Principle

By counting the number of particles in a grid of known volume the number of microorganisms per cm³ can be calculated.

The counting chamber is marked off by double ruled lines into 9 large squares, in a 3 by 3 pattern.

Each large square is sub-divided by single lines into 16 small squares, in a 4 by 4 pattern making a total of 144 small squares      ( 9*16 ).

Equipment

Helber counting slide plus cover slip

Socorex pipette set to 10 ul 

Microscope

Procedure

1.Ensure the slide is clean, wash with methylated spirit if necessary.

2.Drop 10 μl of culture within the engraved circle on the slide.

3.Slide cover slip onto the slide, pressing firmly but gently on the edges.

4.If the cover slip has been correctly positioned, Newtons rings ( colour diffraction ) will be seen in the contact area between the cover slip and the slide.

5.Examine the slide under the  microscope with lowered illumination. Extreme caution when coarsely adjusting the focus, as the cover slip is delicate.

6.Count the number of cells lying within 50 small squares.

7.Cells crossing the lower horizontal or left hand vertical borders are counted as being in that square while those crossing the upper or right hand boundaries are ignored.

Calculation

Each small square has an area of 1/400 mm and with the cover slip correctly in place the depth of liquid is 0.02 mm.

This means the volume of liquid contained within a single square is:-

 = 1/400 x 0.02  mm³      = 0.0025 x 0.02  mm³      = 5 x 10^-5  mm³      = 5 x 10^-8 cm³

If the total count of 50 small squares is ‘200‘ counts then the number of cells

=  200 / ( 50 x 5 x 10^-8 )  cm-³

=  200 x 4 x 10^5  cm-³

=  800 x 10^5  cm-³

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