LABORATORY REPORT
MIC254
FOOD MICROBIOLOGY
LAB 2: GRAM-NEGATIVE ROD-SHAPED
FOOD BACTERIA
DATE EXPERIMENT: 5 APRIL 2023
DATE SUBMISSION: 11 APRIL 2023
STUDENT NAME STUDENT ID
1. LEEYA UMAIRAH BINTI MOHD SHAFIE 2022791815
2. NUR SABRINA BINTI MUHAMMAD SHAH 2022744707
3. ZUHAIRAH BINTI ISHAK 2022318339
4. MUHAMMAD RAIF HANAFI BIN NASURUDDIN 2022132031
TITLE: GRAM-NEGATIVE ROD-SHAPED FOOD BACTERIA
OBJECTIVE:
1) To identify the characteristics of gram-negative rod-shaped bacteria.
2) To expose students to food spoilage characteristics causing by gram-negative rod-shaped
bacteria
INTRODUCTION:
Gram-negative bacteria are those that do not retain the crystal violet stain used in the Gram
staining method of bacterial differentiation and leading them to appear pink under a microscope
due to the decolourization of the crystal violet stain. Gram-negative bacteria are distinguished
by their cell envelopes like cell wall, cell membrane, and outer membrane, which are made up of
a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a
bacterial outer membrane. Only a tiny amount of the crystal violet stain is retained by the thin
layer of peptidoglycan, decolorizing the crystal violet colour and yielding the pink colour.
The bacteria are classified as Gram-negative when stained red or pink during the identification
procedure known as Gram staining. Gram-positive bacteria are stained purple under a
microscope because of a thick layer of peptidoglycan that holds the Crystal Violet stain used in
Gram staining. A thin layer of peptidoglycan is attached to two membranes in gram-negative
bacteria. This appears pink because it cannot contain the stain. Gram-negative bacteria have an
extra membrane called the outer cytoplasmic membrane in addition to a thin peptidoglycan layer.
This results in the requirement for transport mechanisms to cross this membrane and adds
another permeability barrier.
In this experiment, the characteristics of both gram-negative rod-shaped bacterial cultures will be
determined and the students will be exposed to the characteristics of food spoilage brought on by
gram-negative rod-shaped bacteria through the use of three procedures. Firstly, the macroscopic
examination of the cultures on Nutrient Agar plates, second, the motility test and the
biochemistry test and third, the examination of the spoiled food. The table of results includes a
record of every observation.
PROCEDURE/METHODS
A) Macroscopic examination.
1. The cultural characteristics of the given cultures on Nutrient Agar (NA) and
MacConkey Agar (MCA) plates was studied.
2. The picture below was used as a guide.
B) Characterization of
bacteria. Motility test.
3. A loopful of log phase culture broth was placed on the coverslip.
4. A small amount of vaseline was applied at the edge of the coverslip.
5. The cavity slide was inverted over the coverslip.
6. The slide was pressed gently against the coverslip and reverted back.
7. The culture was hung at the center of the cavity slide.
8. The motility pattern of the bacterial culture was observed using the microscope.
Biochemistry test.
1. A loopful of each culture was inoculate into urea broth (1 set/group).
2. A loopful of each culture was inoculated into litmus milk (1 set/group).
3. A loopful of each culture was inoculated into various sugar broth (maltose,
glucose, lactose, sucrose) (1 set/class).
4. Each culture was inoculated into TSI agar slant using inoculating needle by stabbing
through the center of the medium to the bottom of the tube and then streaking the surface
of the agar slant (1 set/group).
5. All broth and agar slant was incubated at 37°C for 24 h.
C) Examination of spoiled food samples.
1. The spoiled food samples was examined.
2. The sample which spoiled under natural conditions and those which were inoculated
with each of the organism was compared.
3. The changes on odor, texture and color was determined.
RESULTS:
A) Macroscopic examination
Bacteria E. coli Klebsiella Salmonella
Texture Nutrient Agar Mucoid Smooth Smooth
MacConkey Smooth Smooth Smooth
Agar
Macroscopic Elevation Nutrient Agar Raised Flat Convex
morphology
MacConkey Convex Draughtsman Convex
Agar colony
Form Nutrient Agar Punctiform Punctiform Punctiform
MacConkey Punctiform Punctiform Round
Agar
Margin Nutrient Agar Entire Entire Entire
MacConkey Scalloped Entire Entire
Agar
B) Characterization of
bacteria Motility test.
Bacteria E. coli Klebsiella Salmonella
Motile / /
Non-motile /
Biochemistry test.
Bacteria E. coli Klebsiella Salmonella
Urea broth Orange Orange Orange
Litmus milk Remain purple White precipitate at Remain purple
the bottom and
light brown colour
on the top
Sugar broth Maltose Pink red Reddish pink Light pink
Glucose Light pink Light pink Light pink
Lactose Light pink Brown chocolate Reddish pink
Sucrose Pale pink Reddish pink Reddish pink
TSI agar Butt color Orange Orange Orange
Slant color Orange Red Orange
Type of Acid/Acid Alkaline/Acid Acid/Acid
reaction
Blackening No There are big dark No
of the spot
medium
Gas Oxygen and carbon Hydrogen sulfide Oxygen and carbon
production dioxide dioxide
c) Examination of spoiled food samples
Criteria Food naturally Food inoculated Food inoculated Food inoculated
spoiled with E. coli with Klebsiella with Salmonella
Odour Putrid smell Sour smell Sour smell Strong putrid
smell
Texture Slimy and Smooth and Juicy and slimy Smooth
mushy slimy
Colour Green and Black spot and Green and White yellowish
yellowish colour the yellowish yellowish colour
colour
DISCUSSION:
A) Macroscopic examination
There were three different procedures used to perform this experiment. The first step is a
macroscopic examination of the E. coli, Klebsiella, and Salmonella cultures that were provided
on Nutrient Agar and MacConkey Agar plates. During this method, the colonial morphology of
gram-negative rod bacteria was seen and investigated. Then, the second step is the motility test
and biochemistry test. During the biochemistry test, the reaction of three cultures with a
particular media was examined and observed after 24 hours of incubation. Lastly, the third step
involved analyzing a sample of spoiled food which is rice had been used. During this technique,
the odour, texture, and colour of rice were all evaluated.
For our observations, the texture on the Nutrient Agar is mucoid and smooth on the MacConkey
Agar when using bacteria E. coli. The elevation shows that E. coli was raised in Nutrient Agar
and convex in MacConkey Agar. For the form, there are punctiform in the Nutrient Agar and the
same thing in the MacConkey Agar. For the margin, we need to see the edge of the agar. In the
Nutrient Agar, the entire E. coli while there are scalloped for MacConkey Agar.
For our observations of Klebsiella, the Nutrient Agar and MacConkey Agar have smooth
textures. Klebsiella has a flat elevation on the Nutrient Agar and Droughtman colony on the
MacConkey Agar. Both the agars have the same form which is punctiform. The margin that
we should see from the edge is entire for Nutrient Agar and Klebsiella.
Lastly, for our observations of Salmonella, the texture of the Nutrient Agar and MacConkey
Agar are smooth. The elevation also has the same thing for both agars which are convex. On the
Nutrient Agar, the form that existed is punctiform and round on the MacConkey Agar. Both
margins, also share the same thing which is entire.
B) Characterization of bacteria
Motility test
E. Coli Salmonella
In our experiment, our E. coli and Salmonella were not moved under a microscope. The theory
about cells moving through some sort of self-propulsion is referred to as motility. E.coli and
Salmonella were the bacteria with the motile during the motility test. There are errors we made
when handling the bacteria. False-negative results could happen if the bacteria are harmed by
heating or shaking. The organism will not move as a result of this environmental shock. For the
solution to solve this problem, we need to handle bacteria extra carefully with make sure not to
heat while inoculating bacteria because it might kill bacteria.
Klebsiella
There are bacteria named Klebsiella which are non-motile. Non-motile bacteria are those types
of microorganisms that lack the capabilities and physical ability necessary to move through their
environment on their own. In our experiment, we cannot see Klebsiella clearly because the lens
was dirty and affected the vision. As a result, the quality of the imagery is not good. To avoid the
problem, we need to clean the lens of the microscope with the lens paper before doing the
experiment.
Biochemistry test
Urea broth
(From left: E. coli, Salmonella and Klebsiella)
A few biochemistry tests were carried out using a particular medium in the same procedure
which is the analysis of the characteristics of both bacteria cultures with the urea broth as the
first medium used. In our experiment, there is nothing changed colour in the urea broth for three
bacteria which are E. coli, Klebsiella and Salmonella. The results of E. coli, Klebsiella and
Salmonella are accurate because E. coli and Salmonella are negative and Klebsiella is weakly
positive and it might take some time to see the change. In theory, the colour of the urea broth
should be transformed into bright pink colour because an alkaline environment is established if
the urea in the broth is broken down and ammonia is produced. So our result is accepted.
E. coli Klebsiella Salmonella
Next, during the litmus milk, our observations show there is a change when Klebsiella is inside
the milk because there is a white precipitate that collects at the bottom of the bottle and the
purple colour turns to light brown. E. coli and Klebsiella for our experiment show negative
results because the colour and consistency remain the same purple. In theory, a pH indicator, the
oxidation-reduction indicator litmus, is introduced into the medium to help differentiate
between the metabolic changes induced in milk. Then, depending on their enzymatic
complement, bacteria can metabolise milk substrates in litmus milk, which makes a great
differential media. Many different metabolic changes take place as a result. So, our observations
were accepted.
E. coli in the sugar broth
(From left: Lactose, Maltose, Sucrose and Glucose)
Klebsiella in the sugar broth
(From left: Lactose, Glucose, Sucrose and Maltose)
Salmonella in the sugar broth
(From left: Sucrose, Maltose, Glucose and Lactose)
After that, we used four sugar broths which are maltose, glucose, lactose and sucrose to react
with these bacteria. For maltose, our observations explained the colour of sugar broths with
Klebsiella is reddish red than E. coli and Salmonella which are pink red colours that produced.
For glucose, our observations show these three glucose sugar broths for E. coli, Klebsiella and
Salmonella produced light pink colours. For lactose, our observations show that lactose produced
brown chocolate colour, E. coli produced light pink colour and Salmonella produced reddish red
colour. For sucrose, E. coli produces pale pink colour, followed by Salmonella and Klebsiella
produced reddish pink colours. So, our results are accepted.
Credit group: Affa group Klebsiella on TSI agar Salmonella on TSI agar
E. coli on TSI agar
Finally, we also used TSI agar to react with these bacteria. A gram-negative bacterium's ability to
produce hydrogen sulphide and ferment a particular type of sugar (carbohydrate) is determined
by the TSI Agar test, a biochemical procedure (Aryal, 2019). The results observed these three
but colours for bacteria E. coli, Klebsiella and Salmonella are yellow and the slant colour was
turned to red colour for Klebsiella while the other two remained. For the type of reaction, our
results show that Klebsiella only had alkaline and acid reaction while E. coli and Salmonella
produced acid and acid reactions. Also, we observed that there is blackening of the medium in
the TSI agar with bacteria Klebsiella and there are no changed TSI agars for E. coli and
Salmonella. Lastly, the gas production for bacteria Klebsiella is hydrogen sulfide, H2S and
oxygen and carbon dioxide for bacteria E. coli and Salmonella. So, our results are accepted.
C) Examination of spoiled food samples
In the experiment, we used rice to investigate samples of spoiled food. Firstly, rice is
naturally spoiled, it releases a putrid smell which is filthy and has a rotten food odour. Rice
naturally spoiled due to heat and moisture, which encourage fungi growth and lead to the
production of mycotoxins, which can result in food poisoning. The rice has a slimy and
mushy texture. Our
observations show that the area around the rice is green and yellowish colour. Next, the odour
released for rice inoculated with E. coli is a sour smell. The texture is so smooth and slimy.
There are a few black spots on the rice and a yellowish colour. Then, after inoculating rice with
Klebsiella, we noticed that the scent was sour. The chemicals that are released from spoiled food as it is
broken down by bacteria or the chemicals that the microorganisms themselves release can both
contribute to the unpleasant odours that are produced. The texture released is slimy and juicy. The colour
is a green and yellowish colour same as rice naturally spoiled. Finally, the odour on rice inoculated with
Salmonella releases a solid putrid smell. The texture looks smooth and the colour is white and
yellowish. You must throw it away if the food appears suspect or if you are unsure whether to eat it or
not. Prevention is always preferable to treatment.
CONCLUSION:
In conclusion, the experiment was successfully conducted using the procedure given. At the end
of the experiment, the characteristic of cultures of gram-negative rod-shaped bacteria which are
Salmonella, E. coli and Klebsiella have been identified in which three of them have different
colonial morphology, they are also motile and non-motile (Klebsiella) but with different patterns
of motility as well as arrangements of their flagella, they also react differently to each of the
biochemistry tests which involves urease test, TSI Agar test, litmus milk test and the
fermentation of sugar broths test. Then, the characteristics of food spoilage caused by
gram-negative rod-shaped bacteria have been exposed to the students in which the spoiled
food is stale rice and the characteristics observed were the odour, texture, and color of the stale
rice. Therefore, all the objectives in this experiment have been achieved successfully.
DISCUSSION QUESTIONS:
1. Discuss why the motility test should be done at the log phase of bacterial growth.
- The motility should be done at the log phase of bacterial growth because it is
optimalphase to test for motility. The bacteria are healthy and this is where
motility occurs.
2. Explain the principle of the urea broth test.
- Urease catalyses the breakdown of urea into ammonia and carbon dioxide. The
test organism is cultured in a medium containing urea and the indicator phenol
red. If the bacterial strain is urease-producing, the enzyme will hydrolyse the urea
to give ammonia and carbon dioxide.
3. Explain the principle of litmus milk test.
- When an organism ferments lactose, lactic acid is produced and the medium
changes to a pink-red color. Some bacteria act on the nitrogenous substrates in the
milk releasing ammonia and yielding a purplish-blue color.
4. Explain the principle of sugar broth test.
- It tests an organism's ability to ferment the sugar glucose as well as its ability to
convert the end product of glycolysis, pyruvic acid into gaseous byproducts. This
is a test commonly used when trying to identify Gram-negative enteric bacteria,
all of which are glucose fermenters but only some of which produce gas.
5. Explain the principle of TSI agar test.
- The presence of fetrous sulphate and sodium thiosulphate fulfils the demand of
sulphur and there is the indicator phenol red for the indication of the change in the
environment of the media due to the production of acid or alkali.
6. Discuss the similarities and differences in food spoilage criteria between naturally
spoiled foods and foods inoculated with microorganisms.
- The similarities of natural food spoiled and foods inoculated with
microorganisms is if you eat or consume spoiled foods either its was spoiled
naturally or was inoculated with microorganisms, you are going to get sick
because of it. But the differences between the two is that although some people
may become physically ill due to smells or disgusting flavors, food spoilage
organisms don’t cause life-threatening infections while food pathogens will,
however, make you sick, possibly even cause death. Other than that, Food
inoculated is when food is contaminated with microorganisms or substances and
eating it could result in foodborne disease. Food spoilage is any undesired change
in the natural color, taste or texture of food items that makes it unfit for
consumption because it has lost its quality and nutritional value.
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