Enumeration Of Microorganisms

Microbiologists have developed a number of techniques suitable for the determination of microbial numbers in a sample. These include:

1. Direct microscopic enumeration of a sample of stained bacteria 2. Cultural techniques 1. Plate counts 2. Most Probable Number (MPN) Techniques 3. Chemical assays and other indirect assays. All of these techniques have their own advantages and limitations. The important concept for you to remember is that none of these techniques is perfect, and in all likelihood none of these techniques gives a completely accurate enumeration of the microorganisms in a sample.

Historically, the most commonly used techniques for microbial enumeration are the cultural techniques. The ability to grow a microorganism in culture still remains the gold standard routine of microbiology. If you can grow it, you know it is viable.

OBJECTIVE: In this laboratory, you will learn one of the cultural techniques for the enumeration of microorganisms - the plate count technique. You will learn the MPN technique in the drinking water laboratory exercise.

The plate count procedure consists of two separate but interrelated manipulations of the culture __ serial dilution and placement of microorganisms in/on a solid medium.

I) SERIAL DILUTIONS Microbial numbers in environmental samples may be extraordinarily high. For example the number of heterotrophic bacteria in a waste water sample may be 3,000,000,000 cells/mL (3x 109 CFU/mL). If you were to take 1 mL of this waste water sample and spread it over the surface of an agar plate, you would in theory get the formation of 3 x 109 colonies (1 cell gives rise to 1 colony). Obviously, you could not count this many colonies. Therefore, in order to have a manageable number of colonies on the plate, it is necessary to dilute the sample. For statistical reasons, the optimal number of colonies on a plate is between 30 and 300 colonies. Therefore, you need some way to dilute your sample so that the number of organisms per unit volume is between 30 and 300 cells.

In order to dilute the microbial solution to the desired concentration of cells (assume 300 cells per mL is the desired concentration unit) it is necessary to dilute the original sample by a factor of 1 x 107. You could do this by transferring 1 mL of your original sample to 999,999 mL (9,999.9L) of sterile dilution water. It is not realistic, however to prepare almost 10,000 L of sterile dilution water every time you need to dilute a sample. In order to arrive at the concentration desired, use serial dilutions instead of making one big dilution. Serial dilutions are simply sequential dilutions. In microbiology, serial dilutions are usually made on a 1:10 and 1: 100 fold basis using small volumes of sample and diluent. By repeating 1: 10 and 1: 100 fold dilutions it is easy to get any order of magnitude dilution you want.

Table 1 summarizes common ways to make 1:10 and 1:100 fold dilutions.

Enumeration of microorganisms is accomplished by first diluting the sample and then spreading a known amount of the diluted sample over the surface of an agar plate. It is important to note that since we are interested in the concentration of microorganisms (cells/mL) spreading of a volume less than 1 mL onto the surface of a plate is the same as diluting the sample. ________________________________________ Table 1: Common Dilutions Dilution Volume sample Into Volume Diluent 1:10 10 uL (0.01 mL) 90 uL (0.09 mL) 100uL 900 uL 1 mL 9 mL 1:100 10 uL 990 uL 100 uL 9.9 mL 1 mL 99 mL Note: Plating 0.1 mL (100 uL) is the same as a 1: 10 dilution. Plating 0.01 mL is the same as a 1:100 diluton. ________________________________________

PRACTICE EXERCISE

With this basic understanding of serial dilutions, let`s work an example problem. Given that the initial solution of microorganisms contains 5 x 108 cells/mL, how should it be diluted to get a final concentration of microorganisms between 30 and 300 cells per mL?

1. Determine the target final concentration: The final concentration should be 50 cells/mL. This is within the 30-300 range. 2. Divide overall dilution needed into a series of 1:10 and 1:100 dilutions. 1. Three 1: 100 fold dilutions in series will give a final dilution of 1: 1 x 106 ? 1 mL of the original culture (5 x 108 cells) into 99 mL of diluent (dilution A) = 5 x 108 (cells)/ 100 mL (final volume = 99 mL + 1 mL) = 5 x 106 cells/mL ? 1 mL of dilution A into 99 mL of diluent (dilution B) = 5 x 106 cells/100 mL = 5 x 104 cells/mL. ? 1 mL of dilution B into 99 mL of diluent (dilution C) = 5 x 104 cells/100 mL = 5 x 102 cells/mL. 3. A 1: 10 dilution of this solution (dilution C) will give a final dilution of 1 mL of dilution C into 9.0 mL of diluent = 500 cells/10 mL = 50 cells/mL

II) Spread Plating (Placement of Microorganisms on the Solid Media)

Once you have diluted your culture to an acceptable range, the next step in microbial enumeration involves spreading the organisms over the surface of a petri dish containing a suitable solid medium (agar). By spreading the inoculum over the plate, you physically separate bacterial cells on the surface. During incubation, each separate cell should replicate and form a colony which you can see with the naked eye.

Microorganisms are spread on the surface of a solid medium using a sterile bent glass rod (disposable sterile plastic rods are now available also). In order to make a spread plate you perform the following steps:

1. Aseptically transfer the desired amount of an appropriated diluted culture to the surface of a plate. 2. Sterilize a bent (L) glass rod 1. Dip the L-rod in alcohol 2. Quickly pass the L-rod through the flame of a bunsen burner 3. Allow the alcohol to burn off 3. Using the sterile L-rod, spread the inoculum over the surface of the plate.

MATERIALS

1. Culture of microorganisms 2. Sterile Pipettors and tips 3. Dilution tubes (test tubes containing premeasured amounts of sterile dilution water) 4. Bunsen burner 5. Petri plates containing Trypticase Soy Agar (6 per student) 6. Inoculation L-rod 7. Alcohol 8. Marking pen 9. Colony counter

METHOD

Class 1 1. If you have not already done so, disinfect your laboratory bench 2. Determine the dilutions you will need to plate to bracket the countable range of 30-300 colonies per plate. To do this you will need to estimate the number of cells/mL in the culture. If the culture appears slightly turbid, there are approximately 1 x 106 cells/mL or greater. You should calculate the dilutions and amounts of inoculum to be plated so that 3. Obtain sufficient dilution tubes to make the serial dilutions needed. Note, the dilution tubes available contain 9.0 mL of sterile dilution buffer. Label the tubes to identify the dilution they will contain. 4. Obtain 6 petri plates with Trypticase Soy Agar. Label the bottom of the plates with your name and today`s date. In addition, label two of the plates with the lowest (least dilute) dilution you have decided to plate, two with the estimated optimum dilution, and two with the highest dilution, 5. Bring a culture to your workbench 6. Vortex the culture tube to mix the microorganisms. 7. Perform the appropriate serial dilutions 8. From the tube corresponding to the lowest dilution transfer 0.1 mL of the contents to the surface of each of the two appropriately labelled plates. 9. Dip the L-rod in alcohol and flame it. 10. Using the sterile L-rod, spread the inoculum over the surface of the plate. 11. Repeat for the next two dilutions. 12. Invert the plates and place them in the incubator. When colonies form after 18-24 hrs, store the plates inside a refrigerator for future use.

Class 2 1. Obtain the plates you inoculated last week. 2. Using a Quebec colony counter, count the number of colonies on each plate. Record the dilution, the amount plated and the number of colonies for each plate. 3. Using the appropriate plates, calculate the geometric mean of the number of colonies. Record this value in your notebook.

Geometric mean is a kind of average of a set of numbers that is different from the arithmetic average. The geometric mean is well defined only for sets of positive real numbers. This is calculated by multiplying all the numbers (call the number of numbers n), and taking the nth root of the total. A common example of where the geometric mean is the correct choice is when averaging growth rates.