Haemocyteter

Question 1

What does a haemocytometer allow?

Answer 1

an estimation of the concentration of cells in a sample to be made

Question 2

What does a haemocytometer resemble?

Answer 2

a microscope slide, but has a grid made up of perpendicular lines (similar to graph paper) etched into the glass.

Question 3

How do you use the haemocytometer?

Answer 3

a cover slide is affixed to it; this creates a chamber with a depth of exactly 0.1 mm.
The cell culture to be counted is gently mixed and 100 µl of the sample is mixed with 100 µl of dye.
Some of this mixture is pipetted under the coverslip and the haemocytometer is placed under a microscope to visualise the grid.

Question 4

Describe what the grid looks like.

Answer 4

The grid has nine ‘large’ squares, each 1 mm × 1 mm

Question 5

How can we calculate the volume of cell culture in each area of the grid?

Answer 5

1 × 1 × 0.1 = 0.1 mm3 (or 0.1 µl)

Question 6

How are the cells within one of the 1 mm × 1 mm areas are visualised?

Answer 6

using a microscope and counted.

Question 7

The cells within one of the 1 mm × 1 mm areas are visualised using a microscope and counted. What does this provide?

Answer 7

an estimate of the number of cells per 0.1 µl of culture medium.

Question 8

What does multiplying by 10 give?

Answer 8

the number of cells per µl

Question 9

What does multiplying by 10,000 give?

Answer 9

the number of cells per cm3

Question 10

What is important to remember when carrying out this calculation?

Answer 10

the cells were diluted in dye as part of this procedure, therefore the final answer should take this into account. A 1:1 dilution gives a dilution factor of 2, therefore the final answer should be multiplied by 2 to give a reliable estimate of cell number per cm3.

Question 11

How can the reliability of the results be improved?

Answer 11

the number of cells in more than one square should be counted and an average calculated.

Question 12

When using a traditional glass haemocytometer, how can the reliability of the results be effected?

Answer 12

by incorrect placement of the coverslip because this provides a chamber with a known volume.

Question 13

To reduce error what do many laboratories use?

Answer 13

plastic disposable haemocytometers which have a coverslip already properly fixed in place, reducing this possible source of error.
Disposable haemocytometers also have the added benefit of limiting exposure to infectious materials