1 Estimate the volume of the specimen and add a equal amount of 10% Sputolysin (6.3mM dithiothreitol).

2 Shake the mixture, wait 5-15min and shake the specimen again. If clumps of mucoid material are still present repeat the treatment.

3 Add an equal volume of 50% ethanol. Allow the mixture to stand for 15min.

4 Centrifuge at 1300g for 5-10min. Remove the supernatant by suction or pouring off.

5 Mix the pellet with the small amount of fluid remaining in the tube.

6 Place 25-50^l in a well of a Teflon-coated slide for immunofluorescence and onto an ordinary microscope slide for Toluidine blue staining.

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