Performing Blood counts using Haemocytometers

University: Biology

The investigation is going to analyse blood samples that had been diluted with phosphate buffered saline to see whether the person it was taken from has a healthy red blood count or not. By analysing the data from this investigation we can compare it with normal red blood cell counts and abnormal red blood cell counts. Previous experience in this line of inquiry may give us a clue into how many blood cells are in the medial range.


Method


To successfully fulfil the aims of the practical investigation, the following methods were adopted.
The blood used for analysis was diluted in order for there to be an accurate red blood cell count in the squares of the haemocytometer. Having too many erythrocytes per square would inevitably lead to inaccurate figures being measured. This seemed the most logical approach to the situation.
In order to get to this stage of dilution a 10µl sample of blood was combined with 90 µl of PBS and mixed gently by flicking the microfuge tubes in which it was contained. This tube was labelled ‘Sample 1’ and was placed in the tube rack.
To extract the correct measurements of blood and PBS, automatic pipettes of different volumes were used. For the blood extraction the 10 µl automatic pipette was used (with a disposable tip to prevent cross contamination for future measurements) and for the extraction of the phosphate buffer saline (PBS), a 100 µl automatic pipette was utilised (with use of disposable tips to prevent cross contaminations between samples.) After each different extraction made with the automatic pipettes the disposable tips were discarded into a sealed sterile fluid. From the microfuge tube which was labelled ‘Sample 1’ an extraction of 10 µl was made using the automatic pipette of the volume 10 µl respectively. This extraction was slowly squeezed from the pipette to prevent air bubbles collating in the sample. At this point it is worthwhile to mention the technique of deposit using an automatic pipette. Initially the plunger is pushed down as far as possible and then it is immersed into the solution. Then plunger is slowly retracted to initial position where the liquid has been sucked up the tube. To deposit the liquid into a microfuge tube, firstly the plunger has to be pushed half way down and then pushed to the fullest extent very slowly. After the extraction of 10 µl was taking from sample one, a further 90 µl of phosphate buffered saline was mixed with it, using the same technique of mixing as described for sample one. This sample was respectively labelled sample two. From this second sample a further 10 µl was extracted and mixed with a further 90 µl of the phosphate buffered saline. From this method of experiment it is notable that the red blood cells have been diluted with the phosphate buffer solution three times. The third sample was labelled ‘Sample 3’. This was done in order for the cells to be counted under a microscope. If too many cells were close together it would have been impossible to get an accurate figure for the count.
A haemocytometer is transparent glass block divided into nine squares. It is used under a microscope to look at cells. A clean and dry coverslip was placed on the central counting chamber of the haemocytometer. This is the part where faint lines can be observed if examined carefully.
The microfuge tube labelled ‘Sample 3’ was then remixed by gently flicking the side of the tube. A pipette was then used (not an automatic pipette) to extract the blood and phosphate buffered saline solution from the tube. The solution was slowly squeezed onto the central edge of the central counting chamber and was drawn up under the coverslip. This is called capillary action.

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