Enumeration by Counting Chamber - Introduction, Principle, Volume, Procedure, Petroff Hauser Chamber, Superior Marienfeld Hemocytometer

Introduction to Enumeration by Counting Chamber

Microbial enumeration by counting chamber is a direct microscopic count in which a counting chamber such as a hemocytometer can be used.

In the enumeration by counting chamber method, the number of bacteria present in a small known volume is placed on the slide that has been marked with precise grids, and the number of cells present in each grid is counted microscopically. That average number is applied to determine the number of bacteria in the larger original sample.

Principle of Enumeration by Counting Chamber

Such hemocytometer counting chamber includes Petroff Hauser Chamber, and Neubauer ruling chamber (Superior Marienfeld Hemocytometer). Both are Neubauer improved and are 5x5 squares in the central grid. Also, only one square field is visible under 10x magnification

The Petroff Hauser Chamber is 0.02mm in depth and is used for bacterial as well as sperm count. The Superior Marienfeld Hemocytometer is 0.1mm deep and is for the all-purpose count.

• both hemocytometers contain 2 chambers

• each camber contains 9 grids measuring 1mm x 1mm

• since each of the 9 grids has a dimension of 1x1mm, the dimension of one chamber is 3x3mm

• The Center grid (grid 5) consists of 25 sub-squares with 0.2x0.2mm dimensions each

• Each of the 25 squares in grid 5 consists of 16 smaller squares each measuring 0.0x 0.55mm

• each medium-sized square is separated by triple lines and the middle one acts as the boundary

• Each grid 1,3,7,9, is divided into 16 squares measuring 0.25 x 0.25 mm in dimension

Formula for Enumeration by Counting Chamber

The total number of cells per mm³ for enumeration by counting chamber is given by the formula:

N = n / v X d.f.

Where,

N = total number of cells/mm³

n = total number of cells counted in one chamber

v = volume of the chamber used

D.f. (dilution factor) = 1 / dilution

Procedure of Enumeration by Counting Chamber

The procedure for the Enumeration by Counting Chamber includes:

1. Take a clean counting chamber slide, put it under a microscope, and make the focus sharp

2. Place a cover slip on the chamber

3. With the help of a dropper or pipette, put the sample inside the engraved grid through the coverslip and platforms of the slide so that no air bubbles are trapped

4. Add a drop of stain to the suspension for better visualization

* For counting later cells such as fungal spores, yeast cells, WBC, etc corner grids 1,3,7,9 is used, and for smaller cells such as RBC, the central grid is used.

* Cells touching any of the triple lines on the upper and left sides are counted while the cells touching the lower and right side of the triple lines should not be counted.

Volume of Enumeration by Counting Chamber

The volume of the hemocytometer depends upon the type of counting chamber used. As the Petroff Hauser Chamber has less depth than the Superior Marienfeld Hemocytometer, it contains less volume.

Superior Marienfeld Hemocytometer

Petroff Hauser Chamber

* nl = nanoliter

* 1nl = 0.001μl

* 1ml = 1000μl