MORPHOLOGY

OF
MICROORGANISMS

CLASSIFICATION SYSTEM
Bergey's Manual
of Determinative Bacteriology the
"bible" of bacterial taxonomy.
There are such levels of microorganisms
organization:
Species Genus Family Class
Division Kingdom

Bacteria are distinguished primarily on:

a) morphological characteristics cell shapes
(rods, cocci, curved, or filament
forming)
b) spore production
c) staining reactions
d) motility
)Other groups are defined based on their
metabolism, or combinations of
morphological and physiological
characteristics.

SHAPE OF BACTERIA
Cocci spherical/ oval shaped

major

groups
Bacilli rod shaped
Vibrios comma shaped
Spirilla rigid spiral forms
Spirochetes flexible spiral forms
Actinomycetes branching filamentous
bacteria
Mycoplasmas lack cell wall

BACTERIA WITH SPORES

Oval central
Spherical central
Oval sub terminal
Oval sub terminal
Oval terminal
Spherical terminal
Free spore

Bulging

BACTERIA WITH FLAGELLA

Polar/ Monotrichous single
flagellum at one pole
Lophotrichous flagella at one
pole
Amphitrichous flagella at
both poles
Peritrichous flagella all
over
Amphilophotrichous flagella
at both ends

STAINING OF BACTERIA
Bacteria cells are almost colorless

and transparent

A staining technique is often applied

to the cells to color them

Their shape and size can be easily
determined under the microscope.

PRINCIPLE OF STAINING
Stains combine chemically with the
bacterial protoplasm.
Commonly used stains are salts:
Basic dyes: colored cation + colorless anion
e.g : methylene blue (methylene blue
chloride)
MB+ + Cl Acidic dyes: colored anion + colorless
cation
e.g : eosin ( Na+ + eosin-).

TYPES OF STAINING
Simple stains
Differential stains
Gram stain
Acid-fast stain
Endospore stain

Special stains
Negative (capsule) stain
Flagellar stain
Fluorescent stains

Staining for electron microscopy

TYPES OF STAINING
Simple staining

(use of a single stain)

For visualization of
morphological
shape & arrangement

Differential staining

(use of two contrasting stains

separated by a decolorizing agent)

Identification
Gram
stain

Acid fast
stain

Visualization
of structure
Spore
stain

Capsule
stain

SIMPLE STAIN
Simple stains provide a quick and easy way to

determine cell shape, size, and arrangement.

1) Perform a bacterial smear.
2) Saturate the smear with basic dye for
approximately 1 minute. You may use
crystal violet, safranin, or methylene
blue.
3) Rinse the slide gently with water.
4) Carefully blot dry with bibulous paper.
5) Observe the slide under the microscope,
using proper microscope technique.

BACTERIAL SMEAR

BASIC SHAPES

Cocci

Bacilli

GRAM STAIN
With Gram stain, bacteria could be divided

into:
Gram positive bacteria: bacteria that
retain crystal violet iodine dye complex
and so appear purple.
Gram negative bacteria: bacteria that
destain with 95% alcohol and appear red
due to counterstaining with carbol fuchsin.
This difference in staining affinity is due to
difference in the permeability of cell wall.

 Interpretation:
1.
2.

Gram positive: dark blue / violet

Gram negative : pink / red

CHAINS OF COCCI

Streptococcus pyogenes

FUNGI IN GRAM STAIN

CLUSTER OF COCCI

Staphylococcus aureus

Sputum smear

BACILLUS SHAPED BACTERIA

Escherichia coli

Pseudomonas aeruginosa

Fusobacterium

BACILLUS CHAIN

Streptobacillus
moniliformis

PALLISADES ARRANGEMENT

Corynebacterium diphtheriae

CURVED / COMMA SHAPE

Vibrio cholerea

SPHIROCHETES

Sphirochetes

Treponema pallidum

ZIEHL NEELSEN STAIN

This stain is used for detection of acid

fast bacteria.
These bacteria are not stained with
ordinary stains but they need exposure to
strong stains with application of heat.
Once stained, they will resist
decolorization with mineral acids such as
H2SO4 or HCL.
This property is due to large amount of
lipids and fatty acids especially mycolic
acid wax in cell wall of these bacteria.

PROCEDURES

 Interpretation:
1. acid-fast bacteria: red
2.

non-acid fast bacteria: blue

Mycobacterium spp.

BURRY-GINZS 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.

ENDOSPORE STAIN

Oscheshko stain
1) Thick layer of sporulating bacteria smeared
on slide.
2) Dried without fixation in normal room
condition.
3) Pour 0.5% HCl for 2mins with heating.
4) Wash and dry.
5) Fixate smear on burner.
6) Next stain as per in Ziehl Neelsen stain.

Schaeffer-Fulton endospore stain

1. Endospore walls are very resistant to
penetration of ordinary stains
2. Malachite green: primary stain- heated
until steam to make endospore walls more
permeable to dye
3. Washing with water will remove the green
dye from all parts of the cell except the
endospore
4. Safranin: counterstain- stain the nonspore-forming or vegetative cells red.

 Genera of bacteria that produce

endospores
1. Bacillus & Clostridium
2.

Other genera:
) Desulfotomaculum
) Sporosarcina
) Sporolactobacillus
) Oscillospira
) Thermoactinomyces

Interpretation:

Spores: green
Vegetative cells: red

ROMANOWSKY-GIEMSA STAIN

To observe bacterial nucleoid

1. Dried smear of pure culture prepared
(B.cereus).
2. Fixate in Karnua Solution,
3. Applicate HCl with heating.
4. Stain the smear with Romanowsky-Giemsa
dye (azur, eosin, and methlyene blue) for
40-60mins.
5. Wash with water and dry the stained slide.

FLAGELLA STAIN
Purpose: To determine the presence
/absence and location of flagella on various
microorganisms.
Principle: Because bacterial flagella are
very thin and fragile a special stain
(flagella stain) is prepared that contains a
mordant. This mordant allows piling of the
stain on the flagella, increasing the
thickness until they become visible. Various
arrangements of flagella are seen on
different cells.

1. Add about 1 ml. of the Flagella Stain

solution (one dropper full) to the smear and
allow to stain for 10-15 minutes. The
solution should not be allowed to flow outside
the waxed area.
2. Flood off the stain by adding tap water to
the slide while it remains on the rack. Do
not tip the slide before this is done.
3. Drain and flood the slide with carbol fuchsin
for one minute. Rinse by flooding. Drain and
air dry. Do not blot.

Bacillus brevis

Proteus - Peritrichous Arrangement of

Flagella

THE END
THANK YOU
PREPARED BY: PATHUMAVATHY RAMANTHAN (21B)