Classification & Staining
Dr. Md. Arifur Rahman
Associate Professor
Department of Microbiology
Classification on different
characteristics
A. Morphological classification
1. Cocci
i) In clusters eg. Staphylococcus
ii) In chain eg. Streptococcus
iii) In pairs with pointed ends eg. S. pneumoniae
iv) In pairs with kidney/bean shape eg. Neisseria
2. Rods (Bacilli)
i) With square ends eg. Bacillus
ii) With rounded ends eg. Salmonella
iii) Club shaped eg. Corynebacterium
iv) Fusiform eg. Fusobacterium
v) Comma shaped [Link]
3. Spirochaetes
i) Relaxed coil eg. Borrelia
ii) Tightly coiled [Link]
4. Coccobacilli eg. Haemophilus influenzae
5. Pleomorphic eg. Corynebacterium diphtheriae
B. On the basis of cell wall
1) Bacteria having cell wall eg. Most of the bacteria
2) Bacteria lacking cell wall eg. Mycoplasma
C. On the basis of habitat
1) Obligate intracellular eg. Rickettsia & Chlamydia
2) Free living eg. All other bacteria
D. On the basis of Gram staining
1) Gram positive
2) Gram negative
E. On the basis of acid fast staining
1) Acid fast eg. Mycobacterium tuberculosis
Mycobacterium leprae
Nocardia
2) Non acid fast
F. On the basis of spore formation
1) Spore forming bacteria eg. Clostridium
Bacillus
2) Non spore forming bacteria
G. On the basis of oxygen requirement
1) Obligate aerobes eg. Pseudomonas
Mycobacterium tuberculosis
2) Facultative anaerobes eg. Enterobacteriaceae
Staphylococcus aureus
Vibrio cholerae
3) Obligate anaerobes eg. Clostridium
Bacteroides
4) Microaerophilics eg. Campylobacter jejuni
Helicobacter pylori
H. On the basis of temperature
1) Psychrophilic (15 – 200 C) eg. Saprophytes
2) Mesophilic (30 -370 C) eg. Most of the pathogenic bacteria
3)Thermophilic (50 – 600 C) eg. Bacillus stearothermophilus
I. Rigid, thick walled cells
A. Free living (extracellular bacteria)
1. Gram positive
a. Cocci Staphylococcus, Streptococcus
b. Rods
i) Spore forming rods
1) Aerobic Bacillus
2) Anaerobic Clostridium
ii) Non spore forming rods
1) Non filamentous Corynebacterium, Listeria
2) Filamentous Actinomyces, Nocardia
2. Gram negative
a. Cocci Neisseria
b. Rods
1) Facultative
a) Straight
i) Respiratory Haemophilus, Bordetella
ii) Zoonotic Brucella, Yersinia
iii) Enteric E. coli, Salmonella, Shigella, Proteus, Klebsiella
b) Curved Campylobacter, Helicobacter, Vibrio
2) Aerobic Pseudomonas
3) Anaerobic Bacteroids
3. Acid fast Mycobacterium
B. Non free living (obligate intracellular) Rickettsia, Chlamydia
II. Flexible, thin walled cells (Spirochaetes) Treponema, Borrelia, Leptospira
III. Wall less cells Mycoplasma
Staining
Some important staining
Gram staining
Ziehl-Neelsen’s staining
Albert staining
Giemsa staining
India ink staining
Leishman staining
Silver staining
Classification of staining
1. Simple stains
Single basic dye is used, such as methylene blue
2. Negative staining
The technique is useful in demonstration of capsule. Bacteria
or fungus are mixed with dye (India ink). The background
gets stained leaving the capsule colourless.
3. Impregnation method
Thin cell wall of some bacteria are thickened by
impregnation of silver to make them visible under light
microscope.
4. Differential stain
They impart different colours to different bacteria or their
structures.
E.g., Gram stain and acid fast stain.
Smear preparation & fixation
1. Use a clean and grease-free slide.
2. Make a thin film, either with a saline suspension of a
colony or with a sample from a liquid suspension on a slide
with a sterilized loop.
3. Dry the film in the air.
4. Fix the film on slide by heat. This is done by slowly passing
the slide three times through a Bunsen flame. Allow the slide
to cool before staining.
Gram staining
1. Primary staining: Cover the film with crystal violet and keep
for 1 minute. It is then washed with tap water.
2. Mordanting: The slide is covered with a solution of Gram’s
iodine for 1 [Link] slide is then washed with tap water.
3. Decolourization: Pour few drops of acetone or alcohol over
the smear, and let it run down. Continue the process till violet
colour comes out from the [Link] not exceeding 30 seconds.
4. Counter staining: Cover the slide with diluted carbol fuchsin
or safranin for 1 [Link] with water and dry in air.
Result
Gram positive bacteria – Violet bacteria against pink background
Gram negative bacteria – Red bacteria against red background
Nice to know:
Violet/Purple/Blue
Red/pink
Mechanism
The crystal violet and the iodine combine in the cytoplasm of
each bacterium and colour it dark violet or purple. Bacteria
that retain this colour after the alcohol has attempted to
decolorize them, are classified as Gram-positive. Bacteria
that lose the dark violet or purple colour after
decolourization, are classified as Gram negative.
As Gram-negative bacteria are colourless after the alcohol
wash, they are no longer visible. This is why the basic dye
safranin is applied. It turns the Gram-negative bacteria pink.
As Gram-positive bacteria retain the original purple stain,
they are not affected by the safranin counter stain.
Explanation
Gram-positive bacteria have a thicker peptidoglycan cell wall
than Gram-negative bacteria.
When applied to both Gram-positive and Gram-negative
cells, crystal violet and then iodine readily enter the cells.
Inside the cells, the crystal violet and iodine combine to form
the crystal violet-iodine (CV- I) complex. This complex is
larger than the crystal violet molecule that entered the cells
and gets trapped within cell. Because of its size, it cannot be
washed out of the intact peptidoglycan layer of Gram-
positive cells by alcohol. Consequently, Gram-positive cells
retain the colour of the crystal violet dye.
In Gram-negative cells, CV- I complex is washed out by
alcohol through the thin layer of peptidoglycan . As a result,
Gram negative cells are colourless until counterstained with
safranin, after which they are pink.
Gram positive bacteria become Gram negative when their
cell wall is ruptured or damaged, and on prolonged
treatment with decolouriser.
Role of mordanting
Purpose of Gram staining
We observe-
1. Gram reaction of the bacteria (whether Gram positive or
Gram negative)
2. Morphology of the bacteria (whether
cocci/bacilli/coccobacilli)
3. Arrangement (whether cluster/chain shaped)
Bacteria that cannot be seen in Gram
stain
Bacteria Reason Alternative microscopic
approach
Mycobacteria Too much lipid in the cell Acid-fast stain
wall, so dye cannot penetrate
Treponema pallidum Too thin to see Dark ground illumination
(DGI)
Mycoplasma pneumoniae No cell wall None
Legionella pneumophila Poor uptake of red counter Prolong time of counter
stain stain
Chlamydia Intracellular Inclusion bodies in the
cytoplasm
Rickettsia Intracellular Giemsa stain
Ziehl-Neelsen’s staining/Acid fast
staining
1. Primary staining: Cover the smear with carbol fuchsin
stain.
2. Mordanting: Heat intermittently short of boiling. Allow
to act for 5 [Link] wash with tap water.
3. Decolourization: It is done by 20% H2SO4 . Wait for 1
minute and then wash with water.
4. Counter staining: Cover the slide with methylene blue
for 30 [Link] with water and dry in air.
Result: Red bacilli against blue background
Role of heat To melt lipid
Appearance- red rod beaded appearance
Why beaded?
Why called acid fast bacilli?
The term “acid fast” refers to an organism’s ability to retain
the carbol fuchsin stain despite subsequent treatment with an
acid.
The high lipid content (Mycolic acid – 60%) of their cell
makes mycobacteria acid fast.
Modified Ziehl-Neelsen’s staining
1. For Mycobacterium leprae 5% H2SO4 is applied
2. For Nocardia 1% H2SO4 is applied
3. For spore 0.5% H2SO4 is applied
Albert staining
1. Cover the slide with ‘solution 1’ and stain for 5 minutes.
[Link] with water.
3. Dry within blotting paper.
4. Cover the slide with ‘solution 2” and stain for 1 minute.
[Link] with water.
6. Dry within blotting paper.
Solution 1: toluidine blue + malachite green
Solution 2: Lugol’s iodine
Result:
The metachromatic granules stain- bluish black
The rest of the bacteria stain- green
Metachromatic granules/Volutin:
Metachromatic granules are intracytoplasmic storage form of
complexed inorganic polyphosphate.
They are called metachromatic granules due to their
displaying of the metachromatic effect; they appear red when
stained with methylene blue.
Example: Corynebacterium diphtheriae