Review: Dilution of Microbial Suspensions

Dilutions are performed in the laboratory in order to make samples (cultures, chemicals)
less concentrated. This is achieved by adding a diluent (water, buffer, etc.) to the
sample. In industrial and research laboratories it is often necessary to monitor bacterial
numbers. Given suitable growth conditions, bacterial cultures can become very
concentrated very quickly. Most methods used to count bacterial cells require that
cultures be diluted first to an appropriate ('countable') concentration. In this exercise, we
will first review some of the main principles of dilutions. You will then perform a
dilution plate count of a bacterial culture.

Dilutions can be expressed in different ways:

For example, a "1 in 10 dilution" may also be expressed as 10-1 or 1/10.

These mean the same thing - one 'part' in a total of 10 'parts'.

General Formula: ________volume of sample________

volume of sample + volume of diluent

A 1/10 dilution, therefore, means 1 'part' of the sample added to 9 'parts' of diluent.
Eg: 1 mL of sample added to 9 mL of diluent.

How would you express each of the following?

a) 0.2 mL of sample added to 9.8 mL of diluent?

b) 3 mL of sample added to 21 mL of diluent?

a) A 2 x 10-2 dilution?

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Large dilutions may prove to be more of a challenge. For example, a 1/8000 dilution
of a bacterial culture could be carried out in a single step by adding 1 mL of the sample
(microbes) to 7,999 mL of diluent. This, however, would require the use of a large volume of
diluent and would be difficult to measure out accurately.

This problem can be dealt with by performing serial dilutions - a series of dilutions
such that the total dilution equals the product of all individual dilutions - ie. multiply
the dilutions together to get the total.

For a 1/8000 dilution?

You could perform the following series of individual dilutions: 1/80 x 1/10 x 1/10

Total diluent used? -

Note that there are several other equally-acceptable dilution series to choose from.

Eg: 1/8 x 1/10 x 1/10 x 1/10 or 1/40 x 1/20 x 1/10

This problem can also be solved by performing equivalent dilutions which use smaller
volumes. A 1/8000 dilution could mean 0.1 mL in a total of 800 mL (0.1/800) or 10 µL
(0.01mL) in 80 mL (0.01/80). Each of these dilutions is equivalent to 1/8000.

The concentration of a culture decreases as it is diluted. If you started with a culture

which initially has a cell concentration of 3.0 x 106 cell/mL and you serially-dilute it
1/20, 1/10, and then 2 x 10-1, what would the concentration be after each dilution?

After 1/20:

After 1/10:

After 2 x 10-1:

Plate Counts: used to determine the concentration (CFU/mL or CFU/g) or the

total CFU in a preparation. Recall: CFU = colony forming unit.

General Formula:

# CFU/mL = # CFU counted x _______1_____________ x ___1____

(or CFU/g) volume plated (in mL) dilution
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 1. 1.5 mL of a bacterial suspension (75 mL total volume) was removed and added to
38.5 mL of sterile diluent. It was then further diluted 1/50. 0.2 mL of the final
dilution was then plated. After incubation, 82 CFUs were counted.

i) Determine the concentration of bacteria in the original suspension.

ii) Determine the total number of bacteria in the original suspension.

Note: 30 - 300 rule - can only use pour plate counts that are between 30 and 300!
- this applies only to the raw data!

- in this particular question, the # CFU = 82 and, therefore, may be used

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 2. A bacterial suspension was diluted and 0.1 mL of each of the dilutions was plated in
duplicate. The following results were obtained.

Dilution # CFU # CFU

10-2 TNTC TNTC
10-3 278 303
10-4 , 36 32

Note: TNTC = too numerous to count

Two ways to solve:

a) Average the plate counts at the beginning. (Note: not always easy to do).

b) "Plug" each valid plate count into the formula and average all results at the end.