Plate Counts

Plate counts measure the number of viable cells in a sample by exploiting the fact that an isolated cell on a nutrient agar plate will give rise to one colony. A simple count of the colonies determines how many cells were in the initial sample (figure 4.13). The two different plating methods, pour-plate and spread-plate, differ in how the suspension of bacteria is applied to the agar plate. As the ideal number of colonies to count is between 30 and 300, and samples frequently contain many more bacteria than this, it is usually necessary to dilute the samples before plating out the cells. The sample is normally diluted in 10-fold increments, making the resulting math relatively simple. The diluent, or sterile solution used to make the dilutions, is generally physiological saline (0.85% NaCl in water). Distilled water can be used, but some bacteria may lyse in this hypotonic environment.

In the pour-plate method, 0.1 to 1.0 ml of the final dilution is transferred into a sterile Petri dish and then overlaid with melted nutrient agar that has been cooled to 50°C. At this temperature, agar is still liquid. The dish is then gently swirled to mix the bacteria with the liquid agar. When the agar hardens, the individual cells are fixed in place and, after incubation, form distinguishable colonies.

In the spread-plate method, 0.1 to 0.2 ml of the final dilution is transferred directly onto a plate already containing a solidified nutrient agar medium. This solution is then spread over the surface of the agar with a sterilized bent glass rod, which resembles a miniature hockey stick.

In both methods the plates are then incubated for a specific time to allow the colonies to form, which can then be counted. By knowing how much the sample was diluted prior to being plated, along with the amount of the dilution used in plating, the concentration of viable cells per milliliter in the original sample can then be calculated. Cells attached to one another form a single colony and are counted as a single cell or colony-forming unit.

Pour plates and spread plates are generally only used if a sample contains more than 100 organisms/ml. Otherwise, few

Transfer 1 ml

Transfer 1 ml

Transfer 1 ml

Transfer 1 ml

Transfer 1 ml

Transfer 1 ml

Total dilution 1:10

Total dilution Total dilution 1:100 1:1000

Total dilution 1:10

Figure 4.13 Plate Counts (a) A sample is first diluted in 10-fold increments. (b) In the pour-plate method, 0.1-1.0 ml of a dilution is transferred to a sterile Petri dish and overlaid with melted, cooled nutrient agar.When the agar hardens, the plate is incubated and distinguishable colonies form on the surface and within the agar. (c) In the spread-plate method, 0.1-0.2 ml of a dilution is spread on a hardened agar plate with a sterile glass rod. After incubation, distinguishable colonies form only on the surface of the agar.

Total dilution Total dilution 1:100 1:1000

(a) Serial Dilutions

(a) Serial Dilutions

Dish is swirled to mix solution; dish is incubated

Rod spreads solution evenly; dish is incubated

Dish is swirled to mix solution; dish is incubated

Rod spreads solution evenly; dish is incubated

(b) Pour Plate

(c) Spread Plate

4.6 Methods to Detect and Measure Bacterial Growth if any cells will be transferred to the plates. In these situations, alternative methods give more reliable results.

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