Report - Practice in Food Microbiology - Group 4

VIETNAM NATIONAL UNIVERSITY HO CHI MINH NATIONAL UNIVERSITY
SCHOOL OF BIOTECHNOLOGY - DEPARTMENT OF FOOD TECHNOLOGY
PRACTICE IN FOOD MICROBIOLOGY REPORT

Semester 1: 2023 – 2024
COURSE CODE: BTFT254IU

INSTRUCTOR: Nguyen Thi Huong Giang
TEACHING ASSISTANT: Bui Thanh Vy
DATE OF SUBMISSION: 30/11/2023
GROUP: 04
MEMBERS:
No. Full name Student ID Contribution (%)

1 Lê Trần Thị Ngọc Châu BTFTIU21150 20
2 Nguyễn Việt Thy BTFTIU21198 20
3 Trương Trần Phương Trinh BTFTIU21203 20
4 Lê Thị Bích Ngọc BTFTIU21178 20
5 Võ Huỳnh Anh Khôi BTFTIU21168 20
International University-VNU HCMC Food Microbiology Laboratory
TABLE OF CONTENT
LABORATORY 1: DETERMINATION OF AEROBIC PLATE COUNT.............................................................

I. INTRODUCTION.......................................................................................................................................
II. MEDIA AND EQUIPMENT.......................................................................................................................
III. EXPERIMENTAL PROCEDURE............................................................................................................
1. Prepare.................................................................................................................................................
2. Procedure.............................................................................................................................................
IV. RESULT AND DISCUSSION..................................................................................................................
1. Resulting..............................................................................................................................................
2. Calculate CFU/ml................................................................................................................................
3. Discussion.............................................................................................................................................
V. CONCLUSION.........................................................................................................................................
VI. REFERENCES........................................................................................................................................
LABORATORY 2: DETERMINATION OF TOTAL COLIFORM........................................................................
I. INTRODUCTION.......................................................................................................................................
1. Prepare medium and dilution............................................................................................................
a. Prepare BPW.................................................................................................................................
b. Prepare BGBB...............................................................................................................................
c. Prepare LST...................................................................................................................................
2. In the process.......................................................................................................................................
1. Result....................................................................................................................................................
2. Calculation...........................................................................................................................................
3. Discussion........................................................................................................................................
V. CONCLUSION.........................................................................................................................................
VI. REFERENCES........................................................................................................................................
LABORATORY 3: DETERMINATION OF SALMONELLA...............................................................................
I. INTRODUCTION.......................................................................................................................................
1. Prepare TSI agar slant.................................................................................................................
2. Streaking the agar slant surface and stabbing the butt.............................................................
a) After 1 day.....................................................................................................................................
b) After 1 week..................................................................................................................................
V. CONCLUSION.........................................................................................................................................
VI. REFERENCES........................................................................................................................................
LABORATORY 4: DETERMINATION OF YEASTS AND MOLDS...................................................................
I. INTRODUCTION.......................................................................................................................................
2
1. Media preparation..............................................................................................................................
2. Procedure.............................................................................................................................................
1. Result....................................................................................................................................................
2. Calculate CFU/ml................................................................................................................................
3. Discussion...................................................................................................................................................
V. CONCLUSION.........................................................................................................................................
VI. REFERENCES........................................................................................................................................
LABORATORY 5: BACTERIAL MORPHOLOGY AND STAINING..................................................................
I. INTRODUCTION.......................................................................................................................................
1. Classification about the bacteria.......................................................................................................
2. Principle of staining method..............................................................................................................
1. Bacteria smear.....................................................................................................................................
2. Gram staining......................................................................................................................................
1. Result.................................................................................................................................................
2. Discussion..........................................................................................................................................
V. YOGURT..................................................................................................................................................
1. Ingredients and method to make homemade yogurt.......................................................................
a. Ingredients.....................................................................................................................................
b. Procedure.......................................................................................................................................
2. The mechanism of the fermenting process.......................................................................................
3. The morphology of yogurt bacteria...................................................................................................
4. The morphology of Kombucha..........................................................................................................
5. Discussion.............................................................................................................................................
VI. CONCLUSION........................................................................................................................................
VII. REFERENCES.......................................................................................................................................
3
LABORATORY 1: DETERMINATION OF AEROBIC PLATE COUNT
I. INTRODUCTION
Bacteria that can only grow and live in the presence of oxygen in their surroundings are
known as aerobic or aerobic bacteria. In an ambient air environment with 21% oxygen and
0.03% carbon dioxide, aerobes can develop and survive. Therefore, if we had to characterize
aerobic bacteria, we could state that they are organisms that require oxygen to survive.
Aerobe is a synonym. Anaerobic microorganisms, in contrast.
The Aerobic Plate Count (APC) estimates bacterial populations in a sample, assuming each
cell forms a visible colony in nutrient-rich agar. It is a generic test for organisms growing
aerobically at mesophilic temperatures and does not differentiate bacterium types. In
addition, APC gauges sanitary quality, organoleptic acceptability, and adherence to good
manufacturing practices, but is a poor safety indicator as it does not correlate to pathogens or
toxins' presence. A low APC does not imply pathogen-free products. Products with high
APCs could be potential health hazards, pending pathogen screening. APC interpretation
considers the product and the expected APC. It is valuable in evaluating food quality, with
large bacterial numbers indicating poor sanitation or process control issues. Fermented
products naturally have a high APC. Low APC does not mean the absence of pathogens.
Assaying foods for specific pathogens or spoilage organisms is often necessary for food
safety or quality decisions.
On the other hand, the APC method is employed in the food sector to assess dairy and poultry
products, ensuring they meet food safety standards. The calculated APC data is instrumental
in forecasting the shelf life of these items. A high APC level in a test sample could signify the
presence of harmful organisms or potential risks. The fact that the APC method, while useful,
has several limitations. It only counts aerobic organisms, which may not represent the total
bacterial population accurately. The counting process, often done by human eyes, can lead to
errors, especially with small colonies that are hard to see. This can result in inaccurate
calculations. Additionally, certain products, like fermented ones, naturally have high APCs,
making it challenging to determine spoilage using this method alone.
Completing this experiment's objectives will require familiarity with the production of plate
count agar (PCA) and buffered peptone water (BPW). Additionally, this laboratory should
compute the colonies in the plates of various dilutions so that the Aerobic Plate Count
technique can be understood.
4
Figure 1.1: Aerobic Bacteria
II. MEDIA AND EQUIPMENT

Table 2.1: Media and equipment used for experimental process
Media Equipment
Glassware and inoculating loop must be Sterile impermeable plastic bag

sterilized Test tube: 4
Buffered peptone water (BPW) 250mL Erlenmeyer flask: 1
Plate count agar (PCA) 1 mL micropipette: 1
⇒ Sterilizing them at 121℃ for 15 minutes Stirring bar: 1
and 1 atm Magnetic bar: 1
Beaker: 500mL & 1000mL
Cylinder: 250mL & 500mL
Alcohol burner: 2
Petri dish: 14
+ Pour - plate technique: 6
+ Spread - plate technique: 6
+ Streak - plate technique: 2
Thermostatic water bath, incubator,
autoclave, heating oven, biological cabinet,
vortex mixer
III. EXPERIMENTAL PROCEDURE

1. Prepare
Step 1: Prepare 7.06g PCA into beaker and 0.8g BPW
5
Figure 3.1: Plate Count Agar and Buffered Peptone Water( Powder)
Step 2: Homogenized sample PCA(300ml distilled water + 7.06g PCA) and PBW(40 ml
distilled water + 0.8g) then heating by using heating oven
Figure 3.2: Plate Count Agar and Buffered Water after heating
Step 3: Take 25 mL of milk and mix well the sample milk( shake 25 times before use to
distribute microorganism)
Figure 3.3: Sample of milk
2. Procedure
Step 1: Put 9ml PBW into each 4 tubes that are labeled 10-1,10-2, 10 -3,10-4 respectively.
6
Figure 3.4: 4 tubes PBW

Step 2: Take 1 ml of milk with a sterile pipette and add 9 ml of BPW
(Shake this primary dilution 25 times to ensure the dilution is mixed together for 5-10s) then
rotate by using a vortex mixer to mix it well.
Step 3: Use a separate sterile pipette, prepare decimal dilutions of 10-2; 10-3, 10-4 of sample:
- Take 1 ml of the initial suspension and add it to the next 9 ml tube of BPW to make
dilution 10-2
- Vortex dilution 10-2 for 5-10 s to obtain 10-2 a dilution.
- Continue further dilutions to obtain 10-3 and 10-4.
(Flame all containers' necks after using them to prevent undesirable microorganisms
around the environment.)
Figure 3.5: Diluted sample Figure 3.6: rotate sample by using vortex mixer
Step 4: Dispense each dilution (1 ml) into labeled PCA petri dishes using pouring, then pour
2/3 media into the petri dish and wait the plates to set (making duplicate for each dilute).
Figure 3.7: Petri dishes is adding dilute Figure 3.8: Petri dishes when adding medium
7
Figure 3.9: Petri dishes after pouring

Step 5: Pour 2/3 media into a petri dish, allow the plates to set, and then dispense each
dilution (0.1ml) into labeled PCA petri dishes using spreading after pouring media (making
duplicate for each dilute).
Figure 3.10: Petri dishes after pouring media Figure 3.11: Petri dishes when adding sample to spread
Figure 3.12: Spreading process

Step 6: Pour 2/3 media into a petri dish, allow the plates to set, and then dispense each
dilution (0.1ml) into labeled PCA petri dishes using streaking after pouring media( making
duplicate for each dilute).
8
Figure 3.13: Petri dishes after pouring media Figure 3.14: Streaking process
Total is 14 dishes 6 pouring (10-2, 10-3, 10-4), 6 spreading(10-2, 10-3, 10-4) and 2 for
streaking(10-2)
Step 7: Incubate the plates at 37oC for 48 ± 2 hrs
Figure 3.15 : Sample 10-2, 10-3,10-4( petri dish 1) after incubating respectively
Figure 3.16: Sample 10-2, 10-3,10-4( petri dish 2) after incubating respectively
9
Figure 3.19: Sample 10-2( petri dish of streaking) after incubating respectively
Step 8: Colonies on Petri dishes were counted. then determine the sample's CFU/ml or
CFU/g.
IV. RESULT AND DISCUSSION

1. Resulting
Counting the number of colonies on Petri plates (spread plating and pour plating)
Table 4.1: Colonies of anaerobic and aerobic (Pouring)

Sample: Milk ( total aerobic microbes)
Dilution Colonies
Petri dish 1 Petri dish 2

−2 223 247
10
−3 162 103
10
−4 110 94
10
Table 4.2: Colonies of anaerobic and aerobic (Spreading)

Sample : Milk ( total aerobic microbes)
Dilution Colonies
10
−2 > 100 > 100
10
−3 > 100 > 100
10
−4 171 172
10
2. Calculate CFU/ml
CFU CFU ¿
Count ( ∨ )=averagenumber of colonies ¿ duplicate plates
ml g dilution factor × volume plated
For pouring:
3
¿ 6.5× 10 6
The colonies of aerobic microbes in plate 10−2 : =6.5 ×10 CFU /ml
0.01× 0.1
132.5 6
0.001×(0.1)
102 7
The colonies of aerobic microbes in plate 10−4: =1.02 ×10 CFU /ml
0.0001×(0.1)
For Spreading:
3
¿ 6.5× 10
The colonies of aerobic microbes in plate 10−2 : =6.5 ×10 6 CFU /ml
0.01× 0.1
3
¿ 6.5× 10 7
0.001× 0.1
171.5 7
The colonies of aerobic microbes in plate 10−4: =1.715 ×10 CFU /ml
0.0001×(0.1)
3. Discussion
Plate Count Agar is a common microbiological medium for counting of viable bacteria in a
sample. When spreading, pouring or streaking, add dilution (including milk and BPW) onto
PCA, it introduces a nutrient source that may encourage the growth of bacteria. Count the
number of viable cells in the plate based on growth after it has been incubated at 37 oC for 48
± 2 hours. A colony forming unit (CFU) is a unit used to estimate the viable colonies that
develop on the PCA.
In the pour plate technique, the sample is added to a solidified medium surface while and the
spread plate technique is sample is mixed with molten agar spilled onto a plate before being
allowed to cool. Reducing the amount of microorganisms in a sample through dilution, makes
it easier to count viable cells.
In 10−2 dilutions, the medium is relatively rich for microorganisms, allowing for the growth
we obtain a plate with greater than 200 colonies. Using a counter with gridlines spaced 1 cm
apart can help choose a representative of the plate to count when there are crowded colonies
on it. But too many colonies can result in overcrowding, making it difficult to count
individual colonies accurately.
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While in 10−4dilution, the medium is comparatively poor for microorganisms because it is

highly diluted, the result in the range of 94 - 171. This dilution is helpful for counting
colonies within a numerable range, resulting in fewer colonies on the agar plate. However,
too few colonies may not provide statistically significant results and report the number as
''estimated''.
When streaking a bacterial sample on PCA agar, the goal is to get isolated colonies, draw a
zigzag line on each quadrant of the plate, and obtain the number of viable bacteria in each
corner. The result after incubating the plates at 37oC for 48 ± 2 hrs. At the first streaking
area, observe a dense growth of bacterial cells in this quadrant, as this is where the highest
concentration while we streak. At last the quadrant should contain well-separated, isolated
colonies cause the streak gradually thins out to the remaining corners.
V. CONCLUSION
After conducting the experiment, It assisted in bringing an obvious understanding of the total
aerobic plate count technique, how to estimate the total bacterial count in samples with the
plate count method, and three culture techniques, including spread-plate, streak-plate, and
pour-plate technique.
The Aerobic Plate Count testing is based on the formation of visible colonies when
introduced to a nutrient-rich substance called agar. Besides, the aerobic plate count plays an
important role in assessing food quality, safety, and shelf-life. Hence, Its measurement could
assist food processors in improving manufacturing processes, ensuring compliance with
regulations, and preventing potential contamination issues
VI. REFERENCES
B.T.Vy (2023). Food Microbiology Laboratory Manual. International University, Vietnam

National University – HCMC
Aubrey Mendonca, ... André Gordon (2020) Plate Count - an overview | ScienceDirect
Topics. Available at:
https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/plate-count
Labmate, I. (2022) What is the aerobic plate count (APC)?, Labmate Online. Available at:
https://www.labmate-online.com/news/mass-spectrometry-and-spectroscopy/41/breaking-
news/what-is-the-aerobic-plate-count-apc/58005
What is the Aerobic Plate Count (APC)? (n.d.). Labmate Online. Retrieved November 28,
2023, from https://www.labmate-online.com/news/mass-spectrometry-and-spectroscopy/41/
breaking-news/what-is-the-aerobic-plate-count-apc/58005
Buffered Peptone Water (BPW) Oxoid CM0509B di Padallean Mart. (n.d.). Tokopedia.
Retrieved November 28, 2023, from https://www.tokopedia.com/padallean-mart/buffered-
12
peptone-water-bpw-oxoid-cm0509b?
utm_source=google&utm_medium=organic&utm_campaign=pdp-seo
LABORATORY 2: DETERMINATION OF TOTAL COLIFORM
I. INTRODUCTION
Coliform bacteria are often harmless and can be found in soil, animal intestines, and other
frequent environmental areas. However, identifying bacteria plays an important role for
detecting bacteria contamination of liquid sources. These bacteria are classified as rod-
shaped, Gram-negative, non-spore-forming, motile, or nonmotile bacteria, at 35–37°C,
lactose ferment and produce gas and acid.
Selective media such as Lauryl Sulfate Tryptose Broth (LST) and Brilliant Green Bile Broth
(BGBB) are used in microbiology, specifically for the identification and counting of coliform
bacteria. LST is a selective medium that promotes the growth of coliform bacteria while
inhibiting the growth of non-coliform bacteria.
Take a few microorganisms from the Lauryl Sulphate Tryptose Broth (LST) culture and
transfer to the Brilliant Green Bile Broth to help them have more nutrients and medium to
grow more by using the Subculture method (subculture of liquid media onto a liquid
medium). Allow the BGBB medium with the inoculated sample to incubate at the appropriate
temperature for the detection of coliforms. The number of positive tubes for each dilution is
determined using the growth (turbidity) and gas formation in the tubes following incubation.
Positive tubes have visible growth, while those without are considered negative.
The Most Probable Number (MPN) technique is a statistical method for estimating the
concentration of viable microorganisms in a sample. After inoculation and incubation,
positive tubes are identified based on growth and gas formation.A table is used to derive the
MPN value.
Figure 1.1: Coliform bacteria
In the laboratory lesson,mainly about how to determine the existence of coliform in food
based on observing the phenomenon (turbidity or gas appearance) in a test tube that is
subcultured from LST to BGBB medium. Obtain some index of the number of coliform
13
bacteria that may be present in food using the MPN technique and calculate based on the
Thomas formula.

MEDIA EQUIPMENT
Buffered peptone water (BPW) Sterile impermeable plastic bag
Brilliant Green Bile Broth (BGBB) Scissor: 1
Distilled water Test tube: 30
Juice (sugar cane) Durham tube: 20
1mL micropipette: 1
Stirring bar: 1
Magnetic bar: 1
Beaker: 500mL & 1000mL
Cylinder: 250mL & 500mL
Bottle: 1500mL & 2250mL
Alcohol burner: 1
Thermostatic water bath, incubator, vortex

mixer, autoclave, heating oven, biological
safety cabinet

1. Prepare medium and dilution
a. Prepare BPW
- Suspend 0.6g in 30ml of distilled water (heat if necessary to dissolve the medium
completely)
- Sterilize by autoclaving at 1 atm pressure (121°C) for 15 minutes
- Mix well and dispense into sterile flasks as desired
b. Prepare BGBB
- Dissolve 4 g in 100 ml distilled water.
- If required, heat to completely dissolve the medium.
- Dispense 10 mL of medium into test tubes containing Durham tubes.
- Autoclave at 121°C for 15 minutes to sterilize (the Durham tubes must be free of air
bubbles after sterilization).
14
c. Prepare LST
- Dissolve 3.56g in 100 ml of distilled water, heating if necessary. Adjust the pH so that
it is 6.8 ± 0.2 at 25°C after sterilization.
- 10 mL of the broth should be dispensed into 9 Durham tubes.
- Place the tubes in an autoclave on a test-tube rack or basket.
- Autoclave at 121°C for 15 minutes to sterilize (the Durham tubes must be free of air
bubbles after sterilization).
Figure 3.1: The medium and dilution need to experiment

2. In the process
Step 1: Take a bottle of juice (sugar cane) into a plastic bottle that will not leak. Flame the
juice container’s neck
Step 2: Take 1 ml of juice with a sterile pipette and add 9 ml of BPW
(Shake this primary dilution 25 times to ensure the dilution is mixed together for 5-10s)
Step 3: Using a separate sterile pipette, prepare decimal dilutions of 10-2; 10-3 of sample:
- Take 1 ml of the initial suspension and add it to the next 9 ml tube of BPW to make
dilution 10-2
- Vortex dilution 10-2 for 5-10 s to obtain 10-2 a dilution.
Figure 3.2: Vortex the dilution

- Continue further dilution to obtain 10-3 dilution.
(Flame all containers' necks after using to prevent undesirable microorganisms around
the workplace).
15
Step 4: Use sterile pipette to transfer 1 ml of each dilution into tube containing 10 ml LST
(making triplicate tubes for each dilution)
Step 5: Incubate at 30°C or 37°C for 24 ± 2 h (Observe after 24 hours; if no gas is formed,
reincubate for further 24 hours)
Figure 3.3: Incubate the dilution

Step 6: Subculture a loopful of incubated LST into tube containing BGBB
Step 7:Incubate at 30oC or 37oC for 24 ± 2 hrs (Observe after 2 hours, if no gas is formed,
reincubate for further 24 hrs)

1. Result
Figure 4.1: BGBB tubes after incubation at dilution of 10-1
16
Table 4.1: Result of BGBB tubes after incubation

Sample: Juice
Dilution Test tube 1 Test tube 2 Test tube 3
10-1 Positive Positive Positive
2. Calculation
Table 4.2: Result of BGBB tubes positive
−1 −2 −3
Aliquot 10 10 10
The number of 3 3 3
17
positive tubes in 3
According to the Most Probable Number (MPN) table from the lab manual, the number of
positive test tubes at each dilution is 3-3-3. This showed the MPN/g or ml accounted for
>1.100
Figure 4.4: Most probable number (MPN) table
3. Discussion
Coliform bacteria are defined as facultatively anaerobic, gram-negative, non-spore-forming
rods that ferment lactose vigorously to acid and gas at 35 ± 2°C within 24 or 48 h.
Additionally, the formation of gas also indicates that lactose fermentation is occurring.
In the experiment, after incubating Lauryl Sulfate Broth (LST) tubes at 37°C for 24 ± 2
hours, we could see the presence of coliforms, which is indicated by the growth and
generation of gas in Durham tubes. Then, we subcultured incubated LST into tubes
containing Brilliant Green Bile Broth (BGBB) in order to provide more nutrients for the
bacteria to grow as there were too many organisms in LST tubes and continued incubating
them at 37°C for 24 ± 2 hours that would create an ideal environment for the bacteria's
growth
In addition, Lauryl Sulfate Broth (LST) was developed to produce a significant amount of gas
and rich growth from a small inoculum of coliform organisms. Tryptose provides essential
growth substances, such as nitrogen and carbon compounds, and Sodium lauryl sulfate (SLS)
inhibits organisms other than coliforms.
Besides, in Brilliant Green Bile Broth 2%, Lactose is a fermentable carbohydrate, and Ox bile
and brilliant green inhibit Gram-positive bacteria and many Gram-negative bacteria,
18
other than coliforms in the sample.
Furthermore, when observing the BGBB test tubes, it is clear that all test tubes at each
dilution had gas formation (considered as positive tubes) and indicated the growth of
coliform bacteria in the juice sample. From the data we have collected, the Most Probable
Number (MPN) value for positive tubes 3-3-3 was >1.100 MPN/ml
V. CONCLUSION
In conclusion, after carrying out the experiment, through some media such as BGBB and
LST, determining whether bacteria are present or not by observing the tubes to see if there
are any signs of bacteria survival, such as containing gas in the Durham tubes, which is a
confirmed test for total target microorganism or cloudy conversion. Furthermore, the purpose
of this lab is to apply the MPN to determine the total number of microorganisms living in the
sample, it is also the way for the tester to evaluate the hygiene of food.
VI. REFERENCES

H.B.D. Halkman, A.K. Halkman (2014) Coliform Bacteria - an overview | ScienceDirect

Topics. Available at:
https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/coliform-bacteria
Figure 1.1 “Coliform bacteria.” Wikipedia, https://en.wikipedia.org/wiki/Coliform_bacteria.

Accessed 29 November 2023.
Erkmen, Osman. “\/.” YouTube, 16 June 2023,

https://www.sciencedirect.com/science/article/abs/pii/B9780323916516000422. Accessed 29
November 2023.
Li, Daoliang, and Shuangyin Liu. “\/.” YouTube, 16 June 2023,

https://www.sciencedirect.com/science/article/abs/pii/B9780128113301000120. Accessed 29
November 2023.
19
LABORATORY 3: DETERMINATION OF SALMONELLA
I. INTRODUCTION
The majority of bacteria are capable of fermenting carbohydrates, especially sugars. Each of
them is limited in the amount of sugars it can ferment; it cannot ferment all of the sugars. As
a result, one of the most crucial criteria for identifying a bacterium is knowing which
carbohydrates it can ferment and which ones it cannot. These unique characteristics are the
basis for differentiating and identifying bacteria based on their biochemical properties.
A microbiological test known as the Triple Sugar Iron (TSI) test is used to evaluate an
organism's capacity to ferment carbohydrates and generate hydrogen sulfide in the
identification of gram-negative bacilli. It is frequently used to distinguish between enteric
bacteria, such as Shigella and Salmonella.
Salmonella infection usually isn't life-threatening. However, in certain people — especially

infants and young children, older adults, transplant recipients, pregnant women, and people
with weakened immune systems — the development of complications can be dangerous.
Various human disorders may be caused by harmful microbes. Thus, it's critical to get rid of
these germs. Food spoiled due to the presence of bacteria. Therefore, it is essential to identify
it in the food industry.
The presence of gas and a discernible shift in the color of phenol red, a pH indicator, are
signs of carbohydrate fermentation. The creation of a black precipitate, which will turn the
medium in the tube's butt black, indicates that hydrogen sulfide is being produced in the
medium. Organisms can ferment glucose, and lactose( or sucrose) the addition of sucrose
increases the sensitivity of the medium by facilitating the detection of sucrose-fermenting
bacilli. In other ways, lactose and/or dextrose fermenters can be observed by the color on the
slant or butt as well as based on other signals such as the crack of the agar or the bubbles. To
enhance the alkaline condition of the slant, free exchange of air in fermentation must be
permitted by closing the tube cap loosely. If the tube is tightly closed, an acid reaction
(caused solely by dextrose fermentation) will also involve the slant.
Figure 1.1: TSI phenomenon

20
An agar test tube with a slanted angle is produced when all of these materials are combined
and allowed to solidify at an angle. This medium's slanted structure offers a variety of
surfaces that may either be completely closed off from the air (an anaerobic environment) or
exposed to it to variable degrees (an aerobic environment), depending on how an organism
ferments.
In the lab practice will prepare the TSI agar use to detect the kind of Salmonella, understand
the biochemical reactions involved in the triple sugar iron agar test and observe the signs of
the presence of bacteria according to the table mentioned above.

EQUIPMENTS MEDIA
5 test tubes 50mL distilled water
1 100-mL glass bottle 3.23g Triple Sugar Ion Agar (TSI)
Magnetic stirrer Nutrient agar plate
1 lab hot-plate
1 inoculating needle
Alcohol burner
1 test tube rack
Biological safety cabinet

In this lab, we will do a slant culture and stab culture with TSI agar (red) into 5 test tubes to
detect Salmonella
1. Prepare TSI agar slant
Step 1: Suspend 3.23 g of TSI agar powder with 50 ml distilled water
Step 2: Mix thoroughly to dissolve the agar completely by heating it to boiling
Figure 3.1: Boiling TSI to dissolved completely
21
Step 3: Dispense into 5 test tubes, a control tube serves as base for comparison, that makes
sure the medium and any reaction in tubes is not affected by the variation in the medium and
other outside factors. The use of replicates for 2 typical and 2 atypical allow for the
consistency and accuracy of reaction in tubes.
Figure 3.2: The TSI agar in slant

Step 4: Autoclave the TSI agar mixture to sterilize at 121°C for 15 min.
2. Streaking the agar slant surface and stabbing the butt
Always put an alcohol lamp while streaking, and work in a biological cabinet that is a sterile
environment
Step 1: Sterilize the inoculating needle by passing it through a flame until it glows red.
Allow it to cool to prevent high temperature from damaging the bacteria.
Figure 3.3:
Step 2: Straight inoculation to pick the well-isolated Salmonella colony in a nutrient agar
plate
Step 3: Lose the cap and pass through the flame, Stab the needle into the agar until it is 2 cm
from the bottom to ensure the colony into the depth of the medium. While lifting the needle
straight up, streak the slant surface with a zigzag line helps to distribute the bacteria across
the slant.
Step 4: Close the cap of the tube. After inoculation, the tube is incubated in 35°C, 18-24 h,
and the reactions are observed.
22
Figure 3.4: TSI agar after inoculation and incubated

Triple sugar iron (tsi) agar test
Figure 4.1: The location determines the identifying signs of bacteria

Alkaline reaction: red color of the medium (K)
Acidic reaction: yellowing of the medium (A)
Gas: medium is separated (+)
H2S: blackening of medium (+) (Trace amount: a bit of blackening)
Table 4.1: Table of identification signs

Salmonella Salmonella Salmonella Shigella spp.
(majority) serovar Typhi serovar Paratyphi
TSI (slant) K K K K
TSI (butt) A A A A
TSI (H2S) + Trace amount Negative Negative
TSI (gas) + No gas + -(most)
23
a) After 1 day
Figure 4.2: Control tube Figure 4.3: 2 typical tubes Figure 4.4: 2 atypical tubes
After 1 day, all 4 test tubes (2 typicals, 2 atypicals) changed TSI agar from the organic color
like control tube to black color in the butt. Based on the table of identification signs listed
above, these microorganisms have the ability to produce hydrogen sulfide (H 2S) gas because
of the enzymatic breakdown of cysteine. H 2S reacts with ferrous sulfate in the medium,
forming a black precipitate called ferrous sulfide.
b) After 1 week
Figure 4.5: TSI was cultured Salmonella after 1 day

After 1 week, the color and status of each test tube inoculated with bacteria display the
following results:
- Typical tube:
In slant: Appear the red color on the surface of TST agar. Salmonella is capable of
fermenting glucose while generating acid, resulting in the creation of an acid product. The
acid generation in the TSI agar causes the slant to turn yellow. However, Salmonella releases
gas during fermentation, which elevates the pH and causes the yellow slant to turn red due to
pH indicator reversion
24
Blackening in the butt: Salmonella can produce hydrogen sulfide (H2S) gas. Salmonella
produces H2S gas when it metabolizes cysteine in the medium. The H 2S gas combines with
the ferrous sulfate in the TSI agar, forming a black precipitate of ferrous sulfide. This
blackening in the agar's butt is a sign that Salmonella is producing H2S.
Cracks or lifting of the agar within the tube: Pressure within the TSI agar tube might be
caused by the formation of gas by Salmonella during fermentation. Pressure accumulation in
the agar medium can cause it to crack or lift, resulting in visible cracks or disruptions.
Figure 4.6: Typical compared with control tube after 1 week

The remaining 3 tubes (1 typical, 2 atypicals): the butt is a constant black color. They still
produce hydrogen sulfide (H2S) gas but there are no sufficient conditions to conclude
Salmonella as shown in the table above. This could be due to the following reasons:
Stronger glucose fermentation compared to lactose: Salmonella

may ferment glucose more effectively than lactose, resulting in a
yellow color shift throughout the TSI agar medium. However,
the capacity of some Salmonella strains to ferment lactose may
be poor or absent, resulting in no red color on the agar slant.
Lack of gas production: While Salmonella can produce H2S, not

all strains release enough gas to cause cracking of the agar
within the tube. This could be owing to a low level of gas
production or a lack of enough to cause this phenomenon.
Reaction time: The reaction time on TSI agar varies depending

on the Salmonella strain. The lack of red color on the slope or
the absence of cracks in the agar can be attributed to a sluggish
or partial reaction during the observation time.
Figure 4.7: The

remaining
3 tubes after 1 week
25
V. CONCLUSION
Through this experiment, learning how to prepare TSI agar and the practical operations of the
stabbing method is extremely essential. Furthermore, It also provides a deeper understanding
of how to identify and differentiate bacteria by their color, shape, moisture, etc. During the
experiment, there were some problems that led to the inability to observe the desirable
microorganism in a typical test tube such as stronger glucose fermentation compared to
lactose, lack of gas production, or reaction time.
The Triple Sugar Iron (TSI) agar plays an important role in differentiating members of the
Enterobacteriaceae family from other gram-negative rods based on their fermentation of
lactose, glucose, and sucrose, and on the production of Hydrogen Sulfide
VI. REFERENCES
Iqbal, B. (2023) Kligler’s iron agar (KIA) test, Microbial notes. Available at:
https://microbialnotes.com/kliglers-iron-agar-kia-test
Tankeshwar, A. (2022) Triple Sugar Iron (TSI) agar: Principle, results, and interpretation,
Microbe Online. Available at: https://microbeonline.com/triple-sugar-iron-agar-tsi-principle-
procedure-and-interpretation/#google_vignette
Smith, M., & Selby, S. (2021, March 19). 3.12: Triple-Sugar Iron Agar. Biology LibreTexts.
Retrieved November 28, 2023, from
https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Mic
robiology_for_Allied_Health_Students%3A_Lab_Manual/3.12%3A_Triple-
Sugar_Iron_Agar
Figure 1.1: Dahal, P. (2023, October 5). TSIA Test: Principle, Media, Procedure, Results,
Uses. Microbe Notes. Retrieved November 28, 2023, from https://microbenotes.com/triple-
sugar-iron-agar-tsia-test/

26
LABORATORY 4: DETERMINATION OF YEASTS AND MOLDS
I. INTRODUCTION
Yeasts and molds are types of fungi that play important roles in various aspects of human
lives. Yeast refers to a microscopic fungus consisting of single, oval cells. They produce
vitamins, minerals, and other nutrients in the fermented food product. In addition, yeasts can
convert carbohydrates to alcohol during fermentation and are hence utilized in the baking and
brewing industries.
Figure 1.1: Yeast

When it comes to molds, they refer to a growth form of fungus that grows in the form of
multicellular filaments called hyphae. Molds are used in the production of cheese
(Penicillium). Molds can cause food spoilage, affecting texture, flavor, and appearance.
Furthermore, a number of molds can produce mycotoxins that are growing on cereals, nuts,
fruits, and vegetables. The most common is aflatoxin, which is the most cancer-causing agent
produced by Aspergillus Flavus and Aspergillus Parasiticus
Figure 1.2: Molds and one type of aflatoxin-Aspergillus Flavus (from left to right)
A serial dilution is a series of sequential dilutions used to reduce a dense culture of cells to a
more usable concentration. Each dilution will reduce the concentration of bacteria by a
27
specific amount. In the sample, there are too many microorganisms, so we have to reduce the
number of them to make it easier to count.
In today’s laboratory class, It provided an obvious understanding of determining the yeasts

and molds by preparing a sample of grapes, making a dilution series, and culturing them to
Dichloran Rose Bengal Chloramphenicol Agar (DRBC) using the spreading technique. After
that, calculate the total colony forming units per milliliter (CFU/ ml) visible colonies in all
the aerobic plate count

Equipment Media
Sterilized plastic bag Grapes
Sterilized blender 225 ml Sterilized Peptone water
Scissor DRBC plates
Erlenmeyer flask
Test tubes
Alcohol burner
Spreader
Incubator

1. Media preparation
- Get the PW and DRBC material ready to use in erlens or bottles. On these lenses or
bottles, the date and the name of the student or group generating the medium should be
recorded. Fill each of the three test tubes with nine milliliters of PW.
a. Prepare DRBC and PW
- Step 1: Prepare 6.3g of DRBC into the beaker and 0.27g of PW
- Step 2: Homogenized sample DRBC( 200ml distilled water + 6.3g DRBC) and
PW( 270ml distilled water + 0.27g PW) then heating by using a heating oven.
Figure 3.1: The DRBC after heating Figure 3.2: The PW after heating
b. Prepare Grapes sample
28
- Step 1: Prepare the sample ( black grapes). Weight 25 grams of sample and 225mL
PW.
Figure 3.3: Weigh 25 grams of grapes
- Step 2: Sterilize the blender with alcohol. Next, put the sample and PW into the
blender. Then, grind the mixture.
Figure 3.4: Grind the mixture

- Step 3: Shake this primary dilution 25 times to ensure the dilution is mixed together
for 5-10s
- Sterilize test tubes holding the medium and the medium itself at 121 °C before use. A
selective supplement containing chloramphenicol (kept at 45°C) should be administered to
sterilize DRBC. Transfer to sterilized petri dishes after fully mixing.
2. Procedure
Step 1: Put 9ml PW into each 3 tubes that are labeled 10-2, 10 -3,10-4 respectively.
29
Figure 3.5: 3 tubes PW

Step 2: Use a separate sterile pipette, prepare decimal dilutions of 10-2; 10-3, 10-4 of
sample:
- Take 1 ml of initial sample of grape (10-1 ) with a sterile pipette and add 9 ml of PW
tp make dilution 10-2
- Rotate by using a vortex mixer to mix it well
- Continue further dilutions to obtain 10-3 and 10-4.
(Flame all containers' necks after using them to prevent undesirable microorganisms around
the environment.)
Figure 3.6: dilution process

Step 3: Pour ⅔ of molten DRBC Agar cooled to 45 oC to each plate, and wait the plates allow
to set.
30
Figure 3.7: Pour DRBC into petri dishes

Step 4: Pipette 0.1 mL each dilution into separate petri dishes. Making duplicate petri dishes
for each dilution. (Incubate at 25oC for 7 days - Lid is uppermost, in an upright position in the
incubator).
Step 5: Count the number of colonies on petri dishes after 7 days. Calculate CFU/g of
sample.
Figure 3.8: Count the number of colonies using colony counter

1. Result
Table 4.1: Colonies of Mold
Sample:Black grapes( Mold)
Dilution Colonies
10-1 28 18
10-2 65 65
10-3 25 39
10-4 1 3( one is green)
Table 4.2: Colonies of yeasts

31
Sample:Black grapes( Yeast)
Dilution Colonies
10-1 128 150
10-2 32 9
10-3 3 6
10-4 none none
2. Calculate CFU/ml
CFU CFU
𝐶𝑜𝑢𝑛𝑡 𝑜𝑟 =
ml g
average number of colonies ¿ duplicate plate ¿
dilution factor x volume plated
23 3
The colonies of molds in plate 10-1: =2.3 x 10 CFU/ml
0.1 x (0.1)
65 4
0.01 x (0.1)
32 5
0.001 x ( 0.1)
2 5
The colonies of molds in plate 10-4: =2 x 10 CFU/ml
0.0001 x (0.1)
139 4
The colonies of yeast in plate 10-1: =1.39 x 10 CFU/ml
0.1 x (0.1)
20.5 4
0.01 x (0.1)
4.5 4
0.001 x ( 0.1)
0
The colonies of yeast in plate 10-4: =0 CFU/ml
0.0001 x (0.1)
3. Discussion
Figure 4.1: Sample 10-1( petri dish spreading) after incubating of group4 and group 3
respectively
32
Figure 4.2 Sample 10-2( petri dish spreading) of group4 and group3 after incubating
respectively
Figure 4.3: Sample 10-3( petri dish spreading) of group4 and group3 after incubating
respectively
Figure 4.4: Sample 10-4(petri dish spreading) after incubating of group4 and group3
respectively
In 8 petri dish plates experiments that contain the grapes were ground in a blender at a
ratio of 25g of sample to 225 ml of distilled water, is added to DRBC Agar and incubates for
a certain time to testing yeasts and molds development.
Results from direct plating reveal the extent of internal infection of samples, mycological
counts obtained on DRBC are detected by using the plating techniques after 48 hrs incubated
it is shown that count plates containing 10- 150 colonies it mainly yeasts are presented, with
the specific data as below:
In 10-1 Petri dish 1 had been found for 128 yeasts and 28 molds
In 10-4 Petri dish 2 had been found for 0 yeasts and 3 molds( one is green)
The data show that the number of molds is quite low, may be in the spreading process not
carefully and mold usually grows more slowly than yeast after being inoculated into the
environment. It explains why mold in the test sample has lower numbers than yeasts.
33
Furthermore, the result also recorded that there is one mold that has a different color with
another [in plate 10-4(2) that is circled] that has a green color. It is because during the
spreading process, the equipment is affected by others in the environment.
In comparison with the test sample of the other group, group 3 has the growth of yeast and
mold really evenly in total Petri dishes because they spreading carefully the suspension
spread evenly in the surface of the medium, and the spreader of group 3 is sterilized better
than our group therefore yeast and mold can growth equally and have the better result with
our group.
V. CONCLUSION
The experiment provides a comprehensive grasp of the process of identifying the presence of
yeasts and molds in food samples, such as grapes, after finishing Laboratory 4. The impact of
microbes on food decomposition and human consumption is also discussed, emphasizing the
importance of exercising caution.
During the experiment, the processes for counting colonies in petri dishes and utilizing the
pour-plate method were also learned. Using data from additional organizations, it was
revealed that yeasts were more frequently detected in the sample than molds. Nonetheless,
yeasts can be counted after three days, whereas molds require 5-7 days incubation in a proper
environment to be identified.
VI. REFERENCES

Lakna (2017) Difference between yeast and mold: Definition, structure, function, similarities,
Pediaa.Com. Available at:https://pediaa.com/difference-between-yeast-and-mold/
Figure 1.1 :Yeast (2023) Encyclopedia Britannica. Available at:

https:/www.britannica.com/science/yeast-fungus
Figure 1.2 : LogicalPosition (2022) The different types of molds and how they 're treated,
Aloha Restoration Co. Available at: https://aloharestorationco.com/2022/07/13/the-different-
types-of-molds-and-how-theyre-treated/
Figure 1.2: Mokobi, F. (2021) Aspergillus flavus- an overview, Microbe Notes. Available at:
https://microbenotes.com/aspergillus-flavus/
BAM Chapter 18: Yeasts, Molds and Mycotoxins. (2022, November 7). FDA. Retrieved
November 28, 2023, from https://www.fda.gov/food/laboratory-methods-food/bam-chapter-
18-yeasts-molds-and-mycotoxins
Pitt, J. I. (n.d.). (PDF) Dichloran-Rose Bengal medium for enumeration of moulds from
foods. ResearchGate. Retrieved November 28, 2023, from
34
https://www.researchgate.net/publication/22657421_Dichloran-
Rose_Bengal_medium_for_enumeration_of_moulds_from_foods
Figure 4.1 in the right taken form group 3

35
LABORATORY 5: BACTERIAL MORPHOLOGY AND STAINING
I. INTRODUCTION
1. Classification about the bacteria
In this investigation, fermented food products (all handmade) were examined for microbial
morphology using a microscope and gram staining techniques.
Microbial species are often classified into two groups: gram-positive bacteria and gram-
negative bacteria:
- With Gram-positive bacteria: they have thick, mesh-shaped peptidoglycan cell wall
capable of absorbing the purple color of crystalline gentian violet dye. Because the wall layer
is thick, alcohol removal will be more difficult, so bacteria retain the purple color of Gentian
purple dye.
- With Gram-negative bacteria: the peptidoglycan wall is thinner and it has an extra
lipopolysaccharide membrane on the outside. When bleached with alcohol, alcohol dissolves
the membrane and because the thin wall is easily erased by alcohol, it cannot retain the purple
color of the dye. Which will stain the dye followed by alkaline Fuchsin solution.
Bacteria come in a variety of morphologies, the most common of which are cocci (spherical),
bacilli (rod-shaped) and spirilla (spiral-shaped). Understanding bacteria's form is critical for
categorization and identification.When bacteria have been adequately dyed, it is typically
simple to determine their overall shape. The most common shapes are presented as belowed:
Figure 1.1: The common shape and arrangement of bacteria
36
2. Principle of staining method
The concept behind staining methods is that bacterial cells have an affinity for various dyes,
allowing the dye to attach to certain cellular components. The premise behind staining
methods is that bacterial cells have an affinity for various dyes, allowing the dye to attach to
certain cellular components.
Crystal violet (20 to 30 seconds staining time), carbolfuchsin (5 to 10 seconds staining time),
and methylene blue (1 minute staining time) are common dyes employed. When bacteria
have been adequately dyed, it is typically simple to determine their overall shape. Summary
of gram staining as follow:
Table 1.1: Summary of gram staining
Application Reagent Cell color
Gram-positive Gram-negative
Primary dye Crystal violet purple purple
Mordant Iodine purple purple
Decolorizer Alcohol/acetone purple colorless
Counterstain Safranin/carbolfuchsin purple pink or red
This experiment entailed learning about the fermentation process, gram staining, evaluating
the quality of fermented food items, and using a microscope to examine the morphology of
bacteria introduced to fermented food products.
The desired outcome of this project would have been proficiency in gram staining
procedures, as well as the research and documentation of the morphology of microorganisms
found in fermented food items by microscopic observation.

CHEMICALS EQUIPMENTS
Crystal violet (1% aqueous solution) Slide glasses
37
Alcohol Microscope: 1
Iodine Alcohol Burner: 1
Safranin Inoculation loop: 1
Distilled water Beaker: 500 mL & 1000 mL: 1
BGBB for Coliforms Test tubes
RVS for Salmonella Clean Microscope slide
PCA for Aerobic bacteria Immersion oil :1
Bibulous paper
Gram staining kit
Wax pencil: 1
Lens paper and lens cleaner
Slide holder or clothespin
Slide warmer
Needle: 1

This lab’s work is carrying out gram staining of Plate count agar (PCA), Brilliant Green Bile
Broth (BGBB), and RVS, as well as observing the morphology of three types of
microorganisms, including aerobic microbes, coliforms, and Salmonella, respectively, under
microscope
1. Bacteria smear
Before conducting the smear, clean the glass slide and coverslips with alcohol. After that,
mark the name of the bacterial culture in the far left corner of each of the slides
- For liquid: (BGBB, RVS)
Step 1: Sterilize the inoculating loop by heating it on the alcohol burner and letting it cool.
Figure 3.1: Heating the loop

Step 2: Shake the culture tube, dip the loop in the sample to transfer 1 or 2 loopfuls of
bacteria to the center of the slide, and spread out the broth culture mixture.
38
Figure 3.2: Put the sample into the glass slide

Step 3: Quickly heat through the fire a few times until dry, then proceed to the gram staining.
Note that while heating, do not let the flame come close to the glass slide, as it can affect the
specimen, and microorganisms could be killed and distort the bacterial cells.
Figure 3.3: Heat-fixation

- For liquid: (PCA)
First, put 1 drop of distilled water in the center of the glass slide. Sterilize the inoculating
needle by heating it in the flame until it turns red, and let it cool before picking 1 needle of
bacterial growth in the plate count agar. Next, we spread out the water-bacteria mixture and
quickly heat it through the fire until it dries.
We have to do heat-fixation for the following reasons:
- To be fixed to the slide
- It is an easy way to kill bacteria without destroying it
2. Gram staining
After completing a bacterial smear, we will perform gram staining using the following steps:
Step 1: A bacterial smear is stained with crystal violet for 60 seconds, then rinse with water.
Do not wash directly to the smear
39
Figure 3.4: Using crystal violet for a minute and rinse with distilled water
Step 2: Treated with an Iodine for 60 seconds to increase the binding of the primary stain,
and rinse with water
Figure 3.5: Using Iodine for a minute and rinse with distilled water
Step 3: Decolorize with 95% Ethanol for 15 seconds to remove the crystal violet from cells
which bind it weekly. Rinse with water
40
Figure 3.6: Using Ethanol for 15 seconds and rinse with distilled water
Step 4: Counterstain with Safranin and wait for about 60 seconds to provide the color
contrast in those cells that are decolorized, and then rinse with water
Figure 3.6: Using Safranin for a minute and rinse with distilled water
Figure 3.7: Procedure of Gram Staining: note the color change after each step
Step 5: Cover the glass slide with a coverslip gently and do not let the formation of bubbles
inside the slide as it is quite difficult to observe the sample under a microscope
41
Figure 3.8: Cover the glass slide

Step 6: Observe the morphology of microorganisms under a 40X microscope
Figure 3.9: Observe the morphology of microorganisms using microscope
42
1. Result
Figure 4.1: Aerobic bacteria under 40x microscope

Gram-positive bacteria with an oxygen-dependent growth mechanism and a rod-like
morphology are known as aerobic Gram-positive bacilli. Their thick cell walls confer Gram-
positive status. From the image above, it is evident that the pink color in the rod is more
dominant, indicating the presence of gram-negative aerobic bacteria in this sample. In
addition, we can see two colors that appear in the glass slide due to not being washed
thoroughly during the staining process, causing confusion when looking under the
microscope.
Figure 4.2: Coliform bacteria from BGBB 10−3 under 40x microscope
Coliform bacteria are rod-shaped, Gram-negative, non spore-forming, motile, or nonmotile
bacteria that, when cultured at 35–37°C, may ferment lactose and produce gas and acid.
Gram-negative, rod-shaped bacteria that do not develop endospores are known as coliform
bacteria. In this case, almost all bacteria are pink, meaning that gram-negative coliform
bacteria predominate in this sample.
43
Figure 4.3: Salmonella from RVS under 40x microscope

Salmonella enterica is a facultative anaerobe that is gram-negative and intracellular pathogen
that affects both humans and animals. It is not a symbiotic commensal. As much of a global
public health risk as foodborne illness is infection. There are an astonishingly high number of
pathogenicity islands (SPI) in the genome-up to 23. During the dyeing process, the step dries
the bacteria on the burning alcohol because the slow operation results in the Salmonella
bacteria almost dying. Therefore, the dyeing process does not achieve theoretical results.
Looking at figure 4.3, we see that the result is that Salmonella bacteria do not "eat" the dye (it
is almost transparent).
2. Discussion
During the staining process to observe bacteria under a microscope with an index of 40x,
unprofessional operations led to results that were not as theoretical. Some incorrect
operations include leaving the bacteria on the alcohol fire for too long, killing the bacteria,
and not rinsing the dye thoroughly with distilled water, so when observing, it is easy to
confuse the bacteria and the dye.
V. YOGURT
In the afternoon class, we would observe the morphology of microorganisms from fermented
products that we prepared at home. Two different types of fermented foods brought to the lab
are kombucha from group 2 and yogurts from the remaining groups. Our group has brought
yogurt
1. Ingredients and method to make homemade yogurt

a. Ingredients
4 milk packets 220ml
2 cartons of no-sugar yogurt
1 carton of condensed milk
Hot water
44
b. Procedure
Figure 5.1: Procedure of making yogurt at home
2. The mechanism of the fermenting process

Yogurt is a rich source of protein and calcium, and the fermentation process makes these
nutrients easier to absorb in our bodies.
During the fermentation, the starter cultures use lactose (milk sugar) to produce lactic acid,
normally Lactobacillus bulgarius and Streptococcus thermophilius. The lactic acid lowers the
pH of milk below 4.6 and makes the milk more acidic, which leads to the coagulation of
proteins and changes the consistency of the milk to form a thicker substance: a yogurt-like
texture. The more lactic acid is produced, the tangier the yogurt will taste. In addition, the
starter cultures produce polysaccharide materials, increasing the thickness and stability of the
yogurt gel. Besides, the types of yogurt starters and the length of incubation time also
contribute to the taste and consistency of the yogurt
3. The morphology of yogurt bacteria

The rod-shaped bacteria Lactobacillus delbrueckii subsp. Lactobacillus delbrueckii is Gram-
positive which can lead to the formation of purple color after gram staining. BBulgaricus
ferments the lactose in milk to form lactic acid, which reacts with the milk protein to give
yogurt its distinctively sour texture and flavor. Streptococcus salivarius subsp. Thermophilus,
Bifidobacterium bifidum, and Lactobacillus acidophilus, also known as casei, are additional
bacteria that can be found in yogurt.
45
Figure 5.2: These are some shapes of different types of bacteria.
Figure 5.3: Yogurt sample under 40x microscope
4. The morphology of Kombucha

A fermented tea beverage made with bacteria and yeast is called kombucha. The flavor of
kombucha can be affected by its fermentation settings. Kombucha flavor is affected by
modifications to the symbiotic culture of bacteria and yeast (SCOBY). The kind of tea used
to make kombucha affects the amount of polyphenols. Acetic Acid Bacteria (AAB) and
Kombucha yeast were identified and their phenotypic characteristics were examined. AAB
are aerobic, rod-shaped, ellipsoidal, non-spore-forming, Gram-negative cells that can exist
singly, in pairs, or in clusters. The width and length of cells vary from 0.4 to 1.0 μm and 0.8
to 4.5 μm, respectively. Most of these bacteria are oxidase negative and catalase positive. The
46
ideal development temperature ranges from 25 to 30 degrees Celsius; however, some strains
that are thermotolerant can reach up to 42 degrees Celsius, and certain AAB strains can thrive
in acidic environments.
Figure 5.4: Kombucha sample under 40x microscope
5. Discussion
Warm milk (110–115°F) is blended with Lactobacillus bulgaricus and Streptococcus
thermophilus bacteria to make yogurt. The mixture is then allowed to settle for several hours.
You can add different kinds of bifidobacteria and lactobacilli. The gram-positive, rod-shaped,
non-motile, non-spore-forming Lactobacillus bulgaricus bacteria is one of the most prevalent
starter bacterial species used in commercial dairy product fermentation. Streptococcus
thermophilus is a nonmotile, spherical to ovoid, Gram-positive coccus with a diameter of
0.7–0.9 μm. It can be found in chains and pairs, some of which can be extremely lengthy. The
ideal temperature range for the bacterium to grow is 40–45. Arginine is not hydrolyzed by
Streptococcus thermophilus.
Figure 5.5: Lactobacillus bulgaricus bacteria
Figure 5.6: Streptococcus thermophilus bacteria

In the yogurt sample observed above, this yogurt sample (figure 5.3) contains many gram-
positive bacteria, which are the characteristic purple-blue color of gram-positive bacteria,
47
with two shapes: bacillus and streptococcus. These are two typical shapes of Lactobacillus
bulgaricus and Streptococcus thermophilus bacteria. So this yogurt sample contains both
types of bacteria.
The probiotic Bacillus coagulans and other fermentation-capable bacteria, such as

Lactobacillus nagelii, Gluconacetobacter, Gluconobacter, and Komagataeibacter species,
predominate in most kombucha beverages.
Figure 5.7: Lactobacillus nagelii bacteria Figure 5.8: Gluconacetobacter bacteria
Figure 5.9: Gluconobacter bacteria Figure 5.10: Komagataeibacter bacteria
In kombucha samples, we can easily observe them under a microscope. The shape of
kombucha is long and straight like a strain of bacillus bacteria. Because the bacteria in
kombucha have almost the same appearance, the kombucha sample (figure 5.4) may contain
four types of bacteria. The most typical is Komagataeibacter bacteria because in figure 5.4
there is an area of bacteria linked into an array like figure 5.10.
VI. CONCLUSION
In conclusion, after this lab provides the knowledge about how to smear and gram staining
bacteria. In staining bacteria as yogurt, kombucha, Coliform from BGBB and Salmonella
from RVS can have a better knowledge in the shape, size as well as the structure of bacteria
based on the color it perform (example bacteria have thick outer membrane will have purple
color , in contrast bacteria have thin layer will give the pink color) therefore it is a condition
to determine kind of bacteria, for instance in yogurt bacteria are determined is Streptococcus
thermophilus and Lactobacillus delbrueckii subsp and Coliform has the rod shape, however,
the shape and structure of Salmonella and Kombucha not clearly.
VII. REFERENCES
Carolina Moyano (2020) Fermentation of yogurt and the chemistry behind it, FoodUnfolded.
Available at:
48
https://www.foodunfolded.com/article/the-chemistry-behind-the-fermentation-of-yogurt
Figure 3.7: Tankeshwar, A. (2022) Gram staining: Principle, procedure, results - microbe
online, Microbe Online. Available at:
https://microbeonline.com/gram-staining-principle-procedure-results/
Figure 5.2: Amanda Gingrasso, A. (2022) Gut Health: Prebiotics and Probiotics, Mayo Clinic
Health System. Available at:
https://www.mayoclinichealthsystem.org/hometown-health/speaking-of-health/good-bacteria-
for-your-gut

Aerobic Gram-Positive Bacilli: Characteristics, Types & Examples (November 28, 2023).
Available at: https://study.com/academy/lesson/aerobic-gram-positive-bacilli-characteristics-
types-examples.html
Salmonella enterica (November 29, 2023). Available at:

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/
salmonella-enterica
Coliform Bacteria (November 29, 2023). Available at:

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/coliform-
bacteria#:~:text=Coliform%20bacteria%20are%20defined%20as%20rod%2Dshaped
%20Gram%2Dnegative%20nonspore,at%2035%E2%80%9337%C2%B0C.
Kombucha: Production and Microbiological Research (October 31, 2022). Available at:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9658962/
Morphology, Different shapes of bacterial cells (July 28, 2023). Available at:
https://byjus.com/neet/important-notes-of-biology-for-neet-shapes-of-bacteria/
Genetic diversity and population structure of Lactobacillus delbrueckii subspecies bulgaricus

isolated from naturally fermented dairy foods (March 04, 2016). Available at:
https://www.nature.com/articles/srep22704#:~:text=bulgaricus%2C%20a%20Gram
%2Dpositive%2C,industrial%20fermentation%20of%20dairy%20products.
Yogurt (February 02, 2023). Available at:

https://www.hsph.harvard.edu/nutritionsource/food-features/yogurt/#:~:text=Yogurt%20is
%20made%20when%20heated,and%20bifidobacteria%20may%20be%20added.
Streptococcus thermophilus (November 29, 2023). Available at:

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/streptococcus-
thermophilus#:~:text=thermophilus%20appears%20as%20spherical%20or,when%20grown
%20in%20liquid%20media.
49
Microbial and Chemical Profiles of Commercial Kombucha Products (February 5, 2022).

Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8838605/#:~:text=Most
%20kombucha%20products%20are%20dominated,%2C%20Gluconobacter%2C%20and
%20Komagataeibacter%20species.
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Report - Practice in Food Microbiology - Group 4

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Report - Practice in Food Microbiology - Group 4

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VIETNAM NATIONAL UNIVERSITY HO CHI MINH NATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY - DEPARTMENT OF FOOD TECHNOLOGY

PRACTICE IN FOOD MICROBIOLOGY REPORT

COURSE CODE: BTFT254IU

No. Full name Student ID Contribution (%)

LABORATORY 1: DETERMINATION OF AEROBIC PLATE COUNT.............................................................

LABORATORY 1: DETERMINATION OF AEROBIC PLATE COUNT

Figure 1.1: Aerobic Bacteria

II. MEDIA AND EQUIPMENT

Glassware and inoculating loop must be Sterile impermeable plastic bag

III. EXPERIMENTAL PROCEDURE

Figure 3.3: Sample of milk

Figure 3.4: 4 tubes PBW

Figure 3.9: Petri dishes after pouring

Figure 3.12: Spreading process

IV. RESULT AND DISCUSSION

Table 4.1: Colonies of anaerobic and aerobic (Pouring)

Petri dish 1 Petri dish 2

Table 4.2: Colonies of anaerobic and aerobic (Spreading)

Petri dish 3 Petri dish 4

While in 10−4dilution, the medium is comparatively poor for microorganisms because it is

B.T.Vy (2023). Food Microbiology Laboratory Manual. International University, Vietnam

LABORATORY 2: DETERMINATION OF TOTAL COLIFORM

Figure 1.1: Coliform bacteria

II. MEDIA AND EQUIPMENT

Buffered peptone water (BPW) Sterile impermeable plastic bag

Brilliant Green Bile Broth (BGBB) Scissor: 1

Distilled water Test tube: 30

Juice (sugar cane) Durham tube: 20

Beaker: 500mL & 1000mL

Cylinder: 250mL & 500mL

Bottle: 1500mL & 2250mL

Thermostatic water bath, incubator, vortex

III. EXPERIMENTAL PROCEDURE

Figure 3.1: The medium and dilution need to experiment

Figure 3.2: Vortex the dilution

Figure 3.3: Incubate the dilution

IV. RESULT AND DISCUSSION

Figure 4.1: BGBB tubes after incubation at dilution of 10-1

Figure 4.2: BGBB tubes after incubation at dilution of 10-2

Figure 4.3: BGBB tubes after incubation at dilution of 10-3

Table 4.1: Result of BGBB tubes after incubation

Dilution Test tube 1 Test tube 2 Test tube 3

10-1 Positive Positive Positive

10-2 Positive Positive Positive

10-3 Positive Positive Positive

Figure 4.4: Most probable number (MPN) table

B.T.Vy (2023). Food Microbiology Laboratory Manual. International University, Vietnam

H.B.D. Halkman, A.K. Halkman (2014) Coliform Bacteria - an overview | ScienceDirect

Figure 1.1 “Coliform bacteria.” Wikipedia, https://en.wikipedia.org/wiki/Coliform_bacteria.

Erkmen, Osman. “\/.” YouTube, 16 June 2023,

Li, Daoliang, and Shuangyin Liu. “\/.” YouTube, 16 June 2023,

LABORATORY 3: DETERMINATION OF SALMONELLA

Salmonella infection usually isn't life-threatening. However, in certain people — especially

Figure 1.1: TSI phenomenon

II. MEDIA AND EQUIPMENT

5 test tubes 50mL distilled water

1 100-mL glass bottle 3.23g Triple Sugar Ion Agar (TSI)

Magnetic stirrer Nutrient agar plate

1 test tube rack

Biological safety cabinet

III. EXPERIMENTAL PROCEDURE

Figure 3.1: Boiling TSI to dissolved completely