LABORATORY EXERCISE 14

MICROBIAL ANALYSIS OF FOOD

December 6, 2017
INTRODUCTION:

The group of microorganisms that use organic carbon sources to grow and can be
found in all types of water are called heterotrophs. The heterotrophic plate count (HPC) or
formerly known as the standard plate count is a method used to measure the colony
formation on a media of heterotrophic bacteria. A high HPC count indicates the presence of
high amounts of bacteria. However, HPC technique does not indicate what specific
heterotrophic bacteria are present; instead, it indicates the presence of microorganisms that
can be cultured. Several factors cam affect what bacterial genera and the level of their
presence that can be recovered by this method. These factors are the following: type of
culture medium, incubation time and temperature, and the source of the sample used (Allen,
et. al., 2004).

A serial dilution refers to any dilution where the concentration decreases by the same
quantity in each successive step. (Kortla, T., n.d.). Since the dilution-fold is the same in each
step, the dilutions are a geometric series, meaning they have a constant ratio between any
adjacent dilutions. In the exercise, a wide series of dilutions were plated because the exact
number of bacteria was unknown. Moreover, greater accuracy can be achieved by plating
duplicates or triplicates of each dilution. The objective of this method is to estimate the
concentration of an unknown sample by counting the number of colonies cultured from serial
dilutions of the sample. The concentration of the sample is expressed by the number of
colonies, organisms, bacteria or viruses that had grown on the medium. (Ben-David, A. &
Davidson, C., 2014)

In the exercise, the food sample was subjected to microbiological analyses using
several culture media in order to detect the presence of certain bacteria. Microbiological
analysis is the use of biological, biochemical molecular or chemical methods for the
detection, identification, or enumeration of the microorganisms present in a material. This is
often applied to disease causing and spoilage microorganisms. One of the most vital parts of
any food manufacturing control strategy is the end – product testing. In products in which
microorganisms can grow, microbiological analysis is an important tool for the confirmation
of the effectiveness of the manufacturing control mechanisms. There are three common
types of microbiological analysis and these are quantitative, qualitative, and semi
quantitative. Quantitative tests refer to those tests in which the results were given in the form
of calculations or counting. Some examples of these tests are the Microbial Limit Test and
colony counting in terms of colony forming units (CFUs). In a semi quantitative test, results
were given either in form of less than or greater than a specified limits. An example of this
type of microbiological analysis is the Bacterial Endotoxin Testing (BET). A qualitative test is
a type of microbiological analysis in which results were only given in the form of either pass
or fail or present or absent. These types of test results are not given in form of counting or
calculations. An example of this type is the sterility test. In the exercise, the students
performed both the quantitative test and the qualitative test (Dhiman, S., 2015).

The exercise was performed to let the students perform a microbial analysis of food.
The food analysis should be done through heterotrophic plate count. This was to let the
students quantify the organisms present in different food samples. After performing HPC, the
students were also expected to be able to test the food samples for the presence of Gram
negative bacterial contaminants using Salmonella – Shigella agar plates, Eosin – Methylene
Blue agar plates, MacConkey agar plates, and Mannitol Salt agar plates.

MATERIALS:

The materials used during the exercise were food samples, Salmonella-Shigella agar
plates, NA plates, Eosin-Methylene Blue agar plates, Mannitol Salt Agar plates, MacConkey
Agar plates, sterile distilled water blanks, hockey sticks, alcohol lamps, micropipette, rubber
aspirator, wire loops, MRVP broth, SIM agar butt, citrate slants.

Salmonella-Shigella agar plates are selective and differential media for the isolation,
cultivation, and differentiation of Salmonella spp. and some strains of Shigella spp. The
nutrient agar plate is a general purpose medium that supports the growth of a wide-range of
non-fastidious organisms. EMB plates are both selective and differential media used to
identify coliforms specifically E. coli. The Mannitol Salt agar plate and the MacConkey agar
plate are used to identify Staphylococcus aureus and other enterics, respectively. Sterile
distilled water blanks is a type of water that has had many of its impurities removed through
distillation. Hockey sticks are used to spread samples on a culture plate. The alcohol lamp is
used to produce an open flame. The micropipette is used to measure and deliver accurate
volumes of liquid by using pipette tips. A rubber aspirator is a device used for removing
liquids or gases by suction. Wire loops are used to retrieve an inoculum from a culture of
microorganisms. MRVP broth is used in the performance of the Voges-Proskauer and
Methyl Red tests as an aid in the identification of enteric Gram negative bacilli. The SIM agar
butt is a medium used for detection of H2S produced by microorganisms. Lastly, citrate
slants are media used to determine whether the microbe can use the compound citrate for
carbon and energy.
PROTOCOL:

Before proceeding to the proper exercise, the laboratory area was disinfected first
using Lysol and all the necessary materials and reagents requested beforehand were
prepared. The first part of the exercise was the sample preparation. Ten grams of the food
sample, the Betamax, bought from “banwa” was placed on a blender. Then, 90ml of distilled
water was disposed on the blender and the mixture was blended until the food sample was
thoroughly shredded and mixed with the water. The resultant mixture was considered as the
101 dilution.

The second part of the exercise was the heterotrophic plate count. The dilution
blanks which were contained in sterile tubes were labelled as follows: 10-1, 10-2, 10-3, 10-4,
10-5, 10-6, and 10-7. Three nutrient agar (NA) plates were also labelled as follows: 10-6, 10-7,
and 10-8. Using a sterile pipette, 1mL of the food sample was transferred into the tube
labelled 10-1 and was mixed thoroughly. A new sterile pipette was obtained and this was
used to transfer and mix 1 mL of the 10-1 tube into the 10-2 tube. From the 10-2 tube, 1mL of
the solution was transferred and was mixed into the 10-3 tube by using a new sterile pipette.
This process of serial dilution was repeated until tube labelled 10-7. A new sterile pipette was
used to transfer 0.1mL of the 10-5 tube to the NA plate labelled 10-6. Then, liquid was spread
thoroughly and evenly over the surface of the agar plate using a sterile spreader. It was
made sure that the spreader did not dig into the agar. From the 10-6 tube, 0.1mL of the
solution was transferred into the NA plate labelled 10-7 using a new sterile pipette. After the
liquid was transferred, it was spread uniformly and carefully over the surface of the plate
using a sterile spreader. For the third plate which was labelled 10-8, 0.1mL of the 10-7 tube
was transferred into it using a new sterile pipette. The liquid was then evenly distributed all
over the surface of the agar plate by using a sterile spreader. After the liquid had absorbed
into the plates, they were tape closed on both tubes and were labelled. All the three petri
plates were then placed inside the incubator having a temperature of 35ºC for 24 hours.
When the incubation period ended, all the petri plates containing between 25 to 250 colonies
were selected. The plates that had more than 250 colonies cannot be counted and they were
designated as too numerous to count (TNTC) while those that had less than 25 colonies
were designated as too few to count (TFTC). The colonies in the plates were counted and
depending on the availability, a colony counter should have been used. The number of
bacteria was calculated in terms of of sample by dividing the number of colonies by the

dilution factor. It was noted that the number of colonies per mL reported should have
reflected the precision of the method therefore; answers with more than two significant
figures were not included. The results obtained were recorded.
 The last part of the exercise was microbiological analysis. In this part, four different
plates were used: EMB, SSA, MCA, and the MSA. Using a new sterile pipette, 0.1 ml of the
10-5 tube was transferred to the SSA plate. The liquid was then spread thoroughly and
evenly over the surface of the surface of the plate using a hockey stick. This process was
repeated for the other three culture media plates. All four plates were incubated at 35ºC for
24 hours. Bacterial growth was observed and recorded for each plate. When the incubation
period ended, a positive result in the SSA was subjected to IMVic testing. After performing
the exercise, the laboratory area was disinfected again with Lysol and all glasswares were
washed returned in the equipment room.

RESULTS AND DISCUSSION:

The heterotrophic plate count is a method that measures colony formation on culture
media of heterotrophic bacteria. This can be done in three ways namely: pour plate method,
spread plate method and the membrane filtration method. In the exercise, the spread plate
method was used.

Betamax, a street food made from coagulated chicken

blood, served as the food sample in the exercise. Ten grams
of which were blended with 90 ml water and underwent a
serial dilution. Once the serial dilution was done, 0.1mL from
tubes 10-5, 10-6, and 10-7 were transferred to plates labelled
10-6, 10-7, and 10-8, respectively. The liquid was evenly
Figure 1: Betamax (Food Sample)
distributed all over the surface of the plate using a sterile
spreader. When the liquid was absorbed by the medium, the plates were incubated at 35ºC
for 24 hours.

For the exercise, only 0.1mL was used in the surface-plating because larger volumes
might not be absorbed by the agar. Plating 0.1mL of a given solution is mathematically
similar to plating a 1mL of a further 1:10 dilution. Because of this reason, the final plate
dilution was given with respect to the size of the inoculum incorporated with the dilution
factor. (Wright, A. & Harding, E., 2010).

Shown on the table below are the results for the heterotrophic plate count method.
The first plate having a dilution factor of 106 and a number of colonies equals to 39 had an
approximate number of bacteria of 3.9x107. The plate which had a dilution factor of 108 and
a 75 counted colonies had approximately 7.5x109 number of bacteria. The 107 plate
however, which had 16 counted colonies had a too few to count colony forming units.
 Table 1. Colony Forming Units (CFUs) After Incubation
Plated dilution Dilution factor No. of colonies CFU
-6
10 106 39
-7
10 107 16 Too Few To Count
-8
10 108 75

The CFUs were obtained by multiplying the dilution factor with the number of counted
colonies. Moreover, the CFUs of the plated dilution having less than 25 colonies and more
than 250 colonies are considered as too few to count and too numerous to count,
respectively. Shown below is the solution in finding the CFUs of the
three plated dilutions.

Plate 1 has a plated dilution of 10-6 therefore its dilution factor

is 106. The number of colonies counted is 39.

Figure 2: 10-6 dilution plate

Plate 2 has a plated dilution of 10-7 thus it has a dilution factor

of 107. However there were only 16 colonies counted resulting to a
colony forming units of too few to count.

Figure 3: 10-7 dilution plate

The third plate has a 10-8 plated dilution and a dilution

factor of 108. The number of colonies counted in this plate is 75.

Figure 4: 10-8 dilution plate

 The TFTC CFU for the second plate (10-7 plate) may be due to some errors
committed while doing the exercise. One of the most common methodological errors that
occur when performing the heterotrophic count method is the insufficient mixing of the
sample with the agar. This error may cause the colonies to clump together thus making the
plate counts inaccurate. Another error that may have occurred was that the spreader used to
evenly distribute the liquid in the agar surface was still hot, killing many of the bacterial cells
in the sample (Sanders, E., 2012).

Moreover, the kind of bacteria in the sample will influence the size and number of
colonies that can develop on a plate. They also noted in there study that the type of medium
used in cultivating the bacteria has an effect to its growth wherein it may favour or inhibit the
growth of the bacteria (Breed, R. & Dotterrer, W., 1916).

For the microbiological analysis, four different media were used and these were the
Eosin-Methylene Blue agar, the Mannitol Salt agar, the MacConkey agar and the
Salmonella-Shigella agar.

In an Eosin-Methylene Blue agar plate, 0.1ml of the 10-5

tube was transferred and spread evenly. This was performed
to determine whether or not Escherichia coli is present in the
food sample. The test resulted negative.

Eosin-methylene blue agar contained the dyes eosin

and methylene blue which generated a sharp distinction
between lactose- and nonlactose-fermenting organisms. When
Figure 5: EMB - test for E. coli E. coli was cultured to EMB, it will produce colonies with
metallic green sheen pigmentations. This metallic appearance of E. coli colonies resulted
from the formation of a dye complex under acidic conditions. Further studies proved the
presence of an amide structure in the dye complex and that these structures along with the
pH of the medium are the key factors in the formation of bonds between the eosin and the
methylene blue to produce the complex (Horvath and
Ropp, 1974).

Figure 6: MAC - test for other enterics

 To detect the presence of other enterics in the food sample, 0.1 ml of the 10-5 tube
was transferred in the MacConkey Agar. The result obtained in this test was negative.

The MAC is used to isolate Gram negative enteric bacteria and to differentiate
lactose fermenting from lactose non-fermenting Gram negative bacteria. Lactose fermenting
strains are indicated by red or pink colonies and may be surrounded by a zone of acid
precipitated bile. This color is due to the production of acid from lactose, the absorption of
the neutral red and a succeeding change in the color of the dye when the pH of the medium
drops below 6.8. On the other hand, lactose non-fermenting strains are colorless,
transparent and do not modify the medium’s appearance. (Aryal, S. 2015)

The Mannitol Salt Agar is used to detect the

presence of Staphylococcus aureus in a sample. The test
was performed by transferring 0.1ml of the 10-5 tube into
the plate. It was observed that there was a single yellow
colony that grew on the medium yielding a positive result.

The MSA contains 7.5% sodium chloride and

mannitol (sugar). The incorporation of sodium chloride
Figure 7: MSA - test for S. aureus
helped to select bacteria that can tolerate high salt
concentrations like S. aureus. This concentration inhibits the growth of other Gram positive
and Gram negative bacteria. Moreover pathogenic staphylococci like S. aureus are positive
in coagulase test therefore it can ferment mannitol. Whenever sugars like mannitol is
fermented, acid is produced, changing the pH of the medium to acidic. Since MSA contains
phenol red as a pH indicator, the medium would turn yellow at pH levels lower than 6.9
(Acharya, T., 2013).

To test for the presence of Salmonella and Shigella in

the food sample, 0.1 ml of the 10-5 tube was transferred to an
SSA plate. After the incubation period, it was observed that
the sample was positive to both Salmonella (black colonies)
and Shigella (clear colonies).

Figure 8: SSA - test for Salmonella-Shigella

 The presence of bile salts mixture and dyes in MSA inhibits the growth of Gram
positive bacteria. Enteric organisms are differentiated by the integration of lactose in the
medium. Those that can ferment lactose produce acid, in the presence of a neutral red
indicator, resulting to the formation of red colonies. Conversely, lactose non-fermenters like
the Salmonella and Shigella uses peptone as a carbon source and form colorless colonies.
Moreover, the medium also contains ferric acid and sodium thiosulfate, which is a reducible
sulfur source. Salmonella species have enzymes that can reduce thiosulfate and produce
hydrogen sulphide, which reacts with the ferric acid in the medium turning the colonies black.
However, Shigella species lacks these enzymes and that is the reason why they remain
colorless and transparent. (Acharya, T., 2016)

Since the food sample yielded positive results to the SSA, it was subjected to IMVic
test for the determination of its biochemical activities. The table below shows the result for
the IMViC test.

Table 2. IMViC Test Results for Salmonella and Shigella

Citrate Indole Methyl Red Voges-Proskauer
Salmonella + - + -
Shigella - - + -
Legend: (+) – positive result (-) – negative result

For the citrate test, Salmonella yielded a positive result while Shigella showed
negative reaction. A positive reaction is indicated by the color of the medium which changed
from green to blue. Citrate catabolism produces products such as alkaline carbonates and
bicarbonates. These by-products would raise the pH of the medium to above 7.6 which
caused the bromothymol blue to shift its color from green to blue. On the other hand, no
trace of any growth was visible on a negative reaction. The color of the medium did not
change and remained green just like an uninoculated agar slant (Acharya, T., 2013).

Both microorganisms are negative in indole production. The indole test is used to
determine the ability of an organism to split the amino acid tryptophan to produce the
compound indole which would react with the 4 (p)-dimethylamino benzaldehyde of the
Ehrlich’s reagent to produce a red colored compound. However, both Salmonella and
Shigella do not have the capability to hydrolyse tryptophan that is why no change in color
was observed after addition of the Ehrlich’s reagent (Acharya, T., 2012)

Salmonella and Shigella are both positive in the methyl red test. This means that
both microorganisms can perform mixed acids fermentation when supplied with glucose.
Since the pH at which methyl red detects acid is lower compared to other indicators, the test
organism should produce large quantities of acid from the carbohydrate substrate used.
These large amounts of acid resulted to a decrease in the medium’s pH ( at or below 4.4)
resulting to a change in the medium’s color to red due to the fermentation of glucose
(Acharya, T., 2014).

Both the test organisms showed a negative reaction in the Voges-Proskauer (VP)
test. Upon treatment with potassium hydroxide, a culture medium would produce a red color.
The active product in the medium which was formed by bacterial metabolism is the acetyl
methyl carbinol. The presence of diacetyl is indicated by the development of a red color. This
means that diacetyl is absent in both microorganisms since no change in color was observed
after the addition of the reagents (Acharya, T., 2015).

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