Making a Bacterial Smear on a Slide  (Atlas, p 27-34)

A bacterial smear on a slide is normally made every time before one stains bacteria for microscopic observation.  Besides placing bacteria on a slide, this procedure dries and fixes the bacteria to the glass, so that the cells are less likely to be washed away during the staining procedure.  The drying, heat fixing and staining normally kill most vegetative bacteria, but one should treat the smear as biohazardous because some cells (especially endospores) may still be alive.  This procedure takes practice, it is very easy to overheat the slide (causing the cells to appear distorted and melted) and very easy to apply too many cells (causing problems with staining and making individual observations difficult).

Procedure:

  1. Appropriately label your slide.
  2. If the culture of bacteria is not growing in a liquid media, add a tiny drop of water to a slide.
  3. Sterilize your loop in an incinerator or flame, cool it, and pick up some bacteria, adding an almost invisible amount of bacteria to your slide.
  4. Spread the bacteria around in the drop of water or liquid media as uniformly as you can to disperse you culture and to speed its evaporation.
  5. If the smear is made correctly, it should look slightly milky (or slightly cloudy).  If it is clear, there are too few bacteria, if it looks milky, there are too many bacteria.  Students usually use too many cells and then cannot view individual cells!
  6. Sterilize your loop before setting it down, and then allow the bacteria on the slide to air dry (smaller drops of water dry faster).  While the smear is wet, the cells are alive and should be treated as a biohazard.  Do not wave the slide around or blow on it as this will disperse bacteria into the air.
  7. Once the smear is dry, hold the slide with you fingers and pass it through a flame three times or touch it to an incinerator passing it over the incinerator three times.  The slide should feel slightly warm on the slide receiving the heat.  If it feels hot, you are overheating it.  If it does not feel slightly warm, repeat the process passiing the slide over the heat a little slower.  Using your fingers instead of a slide holder helps prevent one from overheating the slide!
  8. Allow the slide to cool, and then your bacterial smear is ready for staining.
  9. If your heat fixing step is performed properly and your cells are still washing off the slide, one may use a drop of a protein solution (such a serum, skim milk, or media containing protein) instead of water to help stick your cells to the slide.

Instructor Notes:

If students have not made a bacterial smear before, it is recommended that the procedure be demonstrated.  Students tend to overestimate how many cells they need, resulting in a smear of a few layers instead of a single layer of individual cells.  If the smear is too thick, students will be unable to clearly see their cells.  Overheated cells are deformed and have a melted appearance.