BIOLOGY INTERNAL ASSESMENT

STANDARD LEVEL

Investigating the Microbial Growth in Coke.

How microbial contamination(Bacterial Growth) will increase depending on exposure
of coke in the open environment?
IINTRODUCTION
Soft Drinks comprises one of the largest beverage industries in the world today,
these beverages are segregated into two groups, ie. Carbonated soft drink like
coke, thumbs up and limca and non-carbonated soft drinks like Maaza and minute
maid. The major ingredients of Coke are water, Sugar substitute, carbon dioxide,
flavor emulsion, coloring agents and preservatives.
My experiment was on the bacterial growth. Bacteria’s are the micro-organisms that
grow everywhere. We can collect and grow these bacteria’s on the prepared agar-
agar Petri dish. Nutrient Agar is an excellent medium for growing the bacteria’s in
particular environment. Nutrient Agar with dissolved nutrients are added in water and
then heated and poured into empty Petri dishes, Moreover, the Growth will depend
on the opening of the coke, the amount of Gas in the liquid present will determine the
growth of bacteria according to the time gap of 5 min.
The Lab was conducted in the controlled atmosphere, and transparency and
observance was observed by using specto-photometer, before using the specto-
photometer, the calibration was done using distilled water. There were 8 petri-dishes
with white cloudy solution as Agar Nutrient.The Agar solvent was prepared by
dissolving the Agar in auto clave. The settled wavelength for the specto-photometer
was 620 nm. More the exposure of coke to the open environment,more the
bacteria’s as bacteria’s present around us will enter and thus the bacterial count in
the petri dish increases as time increases.
TOPIC:-
To study the effect of the microbial Growth in Coke with Agar as medium of
measuring the Growth.
RESEARCH QUESTION:-
How microbial contamination(Bacterial Growth) will increase depending on exposure
of coke in the open environment?
HYPOTHESIS:-
More the Gas will have least bacteria’s, hence less the gas more the bacteria’s due
to the opening of Coke in every 5 min.
VARIABLES:-
Independent Variable:- Amount of Agar Nutrient, Amount of Coke,
Dependent Variable:- The Bacterial growth that occurs due to the amount of Coke.
Controlled Variable:- Temperature of Petri-Dishes, Time Period of testing the
samples at an interval of 5 min, Volume of Coke.
MATERIALS:-
Agar Nutrient
Petri-Dishes
Stop-watch
Lid
Swab/ Spatula
Coke
Tweezers and Tongs
Tissues
Beakers(100ml)
Test Tube Racks
Test-tube
Bacteria Incubator
Spectrophotometer
Pipette
Graduated
Cylinders
PROCEDURE:-
1. Coke was given and inoculated at the day of use.
2. Agar Nutrient was prepared in the 7 petri-dishes by school lab technicians.
3. Coke Sampling should be taken in every 5 minutes
4. 0.10ml of coke should be poured in each Petri dishes.
5. Initial Coke sampling should be experimented once.
6. Interval of 5 min should be sufficient to identify the bacterial growth.
7. Put All the 7 Petri Dishes in a bacterial incubator.
8. Set the temperature between 35-40 Degrees.
9. Take them out from Bacterial Incubator and open the Petri dishes gently(so the
agar nutrient don’t get destroyed).
10. Using the swab or Spatula to rub the surface gently.
11. When finished rubbing choose a side where bacterial growth is evident, remove
some Agar nutrient with the bacteria present and pour into a distilled water.
12. When done measuring keep the lid upside down and put the petri dishes back
into bacterial incubator.
DATA PROCESSING
Quantitative Data
 First batch of Sampling got a exposure in controlled temperature, and there were
random colonies of bacteria formed.
 More Bacterial colonies were formed as the time span increases, More the
opening more the Bacterial Colonies formed.
 DAY 1 DAY 2

Time in 25th September

Minutes (m) 2018 Time in 26th September
Minutes (m) 2018
Colonies Formed
Colonies Formed
0 Min 1
5 Min 6
5 Min 4
10 Min 13
10 Min 11
15 Min 19
15 Min 17
20 Min 25
20 Min 22
25 Min 28
25 Min 25 30 Min 29
30 Min 27

DAY 3

Time in 27th September

Minutes (m) 2018
Colonies Formed
5 Min 8
10 Min 16
15 Min 21
The Quantitative Data that is gathered over a period of time is
20 Min
analyse 28 picture.
by the process of above

25Data
The Minwas Very Minute,31hence used above statistical data to
identify the bacterial colonies formed.
30 Min 34

Day 1 25-Sep-18 4:10

Time Transparency Observance

0 min 99.3 2

5 min 95.2 2.021

10 min 94.8 2.023

15 min 93.5 2.029 Qualitative Data

20 min 92 2.036

25 min 91.5 2.038

30 min 91.2 2.04

 Calculating Observance From Transparency…

2 Day
 log(% T)
2 26-Sep-18 1:40

 89.5  Observance
Transparenc
2 Time
 logy 
 100 
5 min 91 2.041
 2.0481
10 min 90 2.045

15 min 90.6 2.0456 Day 3 27-Sep-18 2:40

20 min 90.9 2.04565 Transparen Observanc
Time
25 min 89.9 2.0462 cy e

30 min 89.6 2.0476 5 min 89.5 2.0481

10 min 88.4 2.0535

15 min 87.1 2.0599

20 min 86 2.0655

25 min 85.2 2.0695

30 min 84 2.0757

Processed Data (Graph Formulation)

Bacterial
Bacterial Colonies Bacterial Colonies
Colonies Day
Day 2 Day 3
1

0 min 1 5 min 6 5 min 8

5 min 4 10 min 13 10 min 16

10 min 11 15 min 19 15 min 21

15 min 17 20 min 25 20 min 28

20 min 22 25 min 28 25 min 31

25 min 25 30 min 29 30 min 34

30 min 27

Graph
 Bacterial Colonies Formed Over a Period Of Time (s)
40
35
Time in Minutes (m)

30
25
20
15
10
5
0
0 1 2 3 4 5 6 7 8 9
Bacterial Growth Colonies (ml)

Bacterial Colonies Day 1 Bacterial Colonies Day 2 Bacterial Colonies Day 3

Interpretation of the Graph

The Graph has three lines depicting the upward trend over a period of time. The
readings for day 1 includes the initial reading at 0 min. Black trend shows the Day 1
bacterial growth,Grey trend shows the Day 2 bacterial growth and Red trend shows
the Day 3 bacterial growth. The reason behind increase in bacterial growth is
because of the opening of coke over a period of time. More the opening of coke,
more the bacteria’s enter and thus result to more bacterial colonies to form. All the
sample were in the controlled temperature at 30 *-40* degrees. According to the graph
and the progressed table we can depict that as the transparency decreases,
bacterial level increases.
SAFETY PRECAUTIONS
 Wear a Lab coat
 Be careful with knife and other apparatus (carry a first aid kit)
 Don’t spill the solution
 Avoid Damaging to the apparatus
 Petri Dishes must be rotated at 90 degrees before attracting the bacteria.
 Streak the Agar Petri Dishes gently, do not push it into Bacterial incubator.
 Clean the workstation after the experiment, to ensure cleanliness and
discourage contaminants.
 Do not play with the temperature variation of bacterial incubator.
 Ensure the cotton plug is secure before placing the flask into the autoclave.

CONCLUSION
My Experiment proves my hypothesis, and hence there is a inverse relation between
the transparency and observance, as Transparency decreases observance
increases. The bacterial colonies are formed over a period of time, and its evident
that due to the opening of coke and exposure to the outer atmosphere, brings more
bacteria’s to come into Coke. The data found and analyzed clearly shows that
bacterial growth was dependent on the opening of time. The Hypothesis was proven
by considering the fact of More the gas less the bacteria’s, gas could be a disruptive
for bacteria’s as to an instinct the coke gas is created by high-temperature with dry
distillation, can reduce the bacteria’s. High temperature will kill the bacteria’s for
example: boiling water above 70 degree will kill the microbes and thus its better to
drink boiled water for our body. Hence to recapitulate More the temperature and Gas
less the bacterial colonies, therefore I choose to keep my experiment in a moderate
temperature with the range of 35*-40* degree.
EVALUATION
Strengths of the Experiment
 One of the strength for my experiment is the timing of sampling. I had a very
sharp eye on the timing as I had stop watch and a alarm set for every 5 min.
 No unethical elements or features are used to prepare the experiment.
 Had a Tighter control over the variables; easier to comment on cause and effect.
 Protocols were followed and experiment was ethical in nature.
Limitations (Error) of the Experiment
 Less trials often have more errors and thus by increasing number of trials, we
can improve the reliability of the data.
 Excess amount of Coke
 Power Cutoff
 Human Errors:-
 Human Judgement is not accurate enough in identifying the readings.
 Contamination in incubator:- As the experiments was held in the school and the
equipment are to be shared with others, the incubator might be contaminated
with others experiments, which can lead to the bacterial contamination.
Way(s) of Improvement
 Carry out more trials, so that errors can be minimized and its easier to identify
the bacterial growth.
 Arranging a certain structured to use the Incubator in order to avoid more
complications.
 Improve Measuring Technique
BIBLOGRAPHY/ REFERENCES
“Agar Plate.” Wikipedia, Wikimedia Foundation, 8 Nov. 2018,
en.wikipedia.org/wiki/Agar_plate.
“Blast Furnace, Coke Gas and Converter Gas for Power Production.” Clarke Energy,
14 Oct. 2013, www.clarke-energy.com/steel-production-gas/.
“Bacteria Growing Experiments in Petri Plates.” Bacteria Growing Experiments in
Petri Plates, Science Company, www.sciencecompany.com/Bacteria-Growing-
Experiments-in-Petri-Plates.aspx.
Cooper, A. L., et al. “Factors Affecting the Growth of Bacterial Colonies on Agar
Plates.” Proceedings of the Royal Society of London B: Biological Sciences, The
Royal Society, 5 Nov. 1968, rspb.royalsocietypublishing.org/content/171/1023/175.
“Observing Microbes – Observing Bacteria in a Petri Dish.” Microbiology Online,
microbiologyonline.org/teachers/observing-microbes/observing-bacteria-in-a-petri-
dish.
Owyoung, Palmer. “How to Measure Bacterial Growth in Petri
Dishes.” Sciencing.com, Sciencing, 14 May 2018, sciencing.com/measure-bacterial-
growth-petri-dishes-5837896.html.
https://en.wikipedia.org/wiki/Agar_plate
https://www.clarke-energy.com/steel-production-gas/
https://www.sciencecompany.com/Bacteria-Growing-Experiments-in-Petri-
Plates.aspx
http://rspb.royalsocietypublishing.org/content/171/1023/175
https://microbiologyonline.org/teachers/observing-microbes/observing-bacteria-in-a-
petri-dish
https://sciencing.com/measure-bacterial-growth-petri-dishes-5837896.html

Appendix
One of my Petri- dishes looked like this:-