8/17/2019 Bacteriology_ Determining Bacterial CFU by Miles & Misra Method

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Determining Bacterial CFU :A technique used in Microbiology to determine the exact number of cfu that is colonyforming units in a bacterial suspension or homogenate is known as The Miles and MisraMethod. This is a type of surface viable count technique. In 1 !" Miles and Misrainvented this method. In this method# Miles and Misra used dilution of $.pneumoniae onblood agar plate.

To learn this technique completely# let us understand what is its pre%requirements ofmaterials# its method# its advantages and disadvantages.

Material Pre-requirements:& A calibrated automatic pipette or a dropping pipette # delivering drops of '( )l.& $terili*ed nutrient agar plates& +iluent of ,hosphate -u ered $aline /,-$0& -acterial suspension or homogenate& aminar Air 2low -ench& 2ilter $terili*ed Isop%propyl alcohol

Method& $erial dilution of the inoculums 3 suspension is done in which# the dilution of 1xsuspension is added to x of diluent. In case of unknown sample quantity or unknownbacteria quantity# dilutions should be made to at least 1(4".& 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 '( )l drop is absorbed in the plate after 15%'( minutes of continuous spreading bynatural means or rotation of plates.& All the plates are equally divided into up to eight sectors. ,roper labeling of the platesare done to have traceability.& In each sector# 1 x '( )l of the dilution is dropped onto the surface of the agar andthus 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 !678 for 1" 9 ':hours.& ;ach sector is observed for growth# luxurious grown will be observed at highconcentrations over the area of the drop# or a large number of small colonies which aregenerally merged. 8olonies are counted in the sector where the highest number of full%si*e discrete colonies can be seen.& The following equation is used to calculate the number of colony forming units /82 Average number of colonies for a dilution x 5( x dilution factor.

Advantages& This technique is faster than other methods.& ?ith this technique# less bacterial contamination occurs of the working surface.& This technique is easy to process as compared to other techniques for determiningcolony forming units.

Disadvantages & This technique required high skilled microbiologist to perform serial dilution and alsothe one who are expert in aseptic techniques.

8/17/2019 Bacteriology_ Determining Bacterial CFU by Miles & Misra Method

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& The rate of absorption of drops on to the surface of agar depends upon theenvironmental conditions like temperature and humidity.

Conclusion: 8olony forming units@ determination is a skilled technique and the use ofthis method makes it simple by using sectors and other methods. Any bacteria whengrows on nutrient agar# forms a visible colony. This colony is indication of growth ofbacteria on to the agar surface. This growth of bacteria which is called as colonyforming unit is generally an accumulation of generations of bacteria at certain locationson the nutrient agar surface.

The most important step in this method is uniform absorption of bacterial suspension onto the surface of agar media. nce this is done# the chance of cross contaminationwithin the sectors is reduced. The critical step therefore is to ensure that the dropswithin the sectors do not cross the speciBed superBcial lines. nce this step is over# theplates can be further kept as it is to reduce the chances of micro%droplets of suspensionbeing transferred to other sectors. The inoculated plates are then kept in incubators ininverted condition. These incubators may be either walk in incubators or simple small

incubators. ?alk in incubators are generally used when the sample si*e is huge.

nce incubated in favorable growth conditions like temperature and humidity# thegrowth of bacteria occurs within 1"%': hours of incubation. The temperature used is !6degree 8elsius which is optimum temperature of wide range of bacteria. Thistemperature is very much near body temperature of human being. It is also observedthat this is the adaptation of bacteria to !6 degree 8elsius which was not always there.

This is becauseC many of the bacteria can survive comfortably at lower temperature orsometimes at higher temperature than !6 degree 8elsius.$ince 1 !"# this method is widely used in determining colony forming units of manymicrobial samples.