Direct and Viable Microbial Count Methods

Direct and Viable Microbial Count Methods

Image Source: R-Biopharm AG

1. Direct Counts

Counting Chamber Technique:

  • In the direct microscopic count, a counting chamber with a ruled slide is employed. It is constructed in such a manner that the ruled lines define a known volume.
  • The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation.
  • A culture is placed on the slide marked with precise grids. The number of cells present in each grid is counted and an average determined.
  • Used to count blood cells, Sperm count,
  • Conversion using a formula gives the number of cells per milliliter in the culture.
  • e.g. Petroff Hauser Chamber

Petroff Hauser Chamber/ Neubauer ruling chamber

  • Hemocytometer or ruling chamber is a glass slide with one or two stages for holding the sample. It is a thick glass slide having depression at the center, on both sides of which there are a groove and a coverslip.
  • At the center of the depressed portion, there is a grid of definite area which is divided into 9 squares, each with 1 mm2 area.
  • Each of the squares is divided again into 16 squares each having a 0.05 mm side. Counting is done under a microscope.
  • The distance between the coverslip and the central depression is just 0.1 mm in a hemocytometer and 0.2 mm in the Petroff-Hauser chamber.

Flow Cytometry

  • Mainly it is used for rapid analysis of single-cell or multiple microbes in a mixture and provide information about their distribution within the cell population
  • Microbial suspension forced through a small orifice with a laser light beam.
  • Movement of microbe through orifice impacts electric current that flows through the orifice.
  • Instances of disruption of current are counted.
  • Used in the pharmaceutical industry, environmental sample testing,  the dairy industry, manufacturers of probiotic cultures and products
  • Monitoring pathogens and check no of microbes in fermented food in a short time.

2. Viable Counting Methods

Viable Plate Counting

  • Viable Plate Count (also called a Standard Plate Count) is one of the most common methods, for the enumeration of bacteria.
  • Serial dilutions of bacteria are plated onto an agar plate. Either by spread plate method or pour plate method.
  • The dilution procedure influences the overall counting process.
  • The suspension is spread over the surface of the growth medium. The plates are incubated so that colonies are formed. Multiplication of a bacterium on solid media results in the formation of a macroscopic colony visible to the naked eye. It is assumed that each colony arises from an individual viable cell
  • The total number of colonies is counted and this number multiplied by the dilution factor to find out the concentration of cells in the original sample.
  • Counting plates should have 30-300 colonies at least. Since the enumeration of microorganisms involves the use of extremely small dilutions and extremely large numbers of cells, scientific notation is routinely used in calculations.

Spread plate technique:

  • Make a dilution series from a sample.
  • Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate.
  • Dip the L-shaped glass spreader into alcohol.
  • Flame the glass spreader (hockey stick) over a Bunsen burner.
  • Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petri dish underneath at the same time.
  • Incubate the plates in an inverted position at 37°C for 24 hours.
  • Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/ml in the original sample. Only plates with 30-300 colonies are statistical.

Pour Plate Technique

  • In this method, a fixed amount of inoculum (generally 1 ml) from a broth/sample is further inoculated in the molten agar medium.
  • Molten agar (approx. 15mL) is then poured into the Petri dish containing the inoculum and mixed well.
  • After the solidification of the agar, the plate is inverted and incubated at 37°C for 24-48 hours.
  • Microorganisms will grow both on the surface and within the medium.
  • Colonies that grow within the medium generally are small in size and maybe confluent; the few that grow on the agar surface are of the same size and appearance as those on a streak plate.
  • Each (both large and small) colony is carefully counted (using magnifying colony counter if needed).
  • Each colony represents a “colony-forming unit” (CFU).
  • The number of microorganisms present in the particular test sample is determined using the formula:
    • CFU/mL= CFU x dilution factor  /volume
  • For accurate counts, the optimum count should be within the range of 30- 300 colonies/plate.
  • To ensure a countable plate a series of dilutions should be plated.

Disadvantages of Pour plate method

  • Preparation for the pour plate method is time consuming compared with streak plate/and or spread plate technique.
  • Loss of viability of heat-sensitive organisms coming into contact with hot agar.
  • The reduced growth rate of obligate aerobes in the depth of the agar.

Membrane filter technique

  • Bacteria from aquatic samples are trapped on membranes.
  • Membrane placed on culture media
  • Colonies grow on membrane
  • Colony count determines the number of bacteria in the sample

MPN Method

  • Most Probable Number (MPN) is a method used to estimate the concentration of viable microorganisms in a sample by means of replicate liquid broth growth in ten-fold dilutions.
  • MPN is most commonly applied in the water industry for quality testing of water i.e to ensure whether the water is safe or not in terms of bacteria present in it.
  • MPN test is performed in 3 steps
  1. Presumptive test
  2. Confirmatory test
  3. Completed test

Presumptive test

  •    It is achieved by performing serial dilutions of a bacterial culture, dividing the sample into aliquots or replicates followed by incubation and subsequent visual examination of each sample for growth.

Confirmatory Test

  • Performed when the presumptive test is positive.
  • In this process, a loopful of the sample from each tube showing a positive test (color change with gas) is streaked onto two selective medium like EMB (Eosin Methylene Blue) or Endo’s medium.
  • Incubated at 37°C for 24 hrs.


  • Dark centered colonies with a metallic sheen on EMB agar.
  • Pinkish colonies on Endo’s medium.



Direct and Viable Microbial Count Methods

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