MIC254 Laboratory Report

NAME OF GROUP MEMBERS:

1. ANISYA SAFFIA BINTI ZORHADI (2022477018)

2. SITI AINATASHA BINTI SHA’ARI (2022698778)
3. SORFINA MIRZA BINTI ZAFIZAL (2022841064)
4. SAFFIYAH ATIQAH BINTI SARMADI (2022696148)
5. ANIS NATASHA SHAHROM BINTI SHAHRIN
(2022894238)

NAME OF EXPERIMENT: ISOLATION OF SPORE-FORMING

BACTERIA FROM DRIED FOOD

DATE OF LAB: 12/4/2023

LECTURER’S NAME: DR. HIDAYAH

INTRODUCTION:
Spore-forming bacteria mainly belong to two generals of the Firmicutes phylum, the aerobic
or facultative anaerobic Bacilli and the strictly anaerobic Clostridia. These organisms can
adapt to harsh environmental conditions by producing highly resistant endospores by undergo
a complex developmental cell differentiation process. The dormant spores can survive
extremes of temperature and or and in the absence of water and nutrients. The spore
germinates originating a new vegetative cell able to grow, duplicate and sporulate again as
the environmental conditions return favourable to cell growth.

OBJECTIVES:

 To isolate the spore-forming bacteria from food product.

 To observe the morphology and characteristics of the spore-forming bacteria.

MATERIALS:

 Bacillus subtilis culture

 Nutrient broth
 Litmus milk
 Tryptic soy agar
 Autoclaved distilled water
 Food sample (chili powder, white pepper powder, flour)
 Gram stain set
 Malachite green stain (CARCINOGEN)
 Slide
 70% alcohol
 Universal bottle
 Water bath 80°C
 Micropipette 1000μL
 Micropipette tips 1000μL
 Test tubes
 Microscope
 Test tube rack
METHODS:

A) Microscopic examination

1. A clean glass slide was prepared.

2. A drop of water placed on the slide.
3. Using a sterile loop, a very small sample obtained and transferred to the slide.
4. The sample evenly mixed and spread by circular motion on the slide.
5. The smear was allowed to air-dry and was fixed it quickly by passing the slide over
the Bunsen burner flame. The fixing prevented the smear from washing off during
staining.
6. The slide was flamed not too much as it could alter the cell morphology and induce
the stain to decolorize more rapidly.

For Gram staining, continue to step (7). For spore staining, continue to step (12).

Gram staining

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

Spore staining

12. The smears were 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 steamed for
3 to 5 minutes. The stain was added if it begins to dry.
15. The stain absorbent paper was removed 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. The stain was washed gently with tap water.
 18. The smear was counterstained with safranin and leave it for 1 minute. The stain was
washed gently with tap water.
19. The slide was blot dried before examined it using a light microscope.

B) Characterization of bacteria

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

C) Enumeration of number of spores in dried food sample

1. 1 g of sample was weighed and added with 4.5 ml autoclaved distilled water. The
sample was shaken vigorously for two minutes and allowed to be settling for two
minutes. This preparation produced a sample solution with dilution factor 10^1.
2. The supernatant was transferred to a sterile universal bottle.
3. The bottle was immersed in water bath at 80°C for 30 minutes to kill the vegetative
cells and heat-shock the spores.
4. The bottle was removed from the water bath and the supernatant was mixed
homogenously.
5. Three sterile tubes added with 4.5 ml autoclaved distilled water were prepared. 1 ml
of sample solution was inserted (avoid any food particles) into the first test tubes and
was mixed homogenously. The tube was labelled as T2 (dilution factor 10^2). The
same procedure was done for the next two test tubes and was labelled as T3 and T4
(dilution factor 103 and 104), respectively.
6. 100 μl of the homogenized mixture have been Inoculated (from tubes with dilution
factor 103 and 104) on tryptic soy agar plates and spreaded evenly using L shaped
spreader. Each dilution in duplicated been plated.
7. The agar plate inverted at 37°C for 48 h at aerobic and anaerobic conditions have
been incubated.
RESULTS:

B) Characterization of bacteria

Litmus milk Nutrient broth (37°C) Nutrient broth (55°C)

C) Enumeration of number of spores in dried food sample.

Aerobic condition

Dilution Number of colonies

103

104

Too few to count

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

C = 46
M = 103
V = 0.1 ml
Colony forming unit/ml = (C X M)/V)
= (46 x 103) / 0.1
= 4.6 x 10⁵ CFU/ml

Anaerobic condition

Dilution Number of colonies

103

Too few to count

104

Too few to count

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

C = 19
M = 103
V = 0.1 ml
Colony forming unit/ml = (C X M)/V)
= (19 x 103) / 0.1
=1.9 x 10⁵ CFU/ml
DISCUSSION:

Lastly, the experiment was done to study the enumeration of number of spores using dried
food sample. We used the black pepper as the dried sample. The black pepper was diluted
into several dilution for the serial dilution method and then was spread onto the TSA plate.
The spread was duplicate for aerobic and anaerobic purpose. The agar then was incubated for
3 days, and observation is made. In the procedure, the sample was shaken before transferred
to obtain the maximum supernatant as possible from the result. We observed the macroscopic
morphology of colony form which is dry, irregular, flat and white from this. We can say that
the bacteria form is Clostridium sp. Moreover, we found out that the number of colony
forming unit for 103 aerobic dilutions are 4.6 x 10⁵ CFU/ml while for the anaerobic plate, the
colony forming unit was found out for 103 is 1.9 x 10⁵ CFU/ml. For the first dilution, the
number of colonies form are too many to count. From this. we can see that the number of
colonies form for aerobic is higher than anaerobic. As the number of colony formation in
aerobic is higher, this show that it is the facultative aerobic.

CONCLUSION:

In conclusion, our experiment was successful since we were able to identify and isolate the
bacterium that was growing in dried black pepper. Additionally, we were able to stain the
bacteria using both methods.
DISCUSSION QUESTIONS:

1) Discuss the purpose of shaking the sample before transferring it into a sterile
universal bottle.

The purpose of shaking the sample before transferring it into a sterile universal bottle
is to ensure that our cells are evenly distributed in the tube

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

Several factors can influence bacteria spore, including the presence of nutrients,
moisture, pH, temperature, and the presence of germinant.

3) Predict the results of the experiment if the food sample was not heated before
being inoculated on the agar.

If the food sample was not heated before being inoculated on the agar overgrowth of
non-spore forming bacteria will happen. It is because without heating, the food
sample may contain vegetative cells of non-spore-forming bacteria. These cells can
outgrow spore-forming bacteria on the agar plates, making it difficult to isolate spore-
forming bacteria.

4) Differentiate facultative anaerobe and obligate anaerobe.

Facultative anaerobes are bacteria that can grow in the presence or absence of oxygen,
and they use different metabolic pathways depending on the availability of oxygen
while obligate anaerobes are bacteria that cannot grow in the presence of oxygen, and
they use fermentation or anaerobic respiration to produce energy.
REFERENCES:

 Serial Dilution in Microbiology: Calculation, Method & Technique - Video & Lesson
Transcript | Study.com. (2013). Study.com. https://study.com/academy/lesson/serial-
dilution-in-microbiology-calculation-method-technique.html
 Mannaa, A. (n.d.). food microbiology. Www.academia.edu. Retrieved May 1, 2023,
from https://www.academia.edu/11420479/food_microbiology
 Teknologi, U. (2017). ISOLATION AND CHARACTERIZATION OF SPORE-
FORMING BACTERIA FROM DRIED FOOD MASLIA DEWI BINTI MASDUKI
Final Year Project Reported Submitted in Partial Fulfillment of the Requirements for
the Degree of Bachelor of Science (Hons.) Biology in the Faculty of Applied Sciences.
https://ir.uitm.edu.my/id/eprint/33493/1/33493.pdf
 Mehta, D. (2018). The Influence of Spore-forming Microorganisms on the Quality
The Influence of Spore-forming Microorganisms on the Quality and Functionality of
Cultured Dairy Products and Functionality of Cultured Dairy Products.
https://openprairie.sdstate.edu/cgi/viewcontent.cgi?article=3972&context=etd