LAB REPORT 2 MIC 254

GRAM-NEGATIVE ROD SHAPED FOOD BACTERIA

By:

Muhammad Ilham Nur Hakim Bin Ramli (2022775425)

Muhammad Nasren Hisyam Bin Saifuddin (2022744737)
Muhammad Idham Bin Abu Bakar (2022340509)
Nadia Yasmin Binti Mohd Zaki (2022777181)
Farahin Nursyahirah Binti Mahthir (2022553425)

Lecturer: Madam Syazuani Mohd Shariff

Date of Experiment: 05/03/2023

Date of Submission: 11/04/2023

Group: AS1143A1
TITLE
Gram-negative Rod-shaped Food Bacteria

OBJECTIVES

1) To identify the characteristics of gram-negative rod-shaped bacteria.

2) To expose students to food spoilage characteristics caused by gram-negative
rod-shaped bacteria.

INTRODUCTION

Many people only think of bacteria as "germs," unseen creatures that may infiltrate our
bodies and cause sickness. In the human body, there are about ten times as many bacterial
cells as there are human cells. Few people realise that many bacteria not only cohabit with
humans all the time, but also perform an astounding diversity of useful tasks. Many of these
bacteria cells are found lining the digestive tract to help keep it healthy. Certain bacteria
dwell in the soil or on decaying plant debris, where they, together with fungi, play a vital part
in nutrient cycling. Bacteria may also be employed to produce vitamins and insulin, as well
as to clean up oil spills. Certain bacteria cause food spoilage, food-borne disease, and
agricultural damage, but others are extremely beneficial in the manufacture of fermented
foods like yoghurt and soy sauce. Few bacteria are pathogens or parasites that cause illness in
animals and plants.

Gram negative rods are a kind of bacterium distinguished by their shape and Gram
stain colour. Bacteria are classified into two types based on the structure of their cells which
is rods and cocci. Cocci are spherical bacteria, whereas Rods are rod-shaped bacteria.
Because everyone eats, everyone is at risk of developing a foodborne disease. Meat and dairy
products are the most often implicated foods. Salmonella Typhimurium, E. coli O157:H7, and
Shigella spp. are among the most common foodborne pathogens that harm humans globally.
All pathogens found in food are significant. But, depending on the circumstances, they might
be more or less dangerous. Certain foodborne bacteria, such as Salmonella spp. and E. coli
O157:H7, can cause significant disease with a low infectious dose. Surprisingly,
Gram-negative bacterial pathogens account for around 69% of all infections.
MATERIALS

E. coli on Nutrient Agar

Klebsiella on Nutrient Agar
Salmonella on Nutrient Agar
E. coli on MacConkey Agar
Klebsiella on MacConkey Agar
Salmonella on MacConkey Agar
E. coli in nutrient broth
Klebsiella in nutrient broth
Salmonella in nutrient broth
Urea broth
TSI agar
Litmus milk
Sugar broth (maltose, glucose, lactose, sucrose)
Sample of spoiled food (naturally spoiled and inoculated with culture bacteria)
Cover slip
Cavity slide
Vaseline
Microscope
70% alcohol
Inoculation loop
Inoculation needle
PROCEDURE

A) Macroscopic examination.
1. The cultural characteristics of the given cultures on Nutrient agar and
MacConkey Agar plates were studied.
2. The picture below was used as a guide.

B) Characterization of bacteria.

Motility test.
1. A loopful of log phase culture broth was placed on the coverslip.A small
amount of vaseline was applied at the edge of the coverslip.
2. The cavity slide was inverted over the coverslip.
3. The slide was gently pressed against the cover slip and then was reverted back.
4. The culture was hanging at the centre of the cavity slide.
5. The motility pattern of the bacterial culture was observed using the
microscope.
 Biochemistry test.
1. A loopful of each culture was inoculated into urea broth.
2. A loopful of each culture was inoculated into litmus milk.
3. A loopful of each culture was inoculated into various sugar broths (maltose,
glucose, lactose and sucrose).
4. Each culture was inoculated into TSI agar slant using an inoculating needle by
stabbing through the centre of the medium to the bottom of the tube. Then, the
surface of the agar slant was streaked.
5. All broths and agar slants were incubated at 37°C for 24 hours.

C) Examination of spoiled food samples.

1. The spoiled food samples were examined.
2. The sample which spoiled under natural conditions and those which were
inoculated with each of the organisms were compared.
3. The changes on odour, texture and colour were determined.
RESULTS

A) Macroscopic examination.

Bacteria E. coli Klebsiella Salmonella

Texture Nutrient
Agar Dry Smooth Smooth

MacConkey
Agar Smooth Smooth Dry

Elevation Nutrient
Agar Raised Flat Raised

Macroscopic
morphology MacConkey
Agar Flat Raised Convex

Form Nutrient
Agar Raised and Round and Round
punctiform punctiform

MacConkey
Agar Round Round Round

Margin Nutrient
Agar Entire Entire Entire

MacConkey
Agar Serrate Entire Entire
B) Characterization of bacteria.

Motility test.

Bacteria E. coli Klebsiella Salmonella

Motile ✔ ✔

Non-motile ✔

Picture of E. coli Picture of Salmonella

Picture of Klebsiella

Figure 2.1
Picture of Salmonella in sugar broth Picture of E. coli in sugar broth

Picture of Klebsiella in sugar broth

Figure 2.2
 Biochemistry test

Bacteria E. coli Klebsiella Salmonella

Urea broth
No changes Formation of No changes
white spots on the
wall

Litmus milk
Purple precipitate Precipitate 3 layers formed
formed at bottom formed at bottom with purple
precipitate at
bottom and
formation of
white spots

Maltose Pink-red Reddish pink Light pink

Sugar broth
Glucose Pale pink Light pink Light Pink

Lactose Light pink Brown chocolate Reddish pink

Sucrose
Pale Reddish pink Reddish pink
yellowish-pink

Butt colour
Orange Dark Orange Yellow

Slant colour
TSI agar Orange Dark Orange Yellowish Dark

Type of reaction
Acid/acid Acid/acid Acid/alkaline

Blackening of
the medium No A little dark Darker

Gas production
A lot little A lot
DISCUSSION

This experiment was done by conducting 3 types of experiments. The first experiment
involves examining E. coli, klebsiella, salmonella, by providing cultures on nutrient agar
under a microscope. This approach involved observing and learning about the
gramme-negative bacteria's macroscopic appearance. In this experiment, the macroscopic
morphology of gram-negative bacteria was observed and studied. Next, for the second
procedure was the motility test and Biochemistry test, which are in the motility test, the
motility pattern of movement for E. coli, klebsiella, and salmonella was observed.
Meanwhile, in the biochemistry test, the reaction of both cultures was examined and studied
after 24 hours of incubation at 37°C For the third procedure, was the observation of the
spoiled food sample in which the chosen spoiled food was the rice with E. coli, spoiled rice,
rice with Klebsiella and the rice with Salmonella. It was examined for odour, texture, and
colour. Ultimately, all the observations were recorded in the table 1.1,1.2,1.3,1.4

For the first experiment, in which, macroscopic examination. 3 different types of

bacteria were used in this experiment by using 2 different cultures which are Nutrient Agar
and MacConkey Agar. In Nutrient Agar for E. coli the texture is dry, the elevation was raised,
the form is round and punctiform and the margin is entire. Next, for Klebsiella, the texture is
smooth, the elevation is flat, the form is round and punctiform and the margin is entire.
Lastly, in Nutrient Agar for Salmonella, the texture is smooth, the elevation was raised, the
form is round and the margin is entire. Moreover, in MacConkey agar for E. coli, the texture
was smooth, the elevation is flat, the form is round and the margin is serrate. For Klebsiella,
the texture is smooth, the elevation was raised, the form is round and the margin was entire.
For Salmonella, the texture is dry, the elevation is convex, the form is round and the margin is
entire. Thus, the macroscopic morphology of E. coli, Klebsiella, and Salmonella were
observed.

For the second experiment, in which, motility test. The E. coli and salmonella shows
motility based on theory. However, the E. coli and salmonella motility cannot be observed
under the microscope in figure 2.1. This is due to the personal errors made by students in
which the lens zooming was not properly handled well. Furthermore, some strains of E. coli
are non-motile in addition to its flagellated bacteria with peritrichous flagella arrangement.
For Klebsiella, it does not show motility and is non-flagellated.
 Furthermore, in the same procedure, a few biochemistry tests were conducted by a
specific medium which is urea broth. For both gram-negative bacteria which are E. coli and
Salmonella, it does not show any changes in colour. This is due to the absence of urease
enzymes in urea broth which helps to detect the presence of urease. Decarboxylation of
amino acids results in urea. Ammonia and 𝐶𝑂2 are produced when urea is hydrolysed. The

solution becomes more alkaline due to the creation of ammonia, and the pH change is
indicated by phenol red, which turns from pale orange at pH 6.8 to magenta (pink) at pH 8.1.
Within 24 hours, rapid urease-positive microbes turn the entire medium pink.

For litmus milk in E. Coli, purple precipitate formed at the bottom. For Klebsiella,
precipitate formed at the bottom and for Salmonella 3 layers formed with purple precipitate
formed at the bottom with the formation of white spots. For sugar broth, E. Coli, Klebsiella,
and Salmonella were tested in this experiment.However, this experiment was conducted by 3
different groups which are group 1 , group 2 and group 3 as shown in figure 2.2. In E. coli,
for maltose, pink- red colour is formed. For glucose, pale pink is formed. For lactose, light
pink is formed. For sucrose, pale yellowish-pink is formed. For Klebsiella, In Lactose it
shows brown chocolate colour. In Glucose, it shows light pink colour. For both Maltose and
Sucrose, it shows reddish pink colour. For Salmonella, there are 2 different types of colour
observed which are Reddish-pink colour, and light pink colour. For both maltose and glucose,
it does show light pink colour. Meanwhile, for both lactose and sucrose, it shows reddish pink
colour. Thus, the changes in air bubbles and colour does give a positive result to glucose. For
the TSI Agar test, TSI Agar is a biochemical test used for the identification of the ability of a
gram-negative bacterium to ferment a specific type of sugar and the ability to produce
hydrogen sulphide. For E. coli, the butt colour is orange. For slant colour, it shows orange
colour. For this type of reaction, it is acidic. For blackening of the medium for E. Coli, it
shows no changes.

In the last procedure, there are spoiled food samples. For food naturally spoiled, it
smells odour, sticky and watery texture, and has a black colour. For food inoculated with E.
Coli, it has a sour smell odour, sticky and slimy texture also comes with greenish black
colour. For food inoculated with Klebsiella, it has a sour smell with sticky and mushy texture
along with yellowish green colour. Lastly, for food inoculated with salmonella it has a
pungent smell odour with sticky and wet texture along with no changes in colour.
CONCLUSION

In conclusion, the objectives of this experiment were met. The characteristics of the
gram-negative rod-shaped bacteria E. coli, Klebsiella, and Salmonella have been determined
at the end of the experiment, and it has been discovered that each of them has a different
colonial morphology. According to the motility test, Salmonella and E. coli are both motile,
but Klebsiella is not. Each of the biochemical tests, which include the urea broth test, TSI
Agar test, litmus milk test, and sugar broth test, yields a different response from each of the
three bacteria. The students were then exposed to the features of food spoilage brought on by
gram-negative rod-shaped bacteria, where the spoilt food is stale rice, and the characteristics
noticed were the odour, texture, and colour of the stale rice. The majority of the results were
consistent with theory, though with minor variations.

QUESTION

1) Discuss why the motility test should be done at the log phase of bacterial growth.
The motility should be performed during the log phase of bacterial development
because it is the best time to test for motility. The log phase of bacterial development
is the time when the bacteria are healthy and actively growing. During the log phase,
motility will occur.

2) Explain the principle of urea broth test.

The principle of urea broth test is a differential media termed urease broth is used to
assess an organism's capacity to produce the exoenzyme urease, which hydrolyzes
urea into ammonia and carbon dioxide. The broth includes the pH indicator phenol
red, two pH buffers, urea, very little nutrition for the bacteria, and urea. In an alkaline
environment, phenol red turns fuchsia, while it turns yellow in an acidic environment.
The media turns pink as the urea in the broth is broken down and ammonia is formed,
which also creates an alkaline environment.
3) Explain the principle of the litmus milk test.
Lactose, a milk sugar, and casein, lactalbumin, and lactoglobulin, milk proteins, are
the primary milk substrates that can be transformed. An oxidation-reduction indicator
like litmus is introduced into the medium to serve as a pH indicator to help
differentiate between the metabolic changes induced in milk. Following that, litmus
milk creates a great differential media where bacteria can metabolise milk substrates
based on their enzymatic complement.
When acid is produced and the litmus turns pink as a result, lactose has started
to ferment. If enough acid is produced, milk's casein will coagulate and solidify.
When certain microorganisms are present, the curd thickens and whey forms on the
top. Certain bacteria break down casein, turning milk straw-coloured and making it
resemble turbid serum. The medium can also lose its colour at the bottom of the tube
when certain organisms reduce the amount of litmus.

4) Explain the principle of the sugar broth test.

According to the principle of carbohydrate fermentation, when an organism consumes
a carbohydrate substrate, the medium becomes more acidic, which can be seen by a
pH indicator dye. Microorganisms use the fermentation of carbohydrates to produce
energy. During glycolysis, the majority of bacteria convert glucose to pyruvate;
however, certain species utilise alternative routes. A fermentation medium is made up
of a baseline medium that is fermentable and contains just one type of carbohydrate
(such as glucose, lactose, sucrose, mannitol, etc.). The medium also includes a
number of pH indicators. Each tube also contains a Durham tube to catch gas
produced by metabolism in addition to a pH indicator to track acid generation from
fermentation. Differentiating between bacterial groups or species can be done using
the carbohydrate fermentation patterns that various organisms exhibit.

5) Explain the principle of TSI agar test.

Triple Sugar Iron Agar (TSIA) includes three sugars—glucose (dextrose), sucrose,
and lactose—in a ratio of 1: 10 as the carbon source. Together with this, the media
also includes casein as a source of protein, yeast, beef extract, and peptic digest of
animal tissue. To distinguish between gramme negative enteric, apply this test.
Utilisation of glucose takes place anaerobically on the butt and aerobically on the
slope where oxygen is present. The glucose fermenting bacterium will continue to
metabolise pyruvate through the TCA cycle to produce acid end products once it has
converted all of the available glucose to pyruvate. The pH indicator phenol red
becomes yellow when exposed to the acid in the media. The slant and butt of TSIA
that has been infected with a glucose fermenter will therefore appear yellow after 6
hours of incubation. In the slant region where the condition is aerobic, the byproduct
of protein and amino acid metabolism, NH3, changes the pH environment of the
medium from neutral to alkaline, causing the colour to appear red and the reaction to
be known as alkali/acid, while the colour of the butt is still yellow because of
anaerobic glucose breakdown.
When holes appear or the material is torn into multiple pieces, gas production
can be seen. The medium turns black as a result of H2S generation by the bacterium.
Its colour is a result of the medium's sodium thiosulfate ingredient producing
hydrogen sulphide (H2S), which subsequently reacts with ferrous ammonium sulphate
to generate the insoluble, dark chemical ferrous sulphide.
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