UNIVERSITI MALAYSIA TERENGGANU

FACULTY OF FISHERIES AND FOOD SCIENCES

ADVANCE FOOD MICROBIOLOGY (STM3043)

LABORATORY REPORT (K2)(G1)

BACHELOR OF FOOD SCIENCES (FOOD TECHNOLOGY)

LAB TITLE: IDENTIFICATION OF ENTEROBACTERIA

LECTURER: DR MANNUR ISMAIL SHAIK

DUE DATE 09/05/2023

NAME MATRIC NUMBER

1) SITI NUR HUSNINA NISA BINTI MUSTAZA S61917

2) LEE XIN YEE S61999

3) NUR ALYAA NADHIRAH BINTI MAMAT S62049

4) TOH SHI YIN S62106

5) INTAN NURUL AIN BINTI NORDIN S62204

1.0 INTRODUCTION

Enterobacteriaceae is a large, heterogeneous group of Gram-negative rods that

includes bacteria that naturally inhabit the mammalian gut but also can occur and multiply in
other environments. Enterobacteriaceae have various types of bacteria. These bacteria
generally grow well, aerobically and anaerobically, at temperatures ranging between 20 and
37 °C on general laboratory media at neutral pH (Cooney et al., 2014). It can be
psychrotrophic and mesophilic. But, most Enterobacteriaceae are mesophilic. They
commonly are found in soil, water and sewage. They also are causes of botanical disease.
These organisms are facultatively anaerobic and motile. In the food industry,
Enterobacteriaceae are commonly used as indicator organisms that can indicate poor hygiene
practices or failure of a manufacturing process (“Foodborne Pathogens,” 2016).

Firstly, the oxidase test is a technique for detecting the presence of the terminal
enzyme system in aerobic respiration called cytochrome C oxidase or cytochrome a3.
Usually, the family Enterobacteriaceae gives a negative result, whereas Pseudomonas spp,
Aeromonas spp, Vibrio spp and Neisseria spp give a positive result (Tankeshwar, 2017).
Secondly, MacConkey agar is commonly used for the isolation of Gram-negative enteric
bacteria. MacConkey is a commonly used media to differentiate members of
Enterobacteriaceae. It differentiates between lactose-fermenting and nonfermenting
gram-negative rods by the colour of colonial growth. MacConkey agar is used for the
selective isolation and identification of members of the family Enterobacteriaceae from
faeces, urine, wastewater and foods.

Thirdly, the Indole test is used to determine the ability of an organism to split amino
acid tryptophan to form the compound indole. It is used as part of the IMViC procedures to
differentiate members of the family Enterobacteriaceae. Fourth, the Hydrogen sulphide (H2S)
production test is generally used to detect the production of the concerned gas by any
organism. This test is basically useful to identify the bacteria under family
Enterobacteriaceae. This group of bacteria produce H2S via reduction of sulphur containing
amino acids like methionine and cysteine or of inorganic sulphur compounds like
thiosulfates, sulphates or sulphites. The Urease test is a biochemical test that detects the
alkaline fermentation of urine (urea) with the resultant production of ammonia by
microorganisms. Lastly, the citrate utilisation test is a part of the IMViC test that
differentiates organisms on the basis of their ability to use citrate as a sole source of energy.
2.0 OBJECTIVE

To identify the Enterobacteria isolated from food samples.

3.0 MATERIALS AND METHODS

Materials

Media:

● Tryptone broth
● Lactose fermentation broth
● Citrate slant
● Urea broth

Stock cultures:

● Escherichia coli
● Klebsiella pneumoniae
● Proteus mirabilis
● Salmonella enterica

Reagents:

● Oxidase reagent
● Kovac’s reagent (for Indole test)

Methods

Oxidase Test

1. A strip of filter paper is soaked with a little freshly made 1% solution of the reagent.
2. A speck of culture is rubbed on it with a platinum loop.
3. A positive reaction is indicated by an intense deep-purple hue, appearing within 5-10
seconds and a negative reaction by absence of coloration or by coloration later than 60
seconds.
MacConkey Agar Test

1. 49.53 grams of dehydrated medium is suspended in 1000 ml purified/distilled water.

2. The medium is heated until it dissolves completely.
3. Sterilised by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Let it cool to 45-50°C.
5. Mix thoroughly before putting into sterilised Petri dishes.

Indole Test

1. Take sterilised test tubes containing 4 ml of tryptophan broth.

2. The tube is inoculated aseptically by taking the growth from 18 to 24 hrs
culture.
3. Incubated the tube at 37°C for 24-28 hours.
4. 0.5 ml of Kovac’s reagent is added to the broth culture.
5. The presence or absence of a ring is observed.

H2S Production

1. Inoculate the organism into a labelled tube by means of stab inoculation.

2. Incubate the inoculated tubes at 37°C for 24-48 hours.
3. The formation of black precipitate on the medium is observed.

Urease Test

1. The surface of a urea agar slant is streaked with a portion of a well-isolated colony or
inoculated slant with 1 to 2 drops from an overnight brain-heart infusion broth culture.
2. The cap is leaved on loosely and incubates the tube at 35°-37°C in ambient air for 48
hours to 7 days.
3. The development of a pink colour is examined for as long as 7 days.
Citrate Test

1. The slant back and forth are streaked with a light inoculum picked from the centre of
a well-isolated colony.
2. Incubated aerobically at 35°-37°C for up to 4-7 days.
3. A colour change is observed from green to blue along the slant.

4.0 RESULTS

Table 1: The Oxidase test results.

Negative Negative Positive - Pseudomonas

aeruginosa present

Table 2: MacConkey agar for 2 negative results.

Sample Results Observation

A White colonies
 B Red colonies

Table 3: Indole Test, Urease Test and Citrate Test results.

Test Results

Indole test - Eschericia coli Positive. There is a formation of pink

rings.

Indole test - Urease Test Negative.

 H2S Production Positive. The presence of salmonella
results in blackening.

Citrate Test Positive. There is the presence of Serratia

Marcescens.

5. DISCUSSION & QUESTIONS

Based on this experiment, to determine the enterobacteria in a sample, a few tests

must be used to identify the bacteria that are included in the sample: the Oxidase test, the
MacConkey test, the Indole test, the Urease test and the Citrate test. Firstly, the oxidase test is
essential for identifying between the Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -)
families of bacteria. It is also helpful for identifying and speciating a wide variety of other
bacteria, including those that use oxygen as the ultimate electron acceptor during aerobic
respiration. In the Oxidase test, we know that when the electron donor is oxidised, it will
produce a dark purple colour by cytochrome oxidase, which shows the result was positive.
Thus, the result showed that the organism (Pseudomonas aeruginosa) is oxidase positive, as
are others in the enterobacteriaceae families of bacteria like E. coli, Salmonella spp., and
others.
 When the result is negative, it should transfer the bacteria to MacConkey agar to
identify the bacteria in what colonies. MacConkey agar is used to isolate gramme-negative
enteric bacteria and to distinguish lactose-fermenting gramme-negative bacteria from
lactose-non-fermenting gramme-negative bacteria. It is also usual to use the media to
distinguish bacteria based on their ability to ferment carbohydrates other than lactose. As we
know, lactose-fermenting organisms produce pink colonies and generate acidic byproducts
that lower the pH, turning the pH indicator pink. (E. coli, Enterobacteria, and Klebsiella).
Gramme-negative bacterial species will still form colonies; however, colonies will be white
due to the lack of pH change in the absence of lactose fermentation. Salmonella, Proteus,
Yersinia and Pseudomonas are a few examples. On MacConkey media, gramme-positive
bacteria will not form colonies. Staphylococcus, Enterococcus and Micrococcus are a few
examples. In table (2), the result showed that when we transferred the bacteria from the
control, the result showed the positive control on the MacConkey agar while the bacteria
from the live sample.

The Indole test is a biochemical test used to determine the ability of microorganisms
to decompose the amino acid tryptophan and produce indole. Indole is a heterocyclic organic
compound that can be used as a metabolic end product of the amino acid tryptophan in
certain bacteria to coordinate various forms of behaviour (Melander et al., 2014). This test is
commonly used to identify members of the Enterobacteriaceae family, such as Escherichia
coli and Proteus species through indole production. The presence of the enzyme
tryptophanase in these enteric bacteria can break down tryptophan into indole, pyruvic acid
and ammonia. The indole produced can react with the aldehyde present in Kovac’s reagent to
form a cherry-red ring at the top of the broth, which indicates a positive result. Due to the fact
that amyl alcohol is water-insoluble, the cherry-red coloration will form an oily layer at the
top of the broth (Aryal, 2022). In this lab session, a positive indole test can be observed in
Escherichia coli, which is known to produce indole. As a result, it has been proven that
Escherichia coli is one of the members of the Enterobacteriaceae family.

The Urease test is used to identify those organisms that are capable of hydrolyzing
urea to produce ammonia and carbon dioxide through the production of the enzyme urease.
This test is performed as part of the identification of the Enterobacteriaceae family, including
Klebsiella, Proteus, and many other bacteria that produce the urease enzyme. Christensen's
urea agar or urea broth is used to detect the urease activity of the urease-positive organisms
(Brink, 2010). A positive urease test can be obtained through the production of ammonia by
urease producers. The ammonia produced combines with the carbon dioxide and water to
form ammonium carbonate, which turns the medium alkaline, and the pH shift is detected by
the colour change from the original orange-yellow colour to bright pink. Rapid
urease-positive organisms turn the entire medium pink within 24 hours. Weakly positive
organisms may take several days, and negative organisms produce no colour change or
yellow as a result of acid production (Aryal, 2022). The results showed that the culture
medium still remained a yellowish colour, leading to a negative test result. A negative urease
test indicates that there is no urease activity in Escherichia coli and that it does not produce
the urease enzyme. Therefore, Escherichia coli is a generally urease negative bacterial
species.

The Hydrogen sulphide (H2S) production test is used to assist in the identification of
members of the Enterobacteriaceae family, especially in identifying hydrogen
sulphide-generating bacteria like Salmonella based on their ability to produce H2S gas.
Hydrogen sulphide-generating bacteria are capable of reducing sulphur-containing
compounds to sulphides and producing hydrogen sulphide gas during the process of
metabolism. In diagnostic microbiology, Sulfite indole motility (SIM), Kligler iron agar
(KIA), or Triple Sugar Iron (TSI) agars are commonly used for the detection of H2S
production. With all H2S detection systems, the endpoint is an insoluble heavy metal
sulphide, which produces a black precipitate in the medium (Tankeshwar, 2023). A positive
H2S production test will show a blackening of the medium, indicating that the bacteria are
capable of producing H2S gas. According to the results obtained, the blackening of the
medium denotes H2S production by the action of the Salmonella. Thus, the hydrogen sulphide
(H2S) production test is useful for the identification of Salmonella species.

The Citrate test is used to determine whether an organism is capable of utilising

citrate as its sole source of carbon and energy. This test is particularly useful in identifying
members of the Enterobacteriaceae family. To perform the citrate test, a bacterial culture is
inoculated onto a solid growth medium containing sodium citrate as the only carbon source.
If the bacteria is capable of utilising citrate, it will produce an enzyme called citrate
permease, which converts citrate into pyruvate and other metabolites (Aryal, 2022). The
pyruvate is then converted into carbon dioxide, which increases the pH of the medium. The
shift in pH turns the bromothymol blue indicator in the medium from green to blue.
Otherwise, a negative test is indicated by no growth or a lack of colour change. Based on the
results obtained, the colour change of the medium from green to blue indicates the production
of alkaline compounds by Serratia marcescens as a result of citrate utilisation. Therefore, the
citrate test can be used for the identification of Serratia marcescens based on its ability to
utilise citrate.

QUESTIONS

1. Most of the species are the normal flora of the intestine and other parts. How to
control the contamination of these bacteria in food products?

Cross contamination can cause food poisoning when bacteria is transferred into food
that is ready to eat. For example, if raw meat comes into contact with a sandwich, the
person eating the sandwich will consume the bacteria that was on the raw meat. There
are a few methods that control the contamination of this bacteria which is by heating,
boiling, autoclave and oven drying. For heating, the easiest and effective way to
control microbial growth is to use a flame. While boiling is not to be used to kill the
spora so not as sterile.

Other than that, some ways to help prevent cross contamination include to use
separate utensils or thoroughly wash and sanitise utensils between handling raw and
ready-to-eat foods, keep food covered and off the floor during storage and avoid any
unnecessary touching of food. When we store raw foods, especially meat and fish, on
the bottom shelf of the fridge to prevent raw meat juices dripping onto ready-to-eat
foods. Then, keep cleaning chemicals and other non-food items stored away from
food.

2. Some species are human pathogens. E. coli, Klebsiella spp., Proteus spp., and
Salmonella spp. are among the most common human pathogens. What is the
normal practice when you identify the presence of human pathogens in food
samples?

When human pathogens are identified in food samples, it is important to take

immediate action to prevent the spread of the contamination and protect public health.
 If a pathogen is identified, we need to notify the appropriate regulatory authorities,
such as the Food and Drug Administration (FDA) or the United States Department of
Agriculture (USDA). If contaminated food products have already been distributed, a
recall might be required to take them off the market and prevent further spread of the
contamination. Subsequently, it is crucial to investigate the source of the
contamination to prevent future outbreaks. The investigation can trace the
contamination back to its source, such as a specific batch of raw ingredients, or look
into the production process to identify potential sources of contamination. Based on
the investigation's results, corrective measures may be taken to stop further
contamination, including modifying the production process, conducting more testing
and monitoring, or implementing better sanitation and hygiene practices.

6.0 CONCLUSION

The Enterobacteriaceae that are isolated from food samples are identified. All of the
tests which are oxidase test, McConkey agar test, H2S production, indole test, urease test and
citrate test give different kinds of results but it is the reactions that shows the presence and
types of Enterobacteriaceae.
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