Cell Biology Laboratory Manual Table of Contents

  Techniques Manual Table of Contents

  Lab Safety Sheet

 

  Related Topics

Aseptic Technique

Sampling and Inoculation

Benedict's Test

Petri Dishes

Streaking for Isolation

Exoenzymes

Broth and Semi-solid Media

Staining Bacteria



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Microbiology Techniques

Quantifying Bacteria by Spread Plate

The number of bacteria in solution can be readily quantified by using the spread plate technique. In this technique, the sample is appropriately diluted and a small aliquot transferred to an agar plate. The bacteria are then distributed evenly over the surface by a special streaking technique. After colonies are grown, they are counted and the number of bacteria in the original sample calculated.

The end point of our analysis is the number of colony forming units per mL (CFU/mL) since we are counting the number of colonies rather than the actual number of bacteria. We are assuming that the each viable bacteria in the suspension will form an individual colony, which is a valid assumption if we do all the techniques properly. CFU/mL is actually a more useful determination than counting all the bacteria under a microscope because in many bacterial populations some significant number will be dead cells and thus of no interest.

Diluting the bacteria. Bacteria commonly grow up to densities around 109 CFU/mL, although the maximum densities vary tremendously depending on the species of bacteria and the media they are growing in. Therefore, to get readily countable numbers of bacteria, we have to make a wide range of dilutions and assay all of them with the goal of having one or two dilutions with countable numbers. We do this by making serial 10-fold dilutions (see serial dilutions section if this is an unfamiliar concept) of the bacteria that cover the whole probabey range of concentrations. We then transfer 0.1 mL of each dilution to an agar plate, which in effect makes another 10-fold dilution since the final units are CFU/mL and we are only streaking 0.1 mL.

Inoculating the plate. Streaking in this technique is done using a bent glass rod. 0.1 mL of bacterial suspension is placed in the center of the plate using a sterile pipet. The glass rod is sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through the Bunsen burner flame. The burning alcohol sterilizes the rod at a cooler temperature than holding the rod in the burner flame thus reducing the chance of you burning your fingers. When all the alcohol has burned off and the rod has air-cooled, streak the rod back and forth across the plate working up and down several times. Unlike streaking for isolation, you want to backtrack many times in order to distribute the bacteria as evenly as possible. Turn the plate 90 degrees and repeat the side to side, up and down streaking. Turn the plate 45 degrees and streak a third time. Do not sterilize the glass rod between plate turnings. Cover the plate and wait several minutes before turning it upside down for incubation. This will allow the broth to soak into the plate so the bacteria won't drip onto the plate lid.

Counting bacteria. Colonies are most readily counted using a plate counter. The plate counter has a light source and a magnifying glass making colonies easier to see. If at all possible, you don't want to count plates with more than 300 or less than 30 colonies. In the former case, the colonies will be running together and in the latter there are too few to allow statistically accurate counts. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample.