Enumeration of bacteria

Determination of number of micro-organisms

( Enumeration of bacteria )

The term growth as commonly applied in microbiology refers to the magnitude of the total population . Growth in this sense can be determined by numerous techniques based one more of the following types of the measurement

• Cell count:Directly by microscopy or by using electric particle counter or indirectly by a colony count
• Cell mass:Directly by weighing or by measurement of cell nitrogen or indirectly by turbidometry.
• Cell activity:Indirectly by relating the degree of biochemical activity to the size of population

Sometimes because of growth patterns(Eg filaments,clumps )or of the complexity of the medium,it is impractical to measure the cell mass and no . In this cases , growth can be measured by determining the amount of particular in cell constituents ( eg , proteins, DNA, RNA, or ATP) the medium. Such measurements are often the most practical way to determine microbial mass and growth in a natural environment.

Each of the methods has its particular advantages limitations and no one method can be recommended universally. Certain specific producers will illustrate the application of each type of measurement . The best producers can be selected only after these producers are considered in relation to the problem at hand.

Microscopic count ( no.of cells/ml)

• Direct count:

Eg; breed smear( for direct sample)

• Neubauer's chamber ( for liquid sample)
• Electronic cell counter

Electronic enumeration of cell numbers(no of cells /ml)

Plate counts(CUF/ml)

Measurement of biochemical activity(Eg;acid production by culture)

Nitrogen determination (MgN₂/ml)

Dry weight determination(Mg dry weight of cell/ml)

Indirect count

• Determining cell mass(dry weight)
• Oxygen uptake,CO₂ production
• Turbidimetric measurement(spectrophotometric or colorimetric analysis,optical density/absorbance)
• Membrane filter count(count living cells only)
• Social dilution-agar plate(plate count method)

Bread count method

Bread count technique is both viable and non-viable count method in which no of bacteria or yeast or algae or any organism in a sample of both culture,milk,beer,fruit juices etc. may be determined by direct microscopic organisms.In this method of direct count, a known volume of sample(0.01ml)is uniformly spread over one Cm² area of a slide.The resulting film is dried and stained.The average no. of the organism per microscopic field is counted.The diameter of microscopic field is determined by micrometre.Which is used for calculating the area of the microscopic field.Then,the no. of bacteria in the sample is calculated by using the formula;

N(Tc)=n x F

Where N(Tc)=Total no. of organism in one microscopic field

F=No of microscopic fields.

F=Area of smear x 100/Area of microscopic field(A)

A=π r²(r=radius of microscopic field)This method always gives high no of microorganism than plate counts or any other methods as this includes living as well as death inactive organisms.In other words,it does not distinguish between living and death cell and this is the drawback of direct count other limitations of the direct count are small cells are probability missed.It is not suitable for cell suspension of low density.Measurement of cell concentration by using a counting chamber(Haemocytometer or Neubauer’s chamber)

Measurement of cell concentration by using a counting chamber

The haemocytometer is used for counting the organism in the liquid medium.It is a special microscopic side with a counting chamber 0.1 mm square is 0.1 mm³.The counting chamber has a total of nine squares,each of 1mm x 1mm engraved over it but only 1 square field is visible under 100(times) x magnification.A 1 mm square in the centre is divided into 25 medium sized squares(0.2mm x 0.2 mm each).Each of which is further subdivided into 16 small squares (0.5 mm x 0.05 mm each).Thus a total no of 400 sub-squares in 1mm squares.Each medium sized squares is separated by triple lines,the middle one acts as the boundary.The 1mm squares at 4 corners,each is subdivided into 16 subsquares (0.2 mm×0.2 mm each).Thus a total of 16 subsquares in 1 mm square.Thus the volume of each large square at corners and at the centre is (1×1×0.1 mm³) or 0.1 mm³ or=(1/10×1/10×1/100)Cm³ or =(1/10000)Cm³or 10^-4Cm³.The volume of each square with 16 subsquares is 0.004 mm3.

Counting of bacteria is carried out by the use of Neubauer is chamber or Petroff Hauser counting chamber which is only 0.02 mm deep.The number of cells per unit area can be counted under the microscope giving a measure of the no. of cells per small chamber volume.Converting this value to the no of cells per mm of suspension is easily done by multiplying by a conversion factor based on the volume of the chamber used.The total no. of cells per mm³ is given by;

N=n x D_f/v

Where,N=Total no. of cells per mm³

n=Total no of cells counted in 1 chamber

v=Volume of chamber used

D_f=Dilution factor ie. 1/dilution factor

Counting of organisms by membrane Filtration technique

Plating Technique is simple,sensitive and widely used for viable counts of bacteria and other microorganisms in samples of food water and soil.Microbial enumeration or count are often expressed in terms of colony forming the unit(CFU) rather than the no. of microorganism.

A very useful radiation on the plate count technique is based on the use of molecule or membrane filters.Microbial numbers are frequently determined from counts of colonies growing on special membrane filters.These filters have a known uniform porosity of predetermined the size,small enough to trap the microorganisms.this technique is particularly valuable in determining the number of bacteria in a large sample that has a very small number of viable cells.In membrane filter technique,a sample is drawn through a special membrane filter,usually, has a pore size or diameter of 0.45mm.The membrane with trapped organisms is then placed on an agar medium or on a special plate counting an adsorbent pad soaked with the appropriate liquid medium.Special or selective and dice can be used to make it easier to detect certain types of organisms than with the conventional plate count.Upon incubation at appropriate temperature,colonies develop on the membrane filter which is counted and total no of organisms in the sample is calculated as

No of organisms per ml=No of colonies x D_f/Amount of sample filtered

A fixed amount of sample diluted sample is filtered through the membrane filter with the acid of filtering apparatus by applying negative pressure.The filter paper after enriching on few minutes pad can be transferred aseptically to agar medium and then incubated at appropriate temperature.

Turbidometric method

Bacteria in a suspension absorb and scattered the light passing through them so that a culture of more than 10⁷ to 10⁸ cells/ml appears turbid to the naked eye .A spectrophotometric or colorimeter can be used for turbidimetric measurement of cell mass

Turbidometry is a simple rapid method for the following growth;however the culture tube measure most is dense enough to resist some turbidity on the instrument.However,it may not be possible to measure cultures grown in deeply coloured media or cultures that contain suspended material other than bacteria.It must also be recognised that deep as well as living cells contribute to turbidity.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.