Standard Procedure SP 0003:2005

A method for counting micro-organisms in dairy products

1 Scope

This Standard Procedure is adapted from BS 4285-2.3:1984 Microbiological examination for dairy purposes – Enumeration of micro-organisms by surface plate technique for colony count. The serial dilution procedure is adapted from BS EN ISO 8261:2002 Milk and milk products – General guidance for preparation of test samples. It can be used to estimate the number of live micro-organisms in a sample of a liquid dairy product, such as milk, cream or yogurt.
Note: The adapted procedure is not suitable for solid products, such as butter or cheese.

2 Definitions

micro-organisms
bacteria, yeasts and moulds
Note: This procedure does not identify or distinguish between these organisms.

serial dilutions
a series of sample solutions, each prepared by diluting the previous sample by a fixed amount

decimal dilutions
a set of serial dilutions in which each sample is ten times more dilute than the previous sample

3 Principle

A series of decimal dilutions is prepared from the original sample. A small portion of each is cultured on nutrient agar to produce visible colonies of the micro-organisms. Suitable colonies are counted, and the number of micro-organisms in the original sample is calculated from the count and dilution factor.

4 Apparatus

sterile apparatus:
- sample bottle
- rack of 5 test tubes plugged with cotton wool
- 10 cm³ graduated pipette & filler, or syringe
- 1 cm³ graduated pipette, or syringe
- fine dropper (to deliver about 50 drops per cm³)
- Petri dish of nutrient agar
marker pen to write on glass
5 cm³ or 10 cm³ measuring cylinder
adhesive tape
incubator set at 30 ± 1^oC
sterile saline-peptone solution (0.1% peptone, 0.85% NaCl)
(available commercially as ‘Oxoid’ MRD [Maximum Recovery Diluent] CM0733)

5 Test Specimens

The dairy product should be liquid. It may be an emulsion (such as milk or cream) or a suspension (such as yogurt). Solid products (such as cottage cheese) must be emulsified in a sterilised blender / liquidiser.

6 Procedure

IMPORTANT: Before using this procedure you must be familiar with aseptic techniques, which must be used throughout. Flame and re-plug the mouth of a tube each time it is opened.

Sampling

Mix the dairy product thoroughly by inverting the closed container 25 times. This helps to ensure that the micro-organisms are evenly distributed, and the sample will be representative.
Transfer a few cm³ of the product to a sterile sample bottle. Keep refrigerated and test it as soon as possible.

Decimal dilutions

Label 5 test tubes A (10), B (10²), C (10³), D (10⁴) and E (10⁵). The numbers are the dilution factors for each tube.
Using a sterile 10 cm³ pipette or syringe, measure 9 cm³ of sterile saline-peptone solution into each tube A to E.
Using a sterile 1 cm³ pipette or syringe, transfer 1 cm³ of the sample from the bottle into tube A.
Mix well by filling and emptying the pipette several times. This gives a 1:10 dilution.
Using a new 1 cm³ pipette, transfer 1 cm³ of this dilution from tube A to tube B. Mix to give a 1:100 (or 10²) dilution.
In the same way, transfer 1 cm³ from B to C and mix. Then from C to D and mix. Then from D to E and mix. This gives dilutions of 1:1000 (10³), 1:10 000 (10⁴) and 1:100 000 (10⁵).

Culturing

Without opening the lid, place the Petri dish of agar on the bench lid downwards. On the dish write:
- the name of the dairy product and date, in the centre;
- letters A to E, evenly spaced in a circle about 1 cm in from the edge.
Turn the dish over. Lifting the lid as little as possible, use a fine dropper to add 1 drop of dilution E to the surface of the agar in the area marked E, avoiding the areas with writing. Empty the dropper back into tube E.
Repeat this step with dilutions D, C, B and A in turn.
Allow the drops to be fully absorbed into the agar with no liquid left on the surface.
Meanwhile, using the dropper and a small measuring cylinder, count how many drops it takes to make 1 cm³. Record this number, n.
Tape down the lid at four points at 90^o to each other. (Do not seal it completely, since that would cause anaerobic conditions.)
Incubate the dish, lid downwards, at 30 ± 1^oC for 48 hours.
If possible, count the colonies within 4 hours after incubation. Otherwise, store the culture plate, still lid downwards, in a refrigerator at 0 to 5^oC until you can.

Counting colonies

Do not open the dish. Examine the culture to identify one of the areas A to E containing 10-30 colonies. Note its letter, and thus its dilution factor.
Count and record the actual number of colonies c in this lettered area.
When finished with, dispose of the culture plate as instructed by your teacher / supervisor.

7 Expression of Results

Calculate the number of living organisms per cm³ of the original sample, using the formula:

Number per cm³ = C x n x d

where

C = number of colonies produced by 1 drop of diluted sample

n =3 number of drops in 1 cm of diluted sample

d = dilution factor

Tube	A	B	C	D	E
Dilution factor	10	10²	10³	10⁴	105

8 Test Report

Your test report should include:

(a) reference to this standard procedure;

(b) the identity and nature of the sample;