinfectants rather than formaldehyde or gluteraldehye.3,4 Because
for environmental sampling. Although it contains an indicator dye and has
it counteracts the biocidal activity of enrichment capabilities, a surface must be
all the main sanitizers, it also contains resanitized after sample collection.
Table 2. Composition of D/E Neutralizing Buffer
Enzymatic Digest of Casein 5.0 g
Yeast Extract 2.5 g
Polysorbate 80 5.0 g
Dextrose 10.0 g
Lecithin 7.0 g
Sodium Thioglycollate 1.0 g
Sodium Thiosulfate 6.0 g
Sodium Bisulfite 2.5 g
Bromcresol Purple 0.02 g
94
Neutralizing Buffer, often thought to be in the food industry due to their toxicity).
a generic term, is actually a specified It has the advantage of containing no
formulation commonly used in industry enrichment agents, so re-sanitizing the
for Listeria, Total Plate Count, Salmonella, sampling site after sample collection is
E. coli and other types of testing. 2,4 It not necessary. Note that this formulation
does not effectively neutralize phenolic, contains aryl sulfonate complex and may
mercurial, formaldehyde or glutaraldehyde require dilution of the sample prior to
sanitizers (although these are uncommon testing with a molecular-based method.
Table 3. Composition of Neutralizing Buffer
Aryl Sulfonate Complex 5.0 g
Sodium Thiosulfate 0.16 g
Potassium Phosphate, monobasic 0.0425 g
Buffered Peptone Water (BPW) is often capability. Note that Buffered Peptone
used in abattoirs to collect samples from Water is an enrichment broth, so if used
carcasses as directed by regulations. It for environmental sampling, the surface
is not recommended for use on sanitized should be resanitized after sample
surfaces as it has minimal neutralizing collection.5
Table 4. Composition of Buffered Peptone Water
Peptone 5.0 g
Sodium Phosphate, dibasic 0.16 g
Sodium Chloride 0.0425 g
Potassium Phosphate, monobasic 1.5 g
It should be noted that the effectiveness that specific neutralizing media may or
of different, common neutralizing media may not neutralize specific disinfectants
against common sanitizers can vary and (Table 5).
95
Table 5. Effectiveness of common neutralizing media against common sanitizers2
Letheen D/E Neutralizing Neutralizing Buffered

Sanitizer
Broth Buffer Buffer Peptone Water
Quarternary
Ammonium Yes Yes Yes No
Compounds
Phenols Yes Yes No No
Iodine & Chlorine Yes 6,7

Yes Yes No
Mercurials* No Yes No No
Formaldehyde* No Yes No No
Glutaraldehyde* No Yes No No
Peroxyacetic acid
and Hydrogen Some6,7 Yes8,9 No No
peroxide
Acids Yes6,7 Yes8,9 No No
*Not commonly used in the food industry due to their toxicity
EN 1650 Annex B10 can also be referred Any remaining enrichment broth or
to for examples of neutralizers of residual neutralizing solution residue should be
disinfectants. The effectiveness of removed from the sampled surface after
any disinfectant neutralizer should be sample collection according to user-
validated under real use conditions. established procedures.
96
8.2. Selection of sampling device

Unless defined by specific regulations, the Sponges should be free of inhibitory
primary decision to be made should be the substances. Typical household sponges
type of device (sponge or swab) to be used. are not recommended for environmental
The key things to consider when choosing sampling, as they may contain biocides
the device are what the size of the area which would inhibit microbial growth.
being sampled is, whether the area is readily
accessible and which type of testing will Swabs are smaller sampling devices
be conducted on the sample. An effective consisting of a tip or bud for collecting the
environmental monitoring program will use sample attached to a long flexible stem.
a combination of sponges and swabs. Because of their smaller size, they are better
suited for sampling in hard-to-reach places
Sponges are larger sampling devices and and are typically used for areas of 100
are available in a variety of formats, from square centimeters or less.5,11
individual sterilized portions to portions
attached to a handle to aid aseptic handling. Due to their smaller size and ease-of-use
for sampling a defined area, swabs can
Sponges are preferred if qualitative be particularly useful for quantitative
pathogen testing is to be conducted, as environmental testing (e.g. for indicator
they can be used to sample a larger area, organisms). This is important because the
therefore increasing the likelihood of defined area will be used in the calculation
detection. The area sampled should be of results.
greater than 100 square centimeters (15.5
square inches) and preferably greater than The material used is typically synthetic
or equal to 1,000 square centimeters (155 such as alginate, Dacron or rayon. However,
square inches).5,11 However, in many cases, cotton is also sometimes used. 1,5 Evidence
particularly when swabbing for detection of should also be obtained, either through
pathogens or index organisms (e.g., Listeria supplier documentation or product
spp.), sampling of areas of a specific size validation/verification, that the chosen
is not appropriate or feasible as locations device does not have any bacteriostatic or
likely to harbor pathogens do not represent bactericidal activity.
areas that can be easily assessed (e.g.,
long cracks in floors). In these cases it is Additional consideration should be
important to sample as large an area as given to the quality, strength and type
possible (e.g., multiple meters or yards of a of materials used, as fragments of the
floor crack). device may separate, leading to foreign
object contamination of the facility and the
The material used in the manufacture potential implications. Additional features
of sponges is most commonly cellulose such as blue-colored designs and metal
or polyurethane.11,12 Various studies have detectability may also be of benefit.
looked at the different efficacies of each of
these materials for their ability to collect and
allow improved detection rates. However,
these studies have generally shown no
significant difference.13,14
97
8.3. Sampling methods

The sampling methods employed will surface contact as possible to maximize
vary depending on the type of device the likelihood of detection.
being used and the testing intended to be
conducted. Quantitative sampling may require more
care to be taken. For example, if the test
Moisture is one of the most important result is expressed in CFU/cm2, a specific
factors for bacterial survival on surfaces. sample area size is typically defined and
Therefore, regardless of the device or adhered to. Sampling templates can assist
intended testing, it is recommended to with sampling a defined area, but caution
sample from a moistened surface or with should be taken as their use can lead to
a moistened collection device to improve cross contamination.
recovery.13
It would also not be uncommon, even for
A noteworthy exception can be sampling quantitative sampling, to target an area of
of dry environments where introduction undefined size. For example, testing for
of moisture may be undesirable, as it Total Plate Count may be used to assess
enhances the risk of microbial growth. the efficacy of sanitation on difficult
In these cases, specialized tools (e.g., to reach areas, in which case it may be
spatulas, spoons, scoops) may be be used impossible to sample a defined area. For
to collect dry materials and dust from the these unmeasured surface areas, the
environment. results may be reported based on the
entire sampling site instead of the surface
It is also important to only sample a area measured.
single item or area with each device. This
prevents cross-contamination between When environmental samples are taken, it
items or areas in the facility. is critical that proper aseptic technique is
used to prevent inadvertent contamination
Pathogen sampling should have the of the sample. It is recommended to wash
general intention of sampling as much or sanitize hands prior to opening the
surface area as possible to improve sampling device. Each type of sampling
the likelihood of detection. Although device also has particular techniques
regulations may specify the size of that should be followed along with any
sampling area, these can typically be additional guidance from manufacturers if
taken as minimum sizes. As discussed in using proprietary swab designs.
Chapter 4, training materials often cite
specific sampling areas (12 by 12 inches, Swabs (Figure 1) should be aseptically
or 30 by 30 centimeters, for examples), removed from their container and
but many surface areas are not square or particular attention should be given not to
flat enough to accommodate such surface touch the bud or any area of the stem that
area. will be returned into the container. During
removal, the swab tip should be pressed
If sampling sites are not easily accessible, against the container to remove excess
a swab may be more suitable. Again, the liquid.
intention should be to obtain as much
98
Where possible, particularly for easily of the device that will be inserted back
acceptable areas sampled for quantitative into the container.
analysis, multiple directions should be
used when sampling and the swab should The sponge should be wiped over the
be rotated between thumb and forefinger. sampling surface using firm and even
After swabbing the first direction, the pressure. This will help to dislodge
swab should be returned to the container organisms that may be protected by
and rinsed in the neutralizing solution biofilm. After sampling in one direction,
to remove collected organisms and the sponge should be turned over and
re-moisten the tip. The same procedure used to swab in a perpendicular direction.
should then be repeated in two other The sponge should then be placed in its
directions. The swab is then sealed in its container, aseptically taking care not to
container for transportation. insert any portion that is not part of the
sample (e.g., the handles of some devices).
Sponges (Figure 2) should be aseptically The container should then be sealed for
removed from their container using sterile transport.
gloves or forceps, or by manipulating
the container to access the handle of After any sampling has taken place,
the device. Care should be taken not to surfaces should be cleaned of any
contaminate the sponge or any other part neutralizing solution and resanitized.
Figure 1. Example of sampling technique using 3MTM Swab-Sampler
1 2 3 4
5 6
99
Figure 2. Example of sampling technique using 3MTM Sponge-Stick
Sample transport is the final step in the Upon receipt at the laboratory, the
environmental sampling process and, internal temperature of the cooler
again, particular attention must be given should be verified using a thermometer. 2
to some aspects. Samples should be Additionally, samples should not
delivered for analysis at refrigerated be allowed to freeze under any
temperature as soon as possible, circumstances, as sub-zero temperatures
preferably within 24 hours as detailed in may kill or injure the microbes present.
ISO 18593:2004.
If it is not possible to perform sample
Containers used for transportation should analysis within the recommended time
be clean and sanitized. They should frame or transport samples appropriately,
include ice packs and be able to maintain alternatives should be developed and
refrigeration temperature for the duration validated accordingly to ensure it does not
of transportation. undermine the sensitivity of the method.4

sample collection 3M Food Safety expert
www.3M.com/SurfaceSampling www.3M.com/Connect/SurfaceSampling
100
References:
2. The Compendium of Analytical Methods. Volume 3. Laboratory Procedures for the

Microbiological Analysis of Foods. 2010. MFLP-41: Environmental sampling for the detection
of microorganisms. Health Canada.
3. United States Food and Drug Administration. 2015. Testing Methodology for Listeria
species or L. monocytogenes in Environmental Samples. Version 1. https://www.fda.gov/
Food/FoodScienceResearch/LaboratoryMethods/ucm114664.htm
4. United States Department of Agriculture Food Safety and Inspection Service. 2014. FSIS
Compliance Guideline: Controlling Listeria monocytogenes in Post-lethality Exposed Ready-
To-Eat Meat and Poultry Products. https://www.fsis.usda.gov/wps/wcm/connect/d3373299-
50e6-47d6-a577-e74a1e549fde/Controlling-Lm-RTE-Guideline.pdf?MOD=AJPERES
5. International Organization for Standardization. 2018. ISO 18593:2018. Microbiology of the

food chain - Horizontal methods for surface sampling.
6. 3M Food Safety. 1985. Letheen Broth: A Neutralizing Solution for Iodine, Chlorine,
Quaternary Ammonium and Acid Sanitizers (Internal Data).
7. 3M Food Safety. 2012. Sample Handling Sponges – New Sponge Qualification

Performance Summary. TB.119.00.
8. Ignatovich, I., Podtburg, T., Leishman, O., Steinagel, S. 2017. Comparison of Neutralizing
Buffered Peptone Water and Dey/Engley Broth in the Recovery of Salmonella enterica from
Broiler Carcass Rinsates. J Food Protection. 80 (Supplement A): 163.
9. Park, Y.J., Chen, J. 2011. Mitigating the Antimicrobial Activities of Selected Organic Acids
and Commercial Sanitizers with Various Neutralizing Agents. J Food Protection. 74: 820-
825. https://doi.org/10.4315/0362-028X.JFP-10-447
10. EN 1650:2008. Chemical disinfectants and antiseptics – Quantitative suspension test

for the evaluation of fungicidal activity of chemical disinfectants and antiseptics in food,
industrial, domestic, and industrial areas - Test method and requirements (phase 2 step 1).
11. United States Food and Drug Administration. 2017. Chapter 10: Detection of Listeria
monocytogenes in Foods and Environmental Samples, and Enumeration of Listeria
monocytogenes in Foods. Bacteriological Analytical Manual. https://www.fda.gov/Food/
FoodScienceResearch/LaboratoryMethods/ucm071400.htm
101
12. United States Department of Agriculture Food Safety and Inspection Service.
Microbiology Laboratory Guidebook. 2017. Method 8.10: Isolation and Identification
of Listeria monocytogenes from Red Meat, Poultry and Egg Products, Ready-To-Eat
Siluriformes (Fish) and Environmental Samples. https://www.fsis.usda.gov/wps/portal/
fsis/topics/science/laboratories-and-procedures/guidebooks-and-methods/microbiology-
laboratory-guidebook/microbiology-laboratory-guidebook
13. Keeratipibul, S., Laovittayanurak, T., Pornruangsarp, O., Chaturongkasumrit, Y.,

Takahashi, H., Techaruvichit, P. 2017. Effect of swabbing techniques on the efficiency
of bacterial recovery from food contact surfaces. Food Control. 77:139-144. https://doi.
org/10.1016/j.foodcont.2017.02.013
14. Sheth, I., Li, F., Hur. M., Laasri, A., De Jesus, A. J., Kwon, H. J., Macarisin, D., Hammack,
T., Jinneman, K, Chen, Y. 2018. Comparison of three enrichment schemes for the detection
of low levels of desiccation-stressed Listeria spp. from select environmental surfaces. Food
Control. 84:493-498. https://doi.org/10.1016/j.foodcont.2017.08.022
102
Environmental Monitoring Handbook - About the Contributors
About the
Contributors
Alexandra Belias
Cornell University
Alexandra Belias is a graduate student working toward
a doctoral degree in food science at Cornell University’s
College of Agriculture and Life Sciences. Her current
work is focused on projects associated with tracking
pathogen contamination of produce. Belias received
a Bachelor of Science in food science from Purdue
University.
Christian Blyth
3M Food Safety, Canada
Christian Blyth is a pathogen sales and technical
specialist at 3M Food Safety, based in the company’s
Canada office for more than a decade. A regulatory
expert on food safety, Blyth often speaks about
food safety topics involving pathogens at industry
conferences. His bachelor’s degree in biology was
received from the University of Waterloo in Ontario.
John Butts, Ph.D.

Food Safety By Design
Dr. John Butts is the founder and president of Food
Safety By Design, a private consulting firm that helps
producers of high-risk products learn how to better
prevent and manage food safety risks. Butts is also the
advisor to the CEO of Land O’ Frost, and has served in a
technical role at Land O’ Frost since 1974. He received a
doctorate degree from Purdue University in 1974.
103
Ken Davenport, Ph.D.

3M Food Safety
Dr. Ken Davenport, based in St. Paul, Minn., is the
product development manager at 3M Food Safety. With
over 15 years of experience in the food industry and 20
years of experience with rapid method development
and applications, Davenport has a broad and deep
background in both chemical and microbial food safety.
He earned his bachelor’s degree in chemistry from
Spring Arbor University, Ph.D. from the Department of
Biochemistry at Rice University, and an M.B.A. from the
University of Minnesota.
Jean-Francois David
3M Food Safety, Europe
Jean-Francois David is the scientific advisor to Europe
for 3M Food Safety. Based in France and supporting
3M’s customer implementation of new products
and technologies, David has more than 20 years of
experience in food safety. He received his schooling from
the Brest Graduate School of Microbiology and Food
Security (ESMISAB) in Brest, France.
Michele Fontanot
3M Food Safety, Latin America
Michele Fontanot is a professional consultant services
manager for Latin America at 3M Food Safety. Based in
Peru, Fontanot leverages her expertise on food safety to
assist customers in the region working to implement 3M
products and technologies in ways that effectively meet
their processing and manufacturing needs aligned to
international regulations and standards. She has earned
a bachelor’s degree from Cayetano Heredia University
in Lima and a Master of Science from the University of
Chile in Santiago.
104
Thomas Grace
Bia Diagnostics
Thomas Grace is CEO of Bia Diagnostics, one the
leading food allergen testing laboratories in North
America. Grace holds an honorary position in the
Department of Pharmacology at the University of
Vermont Medical School. He has worked in various
fields from cancer research at Dartmouth College
and the University of Vermont Medical Schools, to
developing cutting-edge methodologies in microarray
and SPR technologies. Grace has co-authored
numerous scientific papers, ranging from cellular
signal transduction and oncogene regulation to SPR
application in quantification of folic acid in foods.
Lone Jespersen, Ph.D.

Cultivate
Dr. Lone Jespersen is principal and founder of Cultivate,
an organization dedicated to helping global food
manufacturers make safe, great-tasting food through
cultural effectiveness. She has significant experience
with food manufacturing, having previously spent 11
years with Maple Leaf Foods leading the execution
of the company’s food safety strategy and operations
learning strategy. Dr. Jespersen holds a Master’s degree
in mechanical engineering from Syd Dansk University,
Denmark, a Master of Science in food science from the
University of Guelph, Canada and a Ph.D. in culture-
enabled food safety with Dr. Mansel Griffiths at the
University of Guelph, Canada.
Gareth Lang
3M Food Safety, Europe
Gareth Lang is the professional services manager of
Western Europe for 3M Food Safety. With experience
in a variety of technical and professional microbiological
service roles, he trains and supports a wide range of
food and beverage customers and distributors, working
closely with their technical teams to address immediate
needs and understand emerging issues that aid in the
development of new products and services that improve
food safety control. Lang studied medical laboratory
sciences at Cardiff University, specializing in medical
microbiology.
105
Cari Lingle
3M Food Safety
Cari Lingle is a global technical service microbiologist
for at 3M Food Safety specializing in 3M™ Petrifilm™
Plate products. Located in St. Paul, Minn., she provides
expert-level technical support and education for 3M Food
Safety’s sales, marketing and manufacturing teams, as
well as local technical and professional services. Lingle
received a Bachelor of Science degree in microbiology
from North Dakota State University and a Master of
Science in cellular and molecular biology from the
University of Missouri-Kansas City.
Gabriela Lopez Velasco, Ph.D.

3M Food Safety
Dr. Gabriela Lopez Velasco, based in St. Paul, Minn., has
been a senior microbiologist at 3M Food Safety since
2015. She works with the global technical services and
application engineer teams leveraging her expertise in
food safety to provide technical training and application
of the 3M Food Safety portfolio. She currently oversees
the 3M™ Allergen Testing platform. She received her
doctorate degree in food science and technology from
Virginia Polytechnic Institute and State University and
worked as a postdoctoral researcher at the University of
California, Davis.
Louise Roberts
Alimenti Food Sciences Ltd
Louise Roberts is the founder and managing director
of Alimenti Food Sciences Ltd, an independent
food technical consultancy. With a background in
microbiology, technical management and supply chain
management, Roberts has worked in the manufacturing,
retail and food service aspects of the food supply chain.
Her professional interests focus on food safety and
compliance as well as food fraud and how businesses
can protect themselves. She studied applied biology
at Plymouth Polytechnic and has an honours degree in
microbiology from Polytechnic of North East London.
She also holds a post-graduate certificate of education
from the University of Worcester and is a Chartered
Scientist.
106
Abigail Snyder, Ph.D.

The Ohio State University
Dr. Abigail Snyder is an assistant professor at The
Ohio State University’s College of Food, Agricultural,
and Environmental Sciences. Her research focuses
on characterization of spoilage microorganisms and
evaluation of methods appropriate for their control.
Snyder also works with industry through OSU Extension
to provide training and technical support. She
completed her doctorate degree at Cornell University.
Kelly Stevens, Ph.D.

General Mills
Dr. Kelly Stevens is the senior manager for the Global
Operations Food Safety and Regulatory team at General
Mills. She is accountable for global food safety and
regulatory compliance, is a FSPCA Trainer of Trainers
and served as General Mills’ FSMA implementation
team lead. Stevens joined General Mills in 2004 and has
held varying roles in the quality organization, including
three field assignments: quality engineering and quality
manager, manager of microbiology and quality manager
of premiums, licensing and sampling activities. She
received M.S. and Ph.D. degrees in food science from
North Carolina State University.
Genevieve Sullivan
Cornell University
Genevieve Sullivan is a graduate student working toward
a doctoral degree in food science at Cornell University.
Her current project involves the development of Listeria
control strategies for produce facilities by designing,
implementing, and evaluating Listeria pathogen
environmental monitoring (PEM) and “seek and
destroy” programs, utilizing data from whole genome
sequencing. Sullivan received a Bachelor of Science in
food science from Cornell University.
107
Martin Wiedmann, Dr. Med.

Vet, Ph.D.
Cornell University
Dr. Martin Wiedmann is the Gellert Family Professor in
Food Safety at Cornell University’s College of Agriculture
and Life Sciences. With training as both a veterinarian
and food scientist, Wiedmann’s academic programs
emphasize a comprehensive and interdisciplinary farm-
to-table approach to food safety. His research focuses
on the transmission and systems biology of bacterial
foodborne pathogens and spoilage organisms, and
involves the application of a variety of disciplines and
collaboration across multiple institutions. He received his
veterinary degree and doctorate in veterinary medicine
from the Ludwig Maximilian University of Munich, and his
Ph.D. in food science from Cornell University.
Randy Worobo, Ph.D.

Cornell University
Dr. Randy Worobo is a professor of food microbiology
at the Cornell University College of Agriculture and
Life Sciences. The primary objective of his research
is alternative approaches to enhance the safety and
quality of food, and includes microbial spoilage of foods
and beverages, non-thermal processing alternatives for
juice and beverages, as well as pathogen transmission
and survival on fruits and vegetables. Worobo also
engages with the industry through his extensive
outreach program, which is accomplished through
workshops, conferences and direct contact with food
processors around the world. He received his Ph.D.
from the University of Alberta.
Burcu Yordem, Ph.D.

3M Food Safety
Dr. Burcu Yordem is a senior global technical service
scientist at 3M Food Safety overseeing technologies
such as the 3M™ Clean-Trace™ Hygiene Monitoring
and Management System. Located in St. Paul, Minn.,
she provides expert-level global technical support for
3M Food Safety’s customers, as well as sales, marketing
and manufacturing teams. Yordem received a doctorate
degree in crop molecular genetics from the University
of Massachusetts, Amherst and held a postdoctoral
position at USDA-ARS Cereal Disease Laboratory.
108
EDITOR
John David
3M Food Safety
John David is the global scientific marketing manager

at 3M Food Safety, based in St. Paul, Minn. David
leads global customer education initiatives and the
creation of impactful scientific content in partnership
with industry experts. He has expertise in molecular
biology and microbiology, and has held previous roles
in diagnostic assay development, systems integration
and new product commercialization. David earned both
a Bachelor of Science in biotechnology and a Master
of Science in molecular biology and genetics at the
University of Delaware.
EDITOR
Scott Egan
3M Food Safety, Australia
Scott Egan is the technical specialist for 3M Food Safety

Australia/New Zealand. In this role, he keeps abreast
of local regulations, emerging trends and industry
best practices to help support the local industry while
also providing expert support for 3M’s portfolio of
food safety products. His undergraduate degree in
biomedical science was received from the University
of Western Sydney, and he has previously worked in
pathology and industrial microbiology laboratories
and held quality control, research and development
and process improvement roles for the manufacture of
diagnostic assays.
109
The 3M Environmental Monitoring Handbook is intended to provide general guidance only. The technical
information, recommendations and other statements contained in this document are based on experience
and information that 3M believes to be reliable, but the accuracy or completeness of such information is
not guaranteed. Such information is intended for persons with knowledge and technical skills sufficient to
assess and apply their own informed judgement to the information, taking into consideration the nature of
their business, existing policies and particular laws and regulations that might apply.
Learn more about environmental monitoring at www.3M.com/EnvironmentalMonitoring
3M Food Safety 3M Canada ©3M 2019. All rights reserved.

3M Center, Building 275-5W-05 Post Office Box 5757 3M, Clean-Trace and Petrifilm are
St. Paul, MN 55144-1000 USA London, Ontario N6A 4T1 Canada trademarks of 3M. Used under license
Phone 1-800-328-6553 in Canada
3M.com/foodsafety Phone 1-800-364-3577 Please recycle. Printed in U.S.A.
70-2011-5169-6

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Environmental

Key Terms and Definitions iii

The Importance of Environmental Sampling 1

ATP and Protein-based Hygiene Monitoring 11

Environmental Monitoring for Indicator Organisms 29

Environmental Monitoring for Pathogens 41

TABLE OF CONTENTS (Cont.)

Environmental Monitoring for Spoilage Organisms 59

Environmental Monitoring for Allergens 73

Driving Meaningful Change in Your Organization 85

Environmental Sampling Guidance 95

About the Contributors 107

Key Terms and Definitions

Microbiota The population of microorganisms found in a specific environment.

An action to eliminate a detected nonconformity. These can be immediate activities to

An action to eliminate the cause of a detected nonconformity or other undesirable situation,

The (often-unsuccessful) approach of repeatedly attempting the same solution on a recurring

Methods, processes, preservations and technologies used in combination to ensure that

An organism or group of organisms whose presence relates to the possible occurrence of

An organism or group of organisms whose presence reflects the general microbiological

An action to eliminate the cause of a potential non-conformity or other undesirable situation

Qualitative test A test that determines presence or absence of an analyte(s) in a sample.

The reading of the amount of light as determined by an individual ATP-based hygiene

A multi-faceted systematic approach to finding sites of persistent strains (niches) in food

The “seek and destroy” process can help to:

Providing scientific evidence that a strategy controls a given hazard. Environmental

3. International Organization for Standardization. 2015. ISO 900:2015. Quality management

5. Codex Alimentarius Commission. 2003. General Principles of Food Hygiene. CAC/RCP

6. Dairy Food Safety Victoria. 2016. Dairy Pathogen Manual. http://www.dairysafe.vic.gov.au/

8. United Fresh Produce Association. 2013. Guidance on Environmental Monitoring and

The Importance of Environmental

1.1. Increasing recognition of the food processing environment 2

1.2. Importance of identifying specific purposes and goals for 5

1.3. Target analytes for environmental monitoring programs 7

1.4. Importance of coordination and integration of 7

1.5. The business needs for environmental monitoring 8

1.1. Increasing recognition of the

Facilities and Prevention

Despite the value of HACCP-based food

With the processing facilities’ association

contamination of a ready-to-eat food with This provision demonstrates the growing

1.2. Importance of identifying specific

While there does not appear to be a Importantly, verification may include

application could be sufficient to verify preventive control), with subsequent

Figure 2. A HACCP-informed approach to environmental monitoring

Identify non-process preventive control strategies needed to control each hazard

Determine the limit required to control the hazard

Develop an enviromental monitoring program (with critical limits) and set a

Determine how to respond if a critical limit is exceeded (for example, re-cleaning

Use environmental monitoring to verify that the non-process preventive controls

Maintain records on the validation, monitoring, verification and corrective actions

1.3. Target analytes for environmental

1.4. Importance of coordination and integration

Coordinating and integrating different consistent designation of sampling sites

1.5. The business

Reduced risks of spoilage issues and

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ATP and Protein-based

2.1. Purpose of ATP or protein-based hygiene monitoring 12

2.2. Principle of the methods 12