OBJECTIVE

The objectives of this experiment are:

To learn how to determine the number of microorganisms in a sample, a process called

enumeration.

To utilize several method of enumerating microorganisms

PROCEDURE
Pour plate method
Tap water or pond water sample
Nutrient agar pours (15 mL/tube)
Sterile dilution water blanks (99 mL)
Sterile Petri plates
Pipettes (1 mL, sterile)
Pipette bulb or mechanical pump
Marking pen
Water bath at 50C
1. Six nutrient agar pours were melted in a boiling water bath. Then, place them in a 50C
water bath, mixed it after they liquefy until ready for use.
2. Three of the 99-mL dilution blanks were labeled with 10-2, 10-4 and 10-6, respectively.
Another six Petri plates were labeled 10-2 through 10-7.
3. The unknown sample was shaken to ensure an even distribution of microorganisms).
Then, remove 1 mL of sample aseptically with a sterile pipette and transferred it to the
10-2 dilution blank.
4. The dilution blank was shaken vigorously to distribute the bacteria evenly.
5. Aseptically transfer 0.1 mL and 1.0 mL from the 10-2 dilution to the agar plates by using
a new sterile pipette then labeled it with 10-3 (0.1 mL) and 10-2 (1 mL) respectively. The
same pipette was used to remove an additional 1.0 mL from the 10-2 dilution blank and
transferred it to the 10-4 dilution blank. The original 1 mL of sample now had been
diluted 1 part in a total of 10,000 parts.
6. Shaking procedure was repeated for the 10-4 blank and transfer 0.1 mL and 1.0 mL
portions from this dilution bottle with a new pipette to the plates labeled 10-5 (0.1 mL)
and 10-4 (l mL).
7. The same procedure was repeat to form a 10-6 dilution blank from which 10-7 (0.1 mL) and
10-6 (1.0 mL) plate was established.
8. A tube of melted agar was poured aseptically (50C) into each Petri plate which already
added a dilution of the sample. The plate was swirled to mix the sample with the agar.
The agar was make does not run over the edges of the plate. The lid was replaced and
allows the agar to cool down and solidity.
9. The inverted plate was incubated at 30C.
10. After incubation, the colonies on each plate were counted. Both the colonies on the agar
surface and the colonies growing within the agar must be counted. The colonies were

counted by marking their position on the back of the Petri plates with a marking pen. This
aid in keeping track of those colonies previously counted and avoids recounts. Record
your counts. If a plate has more than 300 colonies record it as TNTC (too numerous to
count).
11. The plate-count data was used to calculate the concentration of bacteria in the original
sample. For the statistical reasons, only plates with between 30 and 300 colonies in this
calculation. Each colony forming unit (CFU) represents the progeny of a single cell.
Therefore, the number of bacterial cells in the original sample is determined by
multiplying the number of colonies on a dilution plate by the corresponding dilution
factor. Each dilution was counted twice, and the mean count was recorded.

Spread plate method

Tapwater or pond water sample
Nutrient agar plates
Dilution water blanks (9 mL)
Pipettes (1 mL, sterile)
Pipette bulb or mechanical pump
Glass spreaders
Alcohol (in a beaker)
Vortex mixer
Marking pen
1. Three of the 9 mL dilution water blanks was labeled as 10-1, 10-2 and 10-3 respectively.
Another four nutrient agar plates was labeled with 10-1 through 10-4.
2. Pipette 1.0 mL of the water sample aseptically into the 10-1 dilution tube. The 10-1
dilution tube was mixed thoroughly by vortexing or vigorous shaking. The concentration
of bacteria in this tube was 1/ 10 of the original sample.
3. A new pipette was used aseptically to transfer 1 mL from the 10-1 dilution tube to the 10-2
dilution tube. The sample was mixed toughly by vigorous shaking. The concentration of
bacteria in this tube is 1/100 of the original sample.
4. The same procedure was repeated, transferred 1 mL from the 10-2 dilution tube to the 10-3
dilution. The sample was vortexing for it to mix well. The concentration of bacteria in
this tube is 1/1000 of the original sample.
5. A new sterile pipette was used to transfer 0.1 mL from each of the dilution tubes and the
original sample to agar plates. As only 0.1 mL was transferred, the sample was diluted by

another factor of ten. Therefore, the greatest dilution plate is 10-4. To start with the
greatest dilution, the tube must be vortex and pipette 0.1 mL onto its respective agar
plate. Do this for each tube and the original sample so that you now have plates ranging
from 1/10 to 1/10,000 of the original concentration. Because you start with the greatest
dilution you can use the same pipette for each of the tubes.
6. The glass spreader was sterilized by dipping it into the 70% ethanol and flaming it in a
Bunsen burner flame.
7. After the glass rod has cooled, spread the sample over the surface of the plate by touching
the rod to the agar and rotating the plate.
8. Repeat this spreading procedure for each of the plates, making sure to sterilize and cool
the glass rod before spreading each sample.
9. Inverted plate was incubated at 30C for 24-48 hours.
10. After incubation, the colonies were counted. Plates with a range of 30-300 colonies need
to be counted. The count was recorded.
11. The concentration of bacteria in the original sample was counted.

1. Why are counts above 300 and below 30 statistically unreliable?

Plates with more than 300 colonies are may cause undercount to the true
number of colonies because they are too crowded to allow all the bacteria to
form distinct colonies due to overcrowding. Plates with fewer than 30 colonies
give statistically unreliable results.
(Micro eGuide, 2010)

http://www.microeguide.com/lab_skills/viable_counts.asp
2. Did you enumerate all the bacteria using this method?

3. What factors make the pour plate method selective? How can you reduce the selectivity of
this procedure?

The warm or hot agar causes pour plate method selective as the heat-sensitive microbe
will be kill during this process. To reduce the selectivity of this procedure, the
agar must be cool down first but still in molten state before pour into the
Petri dish

4. How can you achieve a 1/100 dilution other than by adding 1mL of sample to a 99mL
dilution blank?
Add the 0.1mLof the sample into a 9.9mL dilution blank or add a 0.1mL of the sample to
the 9.0mL dilution blank.

1. What does the term colony forming unit mean and how does it differ from a colony?
colony-forming unit (CFU) is a unit used to estimate the number of viable
bacteria or fungal cells in a sample. A colony is simply a group of bacteria
that probably grew from one original bacterium. The term CFU is used

because it is impossible to be certain that each colony came from only one
bacterium.

http://study.com/academy/lesson/what-is-a-colony-forming-unit-definition-purpose.html

2. What types of errors would be introduced by using a single pipette for all operations in this
procedure?

3. What are advantages and limitations of the spread plate method compared to other
enumeration procedures?

4. How could you modify the spread plate procedure to enumerate obligate anaerobes?