LAB SHEET 7 Haemocytometer Cell Counts and Viability Studies

FACULTY: ENNGINEERING
EDITION:
TECHNOLOGY
LABORATORY: CELL AND TISSUE
ENG TECH LABORATORY
EXPERIMENT: HAEMOCYTOMETER
REVISION NO:
EFFECTIVE DATE:
18/2/2014
CELL COUNTS AND VIABILITY
FACULTY
AMENDMENT DATE:
STUDIES OF ENGINEERING TECHNOLOGY
DEPARTMENT OF CHEMICAL ENGINEERING TECHNOLOGY
CELL AND TISSUE ENG. TECH. LABORATORY
LABORATORY INSTRUCTION SHEETS

COURSE CODE
BNN 30104
EXPERIMENT CODE
EXPERIMENT TITLE
HAEMOCYTOMETER CELL COUNTS AND VIABILITY STUDIES
DATE
STUDENT NAME & MATRIK NO.
GROUP
GROUP MEMBERS
LECTURER/ INSTRUCTOR/
TUTOR
DATE OF REPORT SUBMISSION
MARKS:
ATTENDANCE/DICIPLINE:
RESULTS:
/25%
DATA ANALYSIS:
/25%
DISCUSSION & CONCLUSION:
/35%
REFERENCE:
/10%
TOTAL:
EXAMINER COMMENTS:
/5%
/100%
RECEIVED DATE AND STAMP
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
REVISION NO:
EFFECTIVE DATE:
KOD ETIKA PELAJAR

AMENDMENT DATE:
18/2/2014

STUDIES
(KEP)
JABATAN TEKNOLOGI KEJURUTERAAN KIMIA
FAKULTI TEKNOLOGI KEJURUTERAAN
Saya dengan ini mengaku bahawa saya telah menyediakan laporan ini dengan daya usaha saya
sendiri. Saya juga mengaku tidak menerima atau memberi sebarang bantuan dalam menyediakan
laporan ini dan membuat ikrar ini dengan kepercayaan bahawa apa-apa yang tersebut di dalamnya
adalah benar.
Ketua
Kumpulan
Nama:
Ahli 1
Nama:
No. Matriks:
(Tandatangan)
No. Matriks:
Ahli 2
(Tandatangan)
Nama:
No. Matriks:
Ahli 3
(Tandatangan)
Nama:
No. Matriks:
Ahli 4
(Tandatangan)
Nama:
No. Matriks:
(Tandatangan)
___________________________
Tandatangan Pelajar
Nama : _______________________________
No. Matrik :____________________________
Tarikh :________________________________
1
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
1.0 OBJECTIVES
The objectives of this experiment is to enumerate the cells in cultures.
2.0 LEARNING OUTCOMES
At the end of this laboratory session student will able to:
a. Demonstrate the counting technique by using haemocytometer cell
chamber.
b. Enumerate the lives/dead cells and differentiate it in viability study.
3.0 INTRODUCTION
In order to ensure that cell cultures have reached the optimum level of growth
before routine subculture or freezing, it is helpful to obtain an accurate cell
count and a measure of the percentage viability of the cell population.The
most common routine method for cell counting that is efficient and accurate
is with the use of haemocytometer. There are several types on the market, of
which the Improved Neubaucer has proved most popular. A thick, flat
counting chamber coverslip rests on the counting chamber at a distance of
0.1 mm above the base of the slide. The base of the slide has rulings
accurately engraved on it, comprising 1 mm squares, some of which are
further divided into smaller squares. When cell suspensions are allowed to fill
the chamber, they can be observed under a microscope and the cell counted
in a chosen chamber of ruled squares. From these counts, the cell counted
per mililiter of suspension can be calculated.
Hybridoma cells and others that grow in suspension may be counted directly.
Cell lines that are attached will need to be removed from the tissue culture
flask by trypsinization. Because accuracy of counting requires a minimum of
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
approximately 105 cell ml-1 it may be necessary to resuspend the cells in a

smaller volume of medium.
To ensure that a cell culture is growing exponentially it is useful to know the
percentage viability and percentage of dead cells and hence the stage of
growth of the cells. This can be estimated by their appearance under the
microscope, because live healthy cells are usually round, refractile and
relatively small in comparison to dead cells, which can appear larger, crented
and non-refractile when in suspension.
The use of viability stain such as Trypan blue ensures a more quantitative
analysis of the condition of the culture. Trypan blue is stain that will only
enter across the chambers of dead/non-viable cell. When a cell suspension is
diluted with Trypon blue, viable cell stay small, round and refractile. Nonviable cells become swollen, larger and dark blue. Both the total count of cells
per mililiter and percentage of viable cells can be determined.
4.0 INSTRUMENTS /APPARATUS / CHEMICAL / REAGENTS

1. 0.4 g of Trypan blue in 100ml of physiological saline; pass through a
0.11m filter to remove any debris.
NOTE: Trypan blue is harmful if ingested or inhaled. It is irritating to the
eyes, harmful by skin contact and has been found the cause cancer in
laboratory animals. Appropriate precautions should be taken when
handling Trypan blue and the use of an extraction hood and gloves is
advised.
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
2. Haemocytometer with cover slip improved Neubauer British standard

for Haemocytometer counting chambers, BS 748:1963
3. Hand held counter
4. Microscope low power, X40 to X100 magnification
5.0 PROCEDURE
1. Thoroughly clean the haemocytometer and coverslip and wipe both with
70% alcohol before use.
2. Moisten the edges of the coverslip or breathe on the chamber to provide
moisture before placing the coverslip centrally over the counting area and
across the grooves.
3. Gently move the coverslip back and forth over the chamber until Newtons
rings (rainbow-like interference patterns) appear, indicating that the
coverslip is in the correct position to allow accurate counting, i.e. the
depth of the counting chamber is now 0.1 mm.
Cells
0.1 ml
0.1 ml
0.1 ml
Table 1 Example of suitable dilutions

Trypan blue
0.1 ml
0.3 ml
0.9 ml
Dilution
2-fold
4-fold
10-fold
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
4. Mix the cell suspension gently and add an aliquot to the Trypan blue
solution (see table 1). The dilution will depend on the cell concentration
and may need to be adjusted to achieve the appropriate tange of cells to
be counted (see step 6). Draw a sample into a Pastuer pipette after mixing
thoroughly and allow the tip of the pipette to test at the junction between
the counting chamber and coverslip. Draw the cell suspension in to fill
the chamber. No pressure is required because the fluid will be drawn into
the chamber by capillarity. Both halves of the chamber should be filled to
allow for counting in duplicate.
5. Using a light microscope at low power, focus on the counting chamber.
6. Count the number of cells (stained and unstained separately) in 1-mm 2
areas (see Figure 2.2.2) until at least 200 unstained cells have been
counted. As a rule, the cells in the left hand and top grid marking should
be include in a square and those in the right hand and bottom markings
excluded (see Figure 2.2.3).
7. Count the viable and non-viablecells in both halves of the chamber.
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
6.0
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
RESULTS & CALCULATIONS

6.1 Result.
Please provide your result.
6.2 Calculation.
Total number of viable cells = A B C 104

Total dead cell count = A B C 104
Total cell count = viable cell count + dead cell count
% Viability
Where A = volume of cells, B = dilution factor in Trypan blue (Table 1),

C = mean chamber of unstained cells (i.e. unstained count divided by
the number of areas counted, D = mean number of dead/stained cells
and 104 is the conversion factor 0.1 mm3 to ml.
7.0 ANALYSIS
Please analyze the data and results obtained in this experiment
8.0
DISCUSSIONS
Discuss your results both on the basis of any theory presented and on their
relevance to practical applications and current industrial practise. Comment
on the variation of your results and compare them with the recommended
standard values
9.0
ADDITIONAL QUESTIONS
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
Will be provide in laboratory session
10.0 CONCLUSION
Conclusion is merely a summary, presented in a logical order, of the
important findings already reported in the discussion section. It also relates
to the objectives stated earlier
EDITION:
TECHNOLOGY
ENG TECH LABORATORY
STUDIES
Prepared by / Disahkan oleh:
Signature/Tandatangan:
REVISION NO:
EFFECTIVE DATE:
18/2/2014
AMENDMENT DATE:
Approved by / Disahkan oleh :
Signature / Tandatangan :
Name/Nama: Dr Norshuhaila Mohamed Sunar
Name / Nama : Prof. Madya Dr. Ishak Baba
Date/Tarikh :
Date / Tarikh :

LAB SHEET 7 Haemocytometer Cell Counts and Viability Studies

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FACULTY: ENNGINEERING

CELL COUNTS AND VIABILITY

CELL AND TISSUE ENG. TECH. LABORATORY

LABORATORY INSTRUCTION SHEETS

HAEMOCYTOMETER CELL COUNTS AND VIABILITY STUDIES

DISCUSSION & CONCLUSION:

RECEIVED DATE AND STAMP

KOD ETIKA PELAJAR

CELL COUNTS AND VIABILITY

approximately 105 cell ml-1 it may be necessary to resuspend the cells in a

4.0 INSTRUMENTS /APPARATUS / CHEMICAL / REAGENTS

2. Haemocytometer with cover slip improved Neubauer British standard

Table 1 Example of suitable dilutions

RESULTS & CALCULATIONS

Total number of viable cells = A B C 104

Where A = volume of cells, B = dilution factor in Trypan blue (Table 1),

Will be provide in laboratory session

Prepared by / Disahkan oleh:

Approved by / Disahkan oleh :

Name/Nama: Dr Norshuhaila Mohamed Sunar

Name / Nama : Prof. Madya Dr. Ishak Baba

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