1. PREPARATION OF A SMEAR GRAM STAIN ACID FAST STAIN
LAB: 3
PREPARATION OF A SMEAR GRAM STAIN ACID FAST STAIN

2. BACTERIAL SMEARS
Before bacteria can be stained a smear must be made and fixed.
A bacterial smear is made by evenly spreading bacteria on a clean slide in a drop of liquid (sterile water or broth).

3. BACTERIAL SMEARS Allow the slide to air dry and then:
Heat or Chemically fix the sample to the slide:
1. Heat fix:
Pass it over the incinerator to gently heat fix
2. Chemically fix:
by flooding the slide with methanol and allow to evaporate (Denatures bacterial enzymes and enhances adherence of bacteria to the slide)
THE SLIDE IS NOW READY TO STAIN

4. GRAM STAIN
Developed by Hans Christian Gram in 1884 when he was studying bacteria from different respiratory diseases.
The Gram Stain is a differential stain used to classify bacteria as Gram positive or Gram negative.
The single most important technique in the Microbiology Laboratory.

5. The uptake and retaining of the basic stains depends on the cell wall:
The Gram Stain
The uptake and retaining of the basic stains depends on the cell wall:
*Gram positive bacteria have many layers of peptidoglycan in their cell wall making it easier to hold onto the CV-I complex.
*Gram negative bacteria have one layer of peptidoglycan. Thus the CV-I complex is not retained in the cell.

6. GRAM STAIN REAGENTS 1. Crystal violet (CV): primary basic stain
2. Grams Iodine (I): not a stain; it forms a crystal violet—iodine complex (CV-I) inside the cell wall.
3. Decolorizer: ethyl alcohol, used to remove the primary stain. It removes the CV-I complex from cells without teichoic acid.
4. Safranin: secondary (counter) basic stain

7. SET UP YOUR SLIDE FOR STAINING

8. GRAM STAIN SUCCESS DO NOT USE TOO MUCH WATER OR
BROTH. A SMALL LOOPFUL IS ENOUGH.
DO NOT USE A HEAVY INOCULUM
(AMOUNT OF BACTERIA).
MAKE AN EVEN, THIN SUSPENSION ON
THE SLIDE. THIS WILL ALLOW FOR
PROPER STAINING AND ESPECIALLY PROPER DECOLORIZATION.

9. GRAM STAIN PROCEDURE 1. Flood with crystal violet for 1 minute. Rinse
2.Flood with iodine for 1 minute. Rinse
3.Decolorize with alcohol until no more purple comes off the slide. Rinse
4. Flood with safranin. Rinse and dry.

10. RESULTS Gram positive Organisms will stain purple. Gram negative
stain pink.

11. RESULTS WHAT WOULD HAPPEN IF WE: 1.) OVER – DECOLORIZE
2.) UNDER – DECOLORIZE

12. WHY DO WE NEED TO STAIN MICROORGANISMS???

14. ACID FAST STAIN Developed by Paul Ehrlich in 1882
Kinyoun published ‘cold-staining’ method in 1915, which is commonly used today
Acid fast positive bacteria have fatty acid in their cell wall (mycolic acid) that makes them ‘acid fast’, i.e. do not decolorize with acid alcohol
Acid Fast Stain is a differential stain used to classify Acid fast positive or Acid Fast negative microorganisms

15. ACID FAST STAIN
The group of microorganisms that are differentiated by the acid fast stain are the mycobacteria.
There are many species of mycobacteria, but the most important organism in this group is Mycobacterium tuberculosis, the causative agent of tuberculosis.

16. ACID FAST STAIN REAGENTS
Carbol fuchsin: fuchsin is the red basic stain and carbol is the acid (carbolic acid)
Acid alcohol: decolorizer (Hydrochloric acid and 95% ethanol)
Methylene blue: a blue counter stain

17. For Acid-Fast Stain Lab your slide should look like this.

18. PROCEDURE – ACID FAST STAIN
COLD KINYOUN METHOD
ACID FAST
STAIN STEPS
Color of
Acidfast + cells
Acidfast – cells
1. Primary stain:
Carbol fuchsin
RED
2.Decolorizing agent:
Acid alcohol
COLORLESS
3.Counterstain:
Methylene Blue
BLUE

19. Acid-Fast Staining: Kinyoun Method
ACID FAST POSITIVE - RED ACID FAST NEGATIVE - BLUE

20. Acid-Fast Staining: Kinyoun Method
ACID FAST POSITIVE – RED
ACID FAST NEGATIVE – BLUE

21. RESULTS
??? OVER – DECOLORIZE
???UNDER – DECOLORIZE

22. WHY DO WE USE THE ACID FAST STAIN????