Miles and misra method (surface viable count)

•  The technique was first described in 1983 by Miles, Misra and Irwin.
• Determination of bacterial cfu.
• Explained about dilution and culture suspension.
• A rapid and simple method for counting viable bacteria.

Materias:

• 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.
• Bacteral 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 - 2.5 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 (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 discreate 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 originalaliquot / sample:

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

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