Lab project

To what extent bacterial diversity and amount of bacterial cells could be

distinguished between public bus and private car

Sultan Makhmetov, Alibek Ysmaiyl

14.11.2016
Lab instructors name: Syed Hamid Ali
Introduction to Microbiology with Lab-Lab
Fall semester/2016
Section 1

Introduction
The reason for choosing this sphere of question is that microbiological investigations on public transport
gaining considerable attention from researchers, since contaminated handrails or steering wheels can act
as pathogenic which could cause minor to major diseases, and people everyday use public transport and
private cars transferring many types of bacteria from place-to-place increasing the risk of dangerous for
mankind.
This experiment consists of several parts, such as serial dilution plate analysis, isolation of pure cultures,
gram-staining and using specialized media. Serial dilution was used in order to count number of
bacterial cells in public bus and private car, also for isolation of pure cultures. Gram-staining was used
in order to determine morphology and with specialized media to determine types of these bacterial cells.
[1]
Hypothesis: First of all, it is expected that diversity and amount of bacterial cells would be higher in the
public bus than in the private car.
Objectives: The main aim of this experiment is to compare the diversity and amount of bacteria
between public bus and private car. Other aims are to determine number of bacterial cells and
morphology in both type of environment.

Materials and method

Equipment: 5 sterilized tubes with sterilized water, sterilized cotton swab, broth media, 12 NA plates,
inoculation loop and needle, Bunsen burner, crystal violet, distilled water, 95% ethanol, grams iodine,
safranin, clean glass slides, staining tray, light microscope, 2 mannitol agars, 2 phenylethyl alcohol
agars, 2 MacConkey agars.
Procedure:
Part 1: Sample collection
1. Sterilized tubes with sterilized water were labeled with marker: Car 1, Car 2, Bus 1, Bus 2 and
Control.
2. Sterile cotton swab was damped into sterile water and excess water was wringed out. Then with
this moistened cotton swab the samples were obtained from steering wheel of Car 1,2 and from
handrails of Bus 1,2. These contaminated swabs were placed back into appropriate tubes with
sterile water.
3. Sterilized cotton swab was placed into tube Control in order to get control sample.
Part 2: Incubation of collected samples
1. 5 tubes with broth media were labeled with marker: C1, C2, B1, B2 and Control
2. 3 ml from each sample tubes was transferred into appropriately labeled tube with 25 ml broth
medium
3. Then these tubes were incubated for 24 hours at 37oC .
4. Then results were observed. All tubes had bacterial growth except control tube.
Part 3: Serial Dilution Agar plates
1. Tubes C2 and B2 were chosen for serial dilution, since these tubes had more precipitated
bacteria.
2. The bottom of each six Petri dishes with NA agar was labeled as 1A Car, 1B Car, 2A Car, 2B
Car, 3A Car and 3B Car respectively.
3. Seven test tubes with 9 ml of sterile water were labeled with numbers from 2 to 8.

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 4. 1 ml from tube C2 was transferred to tube 2, and then 1 ml from tube 2 was transferred to tube
3. This procedure was done for other tubes, so each tube was diluted.Tube 2 was diluted 10
times, tube 3 100 times and so on.
5. 0.1 ml from tube 5 was transferred to 1A Car agar plate and distributed on the whole surface by
bent glass rod.
6. 1 ml from tube 6 was transferred to 1B Car plate and 0.1ml to 2A Car plate and then they were
distributed by bent glass rod.
7. 1 ml from tube 7 was transferred to 2B Car plate and 0.1 ml to 3A Car plate and also distributed
by bent glass rod.
8. Then 1 ml from tube 8 was transferred to 3B Car plate and distributed by bent glass rod.
9. During each transferring and distribution, aseptic techniques were used in order to avoid
contamination.
10. The bottom of each six Petri dishes with NA agar was labeled as 1A Bus, 1B Bus, 2A Bus, 2B
Bus, 3A Bus and 3B Bus respectively.
11. Then step 3-9 were repeated for Tube B2.
12. After all these procedures, plates were incubated in an inverted position for 24 hours at 37oC.
13. Amount of colonies on the surface of agar plates were counted. Counts were obtained from
bacterial cell dilutions that yield between 30 and 300 colonies.

Part 3: Isolation of pure cultures

1. Tubes with broth media were labeled: culture C1, C2, C3 and culture B1, B2, B3.
2. 3 discrete colonies were randomly taken from 1A Car plate and 1B Bus plate with inoculation
needle.
3. Then each discrete colony was transferred into appropriate labeled tube with broth medium and
incubated for 24 hours at 37oC.

Part 4: Gram Staining

1. Smear was prepared on the clean glass slides of each isolated colony sample (culture C1, C2, C3
and culture B1, B2, B3).
2. All smears were gram stained and observed under light microscope.

Part 5: Specialized media

1. Phenylethyl alcohol agar plates, Mannitol salt agar plates and MacConkey agar plates were
prepared.
2. The bottom of the 1st Phenylethyl alcohol agar plate was divided into three sections: Car1, Car2,
Car3 and the 2nd Phenylethyl alcohol plate was divided into three sections: Bus1, Bus2, Bus3.
3. Culture C1, C2, C3 and culture B1, B2, B3 were inoculated with inoculation loop on the divided
into three sections 1st and 2nd Phenylethyl alcohol plates, respectively.
4. Above step 2-3 were repeated for other types of agar plates.
5. After, all plates were incubated for 24 hours at 37oC.
6. The results were observed.

Results

Picture 1: Incubated samples of environments and control in broth media

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 According to above picture, it can be seen that control is transparent, while Bus 2 and Car 2 are turbid.

Table 1: Observations of inoculated plates of B2 sample

Plate Dilution ml of Final Number of Bacterial Average

factor dilution dilution colonies count per ml count per ml
plated on plate of sample of sample
(ml) (CFU/ml) (CFU/ml)
1A Bus 105 0.1 10-4 162 1.62107 1.57107
1B Bus 105 1 10-5 151 1.51107
6 -5
2A Bus 10 0.1 10 TFTC - -
6 -6
2B Bus 10 1 10 TFTC - -
3A Bus 107 0.1 10-6 TFTC - -
7 -7
3B Bus 10 1 10 TFTC - -
TFTC too few to count;
Bacterial count per ml of sample (CFU/ml) = Number of colonies Dilution factor

Example of calculation (1A Bus):

Bacterial count per ml of sample (CFU/ml) = 162 105 = 1.62107 CFU/ml

1A Bus and 1B Bus plates have almost similar number of colonies 162 and 151, respectively. Plates 2A
Bus, 2B Bus, 3A Bus and 3B Bus have too few amounts of colonies to count. Results of plates (2A Bus,
2B Bus, 3A Bus and 3B Bus) couldnt be used in calculation of Bacterial count per ml of sample (initial
number bacterial cells in the sample). Calculated amount of bacterial cells in sample are 1.6210 7
CFU/ml and 1.51107 CFU/ml; and average count is 1.57107 CFU/ml.

Table 2: Observations of inoculated plates of C2 sample

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Plate Dilution ml of Final Number of Bacterial Average
factor dilution dilution colonies count per ml count per ml
plated on plate of sample of sample
(ml) (CFU/ml) (CFU/ml)
1A Car 105 0.1 10-4 127 1.27107 1.23107
1B Car 105 1 10-5 119 1.19107
6 -5
2A Car 10 0.1 10 TFTC - -
6 -6
2B Car 10 1 10 TFTC - -
3A Car 107 0.1 10-6 TFTC - -
7 -7
3B Car 10 1 10 TFTC - -
TFTC too few to count;
Bacterial count per ml of sample (CFU/ml) = Number of colonies Dilution factor

Example of calculation (1A Car):

Bacterial count per ml of sample (CFU/ml) = 127 105 = 1.27107 CFU/ml

1A Car and 1B Car plates have almost similar number of colonies 162 and 151, respectively. Plates 2A
Car, 2B Car, 3A Car and 3B Car have too few amounts of colonies to count. Results of plates (2A Car,
2B Car, 3A Car and 3B Car) couldnt be used in calculation of Bacterial count per ml of sample (initial
number bacterial cells in the sample). Calculated amount of bacterial cells in sample are 1.2710 7
CFU/ml and 1.19107 CFU/ml; and average count is 1.23107 CFU/ml.

Moreover, the discrete colonies in 1A Bus and 1B Bus were significantly larger than in 1A Car and 1B
Car.

Table 3: Observation of gram staining

Culture Shape Gram test Picture

C1 Spherical Positive-blue

C2 Spherical Positive-blue

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C3 Spherical Positive blue

B1 Spherical Positive-blue

B2 Rod Negative-pink

B3 Rod Negative pink

Results show that C1, C2, C3 and B1 are gram-positive bacteria with spherical shape and B2 and B3 are
gram-negative bacteria with rod shape

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 Table 4: Observation and result of inoculated specialized media

Type of Medium Bacterial Amount Appearance of

medium species of medium
Picture
growth
Selective Phenylethyl C1 large -
alcohol C2 nothing -
agar C3 nothing -

Phenylethyl B1 small -
alcohol
agar

B2 small -

B3 small -

Differential/ Mannitol C1 large yellow

selective salt agar

C2 large yellow

C3 large yellow

B1 large no change
Mannitol B2 small no change
salt agar
B3 small no change

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 Macconkey C1 medium Slightly
agar
yellow

C2 small Slightly
yellow

C3 medium Slightly
yellow
Macconkey B1 medium Pink
agar

B2 medium Pink

B3 medium Pink

1. Phenylethyl alcohol agar C2 and C3 did not grow, C1 was grown in large amounts with white
color. All sections of medium didnt change.

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 2. Phenylethyl alcohol agar B1, B2 and B3 were grown in small amounts with white color. All
sections of medium didnt change.
3. Mannitol agar the growth of all colonies (C1, C2, and C3) was large with white color. All
sections of medium form yellow zone.
4. Mannitol agar the bacterial growth of B1 section was large, while other sections shown small
bacterial growth. All of bacterial cells had white color. There was no change in medium.
5. MacConkey agar the bacterial growth of C2 was small with white color, and C3 medium with
white color and C1 medium with yellow color. There was change in medium in all sections to
yellow color.
6. MacConkey agar the bacterial growth of all colonies (B1, B2, and B3) was medium with white
color. The color of medium of all sections did not change.

Discussion

Part 1: Incubation of collected samples.

According to the results of picture 1, the tube Control was transparent while tubes Car 2 and Bus
2 were turbid. It means that tube with sterile water and sterile cotton swab were not contaminated,
therefore other samples also were not contaminated.

Part 2: Serial Dilution

According to the results, the average count per ml of sample of B2 is 1.57107 CFU/ml and C2 sample
is 1.23107 CFU/ml. It means that number of bacterial cells in B2 sample (Bus) is higher than in C2
(Car). Moreover, the size of colonies in 1A Bus and 1B Bus was much larger than in 1A Car and 1B Car
plates. These results support the part of the first hypothesis that amount of bacterial cells in public bus
would be higher than in private car.

Part 3: Gram Staining

According to the table 3, C1, C2, C3 and B1 are gram-positive bacteria with spherical shape, which
means that these bacterial cells could be staphylococci. Meanwhile B2 and B3 are gram-negative
bacteria with rod shape. Probably, these bacterial cells could be bacillus.

Part 4: Specialized media

According to the table 4, all bacterial cultures (C1, C2 and C3) were grown in large amounts in
Phenylethyl alcohol agar. It means that all of them are gram positive bacteria. Since phenylethyl alcohol
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agar has inhibitory effect only on gram negative bacteria. Moreover, B1 and B2 were grown in small
amounts that show inhibitory effect, except B3 that grown in large amounts. Therefore B1 and B2
bacterial cultures are gram negative and B3 is gram-positive bacteria.
The growth of C1, C2 and C3 on the mannitol agar was large. It means that these bacterial cultures
are halophilic bacteria (salt-loving) and probably they are staphylococci. Moreover, the color of all
sections of medium was changed forming yellow zone. It means that these bacteria are capable of
fermenting, and this provides evidence that these bacteria are mannitol-fermenting staphylococci. Since
these bacteria are staphylococci-fermenters, probably type of these bacteria is S.aureus.
The B1 on the mannitol agar was large, but the color of medium did not change. It means that B1
culture is halophilic and mannitol-nonfermenter staphylococci, therefore it could be S.epidermidis.
While B2 and B3 cultures were grown in small amounts, showing inhibitory effect. Therefore, these
bacterial cultures are not halophilic, and not staphylococci. Moreover, the color of medium of these two
bacterial cultures did not change, meaning that they are not mannitol fermenters.
The growth of C1, C2 and C3 culture on MacConkey agar was medium, meaning that there is low
inhibitory effect, so these bacteria are gram-positive. Moreover, the medium of all bacterial cultures
slightly changed to yellow color. It means that these bacterial cultures are nonfermenters and not
Coliform bacilli. It provides additional evidence that these bacteria are S.aureus.
The growth of B1, B2 and B3 on the MacConkey agar was medium, meaning that there is partially
inhibitory effect or normal growth. The color of medium of all sections was pink. It means that these
bacterial cultures are Coliform bacilli. However, this result has contradiction with Mannitol result. The
mannitol agar shows that B1 is probably S.epidermidis, therefore it cannot be grown in MacConkey agar
and be Coliform bacilli. While B2 and B3 do not have contradictions and therefore results show that
they are coliform bacilli and provide evidence that they are E.coli.
Limitations and improvements: First of all, the experiment was very narrow, since only four
environment samples (2 cars and 2 buses) were collected for the study. Moreover, these samples were
collected from only one hand touch place of bus and car. Therefore, the results of the experiment could
not be considered as reliable for investigation of bacterial diversity and amount of bacterial cells in all
public buses and private cars in Astana. The solution of this problem is providing higher investments
and time for the study. In order to collect more environmental samples from public buses and private
cars.
The second limitation of this experiment was in the part of isolation of pure cultures in which small
amounts of discrete colonies (only three discrete colonies from about one hundred possible discrete
colonies from each environment sample) were taken randomly for next investigations. These
investigations consist from gram-staining and specialized media investigations. Since small amount of
discrete colonies were taken for the study, the results of diversity were low, for example bacterial cells
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from car sample were only S.aureus. This part of experiment could be improved, if all isolated colonies
would be used for the study which could be realized also with higher investments and providing more
time. This improvement could be resulted in higher diversity identification and morphology, and finally
the reliable results for other studies.
Conclusion: First of all, the hypothesis of this experiment was supported by results that public bus has
more bacterial cells and more bacterial diversity in comparison with private car. In public bus, it was
determined that there are S.epidermidis and E.coli. Meanwhile, in car there are gram-positive, spherical
S.aureus bacteria. Finally, despite the limitation of this experiment, it could be considered as
successfully performed study.

Reference List:
[1] - Cappuccino, James G and Natalie Sherman. 2011. Microbiology. San Francisco: Pearson/Benjamin
Cummings.

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