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� CS 566: ADVANCED��SOIL MICROBIOLOGY�

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Module 3

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Identification & Enumeration of Soil Microflora

  • For most purposes identification required removal from the soil – isolation.

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  • Isolation – Mechanical e.g. removal of fungal spores or physical followed by cultivation.

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Preservation of microbial cultures

  • For further references
  • Comparisons
  • Re-examination
  • Future usage

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Basic Principle: - Keep the morphological and physiological characteristic of organism intact.

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Why culture?

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Preservation methods

  • Culture tubes (agar slants)

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  • Preservation in soil (3 parts air dry soil + I part dried humus)

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  • Lyophilization

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Detection and measurement of activities in soil

  • General activity measurements
    • Respiration measurements (oxygen uptake or carbon dioxide evolution)
    • Cell division rate
    • Mycelial extension
    • Enzyme activity or content
    • Substrate utilization rate
    • Product accumulation rate
    • Radioisotope cycling studies

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  • CO2 evolved:
        • Milligrams C or CO2 = (B - V)NE
  • Where
    • V = volume (ml.) of acid to titrate the alkali in the CO2 collectors from treatments to the end point,
    • B = volume (ml.) of acid to titrate the alkali in the CO2 collectors from controls to the end point,
    • N = normality of acid, and
    • E = equivalent weight. If the data are expressed in terms of carbon,

E = 6; if expressed as CO2, E = 22.

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Examination of microbes by staining techniques

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  • The morphology of bacteria is examined in two ways:

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  • By observing the living unstained organisms

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  • By observing dead cells stained with dyes

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Thus the aim of staining is to be able to visualize unique microstructures or basic morphological features of microbial cells.

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Simple staining – this requires only one type of dye for coloration of a bacterial smear. Basic dyes used include Methylene blue and Crystal Violet

Differential staining is based on the principle that microorganisms differ from one to another chemically and physically, thus they react differently to a given staining procedure. The most common differential stain is GRAM STAIN.

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�������Hans Christian Joachim Gram� Danish Bacteriologist

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  1. Gram-negative microorganisms e.g. Rhizobium, Azotobacter, Azospirillium and Pseudomonas.

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  • Gram-positive microorganisms e.g. Bacillus

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Gram Staining Procedure

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  • Apply specimen to slide.

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  • Heat fix to slide

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  • Apply crystal violet primary stain for 1 minute

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  • Wash off excess stain.

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  • Apply Gram’s iodine mordant for 1 minute.

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  • Wash off excess mordant.

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Cont’d

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  • Apply acetone/alcohol decolorizing agent for about 30 seconds.

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  • Wash off excess decolorizing agent

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  • Apply safranin counterstain for 30 seconds.

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  • Wash off excess counterstain.

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  • Blot dry.

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The Cell walls differ…

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Colour makes the Difference in Gram staining

  • Bacteria that manage to keep the original purple dye have only got a cell wall - they are called Gram positive.

  • Bacteria that lose the original purple dye and can therefore take up the second red dye have got both a cell wall and a cell membrane - they are called Gram negative.

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Common errors in staining procedure

  • Excessive heat during fixation.
  • Low concentration of crystal violet.
  • Excessive washing between steps.
  • Insufficient iodine exposure.
  • Prolonged decolourization.
  • Excessive counterstaining.

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