Bacterial Identification and
Classification
Methods in bacterial identification
1. Microscopic morphology - Gram Staining, Shapes,
arrangements, motility
2. Macroscopic morphology – colony appearance, motility
3. Physiological / biochemical characteristics – aerobic,
anaerobic, photosynthetic, growth on selective media
4. Chemical analysis – e.g.peptides and lipids in cell
membranes
5. Phage Typing – which phage infects the bacterium
6. Serological analysis – what antibodies are produced against
the bacterium
7. Pathogenicity – what diseases does the bacterium cause.
8. Genetic & molecular analysis
• G + C base composition
• DNA analysis using genetic probes
• Nucleic acid sequencing & rRNA analysis
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1. Microscopic morphology - Gram Staining,
Shapes, arrangements, motility
Bacterial Morphology, structure and Staining techniques
• THE BACTERIAL CELL
• Bacteria cannot be visualized by naked eye. To understand their size,
one needs to revisit various measurement units. The unit of
measurement used in bacteriology is the micron (µ) or also called
micrometer (µm). 1 µm = One thousandth of a millimeter
• A bacterium shall characteristically have a cell envelope which
includes a layered cell wall and external surface adherents. The
appendages of cell wall include flagellae-the organs of locomotion
and fimbriae which help in adhesion of bacteria. Internally the
bacterium has loose arrangement of DNA, i.e. nuclear apparatus
surrounded by an amorphous cytoplasm which contains ribosomes.
Mesosomes and inclusion granules are other structures present in
bacterium. 4
Cell morphology
• Bacteria come in a wide variety of shapes
• Perhaps the most elemental structural
property of bacteria is cell morphology
(shape). Typical examples include:
• coccus (spherical)
• bacillus (rod-like)
• spirillum (spiral)
• filamentous
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Shape and Size of Bacteria
• Bacteria can have any of the following three shapes
• Spheroidal (cocci),
• Cylinderical (bacilli or rods) and
• Spirillar (spirochetes).
• Cocci are true spheres with diameter ranging between 0.75 to 1.25 µm
(and average of 1 µm). Bacillary in length from 2-10 times their width.
• Coccobacilli are very short bacilli. Filaments are long threads of bacilli
which have not separated into single cells. Curved bacterial rods vary
from small, coma shaped or mildly helical shaped organisms with only
one curve such as Vibrio cholerae.
• Spirochetes are long and curved bacteria They may be:
• Rounde d,
• Square cut or
• Sharply pointed.
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The Gram stain
• In Gram staining bacteria fixed to a slide are treated with a basic
dye that binds electrostatically to the negatively charged cells.
• Next, the preparation is treated with a mordant such as iodine to
form an insoluble dye-iodine complex.
• The slide is then washed with alcohol to solubilize and remove the
dye-mordant from Gram negative cells but not Gram positive
ones.
• Differential extraction of the dye-mordant by the decolorizing
agent is the critical step that distinguishes the bacteria.
• A counterstain, safranin, is applied in the final step. Cells that
have been decolorized will take up the second basic dye whereas
those already stained with the first dye will not.
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Microscopic observation of a Gram stain
• Higher magnifications are needed in order to see any detail
at all.
• Bacteria are often concentrated in a ring around the original
smear.
• Bright field oil immersion microscopy is necessary to see an
undistorted image stained bacterial smear..
• After putting immersion oil on a slide, the high dry (40x)
lens can't be used again unless the oil is removed.
• Slides are usually blotted to remove excess oil, then dipped
in xylene several times to dissolve the oil film, and air dried
in the fume hood.
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Bacterial Motility
• Many but not all bacteria exhibit motility, i.e. self-propelled
motion, under appropriate circumstances. Motion can be
achieved by one of three mechanisms:
• Most motile bacteria move by the use of flagella (singular,
flagellum), rigid structures 20 nm in diameter and 15-20 µm
long which protrude from the cell surface (e.g.
Chromatium).
• Spirochaetes are helical bacteria which have a specialized
internal structure known as the axial filament which is
responsible for rotation of the cell in a spiral fashion and
consequent locomotion (e.g. Rhodospirillum).
• Gliding bacteria all secrete copious slime, but the
mechanism which propels the cells is not known
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Colony Morphology: Describing Bacterial Colonies
• 1. Form – The form refers to the shape of the colony. These forms represent
the most common colony shapes you are likely to encounter: CIRCULAR
IRREGULAR FILAMENTOUS RHIZOID
• Size – The size of the colony can be a useful characteristic for identification.
The diameter of a representative colony may be measured. Tiny colonies are
referred to as punctiform.
• Surface – Bacterial colonies are frequently shiny and smooth in appearance.
Other surface descriptions might be: veined, rough, dull, wrinkled (or
shriveled), glistening.
• Texture – Several terms that may be appropriate for describing the texture or
consistency of bacterial growth are: dry, moist, mucoid, brittle, viscous,
butyrous (buttery).
• Color – It is important to describe the color or pigment of the colony. Also
include descriptive terms for any other relevant optical characteristics such as:
opaque, cloudy, translucent, iridescent.
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• 2. Elevation – This describes the “side view” of a colony.
These are the most common :FLAT, RAISED,
UMBONATE(having a knobby
• protuberance) , CRATERIFORM, CONVEX,
PULVINATE (cushion-shaped)
• 3. Margin – The margin or edge of a colony (or any
growth) may be an important characterisic in
identifying an organisms. Several examples are shown
below.
• ENTIRE, UNDULATE(wavy), LOBATE, CURLED,
FILIFORM (filamentous)
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