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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.

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

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. Using the ruling described above the volumes are only 1/5 compared to the standard 0. yeast or bacteria can be counted. Using a special hemocytometer with a depth of 0.02mm smaller particles such as sperm.1mm deep chamber. .

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

 Dead cells are colored.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. .

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

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

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 .

coli  70% Ethanol  Trypan Blue .Materials & Equipment  E.

 Micropippetor and tips  Microscope  Haemocytometer .

Mix with 100 µl of Trypan Blue .Methods 100 µl E. Coli cell suspension .Mix with 100 µl of Distilled water .

Tabulate results .Mount Haemocytometer with cover slip Wipe with 70% Ethanol Introduce mixture on the counting chambers Count number of viable and non-viable cells.

Results and Discussion .

Grid 2 1 5 4 3 .

200.000 .43% 46.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.

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

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 71.000 .200.43% 46.

 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: .

000 .200.43% 46.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.

com/wp-content/uploads/2010/11/bacterial-growthcurve. Time and Log Viable Cells vs.Viable Cells vs. Time Graph Acquired from: http://ebsbiowizard.png .

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

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 .

If you have a total of 170 cells in all 4 corner squares.1. what is the cell count (cells/mL)? .

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.1 mm. When multiplied. the computed cell concentration (ave. Since 1cm3 is equivalent to 1 ml. Count x df) in equation 4-2 are multiplied by 104 to produce their actual values outside the counting chamber. . each square has surface area of 1mm2 and depth of 0. the resulting total volume is mm3 or 10-4 cm3.2.

Cell viability presents the number of living cells in relation to the dead ones and is represented by the percent viability. the nonviable cell count is needed. High percent viability – cells are in proliferative state Low percent viability – cells are in less optimal conditions .3. The data collected can give us information about the current state of the cells in the culture. To compute for this. Why do you have to count the non-viable cells? Living and dead cells are said to be viable and non-viable respectively.

they are more suitable to use. Since their corresponding logarithmic values are smaller and easier to use in graphing.4. why is the log value of the cell concentration used? Cell concentrations are recorded as very high values. . When the data are plotted in a graph.

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.5. . Though being the more tedious option. 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.