lab 17 serial dilution

8/11/2019 Lab 17 Serial Dilution

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Keep in mind that if the organism normally formsmultiple cell arrangements, such as chains, the

colony-forming unit may consist of a chain of bacteria rather than a single bacterium.

In addition, some of the bacteria may be clumped together.Therefore, when doing the plate count technique, we generallysay we are determining the number of Colony-Forming Units(CFUs) in that known dilution.

By extrapolation, this number can in turn be used to calculate

the number of CFUs in the original sample.

bacterial counts by these methods are usually expressed ascolony forming units per milliliter (CFU/mL).


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Normally, the bacterial sample is diluted by factorsof 10 and plated on agar.

After incubation, the number of colonies on adilution plate showing between 30 and 300 colonies is determined.

A plate having 30-300 colonies is chosen because this range isconsidered statistically significant.


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If there are less than 30 colonies on the plate, smallerrors in dilution technique or the presence of a few

contaminants will have a drastic effect on the finalcount. (too few to count (TFTC).

Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. (too

numerous to count (TNTC).


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Procedure

1) Using sterile technique, transfer 1 mL sample to the firstdilution blank. Mix the bottle by inverting it 20 times.Label the bottle "10-1."


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2) Using a fresh pipette, transfer 1 mL from the first blank to the second blank. Mix as before. Label thesecond bottle "10-2."

3) Using a fresh pipette, transfer 1 mL from the first blank to thesecond blank. Mix as before. Label the second bottle "10-2 .

4) Using a fresh pipette, transfer 1 mL from the first blank tothe third blank. Mix as before. Label the second bottle "10-3 .

5) Using a fresh pipette, transfer 1 mL from the first blank to theforth blank. Mix as before. Label the second bottle "10-4 .

6) Using a fresh pipette, transfer 1 mL from the first blank to thesecond blank. Mix as before. Label the fifth bottle "10-5 .

7) Label the Petri dishes: 10-2, 10-3, 10-4, 10-5, and 10-6,respectively.


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Colonies Forming Units {CFU}

Calculate the number of bacteria (CFU) per milliliter or gramof sample by dividing the number of colonies by the dilution

factor multiplied by the amount of specimen added to agarplate.

To compute the number of CFU/mL, use the formula:c = concentration, CFU/mL

n = number of coloniesd = dilution blank factors = volume transferred to plate.


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CFU Calculation Example

You count 46 colonies on your plate You put 1 ml of bacterial culture into 99 ml of saline and plated0.1 mlDilution 1/100

46CFU=1/100 * 0.1

= 46 * 100 * 10 =46000 CFU/ml


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lab 17 serial dilution

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