The Enumeration Of Microorganisms
Enumeration is the determination of the number of individual viable microbes in a sample. Enumeration of microorganisms is performed to determine the number of microorganisms in a sample because the agar method of enumerating visible colonies is time consuming and not accurate. It is essential to measure the number of viable microorganisms accurately because it is considered as a key step in developing new antimicrobial agents, and the determination of minimum inhibitory concentration (MIC). The three enumeration techniques (direct count, viable count, turbidity study) makes the counting of microorganisms easier since the microorganisms are too small to be seen with naked eyes. The direct counting of microorganisms is done through a microscope using a hemocytometer. Hemocytometer is a device used for the direct counting method. It creates a grid in different size cubes which makes the counting of the microorganisms easier. Direct methods are generally more sensitive and precise compared to indirect methods. Microscopy is a prominent technique used for searching direct methods for enumerating microorganisms and it is considered as one of the first used technique in the food industry. However, the disadvantages of microscopic techniques such as lack of sensitivity, operator fatigue after prolonged used of the microscope, and bothersome debris can affect the direct counting since the direct counts are more sensitive and precise. A viable cell is able to divide and form a population (or colony). The viable counting is done by diluting the original sample, plating aliquots of the dilutions onto an appropriate culture medium and then incubating the plates under proper conditions to form the colonies. However, the disadvantages of this technique are that it requires direct count to obtain dilution factor and it is time consuming. It takes 1 to 3 days to obtain the results. The turbidity study is a quick and efficient method for estimating the number of bacteria in a liquid medium. This method is commonly used for counting large number of cultures since it is considered as a fast method of enumeration. According to the study done by the USGS Water Science School, turbidity is the measure of clarity of the liquid. It determines the quality of water and is measured using Spectrophotometer.
For this enumeration technique, a hemocytometer, coverslip, counter and a compound light microscope were obtained. To perform the direct count method, first the hemocytometer grid was focused under 40X total magnification and then the coverslip was applied on it. Next, a drop of yeast culture was added to the groove of the hemocytometer. After that the grid was focused under 100X total magnification to make 25 larger boxes visible (each consisting of 16 smaller boxes in a 4X4 pattern) and then the focus was shifted to 400X total magnification to view an individual large box (4X4 small boxes). Next the five large boxes were counted using the counter and the number of cells in each box were recorded. At last the actual cell number per ml was calculated using the formula, (total cell count per 5 boxes) x 5 x 104 = cells per ml of solution.
For this enumeration technique, clean petri dishes, sterile transfer pipettes, 99 ml sterile DI H2O bottles, 9 ml sterile DI H2O tubes and sterile melt YED agar in liquid form (in water bath with 58-60C) were obtained. To perform the viable count method, first the viable count was calculated from the results of direct count by using serial dilutions so that the total cell count number is from 30 to 300 in the final dilution. Next two 99 ml water bottles were obtained and labeled with (1) 1:100 (10-2) and (2) 1:10,000 (10-4) and one 9 ml water tube was obtained and was labeled with (3) 1: 100,000 (10-5) respectively. After that 1 ml of undiluted yeast culture was added into bottle (1) and was mixed well for few seconds. Next 1 ml from bottle (1) was taken and transferred into bottle (2) and was mixed well. After that 1 ml from bottle (2) was taken and transferred into 9 ml water tube (3) and was again mixed properly. After the serial dilutions of yeast culture, it was ready to be plated on YED medium. Next the YED agar bottles were melted and kept in the water bath with the temperature set at 58C to maintain the liquid form. After that the YED was poured into the dishes until the dishes were half full and then plating of the required amount of culture from tube (3). Next two petri dishes were obtained and labeled as plate A, 1 ml and plate B, 2 ml respectively onto the plates when the medium was still in liquid form and it was swirled gently to mix. Then the plates were left at the room temperature until the plates solidified and then the petri dishes were inverted and after the plates were inverted they were placed into 37C incubator for 2 to 3 days. After 24-48 hours the plates were taken out of the incubator and the colonies on the plates CFU (colony forming units) were counted using the formula, Viable count (cell number/ml) = the plate count x dilution factors. The percent viability was calculated using the formula, Viability = [viable count/direct count] x 100%.
For this enumeration technique, undiluted yeast culture, DI H2O, clean test tubes, spectrophotometer and cuvettes were obtained. To perform the turbidity study, the serial dilutions were made and then the optical density (OD) measurement was taken. To make serial dilutions, first 8 ml of undiluted yeast culture (in DI H2O) was obtained in a clean test tube labeled (1) 1:1. Next 4 ml of yeast culture was taken from test tube (1) and was placed it into 2nd clean test tube and then 4 ml was of DI H2O was added and it was labeled as (2) 1:2 and it was mixed properly before moving onto next step. After that 4 ml of yeast culture was taken from test tube (2) and was placed it into 3rd clean test tube and then 4 ml of DI H2O was added and it was labeled as (3) 1:4 and it was again mixed properly. Next 4 ml of yeast culture was taken from test tube (3) and was placed into 4th clean test tube and then 4 ml of DI H2O was added and it was labeled as (4) 1:8 and was mixed well. At last 4 ml of yeast culture was taken from test tube (4) and was placed into 5th clean test tube and then 4 ml of DI H2O was added and was labeled as (5) 1:16 and mixed properly. At the end, all the test tubes should have approximately 4 ml of yeast solution, except for the final test tube which will have 8 ml of the yeast solution. To measure the optical density (OD), first the spectrophotometer was turned on and the wavelength was set up at 686 nm. After that the spectrophotometer was left to warm up for 20-30 minutes before using it. Next 6 clean cuvettes were obtained and were labeled as “B” (as blank), “1”, “2”, “3”, “4”, and “5” respectively. After that 1 ml of DI H2O was dropped into cuvette “B”. Next 1 ml yeast solution from test tube (1) was dropped into cuvette “1”, 1 ml yeast solution from test tube (2) was dropped into cuvette “2”, 1 ml yeast solution from test tube (3) was dropped into cuvette “3”, 1 ml yeast solution from test tube (4) was dropped into cuvette “4”, and 1 ml yeast solution from test tube (5) was dropped into cuvette “5”. Next the spectrophotometer 20 was zeroed with the blank (cuvette “B”). After that each cuvette was placed into the spectrophotometer 20 one by one to measure the optical density (OD) as % absorbance and was recorded on the data sheet. The spectrophotometer 20 was blanked using cuvette “B” between each sample.
The purpose of this experiment was to determine the number of microorganisms in a given sample using the different enumeration technique which are direct count, viable count, and turbidity study. The enumeration technique makes the counting of the cells easier and further helps in identifying living cells in a particular population.
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