Flow Through Gram Stain

A Gram Stain is usually performed on a smear preparation that has been heat fixed. One function of fixation is to secure (fix) the cells to the slide. In a biofilm, however, the cells are already fixed. Furthermore, a heat fixed slide is dry, but a biofilm is mostly water. Drying alters the biofilm virtually beyond recognition. Described here is a method for obtaining a Gram Stain on a minimally altered biofilm.

Instructions

Preparing the biofilm for staining.
  1. Prepare a biofilm slide (by the micro-fishing technique for example).
  2. Wipe the top and bottom 2 mm of the slide clean.
  3. Thinly smear some petroleum jelly on a sheet of paper. Hold a cover slip at right angles to the petroleum jelly and scrape it in such a way as to build up a thin ridge along one edge of the cover slip.
  4. Repeat the process with the opposite side of the cover slip.
  5. Place the cover slip onto the biofilm slide preparation.

Preparing the Gram stain.

The Gram stain reagents are added at one open end of the slide and are drawn through the space under the slide by means of an absorbent paper towel. The reagent sequence is similar to that in the standard Gram Stain but the timing is somewhat longer to compensate for a possible dilution effect of the aqueous phase of the biofilm. It is important to be certain that sufficient reagent has been drawn under the coverslip so that dilution by the volume of water present or the previous reagent does not occur. The bacteria stain as in a traditional Gram Stain, but because of the thickness of the preparation and the presence of water the slides do look different from a typical Gram Stain.

  1. Add the Crystal Violet dye to one edge of the coverslip. Draw the dye through using an absorbant paper towel applied to the opposite side of the cover slip. Draw the dye through until all the water has been replaced by dye. Stain for 30 seconds.
  2. Wash the slide by drawing water under the coverslip with a paper towel until no further purple color is removed.
  3. Add the Gram's Iodine solution by adding several drops of the reagent to the same edge of the coverslip and drawing it through with a paper towel. About 1.5 minutes.
  4. Decolorize the slide by drawing 95%alcohol under the slide until no further purple due is removed.
  5. Wash the slide as in step two.
  6. In a similar manner, counter stain the slide with Safranin dye for .5 minute.
  7. Wash the slide as in step two.
  8. Observe the slide under high dry or oil immersion microscopy.

The Following is a Diagram of the above instructions