1. Dr. Souki MICRO 200 Cerritos College
5-Simple Stains
Dr. Souki
MICRO 200
Cerritos College

2. Purpose
To determine cell morphology and arrangement using the simple stain method.

3. What is a stain?
Stains contain a solvent (water or ethanol) and colored molecule (chromogen).
Chromogen
Auxochrome: charged part of the chromogen
Chromophore: color
auxochrome
chromophore
Auxochrome does not absorb light, rather it intensifies absorption of light by its conjugate chromophore.
Intensifies the color of the chromophore.

4. What is a basic stain?
Basic stains contain a positively charged auxochrome.
Examples of basic stains:
Crystal violet
Methylene blue
Safranin
Malachite green
Crystal Violet
Methylene Blue
Malachite Green
Safranin

5. How is a bacterial smear prepared?
Flame an inoculating loop. Cool the loop.
Place a small drop of water onto a clean slide using an inoculating loop.

6. How is a bacterial smear prepared?
3. Flame and cool the inoculating loop. 4. Use the loop to grab bacteria from a slant or broth culture. 5. Mix the bacteria into an emulsion onto the slide using the loop. 6. Let it dry.

7. How is the smear fixed to the slide?
7. Heat fix the smear by passing the smear through the flame 3 times at the inner cone of the flame.
Heat fix because:
Kills the cells (denatures proteins.
Denatured proteins fixes the cells to the slide.

8. How is the heat-fixed sample stained?
Prepare a heat fixed bacterial smear.
Flood slide with crystal violet for 1 min.
Rinse with water gently.
Blot dry with Kimwipe.
View under oil (1000X)

9. What shapes do bacteria form?

10. What arrangements do bacteria form?

11. Cell arrangements

12. Answer the following questions in your notebook.
Describe what physical differences distinguish the bacteria? How will you tell them apart?
Explain the difference between streptococci and streptobacilli.
Why is flaming the inoculation loop important?
What part of the flame is best for flaming the inoculation loop. Why?
Explain why working near the flame is important when opening culture tubes.
Explain what is a simple stain and how it is used.
Explain the proper procedure for cleaning an oil immersion lens after use.
Why should you NOT use Kimwipes to clean microscope lenses? What should you use?