Erythrocyte Count by haemocytometry

Erythrocyte Count by haemocytometry

Principle

The blood specimen is diluted (usually 200 times) with red cell diluting fluid which does not remove the white cells but allows the red cell to be counted under high power (400 X) magnification in a known volume of fluid. Finally, the number of cells in undiluted blood is calculated and reported as the number of red cells/µl of whole blood. The whole blood is held inside a micro chamber made by a slide with a grid at the bottom. The number of cells counted in the volume of fluid held in the chamber.

Reagent/ Requirements:

1. Sample: Whole blood (anti coagulated venous blood).

2. Diluting Fluids: Red cell diluting fluid- isotonic to red cells which prevent hemolysis.

              Trisodium citrate                                                        3.13 gm

             Commercial formaldehyde (37% formalin)                1.0 ml

             Distilled water                                                            100 ml

3. Blood sahli pipette 20 µl and 50 µl.

4. Hand tally counter

5. Hemacytometer or counting chamber (Neubauer chamber).

6. Microscope.

7. Test tubes.

Procedure:

1. Take a 25 ml flask or test tube of 20 ml and labelled with sample code.
2. Mixed the sample well (at least for 1 minute by gentle shaking; be care full of lysis of RBC) and dilute the uncoagulated sample (usually to 200 times or as per requirements) with diluting fluid. 200 times dilution can be done by mixing 0.02 ml blood sample with 4 ml diluent (exactly 3.98 ml).
3. Mix the diluted blood sample very well with help of pipette.
4. Take a haemocytometer (clean and dry) and place on the flat surface. Place the coverslip on the counting chamber.
5. Take small amount (about 20 µl) of well mixed blood sample (before this for this fill the pipette and dispense on same flask for 3 times- rinsing).
6. Allow a small drop of diluted blood hanging from the pipette and to seep into the counting chamber by capillary action. Make sure that there is no air bubble and there is no overfilling beyond the ruled area. (If the liquid overflow into the channel between the two chambers, repeat from no.4.
7. Leave the counting chamber on the flat surface for 3 minutes to allow the cells to settle.
8. Observe the slide under 10X and locate the large square in the center with 25 small squares. Examine uniform distribution of RBCs.
9. Switch to 40X (high power) and focus on smaller upper left corner which is divided into 16 smaller squares (3 lines boundary on each sides).
10. Count the number of RBCs on the small squares (0.2 X 0.2 = 0.04 sq. cm) falling within the square and those touching the outside lines on the top and inside margin on the left (discarding those that touch the inside margin at the bottom and free margin on the right).
11. Repeat the counting with four other squares (upper right corner square, lower right corner square, lower left corner square and center square divided into smaller 16 divisions in written order or those marked r as in figures.
12. Make the total of count.

                          

Reference: Mukherjee KL (2008). Medical laboratory Technology- A procedure manual for routine diagnostic test. Volume 1. Tata McGraw-Hill publishing Company limited, New delhi.

Agarose gel electrophoresis of DNA

Principle:

Agarose gels are more porous and have a larger pore size as compared to polyacrylamide gels and, therefore, used to fractionate larger macromolecules such as DNA or RNA. Porosity of a gel is determined by concentration of agarose – higher the agarose concentration, smaller the pore size and vice versa. When an electric field is applied across the gel, DNA molecules that are negatively charged at neutral pH, migrate towards oppositely charged electrode at rates determined by their molecular size and confirmation. Since charge to mass ratio of NA is constant, the rate of migration is inversely proportional to log₁₀MW, i.e., smaller DNA molecules will travel faster as compared to the larger ones. Further, DNA molecules of the same size but with different conformations travel at different rates. The order of the migration velocity in the increasing order of various forms of DNA is: Supercoiled DNA > linear double stranded DNA > open circular DNA.

Table 1: Percentage of gels and range of resolution for DNA electrophoresis

% Agarose	Range of resolution (linear dsDNA, Kbp)	% Acrylamide	Range of    resolution
			dsDNA (bp)	ssDNA (nt)
0.5	30 – 1.0	3.5	100 – 1000	750 – 2000
0.7	12 – 0.8	5.0	75 – 500	200 – 1000
1.0	10 – 0.5	8.0	50 – 400	50 - 400
1.2	7 – 0.4	12.0	35 – 250
1.5	3 – 0.2	15.0	20 – 150
		20.0	5 - 100

Requirements:

1. 1 X TAE buffer at pH 8.0 (50 X, 24.2 gm Tris, 5.71 ml GAA, 11.1 ml of 0.5 M EDTA, 100 ml DW) Autoclave before use
2. Agarose gel in 1 X TAE
3. Loading dye  (6 X, 6.0 ml of 50% Glycerol, 1.0 ml of 2% BPB, 3 ml sterile DW)
4. Ethidiun Bromide (EtBr)*: 10 mg/ml stock
5. λ/HindIII Marker (23.13, 9.42, 6.56, 2.32, 2.07, 0.56 and 0.13 Kb)
6. Plasmid DNA (pUC18, pBR322)

Procedure:

1.  Prepare 0.8% agarose gel in TAE buffer.

2.  Dissolve agarose completely in micro-oven and cool to 60°C.

3. CAREFULLY, add EtBr into the gel solution to final concentration of 0.5 µg/ml.

4. Pour the molten gel into gel-mould. Immediately position a comb in the mould.

5.  Let the gel cool for 30 minutes.

6.  Pour TAE buffer into the gel buffer reservoirs.

7.  Prepare sample taking 20 µl of DNA sample and mix with 4µl blue juice (6 X).

8.  Carefully remove the comb.

9.  Load the DNA (15 - 20 µl) per well, flanking wells with similarly processed DNA size standard.

Note: the amount of the sample that can be loaded in a well depends on the thickness of the gel as well as dimensions and placing of the comb.

10. Put the lid on the gel apparatus, attach the electrodes and adjust voltage to 100 volts.

11. Allow the gel to run until line of blue juice is visible       near the end of the gel.

12. Turn off the current and visualize the gel in UV - Tran illuminator.

13. Interpret the results.

*Note: EtBr is carcinogen, so, handle with gloves.