Methods for enumeration of soil microorganisms

Quantitative analysis of soil microorganisms

Soil may contain up to 10⁸–10¹² bacterial cells per gram of soil.

There are some common (basic) procedures used to measure the number of individuals in the soil: the direct microscopic count, the viable count and the MPN method (Most Probable Number).

The direct microscopic count involves the enumeration of the total number of microbial cells in a soil sample both living and dead, with the aid of a microscope.

The viable count involves counting the number of living cells that form colonies on agar plates or solid media. Each viable microorganism in a suspension will give a single colony after incubation in a suitable media. After incubation the number of colonies is counted. In this case the viable counts are expressed as colony forming units (CFU)/ml.

Determination of CFU can be done with pour plate method and spread plate method.

The pour plate method:

The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air, for example water samples.  In the pour plate method a diluted bacterial sample is mixed with melted agar and then that mixture is poured into a petri dish. The colonies would be counted and the viable cell count calculated.

The steps of the method are the following:

• Melt in boiling water bath the sterile agar media contained in the tubes, then cool the medium at 50–55^oC.
• Make tenfold dilution series from the soil samples in sterile water (10¹–10⁴).
• Pipette 0.1−0.1 ml from dilutions of the soil suspensions into labelled Petri dishes.
• Pour cooled agar media onto the sample suspension within the Petri dish and swirl the plate carefully to mix the sample with the agar.
• After the plates are solidified place the plates into an incubator at 30–37^oC (it depends on the media).
• Incubate the plates for 24–48 hours.
• Count the colonies on plates.

The spread plate method:

With a spread plate one makes serial dilutions in liquid media and then spreads a known volume from the last tube in the dilution series. The colonies on the plate can then be counted and the concentration of bacteria in the original culture can be calculated.

The steps include the following:

• Melt in boiling water bath the sterile agar media contained in the tubes.
• Pour cooled agar media into the Petri dishes. Wait until the media is solidified.
• Make tenfold dilution series from the soil samples in sterile water (10¹–10⁴).
• Pipette 0.1−0.1 ml from dilutions of the soil suspensions onto the surface of the solidified agar.
• Using a glass spreader spread the suspension over the whole surface of the agar.
• Place the plates into an incubator at 30–37 ^oC (it depends on the media).
• Incubate the plates for 24–48 hours.
• Count the colonies on plates.

The MPN method is based on the presence or absence of bacteria using ten-fold dilution in which replicate tubes of general or special (selective) broth are inoculated with 1 ml aliquots of the serial dilution. Growth or positive tests of a specific product (gas formation, acid production etc) are recorded at the end of incubation time. 3–5 parallel tubes per dilution are used, then evaluate the results based on Hoskins table.