THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
PROCEDURE NO:
MOD:
Page:
GLP 079
1st Issue
1 of 7
Procedure Type:
General Laboratory Procedure
Title:
Cell Counts Using the Trypan Blue Exclusion Method
1.
Risk Assessment:
This Risk Assessment is to be used as a general guide and as such, cannot accommodate all
the varying factors that may be encountered when using this procedure. Therefore, personnel
are requested to conduct their own Risk Assessment before using this procedure to include any
extra hazards introduced by the task performed.
TASK PERFORMED
Cell count to determine both cell concentration and viability in a specific sample.
HAZARDS
1. Refer to GLP 102 Working with human, murine and GM cell lines for a risk
assessment covering general aspects of this work.
2. Chemical hazard – Trypan blue
Risk phrases: R45(2) - May cause cancer
RISK ASSESSMENT
1. Refer to GLP 102 Working with human, murine and GM cell lines for a risk
assessment covering general aspects of this work.
2. The risk of chemical exposure is low if the Risk Controls are followed.
RISK CONTROL
1. Wear suitable PPE - lab coat, disposable gloves and safety glasses.
2. Refer to GLP 102 Working with human, murine and GM cell lines for a risk
assessment covering general aspects of this work.
3. Trypan blue Safety phrases:
S01 – Keep locked up
S53 - Avoid exposure - obtain special instructions before use
S40 – To clean the floor and all objects contaminated by this material use water
S35 - This material and its container must be disposed of in a safe way
NAME (signed)
DATE
WRITTEN BY
CHECKED BY
AUTHORIZED BY
Lynn Herd
Leonie Ashman
Leonie Ashman
th
9 February 2005
Distributed To: GLP Master File / GLP Lab File
st
21 February 2006
22nd February 2006
DISCIPLINE OF MEDICAL BIOCHEMISTRY
PROCEDURE NO:
Page:
Title:
GLP 079
2 of 7
Cell Counts Using the Trypan Blue Exclusion Method
4. Training should be provided by personnel experienced in this procedure.
5. Training should be undertaken in General Laboratory Safety.
I have read and understood the Risk Assessment for GLP 079 – Cell Counts using the
Trypan Blue exclusion method.
I agree to abide by the Risk Controls outlined in this Assessment.
Name
Signature
Date
DISCIPLINE OF MEDICAL BIOCHEMISTRY
PROCEDURE NO:
Page:
Title:
2.
GLP 079
3 of 7
Cell Counts Using the Trypan Blue Exclusion Method
Purpose:
2.1.
This document describes the procedure to count cells using the Trypan blue
exclusion method.
3.
4.
5.
Equipment:
3.1.
Microscope
3.2.
Counter
3.3.
Pipettors and associated yellow tips
3.4.
Haemocytometer and glass coverslip
Materials:
4.1.
0.5mL microfuge tubes
4.2.
Kimwipes
4.3.
96-well plate or small plastic disposable tubes
4.4.
0.4 – 0.8% Trypan blue
4.5.
Cell suspension
Set Up:
5.1.
Ensure that you have read and understood the safety precautions (Section 6 of
this SOP) prior to commencing this procedure.
5.2.
6.
Turn on microscope and turn to low objective.
Safety Precautions:
6.1.
Good laboratory techniques are to be used at all times.
6.2.
Any material that has been in contact with human, murine, or GM cell lines is
treated as potentially infectious.
6.3.
Wear gloves and lab gown at all times.
6.4.
Always decontaminate waste as soon as possible.
DISCIPLINE OF MEDICAL BIOCHEMISTRY
PROCEDURE NO:
Page:
Title:
7.
GLP 079
4 of 7
Cell Counts Using the Trypan Blue Exclusion Method
Method:
7.1.
Clean the haemocytometer and glass coverslip by spraying with 70% ethanol
and wiping dry with a kimwipe.
7.2.
Position the haemocytometer correctly with the glass coverslip in the middle of
the haemocytometer.
Haemocytometer
7.3.
Make the appropriate dilution to the cell suspension just prior to counting.
7.4.
A suggested initial dilution is 50μl cell suspension diluted with 50μl 0.4-0.8%
Trypan blue in one well of a 96-well plate or in a disposable plastic tube.
7.5.
The optimal concentration of cells for counting is 5 -10 x 105 cells/ml (50 -100
cells per large square) after dilution in the Trypan blue.
7.5.1. Pipette approximately 10μl of the cells/trypan blue mixture into a
haemocytometer.
7.5.2. It is possible to use both sides of the haemocytometer, pipetting sample
into the chamber on each side, and then counting 2 large squares on
each side. If results from one side are obviously different to the other,
then the sampling should be repeated.
7.6.
The cell suspensions should be allowed to flow under the coverslip until the
grid area is just full and not overflowing into the overflow well.
7.7.
If the chamber is loaded too heavily, clean it and begin again.
7.8.
Allow cells to settle. If greater than 10% of the cells appear clustered, repeat
entire procedure making sure the cells are dispersed by vigorous pipetting in
the original cell suspension as well as the Trypan Blue-cell suspension
mixture.
7.9.
Viable cells exclude the Trypan blue, while nonviable cells take up the dye.
After being stained with Trypan blue, the cells must be counted within three
minutes; after that time viable cells begin to take up the dye.
DISCIPLINE OF MEDICAL BIOCHEMISTRY
PROCEDURE NO:
Page:
Title:
GLP 079
5 of 7
Cell Counts Using the Trypan Blue Exclusion Method
7.10. Count cells on 10x magnification on the microscope or higher if necessary to
clearly visualise the cells. Ideally, count 100-200 cells to ensure accuracy
7.11. Count numbers of both viable and non-viable cells using the counter.
7.12. Select which size squares to count based on the number of cells in each
square. Eg. Smaller cells such as splenetic lymphocytes will need to be
counted in medium squares, using a higher magnification lens on the
microscope, than many cultured cell-lines.
7.13. Refer to the Haemocytometer grid provided below.
1 medium square
XXXXXXXXXXXXX
XXXXXXXXXXXXX
XXXXXXXXXXXXX
XXXXXXXXXXXXX
XXXXXXXXXXXXX
XXXXXXXXXXXXX
XXXXXXXXXXXXX
Large
square
7.14. For many cultured cell lines, the cells in 4 – 5 large squares should be counted
and the counts from each square averaged. eg. Count 5 large squares: the 4
large corner squares and the central square of equal size would each be
counted. If instead you wish to count 2 squares from each side of the
haemocytometer, count the central squares and one of the corner squares for
each grid.
DISCIPLINE OF MEDICAL BIOCHEMISTRY
PROCEDURE NO:
Page:
Title:
8.
GLP 079
6 of 7
Cell Counts Using the Trypan Blue Exclusion Method
POINTS TO NOTE:
8.1.
The haemocytometer grid is divided into 9 large squares. Each of these large
squares can be further divided into 25 medium squares. The central large
square is further divided into 400 small squares. Each large square has a
surface are of 1mm2 and the depth of the chamber is 0.1mm.
8.2.
Cell suspensions should be dilute enough so that the cells do not overlap each
other on the grid, and should be uniformly distributed.
8.3.
When counting cells in any given square the convention is to include all cells
that touch the upper or left boundaries, whereas those that touch the bottom or
right boundaries are excluded
8.4.
For accuracy and reproducibility, counts should be carried out in the same
manner each time. Decide on a specific counting pattern to avoid bias.
8.5.
When the haemocytometer is loaded properly, the volume of cell suspension that
will occupy one large square is 0.1 mm3 (1.0 mm2 x 0.1 mm) or 1.0 x10-4 ml.
8.6.
Total cell concentration in the original suspension (in cells/ml) is then:
Cells/ml = total count (average no cells per large square) x 104 x dilution factor.
8.7.
Cell viability is calculated using the following formula:
% viable cells =
Number of viable cells
x100%
Number of viable cells + number of dead cells
9.
Maintenance:
9.1.
Clean haemocytometer with 70% ethanol and kimwipes and return to storage
in LS3.17.
9.2.
Turn off the microscope.
9.3.
Kimwipes used to clean haemocytometer and other solid waste should be
placed into the infectious waste bags for autoclaving.
10.
Change History:
10.1. Issue Number:
Date Issued:
10.2. Issue Number:
1st Issue
22nd February 2006
DISCIPLINE OF MEDICAL BIOCHEMISTRY
PROCEDURE NO:
Page:
Title:
Cell Counts Using the Trypan Blue Exclusion Method
Date Issued:
Reason for Change:
GLP 079
7 of 7