TITLE

Isolation of Spore-Forming Bacteria from Dried Food

OBJECTIVE
Become familiar with isolation and identification techniques of spore-forming
bacteria for dried food sample.

INTRODUCTION

Bacterial spores are much more resistant than their vegetative counterparts. The
most dangerous spore-former is Clostridium botulinum which produces a potent
neurotoxin that can prove fatal. The most common food poisoning from a spore-
former is caused by C. perfringens. Other food poisoning spore-formers include
Bacillus cereus, B. subtilis and B. licheniformis. There are a number of non-
pathogenic spore-formers including butyric and thermophilic anaerobes that cause
significant economic losses to food producers. Clostridium and Bacillus are Gram-
positive spore forming rods that cause bacterial food poisoning. Clostridium species
are strictly aerobic to aerotolerant bacteria found in soil as well as in normal intestinal
flora of man and animals. The genera contain many species, some of which cause
disease in humans and some are important in thermal canning of food. Bacillus
species are aerobic or facultatively anaerobic bacteria; in some species cultures may
turn Gram-negative with age. These organisms can survive in low acid foods and may
cause spoilage when heat processing fails to meet accepted standards. Control of
spore formers requires an understanding of both the resistance and outgrowth
characteristics of the spores. The main food poisoning spore-formers are Clostridium
botulinum, C. perfringens and Bacillus cereus. Occasionally B. subtilis and B.
licheniformis have been implicated in food poisoning incidents. Non-food poisoning
spore-formers can also produce spoilage in food products resulting in commercial
loss, which can be substantial. Spoilage should be carefully investigated because it
may be an indication that there is a fault in the processing or that hygiene standards
are insufficient. Today's spoilage outbreak may be tomorrow's food poisoning
incident. Because spore-formers are inherently more resistant than vegetative cells,
methods of control need to be chosen carefully. Information on control of spore-
formers by disinfectants is best obtained from the manufacturers of these chemicals.
Formulation of disinfectants is constantly evolving to meet the demands of the food
industry and to meet international disinfectant tests. The most widely used
disinfectant is probably chlorine, but it is only slowly sporicidal and is readily
inactivated by organic matter, although there are organic chlorine release agents
which are more effective in the presence of soiling.

UV light is finding increased use in the food industry for the destruction of
micro-organisms on surfaces and in water and air. UV lamp technology has improved
considerably in the last 8-10 years and it is now possible to obtain very powerful
lamps, which can produce significant reduction of spore-formers. Multi-lamp arrays
are also being developed to fit around conveyor systems so that the surfaces of
product and packaging can be 'sterilised' before transfer to high care areas. Besides
studying the morphology of spore-forming bacteria, a spore count fried sample were
also performed. Before plating, we heated the sample to 80° C for 30 minutes. The
heating steps performed for two reasons which are all the vegetative cells will be
destroyed to assure that will only enumerate spores and “heat shock” enhances the
outgrowth of most spores. The heating step is thought to active enzyme that starts the
breakdown of spore “cortex” or “outgrowth”.

MATERIALS
1. Culture provided
a) Bacillus subtilis
b) Bacillus cereus
c) Clsotridium sporogens
2. Media used
a) Litmus milk medium
b) Cooked Meat medium (for Clostridium growth)
c) Nutrient broth (for Bacillus growth)
d) Tryptic Soy Agar (Is a non selective and nondifferential media containing
tryptone (digested casein) soytone (digested soybean meal) as a protein source,
sodium chloride and agar. Glucose (carbon source) is added to the level of 1%
(10g/Liter).
3. Gram and spore staining reagents
4. Samples: black pepper, sugar, flour, turmeric powder and vinegar.
PROCEDURES

i. Morphological studies
The smears of the organisms were prepared and were stained with gram and spore
stains. The shape and arrangements of spore and vegetative cells were noted.
a) Gram staining

1. A slide was placed on a slide holder or a rack. The entire slide was flooded (cover
completely) with crystal violet. The crystal violet was place for about 60 seconds.
Then, the slide was washed for 5 seconds with water after time has elapsed. The
specimen appeared blue-violet when observed with naked eye.

2. Next, the slide was flooded with the iodine solution and the slide was placed about
a minute as well. The slide was rinsed with water for 5 seconds when the time is
elapsed and immediately proceed to step three. The specimen appeared to be blue-
violet at this point.

3. The addition of the decolorizer which is ethanol involved in this step. By using too
much decolorizer could result in a false Gram (-) result which make this step 3
somewhat objective. The ethanol was added a drop wise until the blue-violet color no
longer emitted from the specimen. After that, the slide was rinsed with water for 5
seconds as in previous steps.

4. Applying the counterstain and safranin were involved in the final step. The slide
was flooded with the dye as did in steps 1 and 2. The slide was left for about a minute
to allowed the bacteria to incorporate the safranin. Gram positive cells was
incorporate little or no counterstain and was remained blue-violet in appearance.
the Gram positives. Then, the slide was rinsed again with water for 5 seconds to
remove t he excess of dye.

5. After have completed steps 1 through 4, the slide was not blot gently with bibulous
paper or allowed it to dry before viewing it under the microscope.

b) Spore staining
1. The smears from a colony of the spore-formers Bacillus spp were prepared
from the culture provided.
 2. A piece of blotting paper was cut the size of the smears, but small enough to
avoid from hang over the edges of the slide.
3. The slide was placed over the boiling streamer and the paper was lay on the top
of the smears.
4. The paper was flooded with the malachite green stain which is carcinogen for 3
minutes. The paper was kept wet with stain whenever it begins to dry.
5. The slide was removed after 3 minutes and the paper was lift off with loop and
discard.
6. Then, the slide was washed gently with water.
7. After that, the slide was flooded with the counter-stain safranin to stain the
vegetative cells.
8. The safranin was washed off after one minute and the blotted slide was dry and
then examined under the microscope.

ii. Growth at mesophiles and thermophiles temperatures:

a) Bacillus spp
i. The culture has been incubated at 37°C and 45°C for 2 to 3 days in the
nutrient broth.
ii. The results has been observed and recorded.
b) Clostridium spp
i. The culture has been incubated at 37°C and 45°C for 2 to 3 days in the
cooked meat medium.
ii. The results has been observed and recorded.

iii. Action on Litmus milk

a) The cultures of Bacillus spp and Clostridium spp were incubated in the
litmus milk for 2 to 3 days.
b) Then, the results such as curd formation, acid, gas, whey and etc were
observed and recorded.
iv. Enumeration of number of spores
a) 1 g of the dried food sample was weigh and it was transferred into 9 mL water
blank. Then, it was shaken vigorously for 2 minutes and the settling was allowed
for 2 minutes.
b) The supernatant was transferred as much as possible to a sterile McCartney
bottle.
c) Then, the bottle was immersed in a water bath at 80°C for 30 minutes. This
would heat shock spores and kill vegetative bacteria.
d) The bottle was removed from the water bath. After that, it was mixed well and
the food particles were waited to settle out of the solution.
e) 1 mL was removed and cooled and 3 serial dilutions was made. Then, 1 mL
portions with Tryptic Soy Agar were plated out.
f) Each dilution was plated twice. Two of these plates were incubated aerobically
(A) at 37°C for 48 hours. The other two plates were placed in an anaerobe jar and
were incubated anaerobically (AN) at 37°C for 48 hours.
g) The CFU/g at each growth condition were calculated after 48 hours.
1) A: 37°C, aerobically
2) AN: 37°C, anaerobically
h) The colony morphology were recorded of any surface growth.
RESULTS
i. Morphological studies
a) Gram staining

Sample Microscopy morphology Illustration

Bacillus subtilis - Rod shaped bacterium

- Streptobacillus

Bacillus cereus - Rod shaped bacterium

- Single arrangement

Clostridium sporogens - Rod shaped bacterium

- Single arrangemnent
b) Spore staining

Sample Microscopy morphology Illustration

Bacillus subtilis -present of endospores

-present of vegetative cells

Bacillus cereus -present of endospores

-present of vegetative cells

Clostridium sporogens -present of endospores

-present of vegetative cells

ii) Growth characteristics

 Table 5.1: Growth of spore forming bacteria in media

Bacteria Nutrient broth (37°C) Nutrient broth (45°C) Litmus milk

Bacillus subtilis Turn cloudy due to No change Peptonization with
optimum temperature alkaline reaction and
turned turbid with
slightly white clot
Bacillus cereus Turn cloudy due to No change Curd and formed white
optimum temperature clot at the bottom of the
tube

Bacteria Cooked meat medium (37°C) Cooked meat medium (45°C) Litmus milk
Clostridium Turned darker with foul smell No change Stormy fermentation and
sporogens acid coagulated casein
producing acid clot at the
bottom of the tube

iii) Enumeration of spores from food sample

Food sample: Flour
(a) A: 37°C, aerobically
Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 6 6
10^-2 4 4
10^-3 0 0
CFU/g = 1x10^-4 CFU/g

(b) AN: 37°C, anaerobically

Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 3 3
10^-2 2 2
10^-3 0 0
CFU/g = 5x10^-5 CFU/g

Food sample: Tumeric powder

(c) A: 37°C, aerobically
Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 TMTC TMTC
10^-2 TMTC TMTC
10^-3 30 30
CFU/g = 3x10^-4 CFU/g

(d) AN: 37°C, anaerobically

Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 TMTC TMTC
10^-2 18 18
10^-3 No colony No colony
CFU/g = 1.8x10^-4 CFU/g

Food sample: Black pepper

(e) A: 37°C, aerobically
Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 TMTC TMTC
10^-2 No colony No colony
10^-3 3 3
CFU/g = 3x10^-5 CFU/g
 (f) AN: 37°C, anaerobically
Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 TMTC TMTC
10^-2 No colony No colony
10^-3 No colony No colony
CFU/g = 0 CFU/g

Food sample: Sugar

(g) A: 37°C, aerobically
Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 TMTC TMTC
10^-2 TMTC TMTC
10^-3 7 7
CFU/g = 7x10^-5 CFU/g

(h) AN: 37°C, anaerobically

Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 TMTC TMTC
10^-2 TMTC TMTC
10^-3 TMTC TMTC
CFU/g = TMTC CFU/g

Food sample: Vinegar

(i) A: 37°C, aerobically
Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 4 4
10^-2 1 1
10^-3 0 0
CFU/g = 5x10^-5 CFU/g

(j) AN: 37°C, anaerobically

Dilution as Plated CFU/Plate Average CFU/Plate
10^-1 0 0
10^-2 2 2
10^-3 3 3
CFU/g = 5x10^-5 CFU/g

DISCUSSION

In this experiment, we were able to conduct an experiment in order to become

familiar with isolation and identification techniques of spore-forming bacteria for
dried food sample. The bacteria that were used in this experiment are mainly from two
genera of Gram-positive bacteria which is Clostridium sporogens, Bacillus subtilis
and Bacillus cereus. The methods included in the experiment were gram staining and
spore staining to study their morphology. Gram staining was done for routine bacterial
identification and endospore staining was used to determine endospores. Next, we
used nutrient broth for Bacillus spp and cooked meat medium for Clostridium spp for
culture revival and growth studies. Lastly, we also performed a spore count of dried
food sample.

As usual, before we started the experiment, it is crucial for us to perform aseptic

technique in order to minimize contamination. Firstly, in gram staining we prepared
smears of these three microorganisms. After preparing the smears of the
microorganisms, the smears were air dried and heat fixed. Then, we stain these three
smears using crystal violet dyes. Crystal violet dyes are basic dyes that have
positively charged particle that helps them to bind to negatively charged molecule like
teichoic acid at the cell wall of the bacteria.

This crystal violet dye can dissociates into cv+ and cv- ions. These ion can
penetrate deeps into the cell wall of bacteria and interacts with the negatively
component on the bacterial cell wall. One minute later, the crystal violet was washed
with tap water and then slides are dried. The next step is to add iodine onto each
smears. Iodine was being added as a mordent to form crystal violet-iodine complex,
CVI complex. This complex enables the dyes to not be easily being removed. Next,
we washed the iodine with tap water and dried off the excess water. After that, ethanol
was being added to acts as a decolorizing agent. It interacts with lipid membrane of
positive bacteria. The addition of alcohol dehydrated the layer of peptidoglycan which
in turn would trap the CVI complex. This cause the gram positive bacteria appeared to
be purple color as the CVI complex are being retained. The addition of alcohol is not
be more than 15 seconds as this would break the cell wall of the bacteria, thus
resulting in no stain to be observed. The slides are washed with water and dried off. In
the next step, safranin was used to counterstain all smears. The gram positive bacteria
does not being stained pink when safranin was being introduced because the
peptidoglycan layer already have CVI complex. Then, the slides was washed using
tap water and dried off. The microscopic morphology of these three bacteria were
observed on their shape and arrangement. The shape for these bacteria are rod-shaped.
The arrangement for Bacillus subtilis is streptobacillus, while Bacillus cereus and
Clostridium sporogens are single arrangement.

The standard endospore stain is vigorously staining procedure using Malachite

green as the main stain and safranin as a counterstain. Endospores are a bacterial
structure for survival that do not readily allow stains to get in or attach to the spore.
Heat or steam was used to get Malachite green into the endospores and the vegetative
cells as well. The slide is then washed with water. As Malachite green is a water
soluble stain, it washes out of vegetative cells, but not out of the endospores. Safranin
is then applied, which stains the vegetative cell redish which it is unable to get into
the endospores. A prepared slide of Bacillus subtilis, Bacillus cereus and Clostridium
sporogens were observed under the microscope. These organisms had green dots
located in the center of the cell which indicted that had nutrient depletion in the
Bacillus subtilis had caused it to produce rod-shaped shaped spores in order to
survive. For Clostridium sporogens, the rod shaped and cigar shaped vegetative cells
were observed with spore staining revealed that spores are sub terminal and oval in
shaped.

Next, we conduct an activity to detect the growth of the bacteria at mesophiles

and thermophiles temperatures. For Bacillus spp, the nutrient broth has been used at
37°C and 45°C where by the culture has been incubated for 2 to 3 days and the result
has been recorded. Meanwhile, for Clostridium spp, the cooked meat medium has
been used to incubate the culture for 2 to 3 days also. Sporeforming bacteria that are
present in foods are important because the formation of the spore by the bacterium
allows it to be resistant to heat, freezing, chemicals, and other adverse environments
that our food undergoes during processing and preparation. Although the vegetative
cell is killed by these conditions, the spores can survive and need harsher conditions
to be inactivated. Some of the bacteria that are important belong to the genus Bacillus,
which are aerobic to facultatively anaerobic rod-shaped microbes. These microbes can
either grow under mesophilic temperatures which means at 35ºC or some grow under
thermophilic temperatures at 55ºC. These Bacillus species can cause food spoilage or
some cause food-borne illnesses. So, based on our result, Bacillus subtilis and
Bacillus cereus are grow under mesophilic temperature at 35ºC because the nutrient
broth turns into cloudy while at 45ºC the nutrient broth was not changed. The other
important group of sporeforming bacteria is the Clostridium species. These are
anaerobic bacteria that can grow at temperatures that are both mesophilic and
thermophilic, depending on the species involved. They are of interest in foods because
they also cause food spoilage and some species cause food-borne disease. Based on
the result obtained, the Clostridium sporogens grow at 37ºC which is mesophilic
temperature that caused the cooked meat medium turned darker with foul smell.

Third activity of this experiment was to detect the action of spore-forming

bacteria on litmus milk. Litmus Milk is a differential medium used to determine
different metabolic functions. Litmus Milk is also useful in the maintenance and
propagation of lactic acid bacteria. Litmus Milk is the most useful medium in dairy
industry as it is a reliable indicator of bacterial action on milk. Litmus is a good
indicator of acidity, alkalinity and its oxidation-reduction potential is useful in milk
media with lower toxicity to microorganisms than bromocresol purple. These three
bacteria were incubated in the litmus milk for 2 to 3 days at 37ºC and observed the
changes in the litmus milk. For Bacillus subtilis, the litmus milk caused peptonization
with alkaline reaction and turned turbid with slightly white clot while for Bacillus
cereus, the litmus milk formed a curd and white clot at the bottom of the tube which
produced acid coagulation lactic acid. In Clostridium sporogens bacteria, the litmus
milk formed stormy fermentation and acid coagulated casein producing acid clot at
the bottom of the tube.

Besides, the last activity for the isolation of spore-forming bacteria from dried
food was the enumeration of number of spores. The food samples that have been used
including black pepper, sugar, flour, turmeric powder and vinegar. The need to assess
the accuracy of total counts arose at the outset of an investigation of factors which
may affect the viability of bacterial spores. When spores are plated on a nutrient
medium production of a colony depends on germination of the spore and emergence
of a vegetative cell, survival and multiplication of the vegetative cell. These two
phases may require different optimal conditions. A determination of the total count of
a spore suspension would enable calculation of the percentage of spores giving
colonies on a nutrient medium. When this percentage is low the reason may be
because a proportion of spores are not viable, they may be viable but fail to germinate
in the growth conditions supplied, or germination may be initiated, but the
environment may fail to support outgrowth and multiplication. Microscope counts of
bacteria in chambers of known depth have generally been accepted and used as the
most accurate total count technique available. Each food samples were weighed with
1 g and were transferred into 9 mL water blank. Then, the samples that have been
cooled after being heated in the water bath at 80ºC for 30 minutes, we proceed with
serial dilutions. The sample was removed by using micropipette with 1 mL into the
Tryptic Soy Agar. These plates were then incubated into aerobically and anaerobically
at 37ºC for 48 hours.
 Then, after two days, the result has been recorded by identifying the colony
morphology of any surface growth on each plates to calculate the CFU/g.
Only replicate plates from the same dilution with 30-300 colonies are counted. Plates
with fewer than 30 colonies give statistically unreliable results, while plates with
more than 300 colonies are too crowded to allow all the bacteria to form distinct
colonies. This results has been shown in each food samples. In food sample of flour
and vinegar, the CFU/g obtained was less than 30 colonies in both aerobic and
anaerobic. In tumeric powder, black pepper and sugar, the total CFU/g in both aerobic
and anaerobic were also less than 30 colonies. There were also dilution that the
colonies were too many too count so it is hard to count. Usually, more than one
dilution in a series is plated, just to be sure that results in a countable range will be
obtained.

QUESTIONS

1. If you had a spore-forming bacterium that could grow under both aerobic and
anaerobic conditions, how would classify it, as a facultative or strict
anaerobic? Why?
- Facultative anaerobic. However, to classify as facultative aerobic or facultative
anaerobic, we need to examine the growth rate in both aerobic and anaerobic
environments and determine which environment leads to better growth rates.

2. What is the purpose of shaking the sample before transferring the sample into a
sterile McCartney bottle (step in 4b)?
- To obtain as much supernatant as possible.

3. What are the factors in determining the germination of spores to occur?

- Free endospores do not show any detectable metabolism, but they retain the
potential capacity to germinate and develop into vegetative cells. They are highly
resistant to heat UV light, and ionizing radiations and many toxic chemicals.
Endospores germinate under favourable conditions. Sufficient nutrients, humidity and
suitable temperature are essential for germination. These condition triggers a series of
metabolic reactions. Spores imbibe water and swell up, loosing their refractivity. The
spore coat rupture and new vegetative cell grow out.

4. Would you expect to have different results if we had not heated the food sample
solution before heating?
- No.

CONCLUSION

In conclusion, we were able to performed the tasks that have been given to
familiarize with isolation and identification techniques of spore-forming bacteria for
dried food sample. These activities included gram staining, spore staining, the growth
at mesophiles and thermophiles temperatures, action spore forming bacteria on litmus
milk and enumeration of number of spores. In gram staining, the shape of these
bacteria are rod-shaped while the arrangement are different whereas for Bacillus
subtilis, the arrangement are streptobacillus while Bacillus cereus and Clostridium
sporogens the arrangements are single. Gram staining and spore staining indicated
that the bacteria of Clostridium sporogens is Gram-positive with sub terminal
endospore. Rod shaped and cigar shaped vegetative cells were observed with Gram
staining, and spore staining revealed that spores are sub terminal and oval in shape.
Next, we were able to detect the growth of the bacteria at mesophiles and
thermophiles temperatures. Nutrient broth and cooked meat medium has been used in
this experiment. Microbes can be roughly classified according to the range of
temperature at which they can grow. The growth rates are the highest at the optimum
growth temperature for the organism. The lowest temperature at which the organism
can survive and replicate is its minimum growth temperature. The highest temperature
at which growth can occur is its maximum growth temperature. In this cases, Bacillus
subtilis and Bacillus cereus grow at 37ºC which is optimum temperature. This shows
they grow at mesophilic temperature. Organisms that grow at optimum temperatures
of 50 °C to a maximum of 80 °C are called thermophiles. They do not multiply at
room temperature. Thermophiles are widely distributed in hot springs, geothermal
soils, and manmade environments such as garden compost piles where the microbes
break down kitchen scraps and vegetal material and the results have shown that
Clostridium sporogens grow at this temperature.

After that, the third activity was to identify the action spore forming bacteria on
litmus milk. For Bacillus subtilis, the litmus milk caused peptonization with alkaline
reaction and turned turbid with slightly white clot while for Bacillus cereus, the litmus
milk formed a curd and white clot at the bottom of the tube which produced acid
coagulation lactic acid. In Clostridium sporogens bacteria, the litmus milk formed
stormy fermentation and acid coagulated casein producing acid clot at the bottom of
the tube. Lastly, the enumeration of number of spores. One of the most fundamental
microbiological techniques is plate counting which is used to determine the number of
viable cells in a sample. There are several steps to the technique and all must be
carried out carefully in order to obtain accurate results. Aseptic technique must be
used throughout. Aseptic technique is the term given to a collection of procedures that
aim to avoid contamination of the sample. This involves holding tubes at an angle,
flaming lids of bottles and using sterile pipettes. Only replicate plates from the same
dilution with 30-300 colonies are counted. Plates with fewer than 30 colonies give
statistically unreliable results, while plates with more than 300 colonies are too
crowded to allow all the bacteria to form distinct colonies. This results has been
shown in each food samples. In food sample of flour and vinegar, the CFU/g obtained
was less than 30 colonies in both aerobic and anaerobic. In tumeric powder, black
pepper and sugar, the total CFU/g in both aerobic and anaerobic were also less than
30 colonies. Overall, we able to performed the experiment well and we become more
familiar with the isolation of spore-forming bacteria from dried food.

REFERENCES

http://shodhganga.inflibnet.ac.in/bitstream/10603/68201/8/08_chapter%203.pdf
http://www.himedialabs.com/TD/M609.pdf
http://www.math.unl.edu/~jump/Center1/Labs/Sporeforming%20Bacteria%20in
%20Foods.pdf
 MIC 500

FOOD MICROBIOLOGY

EXPERIMENT 5

Isolation of Spore-forming Bacteria from Dried Food

NAME: ADREANA HAZEEQAH BINTI NORHISHAM

MATRIC NO: 2017405578

GROUP: AS2463C

LECTURER’S NAME: DR. KHALILAH ABD KHALIL

DATE OF SUBMISSION: 17th MAY 2018