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MP Practicals

The document outlines an experiment to measure bacterial cell count and viability using a hemocytometer. It describes the principle of using trypan blue dye for viability assessment, the calculation methods for total and viable cells, and the step-by-step procedure for conducting the experiment. The results section is left for the user to fill in the number of bacterial cells per mL and the percentage of viable cells.

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6 views

MP Practicals

The document outlines an experiment to measure bacterial cell count and viability using a hemocytometer. It describes the principle of using trypan blue dye for viability assessment, the calculation methods for total and viable cells, and the step-by-step procedure for conducting the experiment. The results section is left for the user to fill in the number of bacterial cells per mL and the percentage of viable cells.

Uploaded by

enochdenis06
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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You are on page 1/ 3

(Please Draw the Diagram in the Left-hand side)

Experiment No.1

BACTERIAL CELL COUNT AND VIABILITY MEASUREMENT –


HEMOCYTOMETER

AIM:
To count the number of microbes in a sample using hemocytometer.

PRINCIPLE:
Hemocytometer is a special slide that can be used to count bacterial cells. Hemocytometer
have two chambers forming square areas of defined size and by placing a coverslip on the
hemocytometer, a defined volume space is formed. This is the counting chamber into which
a known volume of cell suspension (10 µl) can be pipetted. The cells can then be easily
counted by looking at the slide with a microscope. For the viability assessment of bacteria
based on membrane integrity criteria, the dye exclusion assay using trypan blue is one of the
most common and useful techniques. Trypan blue is an anionic hydrophilic azo dye widely
used to stain dead cells. Due to its high negative charge, it is excluded from the bacteria with
intact membranes. In contrast, dead bacteria can take up trypan blue because they lose
membrane selectivity.

Side view of a Hemocytometer


Hemocytometer grid

BACTERIAL CELL NUMBER CALCULATION

Each of the two grids of the hemocytometer (chamber 1 and 2) is a square with a size of 3
mm.

The square is divided by 3 vertical and horizontal equidistant lines that form 9 squares with a
size of 1 mm.

The area of each square is therefore 1 mm2.

Placing a coverslip on the hemocytometer forms a chamber that is 0.1 mm high.

The cell suspension volume included in a 1 mm2 square will therefore be 1mm2 x 0.1mm =
0.1mm3.

To know the number of cells in 1 ml, it will be enough to divide the number of cells counted
in a square by 10-4 ml, i.e. multiply the number of cells x 104.

The formula for calculating cell number using hemocyter is N x 104 x f cells / ml
where N is the average number of cells counted in the different squares.
If the cells are diluted because they are too dense, or with trypan blue for the viability test,
then the number must be multiplied by the dilution factor.
N x 104 x f
where f is the dilution factor
For example: if the cells are diluted 1: 2, putting an equal volume of cells and trypan blue,
then the formula will be Nx 104 x 2

VIABLE CELLS CALCULATION:


The formula for calculating the percentage of viable cells is by dividing the number of viable
cells by the number of total cells and multiplying by 100
% Viable cells = [(Number of colorless cells ÷ Number of total cells)] × 100.

PROCEDURE
(1) Place 100 µl of cell suspension in an Eppendorf tube.
(2) Add equal volume of 0.4% trypan blue dye to the cell suspension to obtain a 1 to 2
dilution (example: 100 µl of cells to 100 µl of trypan blue) and mix by pipetting up and
down.
(3) Incubate mixture for less than three minutes at room temperature. If cells are counted after
approximately five minutes, viability will be inaccurate due to cell death.
(4) With the cover slip already in place, fill one side of a hemacytometer counter with the cell
suspension by placing the tip of the pipette at the notch. Typically, each side will take 10 to
20 µl.
(5) Place the hemacytometer on the stage of a light microscope and focus onto the cells.
(6) Each side of the hemacytometer contains multiple squares. Count all cells (clear and blue)
in each large square in each corner of the hemacytometer. Each large square contains 16
small squares. In each large square count cells that are on the border lines on two sides only.
Keep track of the number of blue cells separately as well as part of the complete number of
cells. Blue cells are the non-viable cells.
(7) Calculate the total number of bacterial cells per mL and percentage of viable cells using
the formula and tabulate the results.

RESULT:
No. of bacterial cells per mL of sample =

Percentage of viable cells =

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