Negative Stains

Background

In contrast to direct stains that bind to bacteria directly, a negative stain colors the background of a smear rather than the bacteria. These stains have negatively charged functional groups so they can't bind directly to negatively charged bacteria. The advantages of negative staining are:

1. bacteria are not heat fixed so they don't shrink, and

2. some bacterial species resist basic stains (Mycobacterium) and one way they can be visualized is with the negative stain.

However, negative staining does not differentiate bacteria, one can only determine morphology.

Procedure

1. Using a flamed inoculating loop, place 2-3 loopfuls of Congo Red in two separate circles on a clean slide. There is no need to add water to the Congo Red.

Slide diagram

2. Using a flamed inoculating NEEDLE, pick up a small amount of Bacillus subtilis and stir it into one drop of Congo Red.

slide with red stain

3. Use a toothpick to scrape material from your teeth near the gumline and stir this into the second drop of Congo Red. Be sure to keep the two drops separate.

slide with red smear

4. Air dry - DO NOT HEAT FIX.
5. Flood the slide with acid-alcohol (95% ethanol, 3% HCl) until it turns blue. This generally takes ~ 2 seconds. Drain the excess acid-alcohol but do not wash the slide.

slide with blue smear

7. Allow the slide to air dry; do not blot.
8. Examine both slides. First focus using the 10X objective. You will not be able to see individual organisms, but you should be able to focus on the stain. Then move to 40X and finally to the oil immersion lens with oil. 

9. Draw a typical microscopic field for each slide in the Results section of this lab.

Results

Examples of Bacillis subtilis and tooth scraping negative stains. Note: Organisms appear white (colorless) against a blue stained background.