LAB REPORT 4 MIC 254

ISOLATION OF SPORE-FORMING BACTERIA FROM DRIED FOOD

By:

Muhammad Ilham Nur Hakim Bin Ramli (2022775425)

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

Lecturer: Madam Syazuani Mohd Shariff

Date of Experiment: 12/04/2023

Date of Submission: 18/04/2023

Group: AS1143A1
TITLE
Isolation of Spore-forming Bacteria From Dried Food

OBJECTIVES

1. To isolate the spore-forming bacteria from food products.

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

INTRODUCTION

The primary technique for dividing various groupings of microorganisms is the isolation of
bacteria. According to their growth patterns, this method enables us to distinguish between
several bacterial groupings. Depending on their needs for growth and other parameters like
temperature, pH, oxygen availability, etc., various bacteria will grow in different ways on
different nutritional media. In order to identify and categorize germs, isolation of the bacteria is a
crucial step. The bacterial isolation approach uses both culture-based and non-culture-based
techniques. From the solid, liquid, and automated liquid culture medium, specimens can be
separated and cultured. Turbidity and the development of distinctive colonies are characteristics
of bacterial growth in both solid and liquid culture media.

Bacterial spores are highly resistant and dormant structures formed in response to adverse
environmental conditions, As bacterial spores are formed within the parent cell, these are called
endospores..Spore-forming bacteria are more resilient than the average microscopic unicellular
organism. These organisms, which belong to the genera Bacillus, Clostridium, and
Sporolactobacillus, can protect themselves with robust protein coatings that enable them to
endure harsh climatic conditions. Bacteria can survive for years as spores because they are
shielded from stimuli including chemicals, heat, radiation, and dehydration. However, when
these bacteria are reactivated, they can cause a multitude of illnesses, such as botulism, anthrax,
tetanus, and acute food poisoning.
 In this experiment of isolation of spore forming bacteria from dried food, students will
conduct 3 types of experiments. First procedure is, gram staining and spore staining method.
Secondly, the characterization of bacteria. Lastly, the enumeration of the number of spores in
dried food samples.

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 80oC
• Micropipette 1000μL
• Micropipette tips 1000μL
• Test tubes
• Microscope
• Test tube rack

PROCEDURE

A) Microscopic examination
1. A clean glass slide was prepared.
2. A drop of water is placed on the slide.
 3. A very small sample was obtained and transferred to the slide by using a sterile
loop
4. The samples are mixed and spread evenly by circular motion on the slide.
5. The smear is allowed to air-dry and fixed by quickly passing the slide over the
Bunsen burner flame. The smears were prevented from washing off during
staining by the fixing.
6. To avoid the alteration of cell morphology and induce the stain to decolorize more
rapidly, slides were not flamed too much.

Gram staining
7. The smears were flooded with a few drops of crystal violet and left for 1 min. The
stains were gently washed with the tap water.
8. Then, the smears were flooded with a few drops of Gram’s iodine and left for 1
min. The stains were gently washed with the tap water.
9. 95% of ethyl alcohol was dropped and the slides were tilted for a few seconds
until the alcohol ran roughly clear. The slides were washed gently with tap water.
10. The smear with safranin was counterstained and left for 45 sec. The stain were
gently washed with tap water
11. The slide was blotted dry and were examined 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, which has a beaker of steaming water.
14. The paper with malachite green stains which are carcinogen was flooded and let it
steam for 3 to 5 minutes. The stain was added as it began to dry.
15. The stained absorbent paper were remove carefully using loop and discarded
16. The slides were allowed to cool for 1-2 minutes.
17. The stains were gently washed with tap water.
18. The smear with safranin was counterstained and left for 1 minute. The stain gently
washed with tap water.
19. The slide was blotted dry and was examined 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 ℃ for 24 hours.
2. A small amount of Bacillus subtilis colony was inoculated into two tubes of
nutrient broth. The test tubes were incubated for 24 hours at 37℃ and 55℃,
respectively.

C) Enumeration of number if spores in dried food sample

1. 1 g of samples were weighed and added with 9 ml autoclaved distilled water. It is
vigorously shaken for two minutes, and allowed to settle for two minutes. This
1
preparation produced a sample solution with a dilution factor 10 .
2. The supernatants were transferred into a sterile universal bottle.
3. The bottles were immersed in a water bath at 80℃ for 30 minutes by killing the
vegetative cells and heat-shocking the spores.
4. The bottles from the water bath were removed and mixed with the supernatant
homogeneously.
5. Three sterile tubes were added with 9 ml autoclaved distilled water. 1 ml of
sample solution was inserted into the first tube and mixed homogeneously. The
2
tubes were labeled as T2 with dilution factor 10 . The same procedure was
repeated for the next two test tubes and were labeled as T3 and T4 with dilution
3 4
factor 10 and 10 respectively.
3
6. 100 µ𝑙 of the homogenized mixture (from the tubes with dilution factor 10 and
4
10 ) were inoculated in tryptic soy agar plates and were spread evenly using an L
shaped spreader. Each dilution was plated in duplicates.
7. The agar plates were inverted and incubated at 37℃ for 48 hours at aerobic and
anaerobic conditions.
RESULT

A) Microscopic examination.

Gram-staining illustration Spore staining illustration

B) Characterization of bacteria.

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

- Purple coloured - Turbid - Not turbid

solution - Cloudy solution
- Formation of
precipitation at the
bottom
 C) Enumeration of number of spores in dried food samples.

Anaerobic condition

Dilution Number of colonies

10
3 475

10
4 52

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

3 4
= (475 × 10 ) + (52 × 10 )/0.5
=199000/2
=995000

Aerobic condition

Dilution Number of colonies

10
3 40

10
4 0

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

3 4
= (40 × 10 ) + (0 × 10 )/0.5
= 80000/2
= 40000
DISCUSSION

In this experiment, four different types of procedures were used to finish the experiment. The
first procedure involved morphological analysis and microscopic examination of the supplied
culture of Bacillus Subtilis. Gram staining and spore staining methods were used to complete the
above procedure. The second method involved monitoring the growth of the Bacillus Subtilis
culture at mesophilic and thermophilic temperatures. For this method, the bacteria were
inoculated into two bottles of nutrient broth for 24 hours at the specified temperature. The third
experimental procedure involved observing the development of Bacillus Subtilis culture on a
litmus milk medium. For this procedure, the bacteria were inoculated into a bottle of litmus milk
and then incubated at 37°C for 24 hours. In addition, the fourth procedure in the experiment was
the enumeration of the number of spores in dried food samples. The dried food that was selected
to complete this method was chili powder. At the end of the experiment, a result data sheet with
pictures of the end result and observations was completed.

For the microscopic examination, a gram staining method was applied to observe
Bacillus Subtilis under the microscope. The results showed that Bacillus Subtilis was purple in
colour, which, according to theory, implies a gram-positive bacteria. This is because
gram-positive bacteria have a thick peptidoglycan layer, whereas gram-negative bacteria have a
thin one. Because of this, the thick peptidoglycan layer forms a greater amount of crystal violet
stain upon the addition of iodine than the thin peptidoglycan layer. Hence, the result yielded the
same outcome as what was anticipated in theory; a purple stain appeared on the Bacillus Subtilis,
indicating that the bacteria observed under the microscope was a gram-positive bacteria.

Next, for the second microscopic examination of Bacillus Subtilis, a spore staining
procedure was performed to observe the appearance of spores under the microscope. The spore
staining method is used to distinguish bacterial spores from other vegetative cells and to
determine the distinction between spore-forming and non-spore-forming bacteria. According to
theory, the malachite green stain will stain the spores and when the decolourizer is introduced,
the vegetative cell will lose its main stain. The safranin’s pinkish-red hue will be absorbed by the
vegetative cell. However, in this experiment, only the vegetative cell of Bacillus Subtilis, which
was pinkish-red in colour was seen and there was no signs of green coloured stain for spores.
This could be as a result of the spores developing into vegetative cells or the culture being a
non-spore forming culture. It could also be due to the culture being contaminated or random
errors done by students when the experiment was conducted. Since Bacillus Subtilis is a
spore-forming bacteria, the results obtained for spore staining of Bacillus Subtilis do not match
the expected results because they are meant to show green staining of the spores.

For the next method which was the characterization of bacteria, Bacillus Subtilis growth
at thermophilic and mesophilic temperatures was also observed. Thermophiles are organisms that
thrive at high temperatures, while mesophiles thrive at moderate temperatures. The turbidity
finding in the nutrient broth was observed after it was incubated at different temperatures (37℃
and 55℃) after 24 hours. Compared to the nutrient broth that was incubated at 55℃, the nutrient
broth that was incubated at 37℃ was more turbid. The nutrient broth that was incubated at 37℃
had a turbid broth compared to the one incubated at 55℃ since the ideal temperature for Bacillus
Subtilis to grow is between 25℃ and 35℃. The optimal temperature reflects the optimal enzyme
activity for the cell, and the growth of living things depends on the activity of enzymes. All
proteins in cells, including enzymes, start to denature at a high temperature, which causes an
overall decline in enzyme performance. As a result, the nutritional broths incubated at 37℃ have
a more cloudy broth than those incubated at 55℃ because the higher temperature denatures the
cell's protein and enzymes, causing a decline in enzyme activity and cell inactivation. As
explained, the outcome of the observations of the characterization of Bacillus subtilis growth at
mesophilic and thermophilic temperatures is consistent with the anticipated outcome,
corresponding to the fact that Bacillus Subtilis is a mesophile.

For the next one which is the observation of the characteristics of the Bacillus Subtilis
culture on the litmus milk medium was the third process. Bacillus subtilis produces an alkaline
pH of the medium, a medium that is purple, and some precipitation development at the bottom of
the bottle, referring to the observed result, which is positive. Bacillus Subtilis was unable to
ferment the lactose in the milk, leaving a small quantity of lactic acid in the medium, which
allowed for the formation of the purple medium. Because Bacillus subtilis produces proteolytic
enzymes and has significant proteolytic activity, the soft curds in Bacillus subtilis imply the
digestive process. Certain species produced by bacteria that make proteolytic enzymes hydrolyze
the milk protein (casein), which leads to the production of clots. This explains the formation of
the precipitate at the bottom of the bottle. Given that Bacillus Subtilis does not digest lactose and
has an alkaline media, the result of its reaction with litmus milk medium is in line with what was
anticipated.

For the last procedure which is the enumeration of the number of spores in dried food
samples. In this experiment, the dried food that was chosen and used is chili powder. For the
3
plates incubated in an aerobic condition at 37℃ for 48 hours, the dilution with a factor of 10
4
the number of colonies formed was 475. Meanwhile, for the dilution factor 10 , the number of
colonies formed on the agar plates was 52. The results obtained for both of the agar plates is
positive as there was growth of bacteria colonies after 48 hours of incubation. The CFU/g for the
aerobic condition is 995000. The result obtained in this experiment matched up as what was
expected, which was there were colonies formed and growth on the agar plates for enumeration
of the number of spores in dried food samples in an aerobic condition. Besides that, the agar
3
plate with dilution factor 10 had the number of colonies of 40. This shows that the bacteria that
was present in the chili powder can grow in anaerobic conditions. However, the plate with
4
dilution factor 10 had no growth of bacteria. This might be due to the fact that the number of
bacteria decreases as the dilution factor increases, resulting in the absence of growth. Overall, the
result of the enumeration of the number of spores in dried food samples complies with the
expected result.
CONCLUSION

In a nutshell, both of the objectives of this experiment were accomplished. Techniques to isolate
the spore-forming bacteria from food products which are learned and used.Then, enumeration of
the spore forming- bacteria from food products samples were done. Also we managed to observe
the morphology and characteristics of the spore-forming bacteria Bacillus subtilis. For example,
how Bacillus subtilis react and grow in different environments like in litmus milk and nutrient
broth with different temperatures. Almost all the results obtained in this experiment follow the
expected results except for spore staining. Some errors and mistakes happen, altering the result a
bit.

QUESTION

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

The first step is to shake or swirl the tube gently. This will ensure that your cells are
distributed evenly throughout the tube. Cells should be passaged, or subcultured, when
they cover the plate or when the cell density exceeds the medium's capacity. This
maintains cell density at an optimal level for continued growth and stimulates further
proliferation. Cell death can occur at high confluency when nutrients in the media run out
or cells compete for space on the culture dish or flask surface. As a result, if cells are
harvested and cryopreserved at a high or critical confluence, many, if not all, of the cells
may die when thawed.

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 result of the experiment if the food sample was not heated before being
inoculated on the agar.

Before sterilization, the agar must be heated and boiled to dissolve completely and evenly
before being dispensed into tubes or conical vials. Otherwise, agar distribution may be
uneven and coagulation may vary after dispensing, heating and boiling before
sterilization of agar medium also helps to speed up heat penetration in the autoclaving
process, improve sterilization effect, and reduce the chance of sterilization failure.
Different bacteria from the air breed on top of the agar with the bacteria we want to
inoculate. This makes determining which bacteria have bred on top of the agar difficult.

4) Differentiate facultative anaerobe and obligate anaerobe.

In the presence of oxygen, obligate anaerobes cannot grow. They rely on fermentation
and anaerobic respiration with an electron acceptor other than oxygen as the final
electron acceptor. Facultative anaerobes grow better in the presence of oxygen, but they
can also grow in the absence of it. Obligate aerobes use oxygen as a terminal electron
acceptor and rely on aerobic respiration. They cannot grow in the absence of oxygen.
Obligate interactions are those in which one or both partners are required to participate in
order to survive. Facultative interactions are those in which the partners may but are not
required to participate.
REFERENCE

1. N, S. (2021, January 7). Isolation of bacteria. Biology Reader.

https://biologyreader.com/isolation-of-bacteria.html#
2. What types of bacteria produce endospores? (n.d.). Sciencing.
https://sciencing.com/what-types-of-bacteria-produce-endospores-13428157.html
3. Aryal, S. (2022, August 10). Litmus milk test - Principle, procedure, uses and interpretation.
Microbiology Info.com. https://microbiologyinfo.com/litmus-milk-test/#
4. Isolation, identification and characterization of novel bacillus subtilis. (n.d.). PubMed Central
(PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880821/#
5. Definition of ENUMERATION. (2023, April 10). Enumeration Definition & Meaning -
Merriam-Webster. https://www.merriam-webster.com/dictionary/enumeration