Cell Counting with Haemocytometry

Introduction

In this experiment, there is requirement of using a haemocytometer for counting cells and thereby presenting within a sample’s set quantity. It is not known that how the cells are concentrated within the broth. The experiment will be carried out with a serial dilution in identifying a dilution with a countable cell number within it. The calculations can be adjusted in identifying the number of cells having presence within 1 ml of the original concentration. Once the serial dilutions have been carried out, the haemocytometer can be used in carrying out the cell counts.

Method

A haemocytometer has been used for counting the yeast cells. The haemocytometer has been used with a microscope for counting the microbes virtually in a liquid and a very small sample. There are chambers in the sides of depth that are known and with an etching of a grid on the surface. In a given area the number of organisms has been counted along with the calculation of the original sample with the use of volume of any dilution factor and the chamber. The haemocytometer grid’s focus has been with the use of x40 followed by x100 lens. Once it has been confronted that the grid can be focused, the sample is obtained from the tube once that uses the pastuer. The sample’s one drop is placed from the tube 1 into the haemocytometer’s grid followed by the placing of the cover slip over it. The grid should be focused upon and the cells would be counted as instructed.

Procedure

Grid Sampling

Usually, there is use of B-squares to count cells within a sample. There is no necessity, however, that all B-squares of 25 types are in grid’s center. The usual practice is counting cells in 5 of the B-squares so that 4 corner squares are counted and so are the central squares. The cells’ mean number in B-square may be determined then and the calculation is possible for the number of cells in 1 cm2 or 1 mm2.

The North and West Rule

There is counting of some cells lying on the triple lines’ boundary between adjacent B-squares. For avoiding the twice counting of these cells touching the middle line on the west and north sides of the squares. The cells that touch the middle line on the east and south sides of the square are ignored.

Requirements

The growth of yeast samples has been at unionculated liquid medium and different temperatures x 5 cuvettes and rack. There is also requirement of transmission or absorbance setting.

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Method

A cuvette is filled till three quarter with uninoculated medium and in the cuvette chamber, it is placed. A green or blue filter is selected and it is ensured that colorimeter is set to Absorbance. As through the sample the light shines, the reading is set to zero by the samples, as there is clarity in sample and there is no absorption. The next step is filling the cuvette till three quarter with the first sample. Then, it is placed in the colorimeter and the same filter is used as before and the absorption is measured. With more absorption, there will be more growth of yeast. The uninoculated sample is used and the colorimeter is re-zeroed by the cuvette. Each yeast sample is repeated.

Results

Red A square = 1 mm3, 100 nl

Yellow B square = 0.04 mm3, 4 nl

Blue C Square = 0.0025 mm3, 0.25 nl

These are recorded at depth 0.1 mm

Estimation of the total cell counts

There have been 2 approaches to determine the cell number per mm3.

B-square volume (0.004 mm3)

12 + 10 + 14 + 9 + 15/5 = 60/5 = 12 cells

Thus, it indicates that in 0.004 mm3, there are 12 cells.

There will be in 1 mm3, 12 divided by 0.004 = 3000 cells.

The dilution factor = 2

Thus, in 1 mm3, the number of yeast cells = 3000 x 2 = 6000 cells.

Blood Agar

Sketch area of the growth

Observations

There are large numbers of streek on both sides and there are many running streeks as well.

Sketch area of the growth

Observations

Both sides do not have any continuation, although the streek marks are on both sides.

Sketch area of the growth

Observations

There is continuation on both sides and there is no streek on either side.

Potato Dextrose Agar

Sketch area of the growth

Observations

The continuation is observed on LB and almost nothing on SF. LB has little streek mark.

Sketch area of the growth

Observations

No streek mark has been observed on either side, although both sides have continuation.

Sketch area of the growth

Observations

SA has been found to have growth although without any continuation. On the other hand, SC has continuation.

Discussion/Conclusion

The yeast cell results that have been described before have occurred because of the growth of e coli that take place between 10 degree and 30 degree Celsius. These are considered to be mesophiles. The least growth possible for the e coli is at 70 degree Celsius. The reason for this is that very high temperature enables it to start to denature. In such condition, the membrane will not be able to function properly.

The blood agar observed in this lab has shown to have hemolysis as the extracellular enzymes are produced by some bacterial species lysing RBC in blood agar. The hemolysin (extotoxin) diffuses radically from the colony outwards that causes partial or complete RBC destruction and the hemoglobin’s denaturation is complete and medium to colorless products (Madigan and Martinko, 2005). The hemolysis is produced by blood agar that are of 4 types Streptococci namely; wide zone or prime alpha hemolysis, gamma hemolysis, Beta hemolysis, and Alpha hemolysis. The observation of homolysis is best when the colonies are examined under inspecting sub-surface colonies or anaerobic conditions.

The 2 types of hemolysis, beta hemolysis and alpha hemolysis, and either of the two types is caused by the colonies that have been observed by the plate. It must be noted that for knowing the blood agar hemolysis, there must be holding of the blood agar plate to the source of light and then be subject to observation with the transmitted light or the light coming from behind.

The potato dextrose agar plates are used in extracting methanol from it and has been produced by standard positive control (Downes and Ito, 2001). There has been studying of cultivation media and cultivation system’s effect in obtaining a fermentation medium that is suitable to produce bioactive compound. The antifungal compound can be produced against S. pombe when cultured on potato dextrose agar. The methanolic extracts of POR 58, on the other hand, from the static liquid culture have shown to have ability of inhibiting F. oxysporum. Apart from this, the Albatrellus sp.’ methanolic extracts grown into malt extract and potato dextrose have given 1 mm inhibition zone against F. oxysporum.

In proteins, there are fungi that are rich in it. Now, there have been characterization and purification of multitude of proteins and several other remains to be isolated. There are certain proteins that have proven to have valuable application e.g. tyrosinase, lignin peroxidases, and laccases.

References

Madigan, M. and Martinko, J., eds. (2005) Brock Biology of Microorganisms (11th ed.), Prentice Hall.

Downes, F. P. and Ito, K., (Eds.) (2001) Compendium of Methods for the Microbiological Examination of Foods [4th Ed.], APHA, Washington, D.C.