Flagella Stain Procedure:


Procedure:

A. Bacterial Suspension
 1. From an agar slant culture:
      a. Suspend a loopful of bacteria in 2 ml distilled water to obtain an opalescent suspension.
      b. Allow the suspension to stand undisturbed for 15 to 20 minutes while flagella are regenerated and extended.

B. Slides
 1. Select new or unscratched slides.
 2. Clean slides with cleaning powder as directed.
 3. Flame one of the slides and allow it to cool.
 4. Make a heavy wax line on the flamed side along the margin of one end, along both sides to within about one inch of the other end, and across the slide to complete the rectangle.  Handle the slide by the unmarked end only.  The wax line creates a retaining wall to allow a pool of stain to surround the cells during the 15 to 15 minute staining period.

C. Preparation of smear
 1. Handling the suspension carefully remove a large loopful of the suspension and place it at one end of the rectangular area.
 2. Tilt the slide to permit the suspension to run down to the other end of the slide.  If the drop fails to run, add another drop.
 3. Air dry the film.  Do not heat.
 4. Place the slide on a horizontal staining rack.

D. Staining procedure
 1. Add about 1 ml. of the Flagella Stain solution (one dropper full) to the smear and allow to stain for 10-15 minutes.  The solution should not be allowed to flow outside the waxed area.
 2. Flood off the stain by adding tap water to the slide while it remains on the rack.  Do not tip the slide before this is done.
 3. Drain and flood the slide with carbol fuchsin for one minute.  Rinse by flooding.  Drain and air dry.  Do not blot.

E. Examination of slide
 1. With the naked eye identify the line made by the drop as it ran down the slide.
 2. Position the slide so that the edge of the run is under the objective.
 3. Focus the edge of the run under low power, as oil, and examine it using the oil immersion objective.
 4. Identify cells as distinguished from dye and debris which will adhere to the slide because of the mordant.  Cells will be small rods and have regular outlines.  They will be more plentiful near the lower end of the run and may look larger than usual because of the mordant.
 5. Once cells are identified follow along the edge of the run, examining cells until some are found with flagella attached.  Flagella are much longer than the cell and often look like faint hairs.
 6. Ideally, cells should be located which are isolated enough to determine unequivocally the arrangement of the flagella and, for the Pseudomonas, the number of flagella per cell.
 7. Draw the cells and their flagella.
 
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Lab Procedures