Manual of basic techniques for a health laboratory - libdoc.who.int

290 Manual of basic techniques for a health laboratoryFig. 9.42 Filling the counting chamberFig. 9.43 Using the improved Neubauer ruledchamberFig. 9.44 Countingleukocytesusing theimprovedNeubauerruled chamberImportant: If the liquid overflows into the channel between the two chambers,you must start again: remove and clean the coverslip; clean the counting chamber;and refill with another drop.6. Leave the counting chamber on the bench for 3 minutes to allow the cells tosettle.7. Place the chamber on the stage of the microscope. Use the ¥ 10 objective andthe ¥ 10 eyepiece. Reduce the amount of light entering the condenser by adjustingthe iris diaphragm. Focus the rulings of the chamber and the leukocytes. Donot mistake pieces of dust for leukocytes.8. Count the leukocytes in an area of 4mm 3 of the chamber, using the corner squaresnumbered 1, 3, 7 and 9 as shown in Fig. 9.43. Include in the count the leukocytesseen on the lines of two sides of each square counted, as shown in Fig. 9.44. Thissquare represents one of the four counted.9. Calculate the number of leukocytes in 1 litre of blood by multiplying the numberof leukocytes counted in the four squares by 0.05. Report the result as “numberby 10 9 /l)”.Example:number of leukocytes counted = 188number of leukocytes per litre = (188 ¥ 0.05) ¥ 10 9Result reported: 9.4 ¥ 10 9 /lExplanation of calculationEach of the four squares in which leukocytes are counted has an area of 1mm 2 ; thetotal area is therefore 4mm 2 . The chamber depth is 0.1mm, therefore the volumein which leukocytes are counted is 4 ¥ 0.1 = 0.4mm 3 . Thus division by four andmultiplication by 10 will give the number of leukocytes in 1mm 3 of diluted blood.Since the dilution is 1 in 20, multiplication by 20 will give the number of leukocytesin 1mm 3 of undiluted blood. Finally, there are 1 million (10 6 ) cubic millimetres in