Classification of bacteria
Bacterial classification depends on the following characteristics.
1. Morphology and arrangement
2. Staining
3. Cultural characteristics
4. Biochemical reactions
5. Antigenic structure
6. Base composition of bacterial DNA
Morphology of bacteria
Bacterial cells are between 0.3 and 5µm in size.
They have three basic forms: cocci, straight rods, and curved or spiral
rods.
When bacteria are visualized under light microscope, the following
morphology are seen.
1. Cocci (singular coccus): Round or oval bacteria measuring
about 0.5-1.0µmb in diameter. They are found in single, pairs,
chains or clusters.
Morphology of bacteria
2. Bacilli (singular bacillus): Stick-like bacteria with rounded, tapered,
square or swollen ends; with a size measuring 1-10μm in length by 0.3-
1.0μm in width.
3. Coccobacilli (singular coccobacillus): Short rods.
4. Spiral: Spiral shaped bacteria with regular or irregular distance
between twisting.
Morphology of bacteria
Morphology of bacteria
Staining of bacteria
Bacterial staining is the process of coloring of colorless bacterial
structural components using stains (dyes).
The principle of staining is to identify microorganisms selectively
by using dyes, fluorescence and radioisotope emission.
Staining reactions are made possible because of the physical
phenomena of capillary osmosis, solubility, adsorption, and
absorption of stains or dyes by cells of microorganisms.
Staining of bacteria
• Individual variation in the cell wall constituents among different
groups of bacteria will consequently produce variations in colors
during microscopic examination.
• Nucleus is acidic in character and hence, it has greater affinity
for basic dyes.
• Whereas, cytoplasm is basic in character and has greater
affinity for acidic dyes.
Factors controlling selectivity of microbial cells
These factors include
1. number and affinity of binding sites
2. rate of reagent uptake
3. rate of reaction
4. rate of reagent loss (differentiation or regressive staining)
Factors controlling selectivity of microbial cells
This is because dyes absorb radiation energy in visible region of
electromagnetic spectrum i.e., “light”(wave length 400-650).
Absorption is anything outside this range it is colorless.
Types of staining methods
1. Simple staining method
2. Differential staining method
3. Special staining method
Simple staining method
It is type of staining method in which only a single dye is used.
Usually used to demonstrate bacterial morphology and
arrangement
Two kinds of simple stains
1. Positive staining: The bacteria or its parts are stained by the
dye.
Eg. Carbol fuchsin stain
Methylene blue stain
Crystal violet stain
1. Positive staining
Procedure:
• Make a smear and label it.
• Allow the smear to dry in air.
• Fix the smear over a flame.
• Apply a few drops of positive simple stain like 1% methylene blue,
1% carbol fuchsin or 1% gentian violet for 1 minute.
• Wash off the stain with water.
• Air-dry and examine under the oil immersion objective.
2. Negative staining: The dye stains the background and the
bacteria remain unstained. Eg. Indian ink stain, Negrosin stain
Differential staining method
Multiple stains are used in differential staining method to
distinguish different cell structures and/or cell types. Eg. Gram
stain and Ziehl Neelson stain
A. Gram staining method
Developed by Christian Gram.
Most bacteria are differentiated by their gram reaction due to
differences in their cell wall structure.
Gram-positive bacteria are bacteria that stain purple with crystal
violet after decolorizing with acetone-alcohol.
Differential staining method
Gram-negative bacteria are bacteria that stain pink with the
counter stain (safranin) after losing the primary stain (crystal
violet) when treated with acetone-alcohol.
Required reagents:
• Crystal violet
• Gram’s Iodine
• Acetone-Alcohol
• Safranin
Gram staining method
Procedure:
1. Prepare the smear from the culture or from the specimen.
2. Allow the smear to air-dry completely.
3. Rapidly pass the slide (smear upper most) three times through
the flame.
4. Cover the fixed smear with crystal violet for 1 minute and wash
with distilled water.
5. Tip off the water and cover the smear with gram’s iodine for 1
minute.
Gram staining procedure cont…
6. Wash off the iodine with clean water.
7. Decolorize rapidly with acetone-alcohol for 30 seconds.
8. Wash off the acetone-alcohol with clean water.
9. Cover the smear with safranin for 1 minute.
10. Wash off the stain wipe the back of the slide. Let the smear to
air-dry.
11. Examine the smear with oil immersion objective to look for
bacteria.
Interpretation:
. Gram-positive bacterium ……………Purple
. Gram-negative bacterium …………..Pink
