Exp 4 Total Count Bacteria

RESULT
Plating Average
Method Colony/plate

Dilution

Total
bacteria/mL

1/10 1/102 1/103 1/104 1/105 1/106

Pour
Plate
Spread
Plate

9.17

14

12

10

5134140

3.83

1272740

CALCULATION
Pour plate method
Average colony/plate =

4+1+14 +12+10+1
6

= 9.17

Total bacteria/mL
( 4 x 10 ) + ( 1 x 102 ) + ( 14 x 103 ) + ( 12 x 104 ) + ( 10 x 105 ) + ( 4 x 10 6 )
5134140 bacteria

Spread plate method

Average colony/plate =

4+7 +2+7+2+1
6

= 3.83

Total bacteria/mL
( 4 x 10 ) + ( 7 x 102 ) + ( 2 x 103 ) + ( 7 x 10 4 ) + ( 2 x 105 ) + ( 1 x 10 6 )
1272740 bacteria

DATA ANALYSIS
The data that we get from pour plate method and spread plate method are different.
For pour plate method, the data shows highest bacteria amount in dilution (1/103); for
spread plate method, the data shows highest bacteria amount in dilution (1/102) and
(1/104) .
Based on the colonies formed in the plates, we can see that some of the plate
contained only one colony. That was maybe caused by we did not scratch the plate
properly for spread plate. For pour plate method, we predict that plate 1 and 3 were
not spread well.

DISCUSSION
The average colony for pour plate is 9.17 while for spread plate is 3.83.The
total bacteria for the pour plate is 51334140 ml meanwhile for spread plate is 1272740
ml. It has differences value for the two plating method. The final plates in the series
should have between 30 and 300 colonies but we get lesser than the actual result.
Fewer than 30 colonies are not acceptable for statistical reasons.

There are some systematic bias error occur during this experiment. Firstly,
some bacteria will remain on the spreader while spreading the agar causing our count
to be too small. Besides, some bacteria were unable to grow more, maybe the bacteria
need more time which is more than one day to grow. Some bacteria may be clumped
together and grow a single colony. Then, as drops differing in size from drop to drop
or some plates being inferior to others. The spreading technique also was not done
properly.

CONCLUSION
There are a variety of ways to enumerate the number of bacteria in a sample. A
viable cell count allows one to identify the number of actively growing or dividing
cells in a sample. The pour plate count method or spread plate relies on bacteria
growing a colony on a nutrient medium. The colony becomes visible to the naked eye
and the number of colonies on a plate can be counted.

QUESTIONS
1. Explain the meaning of phrase two times ten to the eight cells per mL In
your own convenient terminology.
8
The phrases two times ten to the eight cell per mL is defined as 200 x 10 cell per

8
mL. It was a scientific notation which is, since 1 x 10 is equal to 100 000 000

8
hence 2 x 10 is equal to 200 000 000. Due to 200 000 000 cell per mL is difficult

number to write or comprehend, 10

means the same thing.

2. What the meaning of TNTC and the significance amount due to the TNTC?
Give the formula for determining bacteria count.
TNTC was stand as To Numerous To Count and the significance amount due to it is
more than 300 bacteria colonies. In the determination of microorganisms by a
technique in which individual viable units are determined, such as by plate count
assay of bacteria or by plaque count assay of viruses, with insufficiently diluted
samples an overgrowth or dense formation of colonies is noted which is conventially

reported as TNTC that then number of bacterial colonies exceeds 200 on a 46-mm
diameter membrane filter used for coli form detection.
Formula:
Colony Forming Unit = C.F.U. The calculation is performed, thus;
C.F.U/ml original shape = {C.F.U/plate} x {1/ml aliquot plated} x {dilution factor}

3. Design an experiment to compare the bacteria counts in different water

samples (tap water, lake water, swimming pool water and rain barrel water).
Explain the difference of bacteria count for each type of water sample.
Comparing Bacteria Count of Water Sample.
Suggested water samples: tap water,well water, lake water, pond water, river water,
ocean water, swimming pool water, rain barrel water. Sterile tools must used at all
stages analysis.
Materials needed:
1.
2.
3.
4.
5.

Sterile collection container (sterile bottle or test tube) with water sample.
Sterile pipettes
Sterile petri dish
Coliscan Easgyel (Micrology Laboratory)
Incubator set.

Procedure:
1. Put a drop of water of well water on a sterile plate of TGY or other agar.
2. Spread it uniformly over the surface of the agar with any non-absrobent sterile
tool. A glass rod bent into an L-shape is ideal. The bottom of a teaspoon will
also work.

3. Incubate the plate at 30 or room temperature (r.t).

4. Examine the plate as often as you like. At room temperature, it will probably
take a day or two for singles cells to grow into colonies large enough for you
to see. Different species of bacteria grow at different speeds and some species
will take many days.

5. Count the colonies. There are about18 drops per ml the size of drop depends
on the orificie. Small tips make small drops and it can take 30+ drops to make
one mililiter.
6. Compare the Bacteria Count of water samples.

Result :
In this experiment, probability for the city tap water used will got no bacteria, while in
the lake water will got many bacteria. If used water from swimming pool, the bacteria
count would probably many bacteria per drop and it is likely the plate was covered
and could not be counted.

4. In many experiments, there are 2 types of control used which are positive and
negative control. Based on this experiment, what is suitable control? How will
the control affect your findings?
For positive control, inoculate 1 ml of suspension into each of 2 petri dishes. Add
about 20ml of cooled molten YEA to the plates at the same time as the pour plate
method is carried out on test samples. Incubate one control plate with the test plates at
37 and the other plate with the test plates at 22 for the appropriate times.
For Negative control (blank), sterility checks are to be performd for each bottle of
agar. Aseptically pour about 20ml of molten agar, cooled to 45 , into 2 petri
dishes. This should be done at the same time as the test sample is inoculated. Incubate
the control plates with the test plates at 37

REFERENCE

and 22 for the apparatus times.

Master, Gelbert M (1998) Introduction to Environmental Engineering

andScience New Terzey : Prentice Hall

Boundless. Viable Cell Counting. Boundless Microbiology. Boundless, 14

Nov. 2014. Retrieved 13 Mar 2015.

https://www.boundless.com/microbiology/textbooks/boundless-microbiologytextbook/culturing-microorganisms-6/counting-bacteria-63/viable-cellcounting-384-5695/
http://www.disknet.com/indiana_biolab/b038.htm