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.
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.
^Hedges, AJ (Jun 25, 2002). "Estimating the precision of serial dilutions and viable bacterial counts". International Journal of Food Microbiology. 76 (3): 207–14.
doi:
10.1016/s0168-1605(02)00022-3.
PMID12051477.
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.
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.
^Hedges, AJ (Jun 25, 2002). "Estimating the precision of serial dilutions and viable bacterial counts". International Journal of Food Microbiology. 76 (3): 207–14.
doi:
10.1016/s0168-1605(02)00022-3.
PMID12051477.