1. Staining

2. Bacteria are microscopic organism, they tend to be transparent even when they are magnified. The simplest way to make bacteria visible to human eyes is stain them with a visible dye. The best bacterial stains are aniline dyes. These dyes react in either: an acidic , basic, or neutral manner.
Acidic dyes are anions(negative charged)
Basic dyes are cations (+ve charged)
Bacteria are slightly negatively charged so stain very well with basic dyes.

3. Stains and Staining Basic dye stains bacteria
Acidic dye stains background

4. We stain bacteria to study there :
A) Morphology and Arrangement :
Coccus: pairs, chains, clusters
Bacillus:
spiral

5. Bacterial arrangement and Morphology

6. B)Differentiated bacteria to groups according to there biochemical composition of cell wall
C) Study structures of bacteria (capsule, flagella)
Stains are classified into:
Simple stain (methylene blue, crystal violet)
Differential stain(Gram's stain ,acid fast stain).
Special stain (capsule stain, flagella stain).

7. Preparation of smear Clean the slide
Place a loopfull of water in the centre of the slide
Mix a small amount of bacteria using a sterile loop (heating it at Benzen' burner) with the water and spread it out.
Allow the slide to air dry
Heat-fix the smear by passing the slide through the Benzen' burner three times.

8. Simple Stains Prepare the smear
Flood the slide with methylene blue for 3 mins
Wash the slide with tap water genetly, drain off excess water then let the slide dry in air or by using filter paper
Exam it microscopically
The bacteria will appear blue cells.

9. Differential Stains Gram Staining
Basic classification of bacteria is based on the cell wall structure.
Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups:
Gram positive and Gram negative.
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10. Gram-positive cell walls
The characteristic compound found in all true bacterial cell walls
is peptidoglycan. The amount of PPG is among one of the
differences between the GP and GN cell walls.
Gram-positive cell walls
Gram-negative cell walls
Thick peptidoglycan
(90% peptidoglycan)
Teichoic acids
Not many polysaccharides
In acid-fast cells, contains mycolic acid
Thin peptidoglycan
(5-10% peptidoglycan)
No teichoic acids
Outer membrane has lipids, polysaccharides
No acid- fast cells (mycolic acid)

11. The process includes the use of:
a primary stain (crystal violet)
a mordant (helper) iodine solution,
a decolorizer (95% ethanol),
a counterstain (safranin).

12. The Gram stain 1. prepare a smear
2. Flood slide with crystal violet and let stain for 1 minute.
3. Drain off crystal violet and rinse off with distilled water; flood slide with Gram's iodine for 1min,rinse off iodine with distilled water.
  4. Remove excess water from slide and blot, so that alcohol used for decolorization is not diluted. Hold the slide on an angle and drop 95% ethyl alcohol for 10 seconds and wash off with tap water.
5. flood the slide with safranin and let stain for 2-3 minutes.
6. Rinse with distilled water and blot dry with filter paper.
Gram
positive
Gram
negative

15. Mechanism of action
the PPG is found in layers in GP CW and the stain molecules are trapped within these layers, when they form the complex with the mordant Iodine molecules.
Since the GN CWs lack much PPG the amount of stain captured in
those CWs is much lesser.
When the cells are treated with the decolorizer – the ethanol – this
causes denaturation of the proteins in the outer membrane of the
GN CWs resulting in gaping holes in these CWs that lead to the
removal of the crystal violet-iodine complexes easily, leaving these
cells unstained.
The counterstain -safranin- thus is used to make these cells visible.

16. There are 4 conditions to be followed for a valid Gram staining :
Young cultures - must be young within 18-24hrs old
(older cultures lose their Gram staining properties
due to changes in the CWs as the cells get older)
Thin smear- thicker or uneven smears will result in uneven staining and decolorization
Fresh reagents - of proper strength
Control cultures - for a known GP bacterium and GN culture (S.aureus & E.coli)

17. History Hans Christian Gram (born in 1853) studied botany in Denmark
In 1883, he graduated from medical school, he settled in Berlin.
In 1884, while examining lung tissue from patients who had died of pneumonia, Gram had discovered that certain stains were taken up and retained by bacterial cells.Certain bacteria (pneumococci) retained the color (gram-positive), while other species became bleached or de-colorized by the alcohol (gram-negative). His initial work with this staining process was performed on Streptococcus pneumoniae and Klebsiella pneumoniae.

18. Gram did not use a counter stain in his
procedure.
the German pathologist Carl Weigert
( , added a final step of
staining with safranin.
In 1891, Gram became a lecturer in
pharmacology
In 1900 he resigned his Chair in Pharmacology
to become Professor of Medicine.
The king of Denmark awarded him in
1912, and 1924.
He retired in 1923 and died in 1938.

19. Special Stains Capsule stain
Many bacteria, including both gram-positive and gram-negative, may be surrounded by an outer polysaccharide-containing layer termed the glycocalyx

20. When the composition of this layer is tightly bound and remains attached to cells, it is referred to as a capsule. The capsule or glycocalyx is a gelatinous outer layer that is secreted by the microbe and remains stuck to it. Capsules may be polysaccharide, glycoproteins or polypeptides depending on the organism.

21. The capsule functions:
It protects the cell from DRYING.
source of NUTRITION. in times of need.
It helps the cells stick or attach to things because of its sticky (adhesive) nature.
It may be TOXIC or inhibitory to a host's defense system and so aid in the disease process.

22.  Capsule staining methods depend upon revealing the presence of the capsule indirectly.
There are 3 procedure to stain capsule
1. India Ink Method
Anthony’s method
Hiss’s method

23. Anthony’s capsule stain
Anthony’s capsule stain procedure
Prepare a smear from a 12- to 18-hour culture
Allow the smear to air dry. DO NOT HEAT FIX (to avoid destroying or distorting the capsule or causing shrinkage). 
Cover the slide with 1% crystal violet for 2 minutes
Rinse gently with a 20% solution of copper sulfate.
Air dry the slide. DO NOT BLOT. (Blotting will remove the un-heat-fixed bacteria from the slide and/or cause disruption of the capsule.)
Examine the slide under an oil immersion lens
the bacterial cells and the background will be stained by the crystal violet while the unstained capsule will appear transparent.
Mechanism of action
crystal violet is used as the primary stain, interacting with the protein material in the culture broth
20% copper sulfate serves as the mordant stabilize the capsule structure..
the bacterial cells and the background will be stained by the crystal violet while the unstained capsule will appear white.

24. Encapsulated Bacillus anthracis using india ink method
 Nonencapsulated Bacillus megaterium stained using Anthony's capsule stain.
Encapsulated Streptococcus lactis stained using Anthony's capsule stain.

25. India ink staining Staining procedure
Place a loopfull of India ink on the side of a clean slide.
mix a loopful of liquid culture  with the ink.
Place a clean cover slip over the preparation avoiding air bubbles.
Examine microscopically.
demonstrate the capsule which is seen as an unstained halo around the organisms distributed in a black background.

26. Principle: India ink is not absorbed by the cells or capsules of organisms. When encapsulated forms are mixed -with India ink on a glass slide, the dark background of ink particles clearly outlines the colorless capsule surrounding the more dense appearing, centrally situated cell.

27. The flagella stain allows observation of bacterial flagella under the light microscope.  Bacterial flagella are normally too thin to be seen under such conditions. The flagella stains employs a mordant to coat the flagella with stain until they are thick enough to be seen. 
Many bacteria are motile; some accomplish this motility by means of flagella.  Flagella can vary by number and location. Some bacteria only have one flagella; this is called monotrichous.  In most monotrichous bacteria, the flagella is at the end of the cell; this placement is called polar. Some bacteria have a flagella at either end of the cell; this arrangement is called amphitrichous. Many bacteria have multiple flagella; these may all be located in a tuft at one end of the cell, in which case the arrangement is lophotrichous, or they may be all over the cell, in which case the arrangement is peritrichous. 

28. The flagella stain allows observation of bacterial flagella under the light microscope.  Bacterial flagella are normally too thin to be seen under such conditions.
The flagella stains employs a mordant to coat the flagella with stain until they are thick enough to be seen. 
Many bacteria are motile; some accomplish this motility by means of flagella. 
Flagella can vary by number and location.
Some bacteria only have one flagella at the end of the cell this is called monotrichous. 
Some bacteria have a flagella at either end of the cell; this arrangement is called amphitrichous.
Many bacteria have multiple flagella; these may all be located in a tuft at one end of the cell, in which case the arrangement is lophotrichous,
or they may be all over the cell, in which case the arrangement is peritrichous. 

29. Special Stains
Flagella stain

30. Differential Stains Acid-fast stain
For MICR 2909
Lecture 2, 2001
Differential Stains
Acid-fast stain
Used to detect Mycobacterium species
Acid fast stain
Acid fast stain (Ziehl-Neelson) for identifying mycobacteria
The lipid mycolic acid (from mycobacteria) is the determinant of retaining the basic fuchsin in the acid-fast stain
BSc(MolBiol) Lect 2.ppt

31. Special Stains
Spore stain (Schaeffer-Fulton)
Bacillus subtilis

32. Microscopy Measurement Microorganisms are very small Use metric system
Metre (m) : standard unit
Micrometre (m) = 1 x10-6 m
Nanometre (nm) = 1 x10-9 m
Angstrom (Å) = 1 x10-10 m