Direct Microscopic count

Objective:

To determine the concentration of bacterial cell in a given sample by direct microscopic count method and calculating the generation time of the cell.

Principle:

Direct microscopic counts are performed by spreading a measured volume of sample over a known area of a slide, counting representative microscopic fields, and relating the averages back to the appropriate volume-area factors. Specially constructed counting chambers, such as the Petroff-Hauser chambers and Heamocytometer, simplify the direct counting procedure because they are made with depressions in which a known volume overlies an area that is ruled into squares. The ability to count a defined area and convert the numbers observed directly to volume makes the direct enumeration procedure relatively easy. Direct counting procedures are rapid but have the disadvantage that they do not discriminate between living and dead cells.

The ruled area of the hemocytometer consists of several large 1 x 1 mm (1mm² ) squares, which are subdivided in three ways; 0.25 x 0.25 mm (0.0625 mm²), 0.25 x 0.20 mm (0.05 mm²) and 0.20 x 0.20 mm (0.04 mm²). The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05 mm (0.0025 mm²) squares. Hold the cover slip(  0.1 mm)  at the raised edges of hemocytometer, which gives each square a defined volume.

 

	Area	Volume at 0.1mm depth
1 x 1 mm	1 mm2	100 nl
0.25 x 0.25 mm (1/16)	0.0625 mm2	6.25 nl
0.25 x 0.20 mm (1/20)	0.05 mm2	5 nl
0.20 x 0.20 mm (1/25)	0.04 mm2	4 nl
0.05 x 0.05 mm (1/400)	0.0025 mm2	0.25 nl

                                            

Materials required:

-Uniform bacterial cell suspension in peptone/nutrient broth

-Haemocytometer

-Coverslip

-Micropipette

-Microscope

 Procedure:

-Preparation uniform cell suspension:  Take cell suspensions in test tube and pipette out up and down for several times to get accurate cell counting and uniform distribution of the cell.

-Place a coverslip over the calibrated surface of the counting chamber.

- Using a pipette, transfer the suspension to the groove of the counting chamber to fill the chamber by capillary action.

-To observe the cells by using high magnification in the microscope, and count the number of cells in at least 5 of the small squares.

-Do the bacterial cell counting by using above methods, from 0^th hour upto several times for every 30 minutes to find out the generation time of cells.

-Take cell suspension for every time from culture flask which is incubated at 37 ͦ C

-Use the following formula to calculate the number of cells and plot the graph to find out the generation time of particular microbial cells

               Bacteria/mm³ = (bacteria/square) (25 squares) (50) (10³) 

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

Peptone Broth Composition:

Peptone    20g

Sodium chloride    5g

Distilled water   1000ml

Nutrient Broth Composition:

Peptone   5g

Beef extract   2g

Yeast extract    3g

Sodium chloride   5g

Distilled water   1000ml