1. Third lecture for microbiology manual
Smear preparation and staining
Design by: Abed Al Rahman Hamad
M.sc. Biological science

2. Preparation of Specimens for Light Microscopy
A thin film of a solution of microbes on a slide is a smear.
A smear is usually fixed to attach the microbes to the slide and to kill the microbes.

3. Preparing Smears for Staining
Live or unstained cells have little contrast with the surrounding medium. However, researchers do make discoveries about cell behavior looking at live specimens.

5. Preparing Smears for Staining
Stains consist of a positive and negative ion.
In a basic dye, the chromophore is a cation.
In an acidic dye, the chromophore is an anion.
Staining the background instead of the cell is called negative staining.

6. Simple Stains Use of a single basic dye is called a simple stain.
A mordant may be used to hold the stain or coat the specimen to enlarge it.

7. Differential Stains: Gram Stain
Named after Christian Gram, Danish scientist and physician, 1853–1938.
The Gram stain classifies bacteria into gram-positive and gram-negative.
Gram-positive bacteria
Gram-negative bacteria.
The Gram stain technique is most reliable when performed on 24 hrs to 48 hrs or on sample directly taken from patient.

9. Differential Stains: Gram Stain
Color of
Gram + cells
Gram – cells
Primary stain:
Crystal violet
Purple
Mordant Iodine
Make complex with crystal violet and increase retention of stain
Decolorizing agent:
Alcohol-acetone
Colorless
Counterstain:
Safranin
Red

10. Differential Stains: Gram Stain
Figure 3.10b

11. Differential Stains: Acid-Fast Stain
Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast.
Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.
T.B. cell wall consist of wax D and mycolic acid.
Figure 3.11

12. Procedure of acid fast stain:
Color of
Non acid fast bacteria
Acid fast bacteria
Primary stain:
Carbol fuchsin
Red
Heating
Decolorizing agent:
H2SO4
Colorless
Counterstain:
Methylene blue
Blue

13. The kinyoun procedure
Kinyoun’s procedure is often referred to as cold staining the concentration of carbol fuchsin and phenol in the stain has been increase and stain prepared with Tergitol No. 7 (heat is not necessary).

14. The metachromatic granules stain
Metachromatic granules or volutin are storage crystals produced by some bacteria when there excess phosphate and carbohydrate in their growth medium. These crystals are deposits of cellular phosphate and are large enough to see when stained.
Corynebacterium diphtheriae is an example for this stain.
There are two procedure for this stain:
1. Albert’s procedure
2. Loeffler’s procedure

15. Special Stains
Negative staining is useful for capsules (india ink stain).
Malachite green is the stain used for examine endospore formation
Heat is required to drive a stain into endospores.
Flagella staining requires a mordant to make the flagella wide enough to see.
Figure 3.12a-c