Measurement of soil microbial numbers, biomass, and metabolism

Two basic approaches:

1. Direct count procedures

2. Indirect count procedures

Direct count procedure

includes viable and total counts, methods include plate counts, MPN, and direct microscopic counts

Indirect count procedures are used when

It is difficult or undesirable to determine the number of microorganisms

Indirect count procedures

Measures quantifiable microbial cell properties that are directly related to cell growth

Microbial growth properties that can be measured by indirect count procedures include

Change in amount of cell component (measures cell weight) and turbidity (based on light scattering)

A sample has high turbidity when

The solution becomes more cloudy

T or F: Direct count produces the highest estimates of microbial numbers

True

Direct count advantages

• allows for observation of microbes that are bound to particles

• Useful for determining form and arrangement of microorganisms in the soil

Direct count allows for enumeration of phases on:

Bacteria cells, viruses on plants, bacteria in sediments fungal mycelia in soil

Disadvantages of direct count method

• impossible to distinguish living from dead microbes

• Excessive amounts of background debris in sample often makes it difficult to distinguish microbial forms, results in underestimation

• Does not allow further study of microbes (determination of species and physiological forms)

Why is cell suspension important in microbiology?

Necessary to determine cell concentration

How is cell density measured??

spectrophotometrically

T or F: A spectrophotometer allows assessment of cell viability and distinguishes cell types

False, it measures the amount of light absorbed

Hemocytometer

The most widely used type of chamber, originally designed for performing blood cell counts

Counting chamber

A device used to determine the number of cells per unit volume of a suspension. They are good for large microbes such as Protozoa, algae, and fungi.

Methods for direct count

1. Counting chambers (small organisms, bacteria)

2. Thin section technique (soil, sediment, plant and animal cells)

3. Agar film technique (enumeration of fungi)

4. Fluorescence microscopy (counting bacteria and algae)

5. Fluorescent antibody technique (used for direct count, good for auto ecological studies)

6. Scanning electron microscopy (replaces light microscopy for direct count of microorganisms)

Two basic approaches to viable count procedures

1. Plate count techniques

2. Most probable number (MPN) techniques

Main consideration for plate count procedures

Composition of medium, incubation conditions, length of incubation

Advantages of agar plate method

Conditions can be adjusted so that only members of a defined group can be enumerated

Fungi bacterial inhibitors added in the agar plate method

Rose Bengal, streptomycin, neomycin

Disadvantages of agar plate method

• not ideal for fungi (favors non-filamentous and spores)

• Light necessary (photosynthesis)

• Omit carbon sources to prevent heterotrophic bacteria

T or F: in the agar plate method, basidiomycetes are underestimated by plate counts techniques

True

MPN technique

Statistical analysis based on Poisson distribution. Can be used to estimate total microbial populations

T or F: like the plate count method, MPN suffers the same limitations in terms of total microbial population

True

The MPN method has two assumptions:

1. Test substrate must be well dispersed throughout initial and subsequent dilutions

2. One or more organisms contained within an inoculate volume are capable of producing a positive result

Replicate dilution must be used normally _____

3 to 10 replications/dilutions

Advantages of MPN

• permit liquid culture, avoids need for solidifying agent such as agar

• Liquid cultures are superior to plate counts for enumeration of some specific groups of microbes (algae, fungi)