Heat Fixed Microscope Slide of Bacteria

How to Prepare a Microscope Slide of Bacteria
Heat Fixing a Bacterial Smear for Staining
LAB NOTES from Science Prof Online


First, use a wax pencil to draw a circle on the microscope slide to separate each type of bacteria that is going to be sampled. If a simple stain of only one type of bacteria, or single mixed sample of bacteria is being prepared, no separation is required.

Preparing a Bacterial Smear
In order to be able to clearly see individual bacteria, a sample of a bacterial colony must be mixed into water or physiological saline. This helps to evenly spread out the bacterial sample.

Article Summary: In order to view individual bacteria through a light microscope, a bacterial smear must be attached to a slide and then stained. Here is the procedure.
How to Prepare & Heat Fix a Bacterial Smear for Staining
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Prokaryotic Cell, Mariana Ruiz
Heat Fixed and Gram Stained Slide of Bacteria
Bacterial smear that has been heat fixed and stained.
In order to heat fix a bacterial smear, it is necessary to first let the bacterial sample air dry. Then either place the slide in the slide holder of a microincinerator, or pass the dried slide through the flame of a Bunsen burner 3 or 4 times, smear side facing up. Once the slide is heat fixed, it can then be stained.

Staining Bacteria
There are many staining techniques that can be used to view bacteria. Some are considered simple stains, a staining process in which only one dye is used. The use of differential staining procedures is more complex. Differential protocols use a series of dyes to distinguish different types of bacteria based on some chemical or structural attribute of the cell.

Differential staining is often used for general identification of bacteria, but do not allow for identification of the exact species. Examples of differential stains include the Gram stainAcid-fast stain and Endospore stain. When doing a differential stain it is best to use known + & - bacterial controls in addition to the unknown bacteria that is being identified. Controls allow for comparison of the unknown to a sample of bacteria that tests positive for that stain and one that tests negative for the stain.

The following are examples of bacterial controls that could be used in differential stains:
    Gram Stain
  • + controls: StaphylococcusBacillusStreptococcus
  • - controls: Escherichia, Salmonella, Enterobacter 
    Acid-fast Stain
  • + controls: Mycobacteria, Nocardia
  • - controls: any type of bacteria that does not have a waxy cell wall
    Endospore Stain​
  • + controls: Bacillus, Clostridium (Clostridium are pathogens, so not a great choice for a control)
  • - controls:  Any type of bacteria that does not produce endospores

Sources & Resources
  • Schauer Cynthia (2007) Lab Manual to Microbiology for the Health Sciences, Kalamazoo Valley Community College.
  • Bauman, R. (2005) Microbiology. Pearson Benjamin Cummings.
Heat Fixing the Bacterial Sample
Before staining, the sample must be heat fixed. This process accomplishes three things. It functions to:
  • kill the bacteria
  • firmly affix the smear to the microscope slide
  • allow the sample to more readily take up the stain
Slides heat fixing on tray attached to microincinerator
Slides heat fixing on tray attached to microincinerator.
​Page last updated 2/2015

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SPO VIDEO: How to Prepare a Bacterial Smear for Gram Staining
Placing the Bacterial Sample on the Slide
  • Place a drop of water into the wax circle that has been created on the slide.
  • Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony.
  • Gently mix the bacteria into the water drop.

Instructor's Corner

Photographic guides to differential stains 
now available!
1. Gram
2. Acid-fast
3. Endopsore

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