LABORATORY REPORT

MIC254
FOOD MICROBIOLOGY

LAB 4: ISOLATION OF SPORE-FORMING BACTERIA

FROM DRIED FOOD
DATE EXPERIMENT: 12 APRIL 2023
DATE SUBMISSION: 18 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:

ISOLATION OF SPORE-FORMING BACTERIA FROM DRIED FOOD.

OBJECTIVE:
1) To isolate the spore-forming bacteria from food product.
2) to observe the morphology and characteristics of the spore-forming bacteria.

INTRODUCTION:

In reaction to environmental stress, some bacteria types generate spores. Bacterial spores
generally form cells that are far more resistant to heat, dehydration, freezing, and irradiation than
the vegetative forms of the cells. These increased resistances of concern for food spoilage and
food safety reasons in food processing. In nature, soil, dust, and water are all related to spores. A
few spore-forming microbes are also found in both human and animal digestive tracts.
Spore-forming bacteria forms under unfavorable conditions. It can withstand extreme conditions
such as heat, freezing or other environmental variables. This spore-forming bacteria can be found
in the contaminated food. They induce a variety of spoilage that vary in temperature, appearance
and others. For instance, the aerobic bacteria Bacillus spp. can develop in both mesophilic and
thermophilic environments. Some bacteria can even create spores, which help protect food from
external factors that might otherwise spoil it.

PROCEDURE/METHODS:

A) Microscopic examination of
1. A clean glass slide was prepared.
2. A drop of water was placed on the slide
3. Sterile loop was used to obtain a very small sample and it was transferred to the slide.
4. The sample was mixed and spread evenly by circular motion on the slide.
5. Allow the smear to air-dry and fix it by quickly passing the slide over the Bunsen burner
flame. The fixing will prevent the smear from washing off during staining.
 6. Do not flame the slide too much as it could alter the cell morphology and induce the stain
to decolorize more rapidly.

Gram staining

7. The smear was flooded with a few drops of crystal violet and was left for 1 min. Wash the
stain gently with tap water.
8. Then, the smear was flooded with a few drops of Gram’s iodine and left it for 1 min.
Wash the stain gently with tap water.
9. Tilted the slide and dropped the 95% ethyl alcohol for a few seconds until the alcohol
runs roughly clear. Washed the slide gently with tap water
10. Counterstain the smear with safranin and leave it for 45 sec. Wash the stain gently with
tap water.
11. The slide was blot dry before examining it using a light microscope.

Spore staining

12. The smear was covered with a piece of absorbent paper.

13. The slide was placed over a staining rack that has a beaker of steaming water.
14. The paper was flooded with malachite green stain (CARCINOGEN) and let it steam for 3
to 5 minutes. Add the stain if it begins to dry.
15. Removed the stained absorbent paper carefully using loop and discard (DO NOT
DISCARD THE PAPER IN THE SINK)
16. The slide was allowed to cool for 1-2 minutes.
17. Wash the stain gently with tap water.
18. Counterstain the smear with safranin and leave it for 1 minute. Wash the stain gently with
tap water.
19. The slide was blot dry before examining it using a light microscope.
B) Characterization of bacteria

1. A small amount of Bacillus subtilis colony was inoculated into litmus milk and incubated
at 37℃ for 24h.
2. A small amount of Bacillus subtilis colony was inoculated into two tubes of nutrient
broth. The test tubes were incubated for 24 h at 37℃ and 55℃ respectively.

C) Enumeration of number of spores in dried food sample

1. Weigh 1 gram of the sample added with 9 ml autoclaved distilled water. Shake vigorously
for two minutes, and then allow settling for two minutes. This preparation produces a
1
sample solution with dilution factor 10 .
2. Transferred the supernatant to a sterile universal bottle.
3. Immerse the bottle in water bath at 80℃ water bath for 30 minutes to kill vegetative cells
and heat-shock the spores
4. Removed the bottle from the water bath and mixed the supernatant homogeneously.
5. Three sterile tubes were prepared and added with 9 ml autoclaved distilled water. 1 ml of
sample solution was inserted (avoided any food particles) into the first tube and mix
2
homogeneously. Labeled the tube as T2 (dilution factor 10 ). Do the same procedure for
3 4
the next two test tubes and label it as T3 and T4 (dilution factor 10 and 10 ),
respectively.
6. 100 microliter of the homogenized mixture was inoculated on tryptic soy agar plates
3 4
(from tubes with dilution factor 10 and 10 ) and spread evenly using an L shaped
spreader. Plate each dilution in duplicates.
7. The agar plate was incubated inverted at 37℃ for 48 h at aerobic and anaerobic
conditions.
RESULTS:

A) Microscopic examination

Gram-staining illustration Spore staining illustration

B) Characterization of bacteria

Litmus milk Nutrient broth (37℃) Nutrient broth (55℃)

2 layers formed White floating liquid No change

Diagram result:
Left is nutrient broth at
55℃, center is nutrient
broth at 37℃ and last
litmus milk at 37℃.
 C) Enumeration of number of spores in dried food sample.
- Aerobic condition

Dilution Number of colonies

10
3 0

10
4 1

Number of bacteria in original sample (use the formula (C×M)/V)

4
= (1 × 10 )/0. 1

= 100000 CFU/ml

- Anaerobic condition

Dilution Number of colonies

10
3 0

10
4 0

Number of bacteria in original sample (use the formula (C×M)/V)

4
= (0 × 10 )/0. 1

= 0 CFU/ml
DISCUSSION:

There were four different procedures used to perform the experiment. In this procedure, gram
staining and spore staining methods have been used; the first procedure was to conduct
morphological studies on the given culture of Bacillus subtilis. Next, the second procedure was
the examination of the Bacillus subtilis culture growth at mesophilic and thermophilic
temperatures which the bacteria were inoculated into litmus milk and incubated at 37°C and two
tubes of nutrient broth and incubated at 37°C and 55°C for 24 hours. Moreover, the third
procedure was the enumeration of the number of spores in a dried food sample. Last but not
least, all the observations on the result data sheet and followed by pictures of the final result were
recorded.

A) Microscopic examination

Gram staining

In the Bacillus subtilis slide, there was no observation obtained for the gram-staining method
used in our group. It is because there was no growth of bacteria in our agar plate. For the
discussion, we use other group results as reference. The morphological studies of Bacillus
subtilis show that the bacteria is in rod-shaped and pink in color which theoretically, the pink
color in a gram staining method indicates gram-negative bacteria. When ethanol, a decolorizer, is
added to the gram-negative, it loses its ability to retain the colour of crystal violet and instead
takes on the colour of the counterstain safranin. Under a microscope, gram-negative cells appear
pink because of the pinkish-red colour of the counterstain safranin. Theoretically, Bacillus
subtilis should appear crystal-violet because they have a thick layer of peptidoglycan. This will
indicate Gram-positive. When observed under a microscope, Bacillus subtilis is supposed to
maintain its crystal-violet color. However, in the experiment, Bacillus subtilis appears pink rather
than crystal-violet. This could be the result of a human error, such as adding too much
decolorizer while carrying out the experiment, or the bacteria culture provided was contaminated
or accidentally provided the incorrect culture. As a result, the outcome of the gram-staining of
Bacillus subtilis does not match the expected results because it does not appear crystal-violet
when viewed under a microscope.
Spore staining

Then, the observations obtained we got when using the spore staining method during the
second Morphological study of Bacillus subtilis were only the vegetative cell of Bacillus subtilis
that showed was pinkish-red colour and there was no appearance of green stain colour for spores.
Additionally, the shape of the cell that we observed was a combination of coccus and rod shapes
in addition to being rod-shaped. According to theory, the primary stain will be removed from the
vegetative cell when the decolouriser is added and after the malachite green stain has stained the
spores. The pinkish-red colour of safranin will transfer to the vegetative stain. Also, the shape
seen under the microscope should only be rod-shaped since the culture of Bacillus subtilis is
spore-forming and rod-shaped bacteria. The spore-staining method is used to distinguish
bacterial spores from other vegetative cells and distinguish between spore-former bacteria and
non-spore-former bacteria. However, the results for Bacillus subtilis only reveal vegetative cells
and no evidence of spores. There are errors that might have happened that influenced the
experiment such as the culture being contaminated, the culture being a non-spore former culture
or mistakes made by experimenters as a result of the spores forming vegetative cells. Thus, the
result of the spore staining procedure did not turn out as predicted because the spores were meant
to be stained green since Bacillus subtilis is a spore-forming bacteria. To avoid problems that
may affect your result, we must always use the proper aseptic technique to avoid contamination
in the culture. The sterilization must be done properly to avoid the appearance of non-spore
former culture and handling the experiments carefully for the experimenters.

B) Characterization of bacteria

In this part of the experiment, we have successfully observed the characterization of Bacillus
subtilis colony. We used litmus milk and nutrient broth as a medium. As we know, Bacillus-type
bacteria produce spores that provide dormancy at high temperatures because the enzyme proteins
modify the structure of the spore as it dehydrates so it can not survive at higher temperature.
Based on the result, Bacillus subtilis colony was found in litmus milk that has been incubated for
24 h at 37℃. It was observed that two layers were formed in the tube. Other than that in nutrient
broth tubes that were incubated at 37℃, it shows that white floating liquid in the test tube while
nutrient broth at 55℃ has no changes because of high temperature for Bacillus subtilis to grow.
C) Enumeration of the number of spores in dried food samples.

Lastly, the experiment was done to study the enumeration of number of spores using dried
food sample. We used white pepper powder as the dried sample. The pepper was diluted into
several dilutions for the serial dilution method and then spread onto the TSA plate. The spread
was duplicated for aerobic and anaerobic purposes. The agar then was incubated for 48 hours and
observation was made. In the procedure, the sample was shaken before transferred to obtain the
maximum supernatant possible. From the result, we observed in aerobic conditions that no
colony was formed on the agar except for T4, where one colony was formed. For the anaerobic
plate, zero colonies were formed. Our experiment failed.

However this is different from the result that we expected it to be, which is for anaerobic, the
number of colonies formed is higher than aerobic as for the dried food the bacteria is
actively grown in anaerobic condition. This result may be affected due to some contamination
occurring while handling the plate which later results in the inaccurate result. This is proven by
the formation of some structure in the TSA plate that indicates contamination. Next, a few
factors affect spore germination. The most important environmental elements that help spore
germination are suitable environmental temperature, accessible water of moisture, and
sometimes the presence of nutrients transmitted from the host into the water. The experiment
would yield a different result if the food sample solution was not heated before heating. This is
because if the sample was not heated, the bacterium cell may expand, indicating that not only
spore growth but also vegetative cell formation would be detected.
CONCLUSION:

In conclusion, the objective of this laboratory was achieved where the objective was to become
familiar with the methods for isolating and identifying spore-forming bacteria from dried food
samples at the end of this experiment. The identification technique was finished using the
gram-staining method, the spore-staining method, the observations of Bacillus subtilis growth in
mesophilic and thermophilic temperature, and the observation of Bacillus subtilis action on
litmus milk medium and two tubes of nutrient broth. Then, the enumeration of the number of
spores from a dried food sample was also finished. Some of the results were in line with what
was expected, while others were not. Therefore, by following all of the procedures and recording
the results, the goal of the experiment has been effectively accomplished.

DISCUSSION QUESTIONS:

1. Discuss the purpose of shaking the sample before transferring it into a sterile universal
bottle.
- The laboratory sample should be shaken by hand before transferring it into a
sterile universal bottle to ensure that the bacteria are evenly distributed.

2. Describe the factors that contribute to the germination of bacterial spores.

- Several factors, including medium composition, temperature, pH, germinant type
and concentration, and heat treatment, are known to influence spore germination
rate and thus time.

3. Predict the results of the experiment if the food sample was not heated before being
inoculated on the agar.
- The result may form a lot of colonies due to other bacteria will also be inoculated
and cause colonies difficult to count.

4. Differentiate facultative anaerobe and obligate anaerobe.

- Facultative anaerobes show better growth in the presence of oxygen but will also
grow without it.
- Obligate anaerobes cannot grow in the presence of oxygen. They depend on
fermentation and anaerobic respiration using a final electron acceptor other than
oxygen.
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