Firas A.

– Biology Standard Level – IB1-A

Bacteria in

Research Question

This experiment shall focus on finding out how many bacteria are present in one randomly
chosen bottle of Yakult.

Introduction

This experiment solely deals with 65mL Yakult bottles manufactured in Almere, the
Netherlands. Mentioning this is of importance, since Yakult bottles are not of the same size in
all countries. For example in Taiwan and China Yakult comes in 100mL1 bottles. Yakult (ヤク
ルト) is a probiotic yogurt-like beverage made by fermenting a mixture of skimmed milk
and sugar with a special strain of the bacteria Lactobacillus casei. Because L. casei Shirota is
naturally found in the digestive system, Yakult is touted as beneficial for intestinal health.2
According to Yakult Honsha Co., Ltd. (株式会社ヤクルト本社) each mL of Yakult contains
100,000,000 bacteria3 when it is manufactured. This means that each bottle of Yakult contains
6.5 billion bacteria.

Hypothesis

The statement made by Yakult Honsha Co., Ltd. is correct. Each mL of Yakult will contain
more than 100,000,000 bacteria.

Apparatus

1Source: http://www.yakult.com.tw/
2Source: http://en.wikipedia.org/wiki/Yakult
3Source: http://www.yakult.com.au/resources/documents/Yakult_SecondaryKit.pdf
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 Firas A. – Biology Standard Level – IB1-A

 65mL Yakult bottle, made in Almere

 Salt solution, 0.9% sodium chloride
 Alcohol (ethanol)
 Distilled water
 Measuring cylinder
 Pipette
 Test tube
 Stirrer
 pH marker
 Plastic bags
 Petri dish, including MRS agar (jelly)
Procedure4
1. Extract exactly 1mL of Yakult from the bottle and place it in a test tube, using a
dropping pipette.
2. Now it is the intention to produce 10mL of diluted Yakult. This can be done by adding
9mL of distilled water to the existing 1mL of Yakult, placed in the test tube before.
3. Extract 1mL from the test tube to obtain 1mL of diluted Yakult. Take a new test tube.
Further dilute the 1mL of diluted Yakult by adding another 9mL of distilled water to
the new test tube. 10mL of further diluted Yakult should be in the new test tube now.
4. Repeat the above described process 5 times.
5. While conducting this process for the sixth time, extract 1mL of diluted Yakult and
place it in a new test tube. In order to dilute this solution 1,000,000 times, add 9mL of
the prepared salt solution (consisting of 0.9% sodium chloride) to the new test tube.
6. Extract exactly 1mL from the final diluted solution, using a dropping pipette. Place
this 1mL on a MRS agar (jelly substance) on a Petri dish, spreading the solution on the
agar. This shall make sure that the bacteria are kept alive.
7. Now the bacteria shall be given the chance to develop into colonies. This is done by
placing the lid on the Petri dish, while keeping it in a plastic bag for three days. The
plastic bag is important to avoid drying up of the Petri dishes.
8. Now it is time to determine how many bacteria were present in the final solution. This
is done by counting the number of bacteria colonies that developed in the Petri dish.
Results

4Following the method outlines described in the following document:

http://www.yakult.com.au/resources/documents/Yakult_SecondaryKit.pdf
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 Firas A. – Biology Standard Level – IB1-A

Group name Number of bacteria colonies formed

A 25
B 31
C 9
D 7
E 1
F 1

The mean value below shall be used in order to determine the number of bacteria in 1mL of
Yakult.

As visible in the table above, the mean was calculated to be 12.3. This means that the final
Petri dish, after being diluted 10,000,000 times, contained 12.3 bacteria. Thus in order to
obtain the number of bacteria residing in 1mL of Yakult, a simple calculation has to be done.
The factor by which the solution has been diluted multiplied by the obtained mean value shall
give the number of bacteria residing in 1mL of Yakult. The result of the calculation was found
to be 123,000,000.

As we are known to be dealing with a 65mL Yakult bottle manufactured in Almere, the
Netherlands, the calculation in order to obtain the number of bacteria in one bottle looks like
this: 123,000,000 (number of bacteria in 1mL of Yakult) × 65 (the number of mL contained in
one bottle of Yakult sold and manufactured in the Netherlands) = 7,995,000,000 bacteria.

The same method for calculating the number of bacteria in one bottle of Yakult was engaged
in order to find out the results of other experimenting groups:

Group name Number of Number of Amount of bacteria with

bacteria/millions bacteria/millions respect to the company’s
contained in 1mL contained in 1 original statement
of Yakult bottle
A 250 16,250 Larger amount of bacteria
B 310 20,150 Larger amount of bacteria
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 Firas A. – Biology Standard Level – IB1-A

C 90 5,850 Smaller amount of bacteria

D 70 4,550 Smaller amount of bacteria
E 10 650 Smaller amount of bacteria
F 10 650 Smaller amount of bacteria
Mean 123 7,995 Larger amount of bacteria
Yakult company 100 and more 6,500 and more Identical

Conclusion

The conducted experiments confirmed the statement made by Yakult Honsha Co., Ltd.
The statement made by Yakult Honsha Co., Ltd. was that each bottle contains more than
100,000,000 bacteria per ml. The investigation revealed that there were around 123,000,000
bacteria per ml. However, groups E and F were only able to find 10,000,000 bacterial per ml.
While at the same time groups A and B found the number of bacteria per ml of Yakult to be
far above the number stated by Yakult Honsha Co., Ltd. (up to 31 times more). Nevertheless,
the statement made by Yakult Honsha Co., Ltd. remains true as the mean number of bacteria
(7,995 millions) found in the Yakult drinks still agrees with the company’s claim (6,500+)

Evaluation

In general it can be said that the experiment did develop quite successfully. However, the fact
that the number of bacteria vary from bottle to bottle is very natural. Nevertheless, the number
of bacteria was found to vary a bit too strongly, considering the calculated numbers of the
final solution. At this point it cannot be said whether the variations are due to the techniques
used by Yakult to insert the bacteria in the Yakult bottles, or if it is just a mistake done in the
procedure of determining the number of bacteria. Taking several (e.g. up to 4) samples from
the same Yakult bottle might help in determining the source of the variation. If these samples,
obtained from the same Yakult bottle, show such strong variations in the number of bacteria,
then it is very likely that the variations are due to mistakes done while trying to determine the
actual number of bacteria in a bottle of Yakult (my fault). On the other hand, if samples,
obtained from one single Yakult bottle, show no (or only slight) variations in the number of
bacteria, then it is very likely that the variations’ source are productions methods engaged by
Yakult.
Furthermore, unsterile conditions in the school’s lab might have resulted in the alteration of
the results. These bacteria are known to be sensitive to many substances, which makes it even
more difficult to obtain accurate results.