Enumeration of bacteria (from soil): standard plate count

Background

Enumeration of bacteria is defined as the process of determining the number of bacteria in a given sample. Studies involving the analysis of materials, including food, water, milk, and—in some cases—air, require quantitative enumeration of microorganisms in the substances. Many methods have been devised to accomplish this, including direct microscopic counts, use of an electronic cell counter such as the Coulter Counter, chemical methods for estimating cell mass or cellular constituents, turbidimetric measurements for increases in cell mass, and the serial dilution–agar plate method. Serial dilution–agar plate method will be adopted here

Principle

Microorganisms are ubiquitous and can be present in thousands or millions in a sample, making it difficult to count their numbers. However, serially diluting the cultures makes it easier to determine the count. This method involves serial dilution of a bacterial suspension in sterile water blanks, which serve as a diluent of known volume. After serial dilution, the aliquots of the diluted sample are plated on an appropriate culture media. Then, the plates are incubated, after which the number of colonies formed is counted. This technique is also known as plate count or colony counts. This method is based on the fact that, on a solid nutrient medium, the viable cells (a cell which is able to divide and form a colony) grow to form colonies that are visible to the naked eye. The resultant colonies are visible to the naked eye making visualization, selection and counting simpler. Plates suitable for counting must contain between 30-300 colonies. To arrive at a suitable dilution that results in this number, serial dilution method is used. The method involves making a tenfold dilution at a time by taking a small amount of the original sample (For e.g. 1 mL) and making up the volume (to say 10 mL) by adding an appropriate buffer or normal saline. For every dilution, a specific volume is plated on a suitable medium.                                                
Plate count method can be of two types:

Pour plate method: Molten agar, cooled to 45°C, is poured into a Petri dish containing a specified amount of the diluted sample. Following addition of the molten-then-cooled agar, the cover is replaced, and the plate is gently rotated in a circular motion to achieve uniform distribution of microorganisms. This procedure is repeated for all dilutions to be plated. Dilutions should be plated in duplicate for greater accuracy, incubated overnight, and counted on a Quebec colony counter either by hand or by an electronically modified version of this instrument. This technique allows the growth of anaerobes (beneath the surface) as well as aerobes (on the surface of the plate).

Spread plate method: A known volume of the dilution is plated on the nutrient agar plate. The colonies obtained can be selected and sub cultured easily for further use. The bacterial count in the original sample is estimated as CFU/mL (Colony Forming units/mL).                                                     
CFU/mL = CFU/volume of dilution plated × Dilution factor

Materials required

Sample (soil), dehydrated nutrient agar powder, Erlenmeyer flasks, culture tubes, Petri plates, spreader, beakers, 90% ethanol, 0.8% NaCl, micropipettes, test tube stand, autoclave, laminar air flow, bacteriological incubator, water bath set at 50°C.

Methodology Preparations

Prepare 400ml of nutrient agar (in Erlenmeyer flasks), 5 numbers of 9ml saline blanks, a 99ml saline blank (in Erlenmeyer flask), 20 Petri plates and autoclave.

Sample collection

Collect ~2g of garden soil sample in a sterile polyethylene bag using ethanol sterilized spatula. Weigh 1g of soil and keep them inside laminar flow, until further use.

Serial dilution and pour plate

1.  Following autoclaving, cool the molten agar in a water bath maintained at 50°C.                                        
2.  Transfer the sterilized Petri plates, saline, serial dilution tubes and micropipettes to ready-to-work (surface sterilized (using ethanol) and UV sterilized for 15 minutes) laminar air flow.                                        
3.   Cool the sterile 99ml 0.8% saline blank, and add 1g of soil and mix well. This dilution is regarded as 100 times dilution (10-2).
4.  Label the saline blank in flask as dilution 2 and five 9-ml saline blank tubes as dilution 3 through 7.                              
5.  Place the labelled tubes in a test tube rack. Label the Petri dishes in duplicates as P3, P4, P5, P6, P7 (pour plates) and S3, S4, S5, S6, S7 (spread plates).
6. With a sterile pipette, aseptically transfer 1 ml from the 99ml saline blank mixed with soil sample to tube 3. The sample has been diluted 1000 times to 10-3. Discard the tip.  
7.  Using sterile tip, add 0.1ml each from 3rd tube to 2 empty P1 plates and leave it aside
8.  Mix the mixture in the 3rd tube by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 3 to tube 4. Now the sample has been diluted 10,000 times to 10-4. Discard the tip.
9. Using sterile tip, add 0.1ml each from 4th tube to 2 empty P2 plates and leave it aside.        
10.  Using a fresh sterile tip, mix the mixture in the 4th tube by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 4 to the tube 5. Now the sample has been diluted 100,000 times to 10-5.
11.  Using sterile tip, add 0.1ml each from 5th tube to 2 empty P3 plates and leave it aside.                         
12.  Using a fresh sterile tip, mix the tube 5 by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 5 to the tube 6. Now the sample has been diluted 10, 00,000 times to 10- 6.
13.  Using sterile tip, add 0.1ml each from 6th tube to 2 empty P4 plates and leave it aside.  
14.  Using a fresh sterile tip, mix the tube 6 by gently pipetting up and down. Using a fresh pipette tip, transfer 1ml from tube 6 to the tube 7. The sample has been diluted 100, 00,000 times to 10-7. Dilution process is now complete.                                                                   
15.  Using sterile tip, add 0.1ml each from 7th tube to 2 empty P5 plates and leave it aside.    
16.   Check the temperature of the molten agar medium to be sure the temperature is above 45°C. Remove a media from the water bath and wipe the outside surface dry with a paper towel.
17.    Using the pour-plate technique, pour the agar into Plate P1 through P5 1A and rotate the plate gently to ensure uniform distribution of the cells in the medium.       
18.    Once the agar has solidified, incubate the plates in an inverted position for 24 hours at 37°C.
19.   Add and spread 0.1 ml of sterile saline in a nutrient agar plate to act as negative control. Open and leave a nutrient agar plate at the corner of the laminar to act as laminar control. Incubate theses controls along with other plates.

Spread plate technique

The spread-plate technique requires that a previously diluted mixture of microorganisms be used. During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent glass rod while the Petri dish is hold on hand or spun on a lazy Susan/turntable. The step-by-step procedure for this technique is as follows:

1.  Prepare soil sample suspensions as described above and label agar plates accordingly.
2.  Place the bent glass rod into a beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion.
3.  Place an appropriately labelled nutrient agar plate on the turntable. With a sterile pipette, add 0.1 ml of soil suspension on the center of the plate.
4.  Remove the glass rod from the beaker, and pass it through the Bunsen burner flame with the bent portion of the rod pointing downward to prevent the burning alcohol from running down your arm.
5.  Allow the alcohol to burn off the rod completely. Cool the rod for 10 to 15 seconds.
6.  Open the Petri plate lid and touch the agar surface with sterilized L-rod. Move the rod up, down and in side ways to spread the sample.
7.  Turn the plate at 90° angle and repeat the spreading process. Again turn the plate at 90° angle and do the spreading. For the last time, turn again the plate at 90° angle and do the spreading.
8.   Immerse the rod in alcohol and re-flame.
9.  Close the plate and allow the sample to adsorb on the agar surface for 3 minutes. Incubate the plates in an inverted position for 24 hours at 37°C.
10.  Add and spread 0.1 ml of sterile saline in a nutrient agar plate to act as negative control and open and leave a nutrient agar plate at the corner of the laminar to act as laminar control. Incubate theses controls along with other plates.

Result

Following 24hours incubation, sample and laminar control remained sterile. Pour and spread plates contained approximately 106 CFU/g and 107 CFU/g of soil, respectively.