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

272 Manual of basic techniques for a health laboratoryImportant:At the beginning of each day:● Clean the matched spectrophotometer (or colorimeter) cuvettes.● Fill one of the cleaned tubes with fresh Drabkin diluting fluid, which is used tozero the spectrophotometer (or colorimeter).● Read a reference solution (see above).Fig. 9.22 Preparing serial dilutions ofhaemiglobincyanidereference solutionCalibration of the spectrophotometer (or colorimeter) usinghaemiglobincyanide reference solution (or a reference solution previouslycalibrated against haemiglobincyanide reference solution)1. Calculate the haemoglobin value of the reference solution in grams per litre byusing the following formula:aconcentration in mg 100 ml ¥ 10b¥ 251c1000where:a = the factor for converting 100ml to 1 litre;b = the dilution factor when 0.02ml of blood is diluted with 5ml of Drabkindiluting fluid;c = the factor for converting milligrams to grams.Since 10 ¥ 251/1000 is very nearly 2.5, the above formula can be simplified asfollows: 1haemoglobin value of reference solution in grams per litre = concentrationin mg/100ml ¥ 2.5Example:concentration of reference solution = 60mg/100mlhaemoglobin value = 60 ¥ 2.5 = 150g/l2. Prepare a series of dilutions of the reference solution in four test-tubes(labelled 1–4) (Fig. 9.22). Pipette into each tube the amounts shown inTable 9.1.3. Mix the contents of the tubes and allow to stand for 5 minutes (Fig.9.23).4. Read the dilutions in the spectrophotometer (or colorimeter):(a) Set the wavelength to 540 nanometres (nm) or place a green filter inthe spectrophotometer (or colorimeter).(b) Fill a matched cuvette with Drabkin diluting fluid and place in thespectrophotometer (or colorimeter).Table 9.1 Preparing serial dilutions of reference solutionTube number Volume of reference Volume of Drabkin diluting Dilutionsolution (ml)fluid (ml)1 4.0 0.0 undiluted2 2.0 2.0 1 : 23 1.3 2.7 1 : 34 1.0 3.0 1 : 41If a dilution of 1 in 200 is used (i.e. 0.02ml of blood and 4 ml of Drabkin diluting fluid),multiply by 2.0 instead of 2.5.