Determining Bacterial CFU :

A technique used in Microbiology to determine the exact number of cfu that is colony
forming units in a bacterial suspension or homogenate is known as The Miles and Misra
Method. This is a type of surface viable count technique. In 1938 Miles and Misra
invented this method. In this method, Miles and Misra used dilution of S.pneumoniae on
blood agar plate.
To learn this technique completely, let us understand what is its pre-requirements of
materials, its method, its advantages and disadvantages.
Material Pre-requirements:
A calibrated automatic pipette or a dropping pipette , delivering drops of 20 l.
Sterilized nutrient agar plates
Diluent of Phosphate Buffered Saline (PBS)
Bacterial suspension or homogenate
Laminar Air Flow Bench
Filter Sterilized Isop-propyl alcohol
Method
Serial dilution of the inoculums / suspension is done in which, the dilution of 1x
suspension is added to 9x of diluent. In case of unknown sample quantity or unknown
bacteria quantity, dilutions should be made to at least 108.
The average of three plates is calculated. This is required to have greater assurance of
results. All three plates are inoculated with each dilution.
A 20 l drop is absorbed in the plate after 15-20 minutes of continuous spreading by
natural means or rotation of plates.
All the plates are equally divided into up to eight sectors. Proper labeling of the plates
are done to have traceability.
In each sector, 1 x 20 l of the dilution is dropped onto the surface of the agar and
thus the drop spreads. It is important to avoid touching the surface of the agar with the
any tool or even with pipette.
The plates are kept upright to dry before inversion and incubation at 37C for 18 24
hours.
Each sector is observed for growth, luxurious grown will be observed at high
concentrations over the area of the drop, or a large number of small colonies which are
generally merged. Colonies are counted in the sector where the highest number of fullsize discrete colonies can be seen.
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
This technique is faster than other methods.
With this technique, less bacterial contamination occurs of the working surface.
This technique is easy to process as compared to other techniques for determining
colony forming units.
Disadvantages
This technique required high skilled microbiologist to perform serial dilution and also
the one who are expert in aseptic techniques.

 The rate of absorption of drops on to the surface of agar depends upon the
environmental conditions like temperature and humidity.
Conclusion: Colony forming units determination is a skilled technique and the use of
this method makes it simple by using sectors and other methods. Any bacteria when
grows on nutrient agar, forms a visible colony. This colony is indication of growth of
bacteria on to the agar surface. This growth of bacteria which is called as colony
forming unit is generally an accumulation of generations of bacteria at certain locations
on the nutrient agar surface.
The most important step in this method is uniform absorption of bacterial suspension on
to the surface of agar media. Once this is done, the chance of cross contamination
within the sectors is reduced. The critical step therefore is to ensure that the drops
within the sectors do not cross the specified superficial lines. Once this step is over, the
plates can be further kept as it is to reduce the chances of micro-droplets of suspension
being transferred to other sectors. The inoculated plates are then kept in incubators in
inverted condition. These incubators may be either walk in incubators or simple small
incubators. Walk in incubators are generally used when the sample size is huge.
Once incubated in favorable growth conditions like temperature and humidity, the
growth of bacteria occurs within 18-24 hours of incubation. The temperature used is 37
degree Celsius which is optimum temperature of wide range of bacteria. This
temperature is very much near body temperature of human being. It is also observed
that this is the adaptation of bacteria to 37 degree Celsius which was not always there.
This is because; many of the bacteria can survive comfortably at lower temperature or
sometimes at higher temperature than 37 degree Celsius.
Since 1938, this method is widely used in determining colony forming units of many
microbial samples.