Solution A

Mix and let stand overnight at room temperature.

Solution B

Mix solutions A and B. Adjust pH to 5.0 with 1 N NaOH or 1 N HCl, if necessary. Refrigerate 2-3 days before use. Stain is stable l month at 4°C or may be stored frozen indefinitely (50 ml portions).

To use, thaw stain, remix, and store at 4°C. Optimum staining time for each batch varies 5-15 min. To determine staining time (after 2-3 days refrigeration at 4°C), stain a known flagellated organism on 3 or more cleaned slides for various times (e.g., 5, 10, 15 min). Mark best staining time on all containers.

IMPORTANT: Stain will not work unless slides are clean. To clean slides, soak them 4 days at room temperature in cleaning solution (either acid dichromate or 3% concentrated HCl in 95% ethanol). Rinse 10 times in fresh tap water and twice in distilled water. Air-dry at room temperature. Store in covered container.

Staining Procedure

To prepare suspension, pick small amount of growth from 18-24 h plate (equivalent to 1 mm colony). Do not pick up agar. Suspend gently in 3 ml distilled water. (Flagella can be knocked off.) Suspension should be faintly opalescent.

To prepare slide, pass cleaned slide through blue part of burner flame several times to remove residual dirt. Cool slide, flamed side up, on paper towel. Mark wax line across slide to give area 2.5 x 4.5 cm. Place large loopful of suspension in center of slide adjacent to wax line. Tilt slide, letting drop run down center of slide to end. If drop does not run evenly, slide is dirty. Discard it. Air-dry slide on level surface.

Hypertext updated by rim 2000-MAR-29