1) Simple staining: 2) Differential staining:

1) Gram staining 2) Acid fast staining

3) Special staining
1) Negative staining 2) Flagella staining 3) Endospore staining

1)With the wax pencil, mark the name of the bacterial culture in the far left corner on each of three slides. 2) For the broth culture, shake the culture tube and, with an inoculating loop, aseptically transfer 1 to 2 loopfulls of bacteria to the center of the slide. Spread this out to about a inch area

 Stains are salts composed of a positive and a negative ion, one of which is colored and is known as the chromophore. Occasionally, a Basic dyes is in the positive ion
crystal violet, methylene blue, malachite green, safranin. chemical is added to the solution to intensify the slain; such an additive is called a

mordant Acidic dyes. it is in the negative ion.

eosin, acid fuchsin, nigrosin.

1) Place the three fixed smears on a staining loop or rack over a sink or other suitable receptacle 2) Stain one slide with alkaline methylene blue for 1 to 1d minutes; one slide with carbolfuchsin for 5 to 10 seconds; and one slide with crystal violet for 20 to 30 seconds. 3) Wash stain off slide with water for a few seconds 4) Blot slide dry with bibulous paper. Be careful not to rub the smear when drying the slide because this will remove the stained bacteria. 5) Examine under the oil immersion lens and complete the report.

 Ziehl-neelsen Acid-fast staining:

Kinyoun (Cold Stain) Procedure (This may be used instead of or in addition to the Ziehl-Neelsen procedure.) 1) Heat-fix the slide as previously directed. 2) Flood the slide for 5 minutes with carbolfuchsin prepared with Tergitol No. 7 (heat is not necessary) 3) Decolorize with acid-alcohol and wash with tap water. Repeat this step until no more color runs off the slide. 4)Counterstain with alkaline methylene blue for 2 minutes. Wash and blot dry. 5) Examine under oil. Acid-fast organisms stain red; the background and other organisms stain blue.

Negative, indirect, or background staining is achieved by mixing bacteria with an acidic stain such as nigrosin, India ink, or eosin.

 Many bacteria have a slimy layer surrounding them, which is usually referred to as a capsule. The capsules composition, as well as its thickness, varies with individual bacterial species. Polysaccharides, polypeptides, and glycoproteins have all been found in capsules. Often, a pathogenic bacterium with a thick capsule will be more virulent than a strain with little or no capsule since the capsule protects the bacterium against the phagocytic activity of the hosts phagocytic cells

Procedure: Capsule Staining (Anthonys) 1) With a wax pencil, label the left-hand corner of a clean glass slide with the name of the bacterium that will be stained.

2) As shown in figure aseptically transfer a loopful of culture with an inoculating loop to the slide. Allow the slide to air dry. Do not heat-fix! Heat-fixing can cause the bacterial cells to shrink and give a false appearance to the capsule.
3) Place the slide on a staining rack. Flood the slide with crystal violet and let stand for 4 to 7 minutes 4) Rinse the slide thoroughly with 20% copper sulfate 5) Blot dry with bibulous paper

Modified Capsule Stain (Graham and Evans) 1. Thoroughly clean the slide to be used with a household cleanser (such as Bon Ami) and alcohol. 2. Mix two loopfuls of culture with a small amount (1 to 2 drops) of India ink at one end of the slide. 3. Spread out the drop using a second slide in the same way one prepares a thin smear 4. Dry the smear. 5. GENTLY rinse with distilled water so that the bacteria do not wash off the slide. 6. Stain for 1 minute with Grams crystal violet. 7. Rinse again with water. 8. Stain for 1d minutes with safranin stain. 9. Rinse with water and blot dry. 10. If a capsule is present, the pink to red bacteria are surrounded by a clear zone. The background is dark.