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

338 Manual of basic techniques for a health laboratoryTable 11.1 Preparation of dilution series for the anti-streptolysin O testTube number Volume of patient’s Volume of Resulting Volume of reduced Volume of 5%serum (inactivated) streptolysin O serum dilution streptolysin O suspension of sheep(ml), diluted: buffer (ml) buffer (ml) erythrocytes1:10 1:100 1:5001 0.8 — — 0.2 1 : 12.5 0.5 0.52 0.2 — — 0.8 1 : 50 0.5 0.53 — 1.0 — 0.0 1 : 100 0.5 0.54 — 0.8 — 0.2 1 : 125 0.5 0.55 — 0.6 — 0.4 1 : 167 0.5 0.56 — 0.4 — 0.6 1 : 250 0.5 0.57 — 0.3 — 0.7 1 : 333 0.5 0.58 — — 1.0 0.0 1 : 500 0.5 0.59 — — 0.8 0.2 1 : 625 0.5 0.510 — — 0.6 0.4 1 : 833 0.5 0.511 — — 0.4 0.6 1 : 1250 0.5 0.512 — — 0.2 0.8 1 : 2500 0.5 0.513 — — — 1.5 control 0.0 0.514 — — — 1.0 control 0.5 0.511.4.2 Latex agglutinationMaterials and reagents●●●●●Test platesStirring rods, wooden sticks or rotatorTest-tubes, 5mlTest-tube rackMicropipettes, 50ml● Anti-streptolysin O latex reagent: suspension of latex particles coated with streptolysinO● Negative control serum● Positive control sera (strongly and weakly positive)● Sodium chloride, 0.85% solution (reagent no. 53).Method1. Bring the reagents and test and control sera to room temperature.2. Apply one drop of each of the test and control sera to the test plates.3. Shake the anti-streptolysin O latex reagent to mix; add one drop to each of thetest and control sera.4. Mix well with stirring rods or wooden sticks (one per sample) and rotate theplates gently about 10 times, or place on a rotator.5. After 2 minutes, examine the plates and compare the reactions of the test serawith those of the controls. A positive reaction is indicated by the presence ofagglutination. A negative reaction is indicated by the absence of agglutination.6. If any sera are positive, repeat steps 2–5, using a twofold dilution.The highest dilution that causes agglutination is the titre. Most anti-streptolysinO reagents have a detection limit (e.g. 200IU/ml) that is usually multipliedby the dilution factor to give the serum concentration of anti-streptolysin O inIU/ml.