Experiment 4

Cell Counting Using The Dye Exclusion Principle

4 Bio 1 Group 7 & 8

Introduction

Dye Exclusion Test

It is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin, or propidium, whereas dead cells do not. a cell suspension is mixed with dye, examined to determine whether cells take up the dye or not.

Haemocytometer
Hemocytometers are often used to count blood corpuscles, organelles within cells, blood cells in cerebrospinal fluid after performing a lumbar puncture, or other cell types in suspension.

 Using a special hemocytometer with a depth of 0.02mm smaller particles such as sperm, yeast or bacteria can be counted. Using the ruling described above the volumes are only 1/5 compared to the standard 0.1mm deep chamber. As it is difficult to distinguish between living and dead organisms unless particular stains are used to distinguish viable from non-viable cells this results in a 'total count' of the bacteria.

 In case of bigger cells the number of dead (permeabilised) cells in the sample can be obtained by adding Trypan blue. Fluorescent dyes give better discrimination particularly when looking at bacteria.

Trypan Blue
Trypan blue is a vital stain used to selectively colour dead tissues or cells blue.

Live cells or tissues with intact cell membranes are not coloured. Dead cells are colored.

Escherichia coli
Escherichia coli (E. coli) is one of the normal bacteria present in the intestines of warm-blooded animals. E. coli is a rod-shaped bacterium, or bacillus, that grows as a single cell, but may form chains as cells divide and fail to separate completely.

 E. Coli, one of the simplest organisms, is one of the most studied and best understood on a molecular level. E. Coli can also be easily cultured in a large quantity in the laboratory, for years it has been a great source for biochemical analysis of important enzymatic activities and for purification of sub-cellular components.

Objectives

 This experiment aims to utilize the dye exclusion principle to calculate the cell concentration of a culture of E. coli through the use of haemocytometer.

Methodology

Materials & Equipment

E. coli 70% Ethanol Trypan Blue

 Micropippetor and tips Microscope

Haemocytometer

Methods
100 l E. Coli cell suspension

- Mix with 100 l of Trypan Blue

- Mix with 100 l of Distilled water

Mount Haemocytometer with cover slip Wipe with 70% Ethanol Introduce mixture on the counting chambers

Count number of viable and non-viable cells.

Tabulate results

Results and Discussion

Grid
2 1

5 4

Data Table
Square Number Viable Non-viable Total % Viability Cell Concentration

1
2 3 4 5 Total

40
51 32 45 47 220

8
19 20 19 22 88

53
70 52 64 69 308 71.43% 46,200,000

 Cell viability is an evaluation of living or dead cells based on a total cell sample. This is measure through Percent Viability and is calculated by the following formula:

Data Table
Square Number Viable Non-viable Total % Viability Cell Concentration

1
2 3 4 5 Total

40
51 32 45 47 220

8
19 20 19 22 88

53
70 52 64 69 308 71.43% 46,200,000

 High Percent Viability cells are healthy and are continuing to grow and divide

Low Percent Viability cells are starting to decay

 The cell concentration shows the number of cells per 1mL of the cell suspension and this is calculated by:

Where:

Data Table
Square Number Viable Non-viable Total % Viability Cell Concentration

1
2 3 4 5 Total

40
51 32 45 47 220

8
19 20 19 22 88

53
70 52 64 69 308 71.43% 46,200,000

Viable Cells vs. Time and Log Viable Cells vs. Time Graph

Acquired from: http://ebsbiowizard.com/wp-content/uploads/2010/11/bacterial-growthcurve.png

Microbial Growth Curve

Acquired from: http://web.deu.edu.tr/atiksu/toprak/curve.gif

Conclusion

 In conclusion, the group was able to calculate the cell concentration of a culture of E.coli cells by utilizing dye exclusion principle through the use of the haemocytometer.

Post-Laboratory Questions

1. If you have a total of 170 cells in all 4 corner squares,

what is the cell count (cells/mL)?

2. What is the significance of the value (10 ) in equation

4

4-2?

Equation 4-2: Cells / ml = average count x dilution factor x 104 When the coverslip is places, each square has surface area of 1mm2 and depth of 0.1 mm. When multiplied, the resulting total volume is mm3 or 10-4 cm3. Since 1cm3 is equivalent to 1 ml, the computed cell concentration (ave. Count x df) in equation 4-2 are multiplied by 104 to produce their actual values outside the counting chamber.

3. Why do you have to count the non-viable cells?

Living and dead cells are said to be viable and non-viable respectively. Cell viability presents the number of living cells in relation to the dead ones and is represented by the percent viability. To compute for this, the nonviable cell count is needed. The data collected can give us information about the current state of the cells in the culture. High percent viability cells are in proliferative state Low percent viability cells are in less optimal conditions

4. When the data are plotted in a graph, why is the log

value of the cell concentration used? Cell concentrations are recorded as very high values. Since their corresponding logarithmic values are smaller and easier to use in graphing, they are more suitable to use.

5. Which is a more accurate technique in counting cells:

microscopic or spectrophotometric? Why?

The microscopic method requires to manually count each cell in the slide in order to compute for the cell concentration. Though being the more tedious option, it is the more accurate of the two in contrast to the faster and easier spectrophotometric methods that just rely on absorbance readings of the sample.