Enumeration Techniques of Bacteria in Microbiology

                    Enumeration is the estimation of the number bacteria in a sample such as water sample or food sample. Enumeration techniques help to determine the growth rate and death rate of bacteria. It gives the information about the degree of contamination of pathogenic microorganisms in MPN test.  

                Direct methods and indirect methods used for enumeration. Direct enumeration methods include pour plat, spread plate, cell chamber and florescent dyes.

                        Pour plate method uses 1mL of inoculum from the samples. 15mL of molten agar at 45◦ C and inoculum are added to plate and mixed by rotating it on the bench top. It is inoculated for 24-48 hours at 35◦ C. Bacterial colonies can be found both on surface and inside of the agar medium. These bacterial colonies are used to estimate number of bacteria in the initial sample. Pour plate methods help to isolate the microorganisms in the sample to isolate based on the oxygen availability. Aerobic respiratory bacteria can be found on the surface of agar. Microaerophilic bacteria can be found the inside of the agar. Anaerobic bacteria can be found at bottom of the agar. Bit pour plate method have some disadvantages too. The aerobic respiratory bacteria those are trapped inside the agar cannot survive. Heat sensitive bacteria may kill due to the high temperature of melting agar.

                       

                       Spread plate method use relatively lower size of inoculum such as 0.1ml from sample compare to the pour plate method. ImL of inoculum form the water sample is transferred to an agar plate. Then sterilized glass spreader is used to spread the inoculum over the surface of agar. It is also inoculated for 24-48 hours at 35◦ C. Bacterial colonies only grow on the surface of agar in spread plate method. If it is inoculated under aerobic conditions, aerobic respiratory bacteria can be isolated and estimated. If it is inoculated under anaerobic conditions, anaerobic respiratory bacteria can be isolated and estimated.

                              But both pour plate method and spread plate method do not give accurate results because these artificial cultural media are unable to provide extreme environmental conditions and nutrients. So these enumeration techniques do not estimate the accurate number in the samples.

                              In above both pour plate method and spread plate method, colony forming units (CFU) are taken into considered to estimate the number bacteria present in the initial sample. Colony forming units are the single microorganisms that are able to from visible single colony. So it is assumed that all colonies in the plates are made by single bacterium cell. Only plates with colony in-between 30-300 are counted.

            A dilution series is prepared in both methods as follows. Inoculum from the initial sample is transferred into test tube with 9mL of broth by using sterilized pipette. Its concentration is 10^-1. Then 1mL of first dilution is added to another into test tube with 9mL of broth by using another sterilized pipette. Its concentration is 10^-2 . Then 1mL of second dilution is added to another into test tube with 9mL of broth by using another new sterilized pipette. Its concentration is 10^-3 . Then 1mL of third dilution is transferred to another into test tube with 9mL of broth by using another sterilized pipette. Its concentration is 10^-4.. Then 1mL of forth dilution is added to another into test tube with 9mL of broth by using another sterilized pipette. Its concentration is 10^-5.

Streak plate method cannot be used for enumeration because the volume of loomful is known.                                       

                   Cell chamber method uses a petrioff to estimate number of bacteria. It is able to count the bacteria directly within the squares of petrioff slide and estimate according to the initial volume.

                   Fluroscent dye method is used to estimate the total microorganisms in samples such as soil and water. It gives accurate estimation of both living and dead bacteria. Acridine dye is applied to observe and estimate the bacteria.

                     MPN test and turbidity are the indirect methods of enumeration. In turbidly technique, turbidity of sample is measured and transferred this measurement into cell number. A plate series is prepared and measured the turbidity of them.  Standard curve is plotted and takes the number of bacterial cell in the initial sample aid in the graph.  

                     A series of dilution is prepared for MPN test. It is used in the estimate of number E coli present in fecal contaminated water sample. Inoculum of initial water sample is transferred as 1mL, 0.1mL and 0.01mL into spate test tube containing broth culture. Five replications for each dilution are made to increase accuracy. Every tube that shows growth is taken as the positive results. It may be acid production and gas collection in Durham tube. Ratio between positive results of each dilution is taken as MPN ratio.  Then number of bacteria in the initial water sample is taken from a MPN table which is made according to various other testing and based on statistics by using MPN ratio.