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).
to prepare the cells for staining.
- It is suggested that students make three slides: one using 1/4 a loop
full of bacteria, 1/8 a loop full of bacteria, and almost invisible amount of bacteria. Which
bacterial smear is better after it is stained?
- Appropriately label your slide.
- If the culture of bacteria is not growing in a liquid media, add a tiny drop of water to
- 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.
- 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.
- 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!
- 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.
- 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
- Allow the slide to cool, and then your bacterial smear is ready for staining.
- 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.
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