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procedure pdf

For microbiology lab

Uploaded by

thomasshabanie
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
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TOTAL BACTERIAL COUNT

MAKING SERIAL DILUTIONS

BACKGROUND

The purpose of this practical is to quantify the number of bacteria in our samples. In
microbiology research, it is often necessary to be able to quantify the number of living bacteria
in a particular sample. One of the major ways to do this is using viable plate counts, in which
bacteria cells from a liquid culture are spread onto the plate agar.

The plate is incubated, the numbers of colonies that grow on the plate are counted and the
number of original bacterial cells in the culture is determined.

In most cases, however, the liquid culture being quantified contains too many cells to be directly
plated on the agar plates- there would be so much growth that it would be impossible to count
individual colonies. Therefore, the liquid culture needs to be diluted.

MATERIALS

Nutrient agar plates

Food samples

5 dilution banks containing 9mls peptone water

PROCEDURES

1. Label the dilution banks as follows: 10


2. Label the agar plates as follows:
3. Using a sterile pipette, transfer 1ml of the food samples into the tube labelled 10^-1. Mix
thoroughly.
4. Using a new sterile pipette, transfer 1ml of the 10^-1 tube into the tube labelled 10^-2.
Mix thoroughly.
5. Repeat till you reach the 5th dilution.
6. Using a new sterile pipette, transfer o.1ml of the 10^-5 tube to the nutrient agar plate
labelled 10^-6, and spread the liquid thoroughly and evenly over the surface of the plate.
(note: since we are only plating 0.1ml of the 10^-5 dilution, the total plated is 10^-6)
7. Do the same for 10^-6 and 10^-7 and label the plates as 10^-7 and 10^-8 respectively
8. After the liquid has absorbed into the plates, tape them closed on both sides. Make sure
your plates are well labelled (name, date, etc.) and incubate upside down at 25℃.

RESULTS

Examine the nutrient agar plates for growth, and count the number of colonies on each plate.
Remember the number has to be between 30 and 300 in order to be statistically accurate. If your
plate has more than 300 colonies, record the number as “TNTC” for “too numerous to count”

If your plate has fewer than 300 colonies, record the number as “TFTC” for “too few to count”.

Then use the formula below to determine the number of CFU/ml of the original broth culture.

PROCEDURE FOR E. COLI

ISOLATION OF BACTERIA

1. Using a sterile loop, streak cultures (liquid broth or isolated colonies picked from plates)
over one forth of the surfacSe of an agar plate. Then flame the loop.
2. Air cool a flamed loop or cool it by touching an unstreaked portion of the plate.
3. Pass the cooled loop once or twice over the initial Streaked portion of the plate. Streak it,
without overlap, to the next quadrant.
4. Flame the loop and allow it to cool as described in step 2.
5. Pass the loop over the streaked portion of the second quadrant of the plate
6. Repeat step 5 to streak the last quadrant.

Most bacteria do not move appreciably from the sites of inoculation but give rise there to clones
of bacteria called colonies. Isolated colonies should arise in the third and fourth quadrants
depending on the concentration of bacteria in the initial inoculum.(Ng’ong’ola-Manani, Tinna.A
and Mngoli, 2017)
REFERENCE

Ng’ong’ola-Manani, Tinna.A and Mngoli, K. . (2017). FST 221 FOOD MICROBIOLOGY


MANUAL FACULTY OF FOOD AND HUMAN SCIENCES.

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