The Miles and Misra Method (or surface viable count) is a technique used in Microbiology

Microbiology

Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...

 to determine the number of colony forming units in a bacterial suspension or homogenate

Homogenization (biology)

Homogenization is a process that involves breaking apart cells — releasing organelles and cytoplasm.When the purpose is to extract organelles, it is frequently done in two steps; first using a blender to break the tissue up, and then with an ultrasonic or mechanical tissue disruptor. The...

.
The technique was first described in 1938 by Miles, Misra and Irwin who at the time were working at the LSHTM

London School of Hygiene & Tropical Medicine

The London School of Hygiene & Tropical Medicine is a constituent college of the federal University of London, specialising in public health and tropical medicine...

. The Miles and Misra method has been shown to be precise.

Materials

• A calibrated dropping pipette
  Pipette
  A pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...
  
  , or automatic pipette, delivering drops of 20μl.
• Petri dishes
  Petri dish
  A Petri dish is a shallow glass or plastic cylindrical lidded dish that biologists use to culture cells or small moss plants. It was named after German bacteriologist Julius Richard Petri, who invented it when working as an assistant to Robert Koch...
  
   containing nutrient agar
  Agar
  Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...
  
   or other appropriate medium.
• Phosphate Buffered Saline
  Phosphate buffered saline
  Phosphate buffered saline is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The buffer's phosphate groups help to maintain a constant pH...
  
  (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.