Enumeration and Isolation

PRACTICE ENUMERATION AND ISOLATION 4 Calculation of the number of microbes (enumeration) contained in a sample can be done in various ways, including: dilution method, direct calculation method in a counting chamber (hemacytometer), filter membrane method, dry weight and cell volume method, MPN method, etc. In this practicum, you will learn to determine the number of cells contained in a sample using the dilution method and direct counting method using a hemocytometer (Improved New Bauwer). 4.1 Dilution method This dilution method uses a series of dilutions of samples which are then grown in medium. After incubation, the colonies that grow can be counted with the assumption that one colony that grows comes from one cell. Thus, the number of cells in the original sample can be calculated by multiplying the number of colonies that grow by the dilution factor. Dilution is usually expressed to a negative power, for example 10-5for dilution 1: 100,000. By using this method we can calculate the total number of microbes contained in a water sample or soil sample. Tools and materials a. Water and soil samples b. NA media c. Sterile bottle d. Petri dish e. Sterile water in a test tube f. Measuring pipette Ways of working a. Take water samples aseptically using a sterile bottle. b. Melt the NA medium in a water bath and cool to 45oC. c. Mark 6 petri dishes with 10-1, 10-2,…10-6. d. Shake the water sample until homogeneous and take 1 ml aseptically, then put it in a test tube containing 9 ml of sterile water to get a dilution factor of 10 times (10-1). e. Shake this test tube well, and aseptically take 1 ml of the water in it, and put it in a petri dish marked 10-1. From this tube too, take 1 ml of sample and put it in a second test tube containing 9 ml of sterile water to obtain a dilution factor of 100 times (10-2). f. Pour the NA medium into the petri dish marked 10-1which already contains 1 ml of sample water, then shaken until homogeneous. g. Take 1 ml of sample from the second test tube and put it in the second petri dish (10-2). From this tube too, take 1 ml of sample and put it in a third test tube containing 9 ml. sterile water to obtain a dilution factor of 1000 times (10-3). h. Repeat this work until the dilution factor is 1,000,000 times (10-6). i. To calculate the total microbes in a soil sample, the procedure used is exactly the same as above, but the number of samples that must be weighed is 1 gram of solid sample or 1 ml of liquid sample (Pay attention to the attachment!!!!). j. Incubate all plates that have been planted with samples containing microbes at a temperature of 25oC for 1 – 7 days. k. Count the colonies that grow in all Petri dishes where the number of colonies ranges from 30 – 300 colonies. l. The number of microbial cells contained in the sample is calculated by multiplying the number of colonies growing in the medium by the dilution factor.