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

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.

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.

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.

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.

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.

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

Differential Stains: Gram Stain Figure 3.10b

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

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

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).

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

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