1. Observation of bacteria using staining procedures

2. Preparing Smears for Staining
Most initial observations of microorganisms are made with stained preparations.
Staining simply means coloring the microorganisms with a dye that emphasizes certain structures.
Before the microorganisms can be stained, however, they must be fixed (attached) to the microscope slide. Fixing simultaneously kills the microorganisms and fixes them to the slide. It also preserves various parts of microbes in their natural state with only minimal distortion .

3. Preparing Smears for Staining
When a specimen is to be fixed, a thin film of material containing the microorganisms is spread over the surface of the slide. This film, called a smear, is allowed to air dry.
In most staining procedures the slide is then fixed by passing it through the flame of a Bunsen burner several times, smear side up, or by covering the slide with methyl alcohol for I minute.

4. Preparing Smears for Staining
Stain is applied and then washed off with water; then the slide is blotted with absorbent paper.
Without fixing, the stain might wash the microbes off the slide.
The stained microorganisms are now ready for microscopic examination.

5. Preparing Smears for Staining
Stains are salts composed of a positive and a negative ion, one of which is colored and is known as the chromophore.
The color of so-called basic dyes is in the positive ion; in acidic dyes, it is in the negative ion.
Bacteria are slightly negatively charged at pH 7. Thus, the colored positive ion in a basic dye is attracted to the negatively charged bacterial cell. Basic dyes, which include crystal violet, methylene blue, malachite green, and safranin, are more commonly used than acidic dyes.

6. Preparing Smears for Staining
Acidic dyes are not attracted to most types of bacteria because the dye's negative ions are repelled by the negatively charged bacterial surface, so the stain colors the background instead.
Preparing colorless bacteria against a colored background is called negative staining.
It is valuable for observing overall cell shapes, sizes, and capsules because the cells are made highly visible against a contrasting dark background.
Distortions of cell size and shape are minimized because fixing is not necessary and the cells do not pick up the stain .
Examples of acidic dyes are eosin, acid fuchsin, and nigrosin.

7. Preparing Smears for Staining
To apply acidic or basic dyes, microbiologists use three kinds of staining techniques:
Simple
Differential
Special

8. Simple Staining
A simple stain is an aqueous or alcohol solution of a single basic dye. Although different dyes bind specifically to different parts of cells, the primary purpose of a simple stain is to highlight the entire microorganism so that cellular shapes and basic structures are visible.
The stain is applied to the fixed smear for a certain length of time and then washed off, and the slide is dried and examined.
Occasionally, a chemical is added to the solution to intensify the stain; such an additive is called a mordant. One function of a mordant is to increase the affinity of a stain for a biological specimen; another is to coat a structure (such as a flagellum) to make it thicker and easier to see after it is stained with a dye.
Some of the simple stains commonly used in the laboratory are methylene blue, carbolfuchsin, crystal violet, and safranin.

9. Simple Stain Objective: Determine morphology and arrangement
All bacteria will be stained

11. Simple Stain

12. Negative Stain Objective: Determine morphology and arrangement
India ink used to stain background, not cells
Gives a good view of morphology
Not heat fixed, so cells are not distorted

14. Differential stains
Unlike simple stains, differential stains react differently with different kinds of bacteria and thus can be used to distinguish them.
The differential stains most frequently used for bacteria are the Gram stain and the acid-fast stain.

15. Gram Stain
Objective: determine if bacteria is gram positive or negative
Initial procedure to determine unknown bacteria
Takes advantage of differences in cell wall composition (differential stain)
Gram positive has thick layer of peptidoglycan
Gram negative has thin layer of peptidoglycan

16. Gram Stain The primary stain is crystal violet.
Iodine is added as a mordant to enhance crystal violet staining by forming a crystal violet–iodine complex.
Decolorization follows and is the most critical step in the procedure. Gram-negative cells are decolorized by the solution (of variable composition—generally alcohol or acetone) whereas Gram-positive cells are not.
Gram-negative cells can thus be colorized by the counterstain safranin.
Upon successful completion of a Gram stain, Gram- positive cells appear purple and Gram-negative cells appear reddish-pink.

17. Gram Stain

19. Gram Stain
The decolorization step is the most crucial and most likely source of Gram stain inconsistency. It is possible to over- decolorize by leaving the alcohol on too long and get reddish Gram-positive cells. It also is possible to under- decolorize and produce purple Gram- negative cells.

20. Gram Stain ( Gram-positive cocci ) Staphylococcus aureus
Bacillus subtilis ( Gram-positive rod )
Escherichia coli ( Gram-negative bacillus )

21. Staining Techniques Simple Stain Uses only one basic dye
Provides basic information about cell shape and arrangement
Differential Stain
Uses more than one dye
These procedures react differently with different kinds of bacteria
Helps distinguish between different kinds of bacteria
Most common and important differential stain is the GRAM STAIN

22. Acid-fast Stain
The presence of mycolic acids in the cell walls of acid-fast organisms is the cytological basis for this differential stain. Mycolic acid is a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution.
Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium leprae, the causative agent of leprosy.
A variety of acid-fast staining procedures are employed, two of which are the Ziehl-Neelsen (ZN) method and the Kinyoun (K) method.
These differ primarily in that the ZN method uses heat as part of the staining process, whereas the K method is a “cold” stain.

23. Acid-fast Stain
In the acid-fast staining procedure, the red dye carbolfuchsin is applied to a fixed smear, and the slide is gently heated for several minutes. (Heating enhances penetration and retention of the dye.)
Then the slide is cooled and washed with water. The smear is next treated with acid-alcohol, a decolorizer, which removes the red stain from bacteria that are not acid-fast.
The acid-fast microorganisms retain the red color because the carbolfuchsin is more soluble in the cell wall lipids than in the acid-alcohol.

24. Acid-fast Stain
In non-acid-fast bacteria, whose cell walls lack the lipid components, the carbolfuchsin is rapidly removed during decolorization, leaving the cells colorless.
The smear is then stained with a methylene blue counterstain.
Non-acid-fast cells appear blue after application of the counterstain.

25. Special Staining

26. Negative Staining for Capsules
Many microorganisms contain a gelatinous covering called a capsule.
In medical microbiology, demonstrating the presence of a capsule is a means of determining the organism's virulence. the degree to which a pathogen can cause disease.
Capsule staining is more difficult than other types of staining procedures because capsular materials are soluble in water and may be dislodged or removed during rigorous washing.

27. Negative Staining for Capsules
To demonstrate the presence of capsules, a microbiologist can mix the bacteria in a solution containing a fine colloidal suspension of colored particles (usually India ink or nigrosin) to provide a contrasting background and then stain the bacteria with a simple stain, such as safranin. Because of their chemical composition, capsules do not accept most biological dyes, such as safranin, and thus appear as halos surrounding each stained bacterial cell.

28. Endospore Stain
An endospore is a special resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental conditions. Although endospores arc relatively uncommon in bacterial cells, they can be formed by a few genera of bacteria.
Endospores cannot be stained by ordinary methods, such as simple staining and Gram staining, because the dyes do not penetrate the wall of the endospore.

29. Endospore Stain
The most commonly used endospore stain is the Schaeffer Fulton endospore stain.
Malachite green, the primary stain, is applied to a heat-fixed smear and heated to steaming for about 5 minutes. The heat helps the stain penetrate the endospore wall.
Then the preparation is washed for about 30 seconds with water to remove the malachite green from all of the cells' parts except the endospores.
Next, safranin, a counterstain, is applied to the smear to stain portions of the cell other than endospores. In a properly prepared smear, the endospores appear green within red or pink cells.
Because endospores are highly refractive, they can be detected under the light microscope when unstained, but they cannot be differentiated from inclusions of stored material without a special stain .

30. Endospore Stain Primary stain: malachite green Decolarization: water
Counter stain: Safranin
Result: endospores are dark green, and vegetative cells are pink

31. Flagella Stain
Bacterial flaglla are structures of locomotion too small to be seen with a light microscope without staining.
A tedious and delicate staining procedure uses a mordant and the stain carbolfuchsin to build up the diameters of the flagella until they become visible under the light microscope.
Microbiologists use the number an arrangement of flagella as diagnostic aids.

32. Flagella Stain