Miles and Misra method

The Miles and Misra Method (or surface viable count) is a technique used in Microbiology to determine the number of colony forming units in a bacterial suspension or homogenate. The technique was first described in 1938 by Miles, Misra and Irwin who at the time were working at the LSHTM.[1] The Miles and Misra method has been shown to be precise.[2]

Materials

• A calibrated dropping pipette, or automatic pipette, delivering drops of 20μl.
• Petri dishes containing nutrient agar or other appropriate medium.
• Phosphate Buffered Saline (PBS) or other appropriate diluent.
• Bacterial suspension or homogenate.

Method

• The inoculum / suspension is serially diluted by adding 1x of suspension to 9x of diluent. When the quantity of bacteria is unknown, dilutions should be made to at least 10⁻⁸.
• Three plates are needed for each dilution series, for statistical reasons an average of at least 3 counts are needed.
• The surface of the plates need to be sufficiently dry to allow a 20μl drop to be absorbed in 15–20 minutes.
• Plates are divided into equal sectors (it is possible to use up to 8 per plate). The sectors are labelled with the dilutions.
• In each sector, 1 x 20 μl of the appropriate dilution is dropped onto the surface of the agar and the drop allowed to spread naturally. In the original description of the method a drop from a height of 2.5 cm spread over an area of 1.5-2.0 cm. It is important to avoid touching the surface of the agar with the pipette.
• The plates are left upright on the bench to dry before inversion and incubation at 37 °C for 18 – 24 hours (or appropriate incubation conditions considering the organism and agar used).
• Each sector is observed for growth, high concentrations will give a confluent growth over the area of the drop, or a large number of small/merged colonies. Colonies are counted in the sector where the highest number of full-size discrete colonies can be seen (usually sectors containing between 2-20 colonies are counted).
• The following equation is used to calculate the number of colony forming units (CFU) per ml from the original aliquot / sample:

CFU per ml = Average number of colonies for a dilution x 50 x dilution factor.

Advantages

• Faster than other methods.
• Produce less bacterial contamination of the working surface.