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.
• A calibrated automatic pipette or a dropping pipette , delivering drops of 20 μl.
• Sterilized nutrient agar plates
• Diluent of Phosphate Buffered Saline (PBS)
• Bacterial suspension or homogenate
• Laminar Air Flow Bench
• Filter Sterilized Isop-propyl alcohol
• 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 10−8.
• 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 μl 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 μl 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 37°C 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 full-size 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.
• 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.
• 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.