Industrial Biotechnology

Objectives

·  Be able to recognize the three basic shapes of bacterial cells.

·  Be able to differentiate between Gram-positive and Gram-negative organisms.

Introduction

Bacteria have almost the same refractive index as water. This means when you try to view them using a microscope they appear as faint, gray shapes and are difficult to see. Staining cells makes them easier to see. In some cases, staining is unnecessary, for example when microorganisms are very large or when motility is to be studied, and a drop of the microorganisms can be placed directly on the slide and observed. A preparation such as this is called a wet mount. A wet mount can also be prepared by placing a drop of culture on a cover-slip (a glass cover for a slide) and then inverting it over a hollowed-out slide. This procedure is called the hanging drop.

Types of stains

1. Simple stain.

2. Differential stain.

3. Special stain.

How Stains Work

Stains are chemicals containing chromophores, groups that impart color. Their specificity is determined by their chemical structure. For example, a basic dye is a stain that is cationic (positively charged) and will therefore react with material that is negatively charged. The surface of bacteria at neutral pH is somewhat negatively charged and will therefore attract basic dyes. Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue. Acid dyes have negatively charged chromophores and are repelled by the bacterial surface. They stain the background and leave the microbe transparent. Nigrosine and congo red are examples of acid dyes.

Note: Bacteria have three basic shapes or morphological types. Round cells are known as cocci, rod-shaped cells are bacilli, and spiral-shaped cells are spirilla. (Fig. 2.1)

Figure 2.1 Various bacterial shapes

I. Simple stain:

Principle

The simple stain consists of one dye. The dye adheres to the cell wall and colors the cell making it easier to see.

Materials:

Heat-fixed bacterial smears
Methylene blue, Crystal violet, or Safranin to act as simple stain
Bibulous paper or paper towels
Microscope

Procedure:

1. Place one or two loopfuls of water on the slide.

2. Using an inoculating needle, mix a very small quantity of the colony with the water and spread over the slide. It is critical that microbes are separated from each other during this step.

3. Allow the smear to completely air drying.

4. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.

5. Stain the smear by flooding it with one of the staining solutions and allowing it to remain covered with the stain for the time designated below.

Methylene blue- 1 minute.

Crystal violet- 30 seconds.

Carbol fuchsin- 20 seconds.

During the staining the slide may be placed on the rack or held in the fingers.

6. At the end of the designated time rinse off the excess stain with gently running tap water. Rinse thoroughly.

7. Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.

8. Examine under the oil immersion lens.

II. Gram stain:

The Gram stain procedure uses 3 different stains. These are crystal violet, Gram’s iodine, and safranin. The cells are first stained with crystal violet, then Gram’s iodine. Following a rinse in alcohol, to de-colorize the cells, the cells are then stained with safranin.

Principle

The differential technique separates bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria. Four different reagents are used and the results are based on differences in the cell wall of bacteria. Some bacteria have relatively thick cell walls composed primarily of a carbohydrate known as peptidoglycan. Other bacterial cells have thinner cell walls composed of peptidoglycan and lipoplysaccharides.

Gm+ organisms are thought to resist decolorization by alcohol or acetone because cell wall permeability is markedly decreased when it is dehydrated by these solvents. Thus, the dye complex is entrapped within the cell, resist being washed out by the solvents, and Gm+ bacteria remain purple following this differential stain.

In contrast, cell wall permeability of Gm- organisms is increased by ethyl alcohol washing. This allows the removal of the crystal violet-iodine complex from within the cell. The decolorized Gm- cell can then be rendered visible with a suitable counter stain, in this case Safranin, which stains them pink. Pink which adheres to the Gm+ bacteria is masked by the purple of the crystal violet.

Crystal violet acts as the primary stain. This stain can also be used as a simple stain because it colors the cell wall of any bacteria.

Materials:

Crystal violet stain
Safranin stain
Gram's Iodine
Acetone-alcohol decolorizer 95%
Bibulous paper or paper towels
Microscope

Procedure:

1. Transfer a loopful of the bacterial suspension to the surface of a clean glass slide, and spread it over a small area. Allow the slide to air dry. Fix the cells by passing the slide briefly through the Bunsen burner flame.

2. Place slide on staining rack and cover specimen with crystal violet. Let stand for 1 minute.

3. Wash briefly in tap water and shake off excess.

4. Cover specimen with iodine solution and let stand for 1 minute.

5. Wash with water and shake off excess.

6. Tilt slide at 45° angle and decolorize with the acetone-alcohol solution until the purple color stops running.

7. Wash immediately with water and shake off excess.

8. Cover specimen with safranine and let stand for 30 seconds to 1 minute.

9. Wash with water, shake off excess, and gently blot dry.

10. Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.

11. Examine under the oil immersion lens.

Figure 2. 2 Steps for gram staining technique

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