CHAPTER 8

Control of Microbial Growth

To avoid the spread of human disease, it is essential to manage the growth and amount of
microbes in or on different products often used by humans. Toys, towels, and doorknobs are
examples of foomites—inanimate objects that can harbor bacteria and convey disease. Two factors
have a significant impact on the level of cleanliness required for a particular fomite and,
subsequently, the approach chosen to achieve this level. First and foremost is the use that the object
will be put to. For instance, programs that need to be inserted into people's bodies must be far more
hygienic than applications that do not. The level of antibiotic resistance exhibited by possible
infections is the second consideration. For instance, the bacterium Clostridium botulinum, which
creates the neurotoxin that causes botulism, frequently contaminates foods that have been canned.
Due to the ability of C. botulinum to create endospores, extreme temperatures and pressures must
be used to eradicate the endospores. Other species might not need to take such drastic steps and
can be controlled by a process, such as using a washing machine to wash clothes.

Sterilization
Sterilization is the ultimate goal of the most stringent microbial control techniques; it
involves eliminating or destroying all vegetative cells, endospores, and viruses from the targeted
object or environment. In environments such as laboratories, hospitals, manufacturing plants, and
the food business, where it may be essential for specific things to be fully free of potentially
infectious pathogens, sterilization techniques are typically used. Sterilization can be achieved by
physical methods, such as chemical methods, pressure, high heat exposure, filtering through a
suitable filter, etc.

Sterilants are substances that can be used to sterilize objects. Sterilants efficiently eliminate
all bacteria, viruses, and, with the right amount of exposure time, endospores as well.

Aseptic approach is essential for many therapeutic applications to avoid contaminating

sterile surfaces. A variety of protocols are used in aseptic technique to maintain sterility, or asepsis
to avoid exposing the patient to infectious diseases and germs. Many different clinical procedures

FC 115 / N. S. SOLIDUM, MSc. CapSU

may introduce germs into the patient's body if aseptic technique is not used. This increases the
patient's chance of developing sepsis, a systemic inflammatory disease response to an infection
that causes a high temperature, faster heartbeat and breathing, shock, and maybe even death. A
sterile field is a specified area that is kept free of all vegetative microorganisms, endospores, and
viruses so that medical treatments that run the risk of contamination can be carried out there. Sterile
fields are created according to protocols requiring the use of sterilized materials, such as packaging
and drapings, and strict procedures for washing and application of sterilants. Other procedures are
adhered to in order to keep the area sterile while the medical operation is being carried out.
Commercial sterilization is one method of food preservation that uses heat at a temperature
low enough to maintain food quality while destroying common microorganisms that are
responsible for food toxicity like C. botulinum. When endospores are present in soil, they can
easily contaminate crops after harvest, and they can later germinate in the anaerobic atmosphere.
Metal food cans contaminated C. botulinum will bulge due to the microbe’s production of gases;
contaminated jars of food typically bulge at the metal lid. To eliminate the risk for C. botulinum
contamination, commercial food-canning protocols are designed with a large margin of error. They
assume an impossibly large population of endospores (1012 per can) and aim to reduce this
population to 1 endospore per can to ensure the safety of canned foods. For example, low- and
medium-acid foods are heated to 121 °C for a minimum of 2.52 minutes, which is the time it would
take to reduce a population of 1012 endospores per can down to 1 endospore at this temperature.
Even so, commercial sterilization does not eliminate the presence of all microbes; rather, it targets
those pathogens that cause spoilage and foodborne diseases, while allowing many non-pathogenic
organisms to survive. Therefore, “sterilization” is somewhat of a misnomer in this context, and
commercial sterilization may be more accurately described as “quasi-sterilization.”
Other Methods of Control
Sterilization protocols require procedures that are not practical, or necessary, in many
settings. Various other methods are used in clinical and nonclinical settings to reduce the microbial
load on items. Although the terms for these methods are often used interchangeably, there are
important distinctions (Fig.8.0).
FC 115 / N. S. SOLIDUM, MSc. CapSU
 The process of disinfection inactivates most microbes on the surface of a fomite by using
antimicrobial chemicals or heat. Because some microbes remain, the disinfected item is not
considered sterile. Ideally, disinfectants should be fast acting, stable, easy to prepare, inexpensive,
and easy to use. An example of a natural disinfectant is vinegar; its acidity kills most microbes.
Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to clean
nonliving surfaces such as laboratory benches, clinical surfaces, and bathroom sinks. Typical
disinfection does not lead to sterilization because endospores tend to survive even when all
vegetative cells have been killed.

Unlike disinfectants, antiseptics are antimicrobial chemicals safe for use on living skin or
tissues. Examples of antiseptics include hydrogen peroxide and isopropyl alcohol. The process of
applying an antiseptic is called antisepsis. In addition to the characteristics of a good disinfectant,
antiseptics must also be selectively effective against microorganisms and able to penetrate tissue
deeply without causing tissue damage. The type of protocol required to achieve the desired level
of cleanliness depends on the particular item to be cleaned. For example, those used clinically are
categorized as critical, semicritical, and noncritical. Critical items must be sterile because they will
be used inside the body, often penetrating sterile tissues or the bloodstream; examples of critical
items include surgical instruments, catheters, and intravenous fluids. Gastrointestinal endoscopes
and various types of equipment for respiratory therapies are examples of semicritical items; they
may contact mucous membranes or nonintact skin but do not penetrate tissues. Semicritical items
do not typically need to be sterilized but do require a high level of disinfection. Items that may
contact but not penetrate intact skin are noncritical items; examples are bed linens, furniture,
crutches, stethoscopes, and blood pressure cuffs. These articles need to be clean but not highly
disinfected. The act of hand washing is an example of degerming, in which microbial numbers are
significantly reduced by gently scrubbing living tissue, most commonly skin, with a mild chemical
(e.g., soap) to avoid the transmission of pathogenic microbes. Wiping the skin with an alcohol
swab at an injection site is another example of degerming. These degerming methods remove most
(but not all) microbes from the skin’s surface.

FC 115 / N. S. SOLIDUM, MSc. CapSU

 The term sanitization refers to the cleansing of fomites to remove enough microbes to
achieve levels deemed safe for public health. For example, commercial dishwashers used in the
food service industry typically use very hot water and air for washing and drying; the high
temperatures kill most microbes, sanitizing the dishes. Surfaces in hospital rooms are commonly
sanitized using a chemical disinfectant to prevent disease transmission between patients. Figure
7.0 summarizes common protocols, definitions, applications, and agents used to control microbial
growth.
Figure 8.0. Common Protocols for Control of Microbial Growth
Measuring Microbial Control
Physical and chemical methods of microbial control that kill the targeted microorganism
are identified by the suffix -cide (or -cidal). The prefix indicates the type of microbe or infectious
agent killed by the treatment method: bactericides kill bacteria, viricides kill or inactivate viruses,
and fungicides kill fungi. Other methods do not kill organisms but, instead, stop their growth,
FC 115 / N. S. SOLIDUM, MSc. CapSU FC 115 / N. S. SOLIDUM, MSc. CapSU
Bioprocessing making their population static; such methods are identified by the suffix -stat (or -static). For
In this process, food-grade microorganisms are used to produce different types of example, bacteriostatic treatments inhibit the growth of bacteria, whereas fungistatic treatments
fermented food using raw materials from animal and plant sources (known as “starter culture”). inhibit the growth of fungi. Factors that determine whether a particular treatment is -cidal or - static
Besides, microbial enzymes are also being used to produce food and food additives. include the types of microorganisms targeted, the concentration of the chemical used, and the
nature of the treatment applied. Although -static treatments do not actually kill infectious agents,
Food Biopreservation they are often less toxic to humans and other animals, and may also better preserve the integrity
Food biopreservation is a food biological preservative by using antimicrobial metabolites of the item treated. Such treatments are typically sufficient to keep the microbial population of an
(taken from certain microorganism in order to control pathogenic and spoilage microorganisms in item in check. The reduced toxicity of some of these -static chemicals also allows them to be
foods). Beneficial bacteria are used to prevent food spoilage and get rid of harmful pathogens. impregnated safely into plastics to prevent the growth of microbes on these surfaces. Such plastics
Lactic Acid Bacteria (LAB) are the most commonly used due to their unique properties are used in products such as toys for children and cutting boards for food preparation. When used
and because they are harmless to humans. LABs release antimicrobials (such as lactic and acetic to treat an infection, - static treatments are typically sufficient in an otherwise healthy individual,
acid, hydrogen peroxide, and peptide bacteriocins) that stop spoilage and inhibit growth of preventing the pathogen from multiplying, thus allowing the individual’s immune system to clear
potentially harmful pathogens. the infection. The degree of microbial control can be evaluated using a microbial death curve to
describe the progress and effectiveness of a particular protocol. When exposed to a particular
Molecular Biology and Biotechnology in Fisheries microbial control protocol, a fixed percentage of the microbes within the population will die.
Because the rate of killing remains constant even when the population size varies, the percentage
killed is more useful information than the absolute number of microbes killed. Death curves are
often plotted as semilog plots just like microbial growth curves because the reduction in
microorganisms is typically logarithmic (Fig. 8.1). The amount of time it takes for a specific
protocol to produce a one order-of-magnitude decrease in the number of organisms, or the death
of 90% of the population, is called the decimal reduction time (DRT) or D-value.

FC 115 / N. S. SOLIDUM, MSc. CapSU FC 115 / N. S. SOLIDUM, MSc. CapSU

 Figure 10.0. In Situ and Ex Situ Bioremediation
Bioprospecting
Bioprospecting is the exploration of natural sources for small molecules, macromolecules
and biochemical and genetic information that could be developed into commercially valuable
Figure 8.1. Microbial Death Curve
products for the agricultural, aquaculture, bioremediation, cosmetics, nanotechnology, or
pharmaceutical industries.
Several factors contribute to the effectiveness of a disinfecting agent or microbial control
protocol. First, as demonstrated in Figure 8.1, the length of time of exposure is important. Longer
exposure times kill more microbes. Because microbial death of a population exposed to a specific
protocol is logarithmic, it takes longer to kill a high-population load than a low-population load
exposed to the same protocol. A shorter treatment time (measured in multiples of the D-value) is
needed when starting with a smaller number of organisms. Effectiveness also depends on the
susceptibility of the agent to that disinfecting agent or protocol. The concentration of disinfecting
agent or intensity of exposure is also important. For example, higher temperatures and higher
concentrations of disinfectants kill microbes more quickly and effectively. Conditions that limit
contact between the agent and the targeted cells cells—for example, the presence of bodily fluids,
tissue, organic debris (e.g., mud or feces), or biofilms on surfaces—increase the cleaning time or
intensity of the microbial control protocol required to reach the desired level of cleanliness. All
these factors must be considered when choosing the appropriate protocol to control microbial Figure 10.1. Phases of bioprospecting
growth in a given situation.
FC 115 / N. S. SOLIDUM, MSc. CapSU FC 115 / N. S. SOLIDUM, MSc. CapSU
 CHAPTER 10 CHAPTER 9
Food/Fisheries Biotechnology Food-borne Diseases and Spoilage Microorganisms

Biotechnology is a technology based on biology. Biotechnology harnesses cellular and Acute infections known as foodborne diseases are linked to recently consumed food. The
biomolecular processes to develop technologies and products that help improve our lives and the food in question is typically tainted by a toxicant or disease-causing microorganism. Such food
health of our planet. has the required germs or toxicants to make someone ill.

Some Concepts of Biotechnology In the United States alone, it is recorded that there are 4.9 million foodborne illnesses per
Biocontrol year. This means that every year, 600 million or 1 out 10 people the world falls ill after eating
Biocontrol is a method of controlling unwanted organisms (pests) using other organisms. contaminated food. In the Philippines, from 2005 to June 2018, there were 209 outbreaks
It relies on predation, parasitism, herbivory, or other natural mechanisms, but typically also equivalent to 13, 591 cases with 97 mortalities. In 2010, $15 Billion was caused by
involves an active human management role. Campylobacter, E. coli (O157:H7 and Shiga toxin producing Esc. coli), L. monocytogenes,
Three basic strategies : nontyphoidal Salmonella, T. gondii, and Norovirus.
1. classical (importation), where a natural enemy of a pest is introduced in the
hope of achieving control Foodborne diseases are considered a burden due to the following effects:
2. inductive (augmentation), in which a large population of natural enemies
are administered for quick pest control • Health effects to individual:
3. inoculative (conservation), in which measures are taken to maintain natural o cause suffering
enemies through regular reestablishment. o discomfort
o debilitation
Bioremediation o can be fatal (cause death)
Bioremediation is a process used to treat contaminated media, including water, soil and
subsurface material, by altering environmental conditions to stimulate growth of microorganisms • Economic effects:
and degrade the target pollutants. o Medical treatment
o Death
o Lawsuits
o Lost wages and productivity
o Loss of business
o Recall and destruction of products
o Cost in the investigation of the outbreaks

FC 115 / N. S. SOLIDUM, MSc. CapSU FC 115 / N. S. SOLIDUM, MSc. CapSU

 • Low-temperature Preservation (e.g. chilling, freezing)
• Effects to establishments › Reduction in temp, increases lag phase
o Loss of customers › Psychrotrophs will still grow
o Increase insurance premiums  Processing
o Lowered employees’ moral › Processing methods that lowers the water content of fish (i.e. salt addition, dehydration)
o Increase employees’ turn over will make the fish unsuitable for bacterial growth
Figure 9.0. Main causes of Foodborne Diseases
Figure 9.1. Events that lead to foodborne disease by bacteria and virus
FC 115 / N. S. SOLIDUM, MSc. CapSU FC 115 / N. S. SOLIDUM, MSc. CapSU
 o Pinking: caused by a group of halophilic bacteria, Halobacterium and Sarcina
 pinking bacteria are killed by preservatives like sulphur dioxide Classification of Foodborne Diseases
o Dun: brown discoloration of the fish caused by the growth of molds, Sporendonema
spp. Dun molds have a temperature range of 10-370C and an optimum of 250C. A. Food intoxication
 Sporodonema is not so biochemically-active against fish flesh compared  chemical or natural toxin (often produced as a by-product of bacteria present in the food -
with the pinking bacteria which break down the flesh. known as an exotoxin) causes the symptoms or illness
 Refer to diseases caused by the consumption of food containing:
Fermented Products o Biotoxicants- which are found in tissues of certain plants and animals.
• Principle : the presence of organic acids has antimicrobial effect o Metabolic products (toxins)- formed and excreted by microorganisms while they
• Decrease of pH below the growth range multiply in food, or in gastrointestinal tract of man.
• metabolic inhibition by the undissociated acid molecules o Poisonous substances- which may be intentionally or unintentionally added to food
• antimicrobial products: H+, CO2, organic acids, peroxides, antibiotics during production, processing, transportation or storage.

Canned Products  Food borne intoxications have short incubation periods (minutes to hours) and are
Thermophilic Bacteria characterized by lack of fever.
 flat sour spoilage: the ends of the can remain flat and spoilage cannot be detected unless
the can is opened. Causative Agents for Food Intoxication:
o B. coagulans and B. sterothermophilus › Staphylococcus aureus (Staphyloccocal poisoning)
 thermophilic acid (TA) spoilage: production of CO2 and H2 › Escherichia coli O157 : H7 ("hamburger disease.")
o Clostridium thermosaccharolyticum › Clostridium botulinum (Botulism)
 sulphide spoilage: sulphur stinker, H2S produced from putrefying proteins. › Clostridium perfringens (“canteen disease”)
o Clostridium nigrificans
B. Foodborne infection
Smoked Fish Products  the microorganism itself grows inside the body and causes of the symptoms
• Smoke component: bactericidal  caused by the entrance of pathogenic microorganisms contaminating food into the body,
• non-sporeforming rods: survived the process introduced during handling and the reaction of the body tissues to their presence.
e.g. Penicillium sp.; Aspergillus sp. (can grow at refrigeration temperature) o Fungal
o Bacterial
Examples of Spoilage Control o viral
• Prevention of initial Contamination o parasitic
› More microorganisms present = shorter shelf life  have long incubation periods and are usually characterized by fever

FC 115 / N. S. SOLIDUM, MSc. CapSU FC 115 / N. S. SOLIDUM, MSc. CapSU

Causative Agents for Food Infection:
› Salmonella
› Campylobacter jejuni
› Listeria monocytogenes
› Shigella
› Vibrio parahaemolyticus
Food and Spoilage
Microbial Spoilage
Approximately 25% of all food in the world is lost post harvest due to microbial activity. Microorganisms are found on all the outer surfaces (i.e. skin, gills) and in the alimentary
tract of live and newly caught fish in varying numbers. A normal range of 102-107 cfu /cm2 on the
“Spoilage” can be defined as a change in fish or fish products that renders them less skin and between 103 and 109 cfu/g in the gills and intestines has been observed. When fish dies,
acceptable, unacceptable or unsafe for human consumption (Hayes, 1985). its entire body resistance mechanisms breakdown, giving way to microorganisms to invade or
diffuse into the flesh.
The change in organoleptic properties (e.g. appearance, smell, taste) that makes it
unacceptable, but not necessarily harmful to consumers, is due to transformation of fish Spoilage in Fish Products
components to undesirable metabolites. During food spoilage, there is a breakdown of various A. lightly preserved fish products
components and the formation of new compounds. These new compounds are responsible for the • Cold-smoked, pickled, marinated, brined
changes in odor, flavor and texture of the meat. • Microflora is dominated by LAB
• High levels of LAB for several weeks before the product becomes sensorically
Factors Influencing Rate of Spoilage rejectionable.
• LAB may produce off-odours (sour, cabbagey, sulphourus) in cold smoked salmon
1. Handling - Rough handling will result in a faster spoilage rate due to the physical (Hansen, 1995)
damage to the fish, resulting in easy access for enzymes and spoilage bacteria. • Elimination of LAB with nisin cause extension of shelflife of cold-smoked salmon (Gram
2. Initial bacterial load - The microflora on tropical fish often carries a slightly higher and Huss, 1996)
load of Gram-positives and enteric bacteria but otherwise is similar to the flora on • LAB are not known to reduce TMAO to TMA, thus spoilage may be caused by other
temperate-water fish. Basically, bacteria populations on temperate fish are bacteria
predominantly psychrotrophic, while fish from the tropics have largely mesophilic
bacteria B. Salt Curing and Fermentation
3. Methods of capture (i.e. of fish) • Two most important conditions in dried salted fish:
FC 115 / N. S. SOLIDUM, MSc. CapSU FC 115 / N. S. SOLIDUM, MSc. CapSU