Asd201 Practical Report Expt 3

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NANYANG POLYTECHNIC
EXAMINATION ADMINISTRATION DEPARTMENT
SCHOOL OF APPLIED SCIENCE
TITLE PAGE FOR COURSEWORK SUBMISSION
Institution Name: Nanyang Polytechnic Course Name: Food Science and

Nutrition
Class: FS2301 Academic Year: 2024/25
Module Title: Diagnostics in Food Module Code: ASD201

Microbiology
Assessment Practical Report Assessment P1MS2

Component: Component Code:
Date of Submission: 21 July 2024 Word Count: Not Applicable
Title of Coursework: Experiment 3 – Examination of Food Sample (Chicken) for Salmonella.
GROUP PARTICULARS
GROUP INDEX NUMBER Not Applicable
CANDIDATE NAME CANDIDATE NUMBER
Muhammad Daruthman Abrie Masjuri 234951W
This Coursework was conducted under the supervision of:
SUPERVISOR NAME DESIGNATION SIGNATURE
Sze Wee Ping Supervising Tutor
This document consists of 42 pages. Blank pages are indicated.
PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

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i. Table of Contents
Contents
i. Table of Contents................................................................................................................................................ 2
ii. Important Notes................................................................................................................................................... 3
iii. Abstract ............................................................................................................................................................. 4
1. Introduction .......................................................................................................................................................... 5
1.1. Objective(s) of this Project ........................................................................................................................... 5
1.2. Literature Review ........................................................................................................................................... 5
1.3. Context of Project .......................................................................................................................................... 8
2. Methodology ......................................................................................................................................................... 9
2.1. Risk Assessment and Safety Precautions .................................................................................................. 9
2.2. Materials and Apparatus ............................................................................................................................... 9
2.3. Experimental Procedures ...........................................................................................................................10
3. Results .................................................................................................................................................................14
3.1. Visuals of Experimental Results .................................................................................................................14
3.2. Organised and Consolidated Experimental Results ................................................................................22
4. Analyses ..............................................................................................................................................................24
4.1. Analysis of Specimen Negative Control Experimental Results ..............................................................24
4.2. Analysis of Specimen Positive Control Experimental Results ................................................................25
4.3. Analysis of Food Sample Experimental Results.......................................................................................26
5. Evaluations .........................................................................................................................................................28
5.1. Reliability and Accuracy of Experimental Results ....................................................................................28
5.2. Statements of Reliability .............................................................................................................................30
5.3. Statements of Anomaly ...............................................................................................................................30
6. Conclusions ........................................................................................................................................................31
6.1. Determining Presence of Salmonella in Food Sample ............................................................................31
6.2. Assessing Suitability of Food for Consumption and Compliance with Government Regulations .......31
6.3. Suggested Recommendations and Improvements..................................................................................31
7. References ..........................................................................................................................................................33
8. Appendices .........................................................................................................................................................35
8.1. Interpretation of Experimental Results ......................................................................................................35
9. Miscellaneous ....................................................................................................................................................38
9.1. Notes to Readers .........................................................................................................................................38
9.2. Formalities ....................................................................................................................................................38
9.3. Glossary of Terms .......................................................................................................................................40
9.4. List of Additional/Supplementary Documents ..........................................................................................41
9.5. Point(s)–of–Contact .....................................................................................................................................41

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ii. Important Notes
(a) The conventions, nomenclature, scientific notations, and data which is utilised in the Singapore–Cambridge
General Certificate of Education Advanced Level Higher 2 Biology (Syllabus 9744), Chemistry (Syllabus
9729), and Data Booklet are generally adopted.
In particular, the traditional names of sulfur, sulfates, and sulfites will be used. Sulfur, and all compounds of
sulfur, will be spelt with ‘f’ and not ‘ph’.
It is intended that, in order to avoid difficulties arising out of the use of l as the symbol for litre, use of dm3 in
place of l or litre will be made.
The convention ⏣ for representing the aromatic ring is preferred.
In addition, the general practices of science (including the guidance on practical work) as explicated in the
SPA Information Booklet are also adopted. Due to copyright restrictions and intellectual rights issues, copies
of these documents may only be obtained directly from the Singapore Examinations and Assessment
Board.
(b) With the exception of raw data, all processed data/final answers are expressed in 3 significant figures or 2
decimal places, unless a different accuracy is specified.
Where there are intermediary mathematical steps/workings which involve substituting variables with non–
exact answers, an intermediary mathematical step/working will use a numerical value that is one significant
figure/decimal place greater than the answer obtained in the immediately preceding mathematical
step/working.
(c) Any anomalous data is indicated with a right superscript asterisk.
(d) Wherever possible, the exponential notation convention (e.g., g cm–3 to represent ‘grams per cubic
centimetre’), rather than the solidus notation convention (e.g., g / cm3), is preferred and will be utilised in this
Coursework.
(e) Attention is drawn to the following sub–Sections:
• 8.1. – Interpretation of Experimental Results; which provides guidance on how the results were analysed
and interpreted.
• 9.3. – Glossary of Terms; which lists all the abbreviations and notations used.

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iii. Abstract
Preamble
This document explicates the philosophy, intent and design of the investigations conducted on a food sample
(chicken) to detect the presence of Salmonella, the analysis and insights of the experimental results obtained
from the investigations, and the conclusions derived from those experimental results.
General Investigative Framework
A pre–weighed 25 g of chicken sample was homogenised in Lactose broth to allow sufficient resuscitation of
injured Salmonella spp. microbes in the food sample. Extracts from the mixture were then incubated in
Selective–Enrichment media broths RV and TT. Mixtures from each RV and TT broths were then streaked onto
Selective–Differential media agar plates which consists of XLD, HE, and BS agars and any bacterial colony
formed was observed after incubation. Typical and atypical isolated colonies of Salmonella spp. usually consist
of colonies with dark or black centres. If these were present in the Selective–Differential agar plates containing
the food sample extract after incubation, the selected colonies were transferred to and incubated in slant media
consisting of TSI and LIA. The slants were observed after incubation for any sign of typical or atypical Salmonella
microbial growth which usually consisted of blackening of the slant media (hydrogen sulfide production), purple
or yellow butts, and red slants. If the results of these tests were inconclusive, the food sample extract were sent
to the bioMérieux® API® 20E, a type of serological test, which will state the most probable active agent in the
sample as well as the confidence level of that deduction.
Conclusions derived from Experimental Results
Based on all the experimental results observed, it was determined that the food sample contained detectable
levels of Salmonella spp. microbes and, hence, the food sample was deemed unsafe to eat as according to the
regulations set by the SFA, High Risk food and meat products that contain detectable levels of Salmonella spp.
microbes in 25 g of the food sample may not be suitable for preparation of food, sale, or consumption.

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1. Introduction
1.1. Objective(s) of this Project
This Project aims to determine the presence of Salmonella spp. in a food sample (chicken) through the selection
and use of appropriate analytical techniques and assess the suitability of the food sample for consumption using
the experimental results obtained with reference to the regulations and guidelines of official government
authorities in the context of Singapore.
1.2. Literature Review
Importance of Proper Food Hygiene and Safety
Food safety and hygiene are very crucial aspects of the food and beverage industries and often forms the very
broad basis for assessing the suitability of food for consumption. Often, lapses in food hygiene practices lead to
the introduction and proliferation of many food–borne pathogens in consumer foods and, almost inevitably,
development of food–borne illnesses in unsuspecting people consuming such foods.
In an increasingly complex, globalised, and interconnected world, consumers are entitled to expect that the food
they consume is safe and suitable to eat. Stakeholders must greatly appreciate that food–borne illnesses and
injuries can have negative repercussions that may be severe or even fatal to human health, whether it is in the
short or long term. Outbreaks of such food–borne illnesses, especially those of great concern, can hurt the trade
and tourism sectors. Spoilage in foods due to the presence of food–borne pathogens is often very wasteful and
significantly impacts trade, consumer confidence, and, most importantly, food security. All stakeholders in the
line of the food production process, whether directly or otherwise, must endeavour to their best abilities to uphold
food safety and security to produce foods safe for consumption. This is the principle on which many guidelines
developed by government authorities managing food regulations and security, especially the Codex
Alimentarius which builds the foundation of such government regulations, has been anchored and developed1.
Salmonella as a Specimen Pathogen of Great Concern
One such pathogen of great concern is Salmonella. Salmonella, which is a member of the family
Enterobacteriaceae, is a genus consisting of rod–shaped, Gram–negative bacteria. Figure 1.2.(a). below shows
an electron micrograph of a typical Salmonella bacterium.
Figure 1.2.(a). – Coloured Scanning Electron Micrograph of a Salmonella bacterium.
It consists of 2 species – Salmonella bongori and Salmonella enterica – with well over 2500 serotypes2.
Salmonella is one of the commonest pathogens for causing gastroenteritis, with over a million infections and
more than 25 000 hospitalisations per year alone in the United States of America, and these figures only get
worse in less–developed countries3. While all serotypes are disease–causing in humans, very few are ‘host–
specific’ and can thrive in a specific host or in organisms closely–related to their specific host. The 2 serotypes
1 WHO. (2022). Codex Alimentarius. CXC_001e.pdf.

2 Mayo Clinic Staff. (2022, April 29). Salmonella infection. Salmonella infection - Symptoms & causes - Mayo Clinic.
3 CDC. (2024, June 18). Salmonella. Salmonella Homepage | CDC.

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that are specifically pathogenic to humans – S. Typhimurium and S. Paratyphimurium – are of the greatest
concern in terms of the pathogenic strains of Salmonella as individuals infected with these pathogens often
develop ‘typhoid fever’, which includes high fever and excessive vomiting4. If left untreated, it has the potential
to lead to fatality. Other serotypes such as S. Enteritidis, and S. Virchow usually only result in mild gastroenteritis
in infected individuals, though this severity varies with the dynamic physiology of the infected individuals.
The bacteria of this genus are considered as a severe–level hazard in food microbiology as even the presence
of the bacteria in small viable numbers can cause food–borne illnesses in consumers. When ingested with
contaminated food, Salmonella can cause a gastrointestinal illness known as ‘salmonellosis’5.
Prominent Sources of Contamination
Contamination is usually due to poor hygiene practices or by using contaminated raw food materials without
proper washing and/or cooking. Travelling to less–developed countries without potable water and/or proper
sewage disposal raises the risk of contracting salmonellosis. Consuming raw or undercooked meat, poultry, and
egg products also encourages the development of the illness as there are less hurdles for the bacteria to
overcome to proliferate in an environment of excess nutrients.
Symptoms of Illness
While Salmonella is part of the natural microflora in the gastrointestinal tract (more specifically, the large
intestine) of human beings, its presence in other parts of the gastrointestinal tract is very likely to cause
salmonellosis. Infection may also be exacerbated if the infection metastasises and starts impairing the functions
of body parts/organs beyond the gastrointestinal tract (metastatic infection) or if the person infected has an
immunocompromised system. While some people infected with Salmonella are asymptomatic, most of those
infected often develop several symptoms of salmonellosis including, but not limited to, diarrhoea, fever,
abdominal cramps, and vomiting within 8 – 72 hours after exposure, with severe cases involving symptoms such
as septicaemia, bloody faecal discharge, typhoid fever, and coma6. Fortunately, most of those diagnosed with
mild symptoms often recover within a week without the need for any specialised or specific medical treatment.
The fatality rate of the infection is also low, at least in developed countries and if prompt treatment is
administered.
Rationale for Prompt Detection of Salmonella in Consumer Foods and Requiring Analytical Techniques
It is, therefore, imperative that effective detection methodologies and analytical techniques with an appreciable
level of reliability and accuracy are developed and utilised to detect the presence, especially in very small viable
numbers, of Salmonella microbes in food samples. In many countries, including Singapore, government bodies
have strict quality control on food manufactured, imported, or sold in the country, which requires testing food
using an approved analytical technique for pathogens, including Salmonella.
Required Local Regulations in Singapore
According to the SFA, High Risk (food that require minimal or no cooking before serving such as Ready–To–
Eat) foods and meat products that contain amounts of Salmonella spp. that can be detected in 25 g of the food
sample are not fit for use, sale, or consumption in Singapore7, with the exception of raw, agriculture foods such
as fresh fruits and microbiologically processed foods such as cheeses. Raw ingredients that contain Salmonella,
such as poultry, may be used in the preparation of food on the condition that the ingredients are washed
4 CDC. (2024, June 18). Salmonella. Salmonella Homepage | CDC.

5 Mayo Clinic Staff. (2022, April 29). Salmonella infection. Salmonella infection - Symptoms & causes - Mayo Clinic.
6 Ellis, R. R. (2024, April 22). Salmonella (Salmonellosis). Salmonella: Causes, Symptoms, Risks, Treatment, and
Prevention.
7 Singapore Food Agency. (2024, May 31). Sale of Food Act, Food Regulations, Eleventh Schedule (pg. 211 – 212).
Singapore Food Agency – Sale of Food Act.

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thoroughly and cooked properly to reduce the amount of viable Salmonella microbes to levels that are not
infective to humans in the final food product.
General Investigative Framework
Stage 1 Stage 2 Stage 3 Stage 4

Resuscitation Selective Selective- Confirmation
(Non-Selective) Enrichment Differential Test Test
• Homogenised food sample • Accelerate growth of now fully • Typical/atypical (suspicious) • To confirm the presence of
inoculated onto non–selective vegetative Salmonella. colonies plated onto solid Salmonella by conducting
enrichment media. • Also suppresses growth of selective differential media for appropriate serological test.
• Allows maximum background/non–target the recognition and isolation
resuscitation of any sub– microorganisms, increasing of confirmed or suspected
lethally impaired (injured) the probability of isolating salmonellae.
salmonellae present. salmonellae.
Figure 1.2.(b). – Flow of Processes in General Investigative Framework.
Figure 1.2.(b). summarises the flow of processes in the General Investigative Framework which underpins the
investigation. The investigation consists of 4 key stages.
• Stage 1 – Resuscitation
The purpose of this stage is to enable sub–lethally impaired (injured) microbes to recover to become viable
microorganisms again. The media selected, lactose broth, is non–selective so as to reduce as many hurdles
present to allow recovery of these injured microbes and maximise the amount of microorganisms that can
become viable again.
• Stage 2 – Selective Enrichment
This stage aims to accelerate the growth of recovered (fully vegetative) Salmonella microbes while suppressing
the growth of other non–target/background microbes (such as E. coli). This is executed to maximise the amount
of Salmonella microbes in the sample. Also, inhibiting background microbes ensures that the Salmonella
microbes are not competing with these background microbes for nutrients or are poisoned by their metabolic
by–products. The media used were RV and TT broths.
• Stage 3 – Selective–Differential Test
The selective enrichment process would have accelerated the growth of Salmonella such that the amount of the
pathogen present should be in large numbers, along with other, restricted, microbial flora. At this stage, the
sample was transferred and streaked onto selective–differential media, namely XLD, HE, and BS agars, which
would show distinct observable physical characteristics (appearance) of isolated typical and atypical
Salmonella colonies growing on these media. Additionally, TSI and LIA slant agars were used to help deduce
presumptive Salmonella colonies. A suggested Interpretation of Experimental Results is included in sub–
Section 8.1. to help analyse the results obtained.
• Stage 4 – Confirmation (Serological) Test
While there may be colonies present in the streaked plates obtained in Stage 3 that are typical of Salmonella
colonies, these may not all be true Salmonella colonies, resulting in false–positives in the experimental results
obtained. A serological test, or more commonly known as a ‘confirmation test’, is executed to confirm that the
colonies formed were Salmonella. The serological test was conducted using the bioMérieux® API® 20E. The
test would indicate what active/infective agent is most probably present in the sample being examined, along
with the percentage of confidence (confidence level) of the results. The higher the confidence level, the more
reliable and accurate the results are.

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1.3. Context of Project
In this Project, the food sample provided for analysis was chicken. Thus, the aim of the Project is to determine
the presence of any Salmonella spp. in the food sample through the selection and use of the appropriate
analytical techniques as indicated in Figure 1.2.(b). earlier on and, subsequently, assess the safety of
consuming the food sample with regards to the guidelines enacted by the SFA.
Summary of Key Experiment(s)
• Resuscitation
A pre–weighed sample of chicken (25 g) was made fully amorphous by dissolving it in approximately 225 cm3 of
lactose broth and then homogenised in a stomacher. The mixture was then incubated at a temperature of 35 °C
for at least 24 hours.
• Selective Enrichment
Extracts from the homogenate food mixture were extracted and transferred into separate RV and TT broths to
allow fully vegetative Salmonella spp. microbes to proliferate while supressing the growth of non–target/
background microflora.
• Selective–Differential Test
Extracts from the food–RV or –TT broth mixture were extracted and streaked onto separate agar plates that
composed of differential media to identify Salmonella microbes. The agar media plates used were XLD, HE,
and BS media. After incubation, bacterial colonies that resembled typical or atypical Salmonella colonies were
identified and isolated. These colonies were then further inoculated into separate tubes containing another set
of selective–differential media of TSI and LIA by stabbing butt and streaking slant. The experimental results
obtained were used to further validate that Salmonella colonies were present.
• Confirmation (Serological) Test
Typical or atypical Salmonella colonies isolated in the streaked plates obtained in the previous stage were then
subjected to serological tests using the bioMérieux® API® 20E. The results of the test were used to determine
if bacterial colonies growing in the selective–differential agar plates were of any Salmonella strain.
The experimental results obtained from Stages 3 (Selective–Differential Test) and 4 (Confirmation (Serological)
Test) were then analysed together and used to determine whether there was any detectable presence of
Salmonella, regardless of any strain.
According to the SFA, if a detectable presence of Salmonella was observed in 25 g of the food sample, the entire
food product is deemed unsuitable for consumption. The converse is also true.

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2. Methodology
This Section provides the risk assessment and safety protocols that should be adopted, the list of the materials
and apparatus required, and the procedures of the experiments conducted to execute the investigation.
2.1. Risk Assessment and Safety Precautions
Scientific subjects are, by their nature, experimental. It is, therefore, unsurprising that in many educational
institutions, the delivery of the theory content and execution of the practical/coursework component of the
subject are often developed synergistically in the scheme of work to promote the development of scientific
literacy and investigative skills in the candidates that they teach.
Regrettably, risks to personal health and/or property are an innate part of conducting practical work and
investigations. It cannot be overstated that educational institutions carry out the required risk assessments,
identify potential hazards, and ideate protocols to mitigate or reduce such hazards.
Proper use of Personal Protective Equipment is expected, as with the operational readiness and accessibility
of safety contingency/emergency equipment such as first–aid kits, safety showers, and fire extinguishers.
Responsibility for safety matters rests with educational institutions.
When planning practical work, Supervisors should ensure that they do not infract any school, education
authority, or government regulations which restrict the sampling, in the context of educational establishments,
of bodily secretions/fluids and tissues. Candidates should also be aware of the need to take simple precautions
for safety and/or accuracy.
2.2. Materials and Apparatus
Unless otherwise noted, the rate of allocation is ‘per candidate group’.
Materials Apparatus
• Food sample • Pipette Gun and disposable pipettes

• Culture Media: • 1 ml pipette and tips
o 1.00 dm3 Lactose broth • 200 µl pipette and tips
o 5 × 10.0 cm3 Rappaport–Vassiliadis (RV) • Inoculating loops
broth
o 5 × 10.0 cm3 Tetrathionate (TT) broth • Stomacher and bags
o 100 cm3 (approximately 5 plates) Xylose • Incubator at 35.0 °C
Lysine Deoxycholate (XLD) agar
o 100 cm3 (approximately 5 plates) Bismuth • Water baths of adjustable temperatures
Sulfite (BS) agar
o 100 cm3 (approximately 5 plates) Hektoen • bioMérieux® API® 20E

Enteric (HE) agar
o 50 cm3 (approximately 5 tubes) Triple Sugar • Bunsen burner with lighter

Iron (TSI) agar
o 50 cm3 (approximately 5 tubes) Lysine Iron • Alcohol (spray or bottle) for disinfection
(LIA) agar
o 100 cm3 (approximately 5 plates) Tryptone • Inoculating needles

Soya (TSA) agar

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2.3. Experimental Procedures
This sub–Section lists all the experimental procedures that were taken to carry out the investigation.
Media Preparation
1 Technical information and full instructions on preparing the required media as listed in sub–Section 2.2.
may be obtained directly from the manufacturer(s).
Resuscitation
2 The following Replicates were prepared as according to Figure 2.3.(a). that follows.
Amount of Amount of Lactose

Description of Sample/Active
Replicate Sample/Active broth added
Replicate Agent
Number Agent
/ cm3
Food Sample
1 Chicken 25.0 g
(Fully Amorphous)
Specimen Positive Salmonella
2 0.1 cm3
Control Typhimurium 225.0
Specimen Negative
3 Escherichia coli 0.1 cm3
Control
4 Sterility Control
Figure 2.3.(a). – Decipher Key 1 – Table for preparation of the different Samples for testing.
Note: • Sterility control is only to test for any presence of contamination and that no active agent, apart
from Salmonella Typhimurium, is giving a positive test result.
• As this investigation is only qualitative in nature, the actual amount of Lactose broth added to
each Replicate is approximate and was estimated, rather than measured.
3 The constituents of the Replicates were then placed into separate, labelled stomacher bags and
homogenised for 1 minute in the stomacher.
4 The Replicates were then incubated in an incubator set at a temperature of 35.0 °C for at least 24 hours.
Selective Enrichment
5 The Replicates were removed from the incubator and mixed thoroughly, ensuring homogeneity. Replicate
4 was inspected for any contamination or irregularity before proceeding with the investigation. If Replicate
4 did not appear clear, the investigation was stopped as it indicated that there was contamination.
Replicate 1 Replicate 2 Replicate 3 Replicate 4
Figure 2.3.(b). – Inspection of the stomacher bags for any irregularities.

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6 With the exception of Replicate 4, using a micropipette, extracts of all Replicates were added to separately
labelled Tubes each containing 10.0 cm3 of RV broth as indicated in Figure 2.3.(c). that follows.
Replicate mixture Amount of Replicate mixture extracted

Tube Number
/ cm3
1
1
(Food Sample)
2
2 0.1
(Specimen Positive Control)
3
3
(Specimen Negative Control)
Figure 2.3.(c). – Decipher Key 2 – Amount of Replicate mixture added to separate RV broth tubes.
7 With the exception of Replicate 4, using a micropipette, extracts of all Replicates were added to separately
labelled tubes each containing 10.0 cm3 of TT broth as indicated in Figure 2.3.(d). that follows.
Replicate mixture Amount of Replicate mixture extracted

Tube Number
/ cm3
1
4
(Food Sample)
2
5 1.0
(Specimen Positive Control)
3
6
(Specimen Negative Control)
Figure 2.3.(d). – Decipher Key 3 – Amount of Replicate mixture added to separate TT broth tubes.
8 All Tubes obtained in Steps 6 and 7 were mixed using the Vortex to ensure homogeneity.
9 The Tubes were incubated in separate water baths with specific temperatures as stated in Figure 2.3.(e).
for at least 24 hours.
Medium Incubation Temperature of Tube

Tube Number
/ °C
1
2 RV 42.0
3
4
5 TT 43.0
6
Figure 2.3.(e). – Incubation temperatures for the Tubes.
Note: The temperatures as stated in Figure 2.3.(e). may only have a maximum deviation of ± 0.2 °C.

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Plating onto Selective–Differential Agar Media
10 A loopful of inoculum from Tube 1 was extracted and streaked onto XLD agar, ensuring that the inoculating
loop was filled with the broth when transferring to the agar plate. This Step was repeated onto HE agar and
BS agar plates.
11 Step 10 was then repeated for the other Tubes. All streaked plates were labelled as indicated in Figure
2.3.(f). that follows.
Tube Broth in Media of Agar that Tube contents were streaked on

Number Tube XLD HE BS
1 T1/RV/XLD T1/RV/HE T1/RV/BS
2 RV T2/RV/XLD T2/RV/HE T2/RV/BS
3 T3/RV/XLD T3/RV/HE T3/RV/BS
4 T4/TT/XLD T4/TT/HE T4/TT/BS
5 TT T5/TT/XLD T5/TT/HE T5/TT/BS
6 T6/TT/XLD T6/TT/HE T6/TT/BS
Figure 2.3.(f). – Decipher Key 4 – Streaked Plate labels corresponding to the Tube labels.
12 All Streaked Plates were incubated at 35 °C for at least 24 hours. After incubation, the Streaked Plates
were examined for colonies typical or atypical of Salmonella. The experimental results obtained were
recorded in Figure 3.1.(a). (see sub–Section 3.1.). Figure 2.3.(g). shows some specimens of typical
Salmonella colonies on the different selective–differential agar media used.
Typical colony
Typical colony
Typical colony
XLD HE BS
Figure 2.3.(g). – Streaked Plates containing typical and atypical colonies for the different agar media.
Selection of Typical/Atypical Colonies (for confirmation of presumptive Salmonellae)
13 For Tube 1, a well–isolated colony from one of its selective–differential Streaked Plates showing colonies
typical of Salmonella was selected. If typical colonies were not present, atypical colonies were chosen
instead.

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14 A sterile inoculating needle was used to extract a selected colony. The inoculating needle lightly touched the
centre of the colony and inoculated in the TSI slant by stabbing butt and streaking slant.
15 With the absence of flaming the same inoculating needle used in Step 14, the same selected colony was
inoculated in LIA slant by stabbing butt and then streaking slant. The LIA slants were ensured to have deep
butts (at least 4 cm in depth).
16 Steps 13 – 15 was repeated for the remaining Tubes. All the slants obtained were labelled as indicated in
Figure 2.3.(h). that follows, if applicable – that is, if there was a need to create such slants due to preliminary
positive/presumptive results.
Tube Number TSI slant label LIA slant label
1 T1/TSI T1/LIA
2 T2/TSI T2/LIA
3 T3/TSI T3/LIA
4 T4/TSI T4/LIA
5 T5/TSI T5/LIA
6 T6/TSI T6/LIA
Figure 2.3.(h). – Decipher Key 5 – TSI and LIA Slant labels corresponding to the Tube labels.
17 The Slants obtained in Step 16 were incubated at 35 °C for 24 hours. The tubes of the Slants were capped
loosely to maintain aerobic conditions during incubation to prevent excessive hydrogen sulfide production.
18 After incubation, the Slants were examined and the results were recorded in Figure 3.1.(b). (see sub–
Section 3.1.).
Confirmation of Salmonellae
19 TSI Slants which have presumptive Salmonellae were re–streaked on TSA agar plates and incubated at
35 °C for 24 hours.
20 After incubation, the bioMérieux® API® 20E was performed, following the manufacturer’s technical details
and full instructions. The results were recorded in Figure 3.1.(c). and (d). (see sub–Section 3.1.).

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3. Results
This Section provides the experimental data obtained from the investigation conducted.
3.1. Visuals of Experimental Results
For all results provided herein, reference to the Decipher Keys (provided in Figure 2.3.(a)., (c). – (d)., (f)., and
(h).) are expected. It is also worthwhile to note that due to logistical limitations, certain experimental results are
not available, and this will be prominently indicated in the relevant sections. The absence of such experimental
results, however, does not undermine the efficacy of the investigation.
Step 12 – Stage 3 (Selective–Differential Test) (XLD, HE, and BS Streaked Plates)
Figure 3.1.(a). – Visuals of Experimental Results obtained for Step 12.
Specimen Visuals of Experimental Results Remarks

Identity
Contents:
Food sample inoculated in RV broth.
Observations:
T1/RV/XLD Presence of bacterial colonies with very
dark/black centres. Orange agar medium
turned red.
Contents:
Specimen Positive Control inoculated in
RV broth.
T2/RV/XLD Observations:
Presence of bacterial colonies with very
turned red.
Contents:
Specimen Negative Control inoculated in
RV broth.
T3/RV/XLD Observations:
No presence of any bacterial colony or
growth. No colour change of orange agar
medium.
Contents:
Food sample inoculated in TT broth.
Observations:
T4/TT/XLD
turned red.

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Identity
Contents:
TT broth.
T5/TT/XLD Observations:
turned red.
Contents:
TT broth.
T6/TT/XLD Observations:
growth. No colour change of orange agar
medium.
Contents:
Observations:
T1/RV/HE Presence of dark blue bacterial colonies
with very dark/black centres. Pale orange
agar medium turned blue in colour.
Contents:
RV broth.
T2/RV/HE Observations:
Presence of blueish–green bacterial
colonies with very dark/black centres. Pale
orange agar medium turned blue in colour.
Contents:
RV broth.
T3/RV/HE Observations:
growth. No colour change of pale orange
agar medium.

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Identity
Contents:
Observations:
T4/TT/HE Presence of blueish bacterial colonies with
very dark/black centres. Pale orange agar
medium turned blue in colour.
Contents:
TT broth.
T5/TT/HE Observations:
Presence of blueish bacterial colonies with
very dark/black centres. Pale orange agar
medium turned blue in colour.
Contents:
TT broth.
T6/TT/HE Observations:
growth. No colour change of pale orange
agar medium.
Contents:
Observations:
T1/RV/BS Presence of dark green bacterial colonies
with metallic sheen.
T2/RV/BS Data not available
Contents:
RV broth.
T3/RV/BS Observations:
growth. No colour change of agar medium.

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Identity
Contents:
Observations:
T4/TT/BS*
Contents:
TT broth.
T5/TT/BS Observations:
Presence of dark green bacterial colonies
with metallic sheen. Area of agar where the
colonies were found appears to have
darkened.
Contents:
TT broth.
T6/TT/BS Observations:

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Step 16 – Stage 3 (Selective–Differential Test) (TSI and LIA slant agar – Select Specimens only)
Figure 3.1.(b). – Visuals of Experimental Results obtained for Step 16.

Identity
Contents:
Food Sample from XLD agar inoculated in TSI
medium.
Observations:
T1/TSI
Blackening of TSI medium indicating presence
of production of hydrogen sulfide. The slant and
butt could not be examined for its colour due to
the intense blackening arising from hydrogen
sulfide production.
T2/TSI Data not available
Contents:
Specimen Negative Control inoculated in TSI
medium.
T3/TSI Observations:
No blackening of TSI medium indicating
absence of production of hydrogen sulfide. The
slant is yellow in colour. The butt is slightly
discoloured.
Contents:
Food Sample from HE agar inoculated in TSI
medium.
Observations:
T4/TSI
Blackening of TSI medium indicating presence
of production of hydrogen sulfide. The slant and
butt could not be examined for its colour due to
the intense blackening arising from hydrogen
sulfide production.
Contents:
Food Sample from XLD agar inoculated in LIA
medium.
T1/LIA
Observations:
Blackening of LIA medium indicating presence of
production of hydrogen sulfide. The butt could
not be examined for its colour due to the intense

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Figure 3.1.(b). – Visuals of Experimental Results obtained for Step 16.

Identity
blackening. The slant is shrouded by blackening
due to hydrogen sulfide production.
Contents:
Specimen Positive Control inoculated in LIA
medium.
T2/LIA Observations:
Blackening of LIA medium indicating presence of
production of hydrogen sulfide. The butt is purple
in colour. The slant is purple in colour.
Contents:
Specimen Negative Control inoculated in LIA
medium.
T3/LIA Observations:
No blackening of LIA medium indicating absence
of hydrogen sulfide production. The butt is yellow
in colour. The slant is purple in colour.
T4/LIA Data not available

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Step 20 – Stage 4 (Confirmation (Serological) Test) (bioMérieux® API® 20E – Select Specimens only)
Figure 3.1.(c). – Visuals of Experimental Results obtained for Step 20.
Specimen
Visuals of Experimental Results
Identity
Replicate 1
(Food
Sample –
Duplicate 1)
Replicate 1
(Food
Sample –
Duplicate 2)
Replicate 2
(Specimen
Positive
Control)
Replicate 3
(Specimen
Negative
Control)

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Figure 3.1.(d). – bioMérieux® API® 20E Results obtained for Step 20.

Identity
Most Probable
Identity of Active
Replicate 1 Agent:
(Food Salmonella spp.
Sample –
Confidence:
Duplicate 1)
99.7 %
Most Probable
Identity of Active
Replicate 1 Agent:
(Food Salmonella spp.
Sample – Confidence:
Duplicate 2) 99.7 %
Most Probable
Identity of Active
Replicate 2 Agent:
(Specimen Salmonella spp.
Positive
Control) Confidence:
99.7 %
Most Probable
Identity of Active
Replicate 3 Agent:
(Specimen E. coli
Negative
Control) Confidence:
93.8 %

Medium of Streaked Results
Presumptive Test Slants TSI and LIA (D/ND) and
Plate
Sample Remarks 3.2.
Identity
XLD HE BS XLD HE BS
Presumptive Presumptive Presumptive

(AT/T/NG) TSI LIA TSI LIA TSI LIA
(+/–) (+/–) (+/–)
PROPERTY OF NYP/234951W
TT NG NG NG ND
Specimen
Negative AAN AAN –
Control
SC/RV NG NG NG ND
TT T T T D
Specimen
Positive KAP KAP +
Control
SC/RV T T D
TT T KAP KKP + D
22
Food
Sample
Duplicate 1
Organised and Consolidated Experimental Results
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SC/RV T AAP KAP – D
Annotation meanings (Key) are provided on the next page.

TT T ??P KAN D
Food
ASD201/P1MS2_EXPERIMENT_3/2024/25/S1
Sample
Duplicate 2
SC/RV T AAN KKP D
TT NG AAP KKP + D
Food
Sample
Duplicate 3
SC/RV T KAP KKP + D
Figure 3.2.(a). – Organised Tabular form of Experimental Results.
[Turn over
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Key (for results obtained in Figure 3.2.(a).):
• For Streaked Plates (XLD, HE, BS):
Annotation Meaning
AT Atypical Salmonella colonies
T Typical Salmonella colonies
NG No growth / Negative (No presumptive Salmonella colonies observed)
• For Slants (TSI, LIA):
TSI medium
Type Annotation Meaning
A Yellow (Acidic)
Slant
K Red (Alkaline)
A Yellow (Acidic)
Butt
K Red (Alkaline)
Hydrogen P Blackening (Production of hydrogen sulfide)
sulfide
production N No blackening (Absence of production of hydrogen sulfide)
LIA medium
Type Annotation Meaning
A Yellow (Acidic)
Slant K Purple (Alkaline)
R Red (Oxidative deamination)
A Yellow (Acidic)
Butt
K Purple (Alkaline)
Hydrogen P Blackening (Production of hydrogen sulfide)
sulfide
production N No blackening (Absence of production of hydrogen sulfide)
• For Remarks:
Annotation Meaning
D Detected (Salmonella spp. colonies present)
ND Not Detected (Salmonella spp. colonies absent)
• For all observations: “?” means Indeterminable.

• For Presumptive Test: “+” means Positive result, “–” means Negative result.
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4. Analyses
This Section provides the required methods of analysing and processing the experimental results obtained.
Reference should be made to sub–Section 8.1., which includes the suggested Interpretation of Experimental
Results.
4.1. Analysis of Specimen Negative Control Experimental Results
Analysis of Selective–Differential Streaked Plates
Reference to the following labelled Streaked Plates • T3/RV/XLD

should be made: • T3/RV/HE
• T3/RV/BS
• T6/TT/XLD
• T6/TT/HE
• T6/TT/BS
These Streaked Plates indicated above contained the Specimen Negative Control (E. coli) mixture. For all of
the Streaked Plates obtained, none of the plates showed any growth of bacterial colony typical or otherwise of
any Salmonella strain. In fact, none of the Streaked Plates showed signs of any bacterial growth at all. These
results strongly indicated the absence of Salmonella spp. microbes in the mixture.
Analysis of Presumptive Test (Slants and Butts)
Reference to the following labelled Slants should be • T3/TSI

made: • T3/LIA
These Slants indicated above contained the Specimen Negative Control (E. coli) mixture.
TSI medium
For T3/TSI, the TSI slant was observed to be yellow in colour, which is different from those produced by typical
Salmonella spp. colonies that are usually red in colour. Thus, the characteristics of the slants implies that the
active agent could not be Salmonella spp. microbes. The TSI butt was observed to be slightly discoloured. As
most bacteria colonies that were not Salmonella spp. would produce a red butt, the observed TSI butt could not
confidently confirm that Salmonella spp. was absent in the mixture. There was no blackening in the slant medium
indicating that hydrogen sulfide production was absent. Therefore, even though at least one of the
characteristics of the Tube indicated absence of Salmonella spp., it could still be considered presumptive for
the presence of atypical Salmonella spp. microbes in the mixture.
LIA medium
For T3/LIA, the LIA slant was observed to be purple in colour. However, as the colour of the slant is arbitrary,
this observation was not very useful in determining the presence of Salmonella spp. in the mixture. The LIA butt
was observed to be yellow in colour. This implies that the microbes in the mixture were most likely not Salmonella
spp. as typical Salmonella spp. microbes would result in a purple butt. There was also no blackening in the slant
medium, indicating that there was no hydrogen sulfide production. This, in turn, further validates that the
microbes in the mixture were not Salmonella spp. as Salmonella spp. microbes growing in LIA medium would
result in the blackening of the slant medium due to hydrogen gas production. Thus, the results of this Tube
alone strongly indicated that Salmonella spp. was absent in the mixture.

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Analysis of bioMérieux® API® 20E
The results obtained from the API® database indicate that the active agent in the mixture is E. coli, with a
confidence of 93.8 %. These results were expected as the identity of the active agent was known before the
experiment to be E. coli, thus giving the expected negative results.
4.2. Analysis of Specimen Positive Control Experimental Results
Analysis of Selective–Differential Streak Plates

• T5/TT/XLD
• T5/TT/HE
• T5/TT/BS
These Streaked Plates indicated above contained the Specimen Positive Control (Salmonella Typhimurium)
mixture. For all of the Streaked Plates obtained, there were isolated bacterial colonies on the agar media that
had observable physical characteristics that were usually congruent with those of isolated typical and/or atypical
Salmonella spp. colonies.
XLD medium
For both T2/RV/XLD and T5/TT/XLD, the isolated bacterial colonies present on their agars often have dark or
black centres. This observable physical characteristic is consistent with the isolated colonies of typical
Salmonella spp. growing in XLD medium which also have such black centres in their isolated colonies. Thus,
these results strongly indicated that Salmonella spp. microbes were present.
HE medium
For both T2/RV/HE and T5/TT/HE, the isolated bacterial colonies present on their agars were often blueish–
green in colour and having dark or black centres. This observable physical characteristic is consistent with the
isolated colonies of typical Salmonella spp. growing in HE medium which are usually blueish–green with
dark/black centres. Thus, these results strongly indicated that Salmonella spp. microbes were present.
BS medium
For T5/TT/BS, there were isolated dark green colonies with a huge metallic sheen surrounding the colonies.
This observable physical characteristic is consistent with the isolated colonies of typical Salmonella spp.
growing in BS medium which are usually dark green or black colonies with a metallic sheen. Thus, these results
strongly indicated that Salmonella spp. microbes were present.
Reference to the following labelled Slants should be • T2/LIA

made:
These Slants indicated above contained the Specimen Positive Control (Salmonella Typhimurium) mixture. For
all of the Slants obtained, there were observable physical characteristics on the slants and butts of the agar
media that were usually congruent with those of typical and/or atypical Salmonella spp. microbes growing in
such media.
LIA medium

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For T2/LIA, the butt was observed to be purple, albeit slightly discoloured. There was also blackening of the LIA
medium, indicating the production of hydrogen sulfide. These observable physical characteristics were typical
of Salmonella spp. microbes growing in LIA medium. Thus, this strongly indicated that Salmonella spp. microbes
were present.
The results obtained from the API® database indicate that the active agent in the mixture is Salmonella spp.,
with a confidence of 99.7 %. These results were expected as the identity of the active agent was known before
the experiment to be Salmonella spp., thus giving the expected positive results.
4.3. Analysis of Food Sample Experimental Results
Analysis of Selective–Differential Streak Plates

• T1/RV/BS
• T4/TT/XLD
• T4/TT/HE
• T4/TT/BS
These Streaked Plates indicated above contained the Food Sample (Fully Amorphous) mixture. For all of the
Streaked Plates obtained, there were isolated bacterial colonies on the agar media that had observable
physical characteristics that were usually congruent with those of typical and/or atypical Salmonella spp.
colonies.
XLD medium
For both T1/RV/XLD and T4/TT/XLD, the isolated bacterial colonies present on their agars often have dark or
black centres. This observable physical characteristic is consistent with the isolated colonies of typical
Salmonella spp. growing in XLD medium which also have such black centres in their isolated colonies. Thus,
these results strongly indicated that Salmonella spp. microbes were present.
HE medium
For both T1/RV/HE and T4/TT/HE, the isolated bacterial colonies present on their agars were often blueish–
green in colour and having dark or black centres. This observable physical characteristic is consistent with the
isolated colonies of typical Salmonella spp. growing in HE medium which are usually blueish–green with
dark/black centres. Thus, these results strongly indicated that Salmonella spp. microbes were present.
BS medium
For T1/RV/BS, there were dark green isolated colonies with a huge metallic sheen surrounding the colonies.
This observable physical characteristic is consistent with the isolated colonies of typical Salmonella spp.
growing in BS medium which are usually dark green or black colonies with a metallic sheen. Thus, these results
strongly indicated that Salmonella spp. microbes were present.
However, for T4/TT/BS, no comments or relevant deductions could be made as there were no bacterial colonies
or growth present on the agar medium. The absence of any bacterial growth for T4/TT/BS despite using a
somewhat strong concentration of bacterial solution and with T1/RV/BS showing bacterial colonies or growth
despite using the same agar medium might indicate that the results obtained for T4/TT/BS may be anomalous.

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Reference to the following labelled Slants should be • T1/TSI

made: • T1/LIA
• T4/TSI
These Slants indicated above contained the Food Sample (Fully Amorphous) mixture. For all of the Slants
obtained, there were observable physical characteristics on the slants and butts of the agar media that were
usually congruent with those of typical and/or atypical Salmonella spp. microbes growing in such media.
TSI medium
For both T1/TSI and T4/TSI, the tubes showed blackening of the TSI medium, which indicated the presence of
hydrogen sulfide production and may be characteristic of typical Salmonella spp. microbes growing in TSI
medium as most of these microbes do indeed produce hydrogen sulfide in TSI medium. Regrettably, for both
tubes, the excessive blackening due to the hydrogen sulfide production made it practically impossible to
examine, with acceptable accuracy, the colours of the slants and butts of the tubes. However, based on the
blackening of the medium alone, it is presumptive that Salmonella spp. microbes were present. The results of
these tubes should be considered in the light of the other experimental results.
LIA medium
For T1/LIA, although the butt was shrouded by intense blackening, observation under a strong light source
showed that the butt had an observable purple colouration. This is consistent with typical Salmonella spp.
microbes growing in LIA medium. Regrettably, the excessive blackening made it practically impossible to
examine, with acceptable accuracy, the colour of the slant of the tube. The tube also showed blackening of the
LIA medium, indicating the presence of hydrogen sulfide production. This is also consistent with typical
Salmonella spp. microbes growing in LIA medium. Thus, the results of this tube strongly indicated that
Salmonella spp. microbes were present.
The results obtained from the API® database indicate that one of the active agents in the mixture is Salmonella
spp., with a confidence of 99.7 %. The results from the API® database are in agreement with the experimental
results obtained from the Selective–Differential Test (Streaked Plates) and Presumptive Test (Slants).

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5. Evaluations
5.1. Reliability and Accuracy of Experimental Results
This sub–Section includes the evaluation of the reliability and accuracy of the experimental results obtained.
Replicate 1 (Food Sample)
Precision and Reliability
Streaked Plates
The results of the Streaked Plates obtained were generally very precise, resulting in an appreciable reliability
of the results obtained. Most of the Streaked Plates obtained have about the same type, in terms of observable
physical characteristics, of isolated colonies growing on the media used. These colonies were typically colonies
that were dark in colour or have dark or black centres similar to typical Salmonella spp. colonies. However, there
is one exception, and this was T1/RV/BS. For T1/RV/BS, it was observed that there were no signs of any
isolated bacterial colony or growth despite having an active agent in its mixture. This contrasted with the other
Streaked Plates obtained for Replicate 1, indicating that the results for T1/RV/BS could be anomalous.
However, as there is only one anomalous Streaked Plate, this indicated that the results of the remaining
Streaked Plates did not differ much from each other and, thus, the results were considered precise and reliable.
Slants
The results of the Slants obtained were generally very precise, resulting in an appreciable reliability of the
results obtained. In all of the Slants obtained, there were blackening of the TSI and LIA media. However, due
to excessive blackening of the media, there is no comment on the comparison of the colours of the slants and
butts between all the Slants. However, with a common observable characteristic (blackening of media) for all
the Slants obtained, it can be positively considered that the Slants did not differ much from each other and,
thus, the results were considered precise and reliable.
Accuracy
Streaked Plates
The results obtained were generally accurate. The expected results were that most of the colonies formed on
the agar plates would have dark or black centres, indicative of Salmonella spp. colonies. The results actually
obtained did not deviate far from this expectation.
Slants
The results obtained were generally accurate. In all of the Slants obtained, there were blackening of the TSI
and LIA media. This is very crucial for T1/LIA as blackening of the LIA medium indicated hydrogen sulfide
production which were typical of Salmonella spp. microbes growing in the medium. This result was also further
substantiated by the bioMérieux® API® 20E test results, which indicated that the Food Sample contained
Salmonella spp. microbes with a confidence level of 99.7 %. However, no comment on the accuracy of the
results based on the colours of the slants and butts was possible due to excessive blackening of the media used
in all the Slants obtained.
Replicate 2 (Specimen Positive Control)
Streaked Plates
of the results obtained. As all of the agar media used in the Streaked Plates were selective in enhancing the
growth of Salmonella spp. microbes only, it was expected that there would be isolated typical or atypical
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Salmonella spp. colonies forming in each of the Streaked Plates. This was observed as all of the Streaked
Plates obtained have colonies with dark or black centres with some having a metallic sheen, indicating the
presence of Salmonella spp. microbes. As the results of each Streaked Plate did not differ much from each
other, the results were considered precise and reliable.
Slants
There is no comment on the precision and reliability of the results obtained as there were too few samples to
generate any useful comparison between the results. However, it is pleasing to note that based on the limited
experimental results obtained, all results were considered accurate.
Accuracy
Streaked Plates
The results obtained were generally accurate. The expected results were that most of the colonies formed on
the agar plates would have dark or black centres, indicative of Salmonella spp. colonies. The results actually
obtained did not deviate far from this expectation.
Slants
While there was only one Slant (T2/LIA) results obtained, those results are deemed accurate. For T2/LIA, it
was observed that there was blackening of the LIA medium, indicating hydrogen sulfide production. The colours
of both the slant and butt are also purple. All these observable physical characteristics are typical of Salmonella
spp. microbes growing in the LIA medium and this shows that the results actually obtained did not deviate much
from the expectation of positive results.
Replicate 3 (Specimen Negative Control)
Streaked Plates
of the results obtained. As all of the agar media used in the Streaked Plates were selective in enhancing the
growth of Salmonella spp. microbes only, it was expected that none of the plates would have any isolated
bacterial colonies forming after incubation as E. coli cannot thrive in such selective agar media. As the results
of each Streaked Plate did not differ much from each other, the results were considered precise and reliable.
Slants
The results of the Slants obtained were generally very precise, resulting in an appreciable reliability of the
results obtained. For all of the Slants obtained, none of them showed any blackening of the media, indicating
that all of them did not have any hydrogen sulfide production. The colours of the slants and butts of all tubes
indicated that Salmonella spp. microbes were absent in the media used. As the results of each Slant did not
differ much from each other, the results were considered precise and reliable.
Accuracy
Streaked Plates
The results obtained were generally accurate. The expected results were that none of the plates would have any
isolated bacterial colony forming. The results actually obtained did not deviate far from this expectation.
Slants
The results obtained were generally accurate. As all Slants did not have any blackening and that the colours of
their slants and butts were indicative of the absence of any Salmonella spp. growing in their media, this shows
that the results actually obtained did not deviate far from the expectation of negative results.
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5.2. Statements of Reliability
The results of all Streaked Plates and Slants obtained were generally very reliable and accurate. The following
precautions were taken to reduce any errors in the experiment:
• Sanitising workplaces/areas, instruments and apparatuses, and wearing gloves to prevent contamination.
• Ensuring aseptic techniques at crucial points in the experiment (such as transferring broths or bacterial
culture).
• Working near an open, non–luminous flame.
• Ensuring homogeneity of solutions (such as bacterial culture solutions and broth mixtures) by mixing them
thoroughly before any subsequent actions (such as extraction or dilution) was done to the solutions.
These precautions taken reduce the occurrence of experimental errors, leading to the high reliability and
accuracy of the results obtained and, subsequently, derived conclusions.
5.3. Statements of Anomaly
This sub–Section provides the possible sources of errors in the experiment and the suggested improvements.
T4/TT/BS
The experimental results of this Streaked Plate are anomalous. This is because there are no isolated bacterial
colonies or growth seen on the agar medium despite using Replicate 1 (Food Sample) mixture that contains
resuscitated active agents (microbes).
As the Food Sample used was fully amorphous, a possible source of experimental error was that there could be
other microbes, such as Citrobacter, that were competing with the Salmonella spp. microbes for growth on the
agar medium. Moreover, those microbes (including Salmonella spp. microbes) may not have been fully
resuscitated and capable on growing on the BS agar medium to form isolated colonies. With competition for the
nutrients in the agar as well as being injured to proliferate freely, the hurdles present may have suppressed the
growth of all the microbes in the agar medium, including Salmonella spp. microbes, resulting in no isolated
bacterial colony or growth observed for this Streaked Plate.
One possible solution for this would be to increase the resuscitation time in the Lactose broth as well as the
incubation time for the Streaked Plate. Increasing resuscitation time would allow more Salmonella spp.
microbes to become fully vegetative and a longer incubation time would allow the more competent Salmonella
spp. microbes to outgrow other microbes in the agar medium to form isolated typical or atypical Salmonella spp.
colonies. This would lead to more reliable and accurate results.

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6. Conclusions
6.1. Determining Presence of Salmonella in Food Sample
Based on the experimental results obtained for the Food Sample, it can be confidently concluded that there are
Salmonella spp. microbes present in the Food Sample provided.
For all the Streaked Plates obtained in the Selective–Differential Test, all the Streaked Plates showed
bacterial colonies that had observable physical characteristics (such as colours of colony centres and presence
of metallic sheens) that were consistent with isolated typical Salmonella spp. colonies. These observations for
the experimental results obtained for the Food Sample include most of the colonies having dark coloured or
black centres for all of the Streaked Plates and presence of metallic sheens on colonies growing on BS agar
medium.
For the Slants obtained in the Presumptive Test, all tubes showed blackening in their media indicating
production of hydrogen sulfide. This is an observable physical characteristic typical of Salmonella spp. microbes
growing in such media. Although the excessive blackening made it practically impossible to carefully examine
the colours of the slant and butt of each tube, at least one of the tubes could be examined for the colour of the
butt under a strong light source. This was T1/LIA, and the colour of the butt, which was purple, matches those
of typical Salmonella spp. microbes growing in LIA medium.
For the bioMérieux® API® 20E results obtained in the Confirmation (Serological) Test, the serological test
showed that Salmonella spp. microbes were present in the Food Sample with a confidence level of 99.7 %.
Therefore, after considering all of the experimental results holistically, it can be positively concluded that the
Food Sample did have Salmonella spp. microbes in its contents.
6.2. Assessing Suitability of Food for Consumption and Compliance with Government Regulations
As the mass of Food Sample used was 25 g and that Salmonella spp. microbes were detected using the
detection methodologies and analytical techniques in this investigation, the successful detection of Salmonella
spp. microbes in the Food Sample infracts the regulation imposed by the SFA, which states that cooked/Ready
–To –Eat food and meat products must not contain detectable amounts of Salmonella spp. in 25 g of that food8.
Regrettably, the Food Sample provided is deemed not fit for consumption especially when it was a
cooked/Ready–To–Eat food and a meat product.
The detectable presence of Salmonella spp. microbes indicates that there could be lapses in sanitary standard
operating procedures and hygiene standards. Possible sources of contamination could include the storage of
the final food product at incorrect temperatures (not below 5 °C and not above 75 °C), use of unsafe ingredients
or non–potable/unsanitary water, cross–contamination with other food ingredients containing Salmonella
microbes, and poor hygiene levels of workers and food handlers. The food company or vendor that prepared
the Food Sample should do a thorough inspection of their premises and operating procedures to identify lapses
in hygiene and food safety standards. Such contaminated food should also be discarded immediately. If any of
such food has been sold, a recall should be promptly initiated and alerted to all affected consumers. They should
also inform the relevant government authorities of such discovery of contamination.
6.3. Suggested Recommendations and Improvements
While the methodology provided in this investigation have a high level of efficacy, robustness, and accuracy,
regrettably, there are points of compromise within its framework that would allow the prevalence of false–positive
results, which might render the conclusions derived from such experimental results misleading.
8Singapore Food Agency. (2024, May 31). Sale of Food Act, Food Regulations, Eleventh Schedule (pg. 211 – 212).
Singapore Food Agency – Sale of Food Act.
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One area of concern is that there are other types of bacteria, such as Citrobacter and Proteus which all belong
to the Enterobacteriaceae family of bacteria, that could give observable physical properties or isolated colonies
similar to Salmonella spp. colonies. Thus, the presence of these non–target but similar class of family of bacteria
could lead to the misidentification of Salmonella spp. microbes, even if the latter is absent from the food sample.
Therefore, the use of qualitative results should be complemented with quantitative serological tests that
objectively measure the genomic differences between the microbes present in the food sample and Salmonella
spp. bacteria, resulting in a more objective and reliable approach.
One suggested improvement would be the use of the PCR9. The use of PCR would result in a more objective
approach as well as obtaining more reliable, and accurate experimental results and conclusion as the principle
of its mechanism is based solely on the genetic material of the microbes or biological agent that is being
examined on. The PCR works by amplifying the genetic material of the specific/target microbes by using specific
primers that bind to specific genes or DNA sequence in the genetic material and then synthesising many new
copies of the genome of the target microbe while the genome of the non–target microbes are not amplified. The
amplified genome is then digested into smaller fragments through the use of specific restriction enzymes that
cut the genome at specific restriction sites. The fragments obtained could then be compared against a DNA
ladder to determine the sizes of fragments obtained. This data is then compared against the data obtained for
Salmonella spp. microbes to determine if there are great similarities. If the similarity is high, there is a high
probability that the target microbe is Salmonella spp. microbes. The converse is also generally true.
However, there are a few drawbacks that accompany the use of the PCR technique10. Firstly, the PCR is a very
expensive analytical technique that requires the use of sophisticated laboratory equipment and hard to obtain
chemicals and genomic (DNA) ladder fragments. This may not be feasible for laboratories that do not have such
financial resources to support the PCR requirements. Secondly, certain chemicals used, such as ethidium
bromide, for staining the genomic fragments are toxic and should be handled and disposed off properly. Thirdly,
the PCR technique requires the experimenter to have very competent laboratory techniques and execution as
even minute genome contamination may render the results obtained to be inaccurate and useless. Fourthly,
concentrations of chemicals, reagents, genomic templates, and conditions of the reaction must be carefully
calibrated and controlled to prevent obtaining undesirable results such as the smearing of the PCR products.
The enzyme used in the PCR (Taq polymerase) lacks proof–reading capabilities and so the PCR genetic results
used to deduce the presence of Salmonella microbes may have low fidelity, leading to inaccurate conclusions.
These drawbacks, along with others not mentioned herein, make the use of the PCR technique a commitment–
intensive decision and expensive investment by laboratories.
Despite all these drawbacks, the benefits of using the PCR technique11 include, but are not limited to, a faster
generation of experimental (PCR) results as little genomic sample and shorter incubation is required, and more
reliable and accurate results. As the PCR amplifies the genome of the target microbes in the sample, it may be
possible to use smaller amounts or undiluted concentrations of these samples, possibly translating into lowered
costs for obtaining dilution and broth chemicals, increased sensitivity to detecting these microbes, and less
contamination of non–target microbes as only the genome of target microbes is amplified through the use of
specific primers that bind to specific genes or DNA segments on the genome of the target microbes.
In summary, it is strongly suggested that the detection methodologies and analytical techniques utilised in this
investigation is complemented by the use of serological tests such as the PCR technique.
9 National Human Genome Research Institute. (2020, August 17). Polymerase Chain Reaction (PCR) Fact Sheet.
10 faCellitate. (2023, April 12). Advantages and disadvantages of PCR technology. Advantages and disadvantages of PCR
technology - faCellitate.
11 faCellitate. (2023, April 12). Advantages and disadvantages of PCR technology. Advantages and disadvantages of PCR
technology - faCellitate.
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7. References
This Section lists all the sources of information referenced in the generation of this Coursework. The sources
were active and accurate at the time this Coursework was published. Each individual source of information is
recorded according to the APA citation format12.
For online sources (such as websites) that are no longer accessible, the use of the Wayback Machine13 may be
of relevance to access such defunct sources. The Group is unable to guarantee that such sources may be
accessible even through the prescribed method.
To avoid the issue of formatting errors due to the inclusion of online sources with excessively long web
addresses, all web addresses will be hyperlinked in the “URL” of the online source14.
For Written Text(s):
S/N Provenance
1 Ministry of Education, Singapore. (2021). SINGAPORE–CAMBRIDGE GCE A–LEVEL PROJECT

WORK (8808): INSTRUCTIONS TO CANDIDATES. Singapore: Singapore Examinations and
Assessment Board.
2 Singapore–Cambridge GCE N(T), N(A), O, & A–Level Examinations–Examination Rules and

Regulations For School Candidates. (2022). Singapore: Singapore Examinations and Assessment
Board.
3 School–based Science Practical Assessment (SPA) Information Booklet. (2014). Singapore:

Singapore Examinations and Assessment Board. URL.
4 Singapore–Cambridge General Certificate of Education Advanced Level Higher 2 Chemistry (9647)

and Biology (9648) syllabuses. (2014). Singapore: Singapore Examinations and Assessment Board.
5 Student Handbook. (2023). Nanyang Polytechnic.
6 Burdass, D., Grainger, J., & Hurst, J. (2016). Basic practical microbiology. Basic Practical
Microbiology; A MANUAL. URL.
7 Urry L, Cain M, Wasserman S, Minorsky P, Reece J. Campbell biology. 11. New York: Pearson;
2017.
8 Mayo Foundation for Medical Education and Research. (2022, April 29). Salmonella infection.
Salmonella infection - Symptoms & causes. URL.
9 Ellis, R. R. (2024, April 22). Salmonella (Salmonellosis). Salmonella: Causes, Symptoms, Risks,
Treatment, and Prevention. URL.
10 AOAC Official Method 967.25 Salmonella in Foods, Preparation of Culture Media and Reagents.
11 AOAC Official Method 967.26 Salmonella in Processed Foods, Detection.
12 AOAC Official Method 967.27 Salmonella in Foods, Identification
13 AOAC Official Method 967.28 Salmonella in Foods, Serological Tests.
12 Citations generated automatically by Citation Machine; a service provided by Chegg Inc. Available at
https://www.citationmachine.net/.
13 An internet archiving service. Available at https://web.archive.org/.
14 To access the website of the online source, click on the “URL” portion of the citation.

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14 AOAC Official Method 978.24 Salmonella spp. in Foods, Biochemical Identification Kit Method.
15 FDA BAM Chapter 5 Salmonella, Jul 2020.
16 Singapore Food Agency. (2024, May 31). Sale of Food Act, Food Regulations. Singapore Food
Agency – Sale of Food Act. URL.
17 Mayo Clinic Staff. (2022, April 29). Salmonella infection. Salmonella infection - Symptoms & causes
- Mayo Clinic. URL.
18 Ellis, R. R. (2024, April 22). Salmonella (Salmonellosis). Salmonella: Causes, Symptoms, Risks,
Treatment, and Prevention. URL.
19 CDC. (2024, June 18). Salmonella. Salmonella Homepage | CDC. URL.
20 WHO. (2018, February 20). Salmonella (non-typhoidal). URL.
21 WHO. (2022). Codex Alimentarius. CXC_001e.pdf. URL.
22 Xie, J., Yi, S., Zhu, J., Li, P., Liang, B., Li, H., Yang, X., Wang, L., Hao, R., Jia, L., Wu, Z., Qiu, S., &
Song, H. (2015, October 2). Antimicrobial Resistance and Molecular Investigation of H2S-Negative
Salmonella enterica subsp. enterica serovar Choleraesuis Isolates in China. URL.
23 faCellitate. (2023, April 12). Advantages and disadvantages of PCR technology. Advantages and
disadvantages of PCR technology - faCellitate. URL.
24 Diep, B., Barretto, C., Portmann, A.-C., Fournier, C., Karczmarek, A., Voets, G., Li, S., Deng, X., &
Klijn, A. (2019, October 22). Salmonella serotyping; comparison of the traditional method to a
microarray-based method and an in silico platform using whole genome sequencing data. Frontiers.
URL.
25 Fujihara, M., Hayashi, M., Hara, K., Sakazume, N., Tsukuda, T., & Tagaino, Y. (2023, October 19).
Verification of different methods used for isolating Salmonella enterica serovar Dublin from cattle
feces. The Journal of veterinary medical science. URL.
26 Salmonella detection and identification methods for food. RMB. (n.d.). URL.
27 National Human Genome Research Institute. (2020, August 17). Polymerase Chain Reaction (PCR)
Fact Sheet. URL.
For Visual(s)15 and Visual Schematic(s)16:
S/N Figure Provenance

Number
1 1.2.(a). Carr, J. H. (2014). Scanning Electron Micrograph of Salmonella Typhimurium

Undergoing Binary Fission. Biol 230 Lecture Guidel: Electron Micrograph of
Salmonella. URL.
2 2.3.(g). Aadya Agarwal, 234861Q. 2024. [Internal Use only]
15Visuals refer to elements such as pictures, moving pictures (i.e., videos), graphs, diagrams, and other related items.
16Visual schematics refer to a representation of the elements of a system using abstract, graphic symbols rather than
realistic pictures.
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8. Appendices
8.1. Interpretation of Experimental Results
This sub–Section provides the expected results and suggested interpretation of the results obtained.
(a) Selective–Differential Streak Plates Results
TYPICAL Salmonella COLONY MORPHOLOGY
(i) Hektoen Enteric (HE) agar

Blueish–green to blue colonies with or without black centres. Many cultures of Salmonella may produce
colonies with large, glossy black centres or may appear as almost completely black colonies.
(ii) Xylose Lysine Deoxycholate (XLD) agar

Pink colonies with or without black centres. Many cultures of Salmonella may produce colonies with
large, glossy black centres or may appear as almost completely black colonies.
(iii) Bismuth Sulfite (BS) agar

Brown, grey, or black colonies; sometimes they have a metallic sheen. Surrounding medium is usually
brown at first, but may turn black in time with increased incubation, producing the so–called ‘halo’
effect.
ATYPICAL Salmonella COLONY MORPHOLOGY
(iv) HE and XLD agars

Atypically, a few Salmonella cultures produce yellow colonies with or without black centres on HE and
XLD agars.
In the absence of typical Salmonella colonies on HE or XLD agars after 24 hours of incubation, then
pick 2 or more atypical Salmonella colonies.
(v) BS agar
Atypically, some strains produce green colonies with little or no darkening of the surrounding medium.
If typical or suspicious colonies are not present on BS agar after 24 hours of incubation, then do not
pick any colonies but re–incubate an additional 24 hours.
If typical or suspicious colonies are not present after 48 hours of incubation, then pick 2 or more atypical
colonies.

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Summary of Examination of Salmonella Colonies using the Selective–Differential Streak Plates
Appearance of Salmonella Colonies on Streak Plate

Media
Typical Atypical
Black/blueish–green to blue colonies with Any one of the following observations:

black centres.
(a) Blueish–green to blue colonies without
HE black centres.
(b) Orange colonies with or without black
centres.
Black/pink colonies with black centres. Any one of the following observations:
XLD (a) Pink colonies without black centres.

(b) Yellow colonies with or without black
centres.
Dark green to black colonies that produces Dark green to grey colonies without metallic
BS metallic sheen. There may also be darkening sheen or darkening of medium.
of medium underneath colonies
(b) Presumptive Test Results
TSI Medium Results
(i) Slant Results

Typical Salmonella colonies in TSI slants would produce a red (alkaline) slant.
pH of Medium
Acidic Alkaline
Colour of Slant Yellow Red
(ii) Butt Results

Typical Salmonella colonies in TSI butts would produce a yellow (acidic) butt.
pH of Medium
Acidic Alkaline
Colour of Butt Yellow Red

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(iii) Hydrogen Sulfide Results
The production of hydrogen sulfide is arbitrary in the determination of the presence of any Salmonella
colonies.
Production of Hydrogen Sulfide
Absent Present
Colour of Slant No blackening Presence of blackening
LIA Medium Results
(iv) Slant Results

The colouration of the slants is arbitrary in the determination of the presence of any Salmonella
colonies.
Medium pH Side Reaction
Acidic Alkaline Oxidative deamination
Colour of Slant Yellow Purple Red
(v) Butt Results

Typical Salmonella colonies in LIA butts would produce a purple (alkaline) butt.
Medium pH
Acidic Alkaline
Colour of Slant Yellow Purple
(vi) Hydrogen Sulfide Results

Typical Salmonella colonies in LIA media would produce hydrogen sulfide.
Production of Hydrogen Sulfide
Absent Present
Colour of Slant No blackening Presence of blackening

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9. Miscellaneous
The entirety of this Section is severable from the Coursework if it is determined by the Group to be required.
9.1. Notes to Readers
(a) Utilising this Coursework
To ensure that the Coursework is not misconstrued or incompletely assessed, it is advised that the Coursework
be read in its entirety, including all additional/supplementary documents and inserts. Please refer to sub–Section
9.4. for the full list of additional/supplementary documents.
(b) Original Authors/Owners of this Coursework
Unless otherwise noted, the Group shall assume to be the originators/owners of this Coursework. All copyright
and master rights to this Coursework (including all related/associated Works) shall vest in and belong to the
Group.
(c) Citing and Referencing this Coursework
Any external Work(s) not authored or owned by the Group that would contain or use any information, part(s), or
idea(s) from this Coursework should provide a proper citation/referencing to this Coursework using the citation
listed below:
Coursework Citation Citation Format

No citations available. APA
Omission of peripheral information (such as page number(s), section heading(s), figure, or table number(s))
when citing/referencing this Coursework does not constitute plagiarism or incomplete citation.
9.2. Formalities
Academic Integrity, Academic Misconduct, and Copyright Notice
Substantial portions of sub–Sections 9.2.(a) – (d) are heavily adapted from the following documents:
• Ministry of Education, Singapore. (2021). SINGAPORE–CAMBRIDGE GCE A–LEVEL PROJECT WORK

(8808): INSTRUCTIONS TO CANDIDATES. Singapore: Singapore Examinations and Assessment Board.
• Singapore–Cambridge GCE N(T), N(A), O, & A–Level Examinations–Examination Rules and Regulations
For School Candidates. (2022). Singapore: Singapore Examinations and Assessment Board.
• Student Handbook. (2023). Nanyang Polytechnic.
Such adaptations include the use of phrases and keywords. Copies of these documents may be obtained directly
from the respective publishers.
(a) Academic Integrity
Academic integrity refers to the acts and practices of candidates and examination personnel to adhere to the
examination and assessment regulations and rules of the examining authority.
It also refers to these acts and practices which safeguard the integrity of the assessment by ensuring no
candidate being assessed is given or attempts to give away any unfair advantages to themselves or to another
candidate. This ensures that the assessment is authentic and, subsequently, the results of the assessment are
accurately reflective of the candidate’s performance and capabilities.
Examples of academic integrity include, but are not limited to:

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• not copying another candidate’s answers or allowing their answers to be copied by another candidate in an
assessment that does not allow any collaboration or communication.
• ensuring that they acknowledge all sources of information referenced in the generation of their works (such
as coursework), where applicable.
• discouraging others from committing acts or practices which contravene any examination and assessment
regulations and rules.
• informing the examining authority of any candidate and/or examination personnel that have a suspicion to
infract any examination and assessment regulations and rules.
Candidates and examination personnel should be aware of and comprehend the examination and assessment
regulations and rules as stated in the latest official NYP Student Handbook that is available.
(b) Academic Misconduct
Academic misconduct is any act or practice which contravenes the examination and assessment regulations
and rules. Such acts or practices may jeopardise the integrity and authenticity of the examination results of the
candidates to be published.
In short, academic misconduct is the antithesis of academic integrity.
Examples of academic misconduct include, but are not limited to:
• collusion or attempted collusion with other person(s).

• attempts to obtain or offer unfair assistance or advantage.
• committing or aiding the offence of plagiarism17.
• sabotaging another candidate’s work or performance.
• any other acts or practices which violates the examination and assessment regulations and rules.
Academic misconduct is not tolerated by the Polytechnic.
(c) Penalties for committing or aiding Academic Misconduct
The Polytechnic adopts a strict stance against any candidate and/or examination personnel who have or are
suspected of committing or aiding the offence of academic misconduct. An investigation will be conducted when
an act committed by a candidate and/or an examination personnel has a cause for suspicion of academic
misconduct. Where clear and convincing evidence exists, disciplinary action by the examining authority will be
taken against any candidate and/or examination personnel found to have committed or aided the offence of
academic misconduct.
Such disciplinary actions, depending on the severity of the infraction committed, that may be taken by the
examining authority include, but are not limited to:
• expelling the candidate from the examination room and/or refuse entry to the candidate for subsequent
examination assessment components.
• annulling the candidate’s grade(s) for the affected examination.
• terminating or imposing a grade penalty to the candidate’s results.
• disqualifying the candidate from the entire examination.
• expelling or dismissing the candidate and/or examination personnel from the Polytechnic.
These disciplinary actions may come into immediate effect or otherwise.
(d) Acknowledgement of the Penalty for Academic Misconduct
17Plagiarism is utilising another entity’s work without acknowledging the provenance of that work and then, with or without
consent, passing it off as an entity’s own.
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The Group has been briefed and informed about the NYP penalty for committing or aiding the offence of
academic misconduct.
The Group has been made aware that committing or aiding the offence of academic misconduct may result in
disciplinary actions (such as a grade penalty), up to and including academic expulsion from the Polytechnic.
The Group has read, understood, and agreed to the examination and assessment regulations and rules as
imposed by the Polytechnic.
The Group is also aware that NYP lecturers are at liberty to screen the Group’s Coursework for academic
misconduct by any means possible in NYP.
By submitting this Coursework, the Group declares that this Coursework submitted is original and, to their best
of knowledge, does not contravene any examination and assessment regulations and rules. The Group has
acknowledged all sources of information referenced.
(e) Use of Copyright Materials under the “Fair Use” Policy
This Coursework may contain copyrighted materials for the sole purpose of drafting and generating this
Coursework. Such materials shall not, in connection with this Coursework, be used for any personal gain or
profit or any other use not specified by the “Fair Use” policy18.
The Group declares no ownership of the copyrighted materials used herein.
Every reasonable endeavour has been made by the Group to trace copyright holders and include them in this
Coursework’s References, but if any items requiring clearance have unwittingly been included, the Group will
be pleased to make amends at the earliest possible opportunity.
9.3. Glossary of Terms
This sub–Section contains commonly used terms utilised throughout this Coursework. The terms used include,
but are not limited to:
• notations.
• abbreviations and acronyms.
• symbols.
• technical/specialised terms, phrases, and keywords.
The terms used and their corresponding meanings are listed in Figure 9.3. and are arranged in alphabetical
order.
18Legislation Division of the Attorney–General’s Chambers of Singapore, Government of Singapore. (2021). Copyright Act
2021–PERMITTED USES OF COPYRIGHT WORKS AND PROTECTED PERFORMANCES. Retrieved September 23,
2022, from https://sso.agc.gov.sg/Act/CA2021?ProvIds=P15-.
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Terms Meaning Remarks

APA American Psychological Association The official, standard citation
format used in this Coursework.
BS Bismuth Sulphite Agar media
DNA Deoxyribonucleic acid
HE Hektoen Enteric Agar media
LIA Lysine Iron agar
NYP Nanyang Polytechnic Examining Authority
PCR Polymerase Chain Reaction A biological analytical technique
used to identity genomic
identities of microbes by
amplifying the DNA sample
present using specific primers,
digesting the fragments by using
specific restriction enzymes, and
then separating the DNA
fragments using gel
electrophoresis. The results are
then compared against a DNA
standard (DNA ladder) to
examine the size of the DNA
fragments obtained.
RV Rappaport–Vassiliadis Agar media
TSA Tryptone Soya agar
TSI Triple Sugar Iron
TT Tetrathionate
XLD Xylose Lysine Deoxycholate
Figure 9.3. – Glossary of Terms.
9.4. List of Additional/Supplementary Documents
The Figure 9.4. lists all the additional/supplementary documents that accompany this Coursework. The
documents are compiled and attached separately.
While every reasonable effort has been made to ensure preparation of all supplementary documents prior to the
official publication of this Coursework, inevitably, there arises circumstances that requires the Group to publish
additional supplementary documents19 past the publication of this Coursework. Therefore, such additional
supplementary documents will not be reflected in Figure 9.4. below. However, such documents will be indicated
on their title pages that they will form part of this Coursework after approval from the Group.
Document Title of Document Remarks

Number
There are no additional/supplementary documents.
Figure 9.4. – List of additional/supplementary documents.
9.5. Point(s)–of–Contact
Any queries or issues arising (whether directly or indirectly) from this Coursework (including all
related/associated Works) should be directed to the person(s) as provided in Figure 9.5. for enquiries.
19Examples of such documents include errata, amendments, and addendum. This footnote is not meant to be exhaustive.
These documents form part of the Coursework.
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All contact information provided in this sub–Section was active and accurate at the time this Coursework was
published. Please allow some time for the Point(s)–of–Contact to respond to any queries or issues.
Name Designation Contact Information

Muhammad Daruthman Abrie Project Team Member [email protected]
Masjuri
Figure 9.5. – List of Point(s)–of–Contact.

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TITLE PAGE FOR COURSEWORK SUBMISSION

Institution Name: Nanyang Polytechnic Course Name: Food Science and

Class: FS2301 Academic Year: 2024/25

Module Title: Diagnostics in Food Module Code: ASD201

Assessment Practical Report Assessment P1MS2

Date of Submission: 21 July 2024 Word Count: Not Applicable

Title of Coursework: Experiment 3 – Examination of Food Sample (Chicken) for Salmonella.

GROUP INDEX NUMBER Not Applicable

CANDIDATE NAME CANDIDATE NUMBER

Muhammad Daruthman Abrie Masjuri 234951W

This Coursework was conducted under the supervision of:

SUPERVISOR NAME DESIGNATION SIGNATURE

Sze Wee Ping Supervising Tutor

This document consists of 42 pages. Blank pages are indicated.

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

The convention ⏣ for representing the aromatic ring is preferred.

(c) Any anomalous data is indicated with a right superscript asterisk.

(e) Attention is drawn to the following sub–Sections:

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

General Investigative Framework

Conclusions derived from Experimental Results

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

1.1. Objective(s) of this Project

1.2. Literature Review

Importance of Proper Food Hygiene and Safety

Salmonella as a Specimen Pathogen of Great Concern

Figure 1.2.(a). – Coloured Scanning Electron Micrograph of a Salmonella bacterium.

1 WHO. (2022). Codex Alimentarius. CXC_001e.pdf.

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

Prominent Sources of Contamination

Required Local Regulations in Singapore

4 CDC. (2024, June 18). Salmonella. Salmonella Homepage | CDC.

Singapore Food Agency – Sale of Food Act.

General Investigative Framework

Stage 1 Stage 2 Stage 3 Stage 4

Figure 1.2.(b). – Flow of Processes in General Investigative Framework.

• Stage 2 – Selective Enrichment

• Stage 3 – Selective–Differential Test

• Stage 4 – Confirmation (Serological) Test

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

Summary of Key Experiment(s)

• Confirmation (Serological) Test

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

2.1. Risk Assessment and Safety Precautions

2.2. Materials and Apparatus

Unless otherwise noted, the rate of allocation is ‘per candidate group’.

• Food sample • Pipette Gun and disposable pipettes

o 100 cm3 (approximately 5 plates) Hektoen • bioMérieux® API® 20E

o 50 cm3 (approximately 5 tubes) Triple Sugar • Bunsen burner with lighter

o 100 cm3 (approximately 5 plates) Tryptone • Inoculating needles

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

Amount of Amount of Lactose

Replicate 1 Replicate 2 Replicate 3 Replicate 4

Figure 2.3.(b). – Inspection of the stomacher bags for any irregularities.

PROPERTY OF NYP/234951W ASD201/P1MS2_EXPERIMENT_3/2024/25/S1 [Turn over

Replicate mixture Amount of Replicate mixture extracted

Replicate mixture Amount of Replicate mixture extracted

Medium Incubation Temperature of Tube