Download Industrial Biotechnology Badr Objectives Be able to recognize the

Industrial Biotechnology
Badr
Objectives
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Be able to recognize the three basic shapes of bacterial cells.
Be able to differentiate between Gram-positive and Gram-negative organisms.
I. 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.
II. Types of stains
1. Simple stain.
2. Differential stain.
3. Special stain.
II.1 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 3.1 Various bacterial shapes
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II.1.1 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.1.1 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.
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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.
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Figure 3. 2 Steps for gram staining technique
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