Bacterial classification and

identification

By
Professor Dina Moustafa Abou Rayia
Medical Microbiology and Immunology Department

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• Taxonomy:
- It is the science dealing with the
classification and nomenclature of living
things.
• Bacterial classification:
- It can be defined as the arrangement of
bacteria into taxonomic groups based on
similarities or relationships.
- The taxonomic groups used in classification
are:
Domain, kingdom, phylum, class, order,
family, genus, and species
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 Methods of bacterial identification

1.Microscopic examination 5. Animal pathogenicity

2. Cultivation (culture) 6.Molecular identification

3. Biochemical reactions 7. Antibiotic sensitivity

4. Serological identification 8. Phage typing

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 1. Microscopic examination
❖ Wet fresh unstained: To detect organism motility.
❖ Stained sample: To study the organism's shape, size, arrangement, and staining reaction.
❖ Methods of staining:
A. Simple stains: e.g. methylene blue.
B. Differential stains: first stain (has a color)-decolorizing agent- counter stain (has
another color). Two common differential stains are used:
- Gram stain: methyl violet iodine (first stain violet in color)- alcohol (decolorizing
agent)- basic fuchsine or safranine (counter stain red in color). So, the organisms can
be gram-positive (violet) or gram-negative (red).
- Ziel-Neelsen’s stain: Strong basic fuchsine (first stain red in color)- HCL or H2So4
acids (decolorizing agents)- methylene blue (counter stain blue). So, the organisms
can be acid-fast which resist decolorization (red) or non-acid fast (blue).

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 • Gram stain steps
1. Violet 2. Alcohol 3. Red

Gram -positive
• Zeil-Neelsen Stain steps Gram -negative

1. Red 2. Acid 3. Blue

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Acid -fast Non-acid fast
 2. Cultivation
• It is a procedure in which nutritive media with known
chemical composition are used to allow for bacterial
growth and multiplication under controlled conditions
in the laboratory.
• The culture is examined in the following ways:
- Naked eye appearance of the growth (size, shape,
color on solid media, pigment production etc
- Film preparation wet and stained
- Source for other identification methods.
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 Exopigment of Pseudomonas changing the
color of the media

Golden yellow colonies of Staph aureus 7

 3. Biochemical reactions
• It is a method used in bacterial identification based on the changes they

produce in different substrates as a result of their metabolic activity.

• The most common tests used in the laboratory are:

A. Action on sugar:

- Bacteria are cultured on peptone water media containing sugar to be tested

for sugar fermentation. If the bacteria ferment sugar with the production of

acid only or acid and gas or don’t ferment sugars. The medium contains a PH

indicator which changes its color upon acid production and a small inverted

tube is introduced into the medium to collect any gases liberated above the

fluid level. A set of standard sugars is commonly used (glucose, lactose,

maltose, mannite, sucrose etc. Any other sugar can be tested as required.
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 3. Biochemical reactions
B. Indole production:
- Indole is produced from the amino acid tryptophane present in
peptone. For testing, if the bacteria produce indole or not, a few
drops of Ehrlich’s reagent are added to the peptone water culture
of the suspected organism, if indole is present, a purple color is
obtained.

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 3. Biochemical reactions
C. Voges Proskauer’s (V.P) test:
- Coliform bacteria of non-animal origin
produce acetyl methyl carbinol as a side
product of glucose utilization. When
grown on glucose phosphate peptone
medium and the addition of
concentrated KOH, a pink color will
appear. Coliform bacteria of animal or
human origin give negative tests. This
test is of value in the diagnosis of fecal
water pollution. A negative test in
Non-animal origin Animal or human
drinking water is indicative of fecal
pollution. 11
 3. Biochemical reactions
D. Methyl red test (M.R.):
- Coliform bacteria of non-animal origin
ferment glucose with less amount of acid
so, the PH is above 4. Coliform bacteria of
animal origin ferment glucose with the
production of a large amount of acid
lowering PH below 4. The test is done by
growing the organism on glucose
phosphate medium containing methyl
red indicator. The indicator is red below 4
Animal origin Non-Animal origin
and yellow above 4.
So, fecal water pollution is suspected
when V.P test is negative and M.R test is
positive.
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 3. Biochemical reactions
E. Urease test:
- Some bacteria e.g. Proteus and
Helicobacter pylori produce urease
enzyme which can be detected by growing
the organism on a medium containing
urea and phenol red indicator. Urease
splits urea with the release of ammonia
that changes the medium PH to alkalinity
so the medium changes into a deep red
color.
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 3. Biochemical reactions
F. Catalase test:
- Some organisms as Staphylococci
produce catalase enzyme which
can be detected by immersing the
bacterial colonies in a few drops of
hydrogen peroxide. A rapid
effervescence indicates oxygen
production and a positive test.

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 3. Biochemical reactions
G. Oxidase test:
- Some bacteria e.g.
Pseudomonas produce oxidase
enzyme. This can be detected by
smearing bacterial colonies on a
filter paper impregnated with
oxidase reagent. An immediate
development of a deep purple
color indicates a positive test.
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 4. Serological tests
• Serologic tests are used to determine if a person has
antibodies against a specific pathogen, or to detect antigens
associated with a pathogen in a person's sample.
• Types:
- ELISA
- RIA
- Immunofluorescence

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 5. Animal Pathogenicity

• The use of laboratory animals is mainly in research for

the following reasons:
- Distinguish between pathogenic and non-pathogenic
strains of bacteria.
- To determine toxin production.
- For growing organisms that do not grow in culture like
Lepra bacilli and some viruses.

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6. Molecular identification
• Detection of microbial nucleic acid.

7. Antibiotic sensitivity
• The susceptibility of bacteria isolated
from pathological samples to different
types of antibiotics to determine the
effective drug to be used in treatment.

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 8. Phage typing
• Determining the susceptibility of a bacterial isolate to
the lytic action of a bacteriophage or a series of
phages.

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