This document provides information on identifying bacteria. It discusses the hierarchy of biological classification and describes methods for bacterial identification including microscopic morphology, macroscopic morphology, physiological/biochemical characteristics, and genetic/molecular analysis. Steps for diagnostic isolation and identification are outlined beginning with streaking samples on culture plates and observing colony characteristics. Methods for examining bacterial cells like Gram stain, flagella, capsules, and spores are covered. Biochemical tests for identification like indole, methyl red, Voges-Proskauer, citrate, and lactose fermentation are also discussed.

1. Basics in
Identification of Bacteria
Dr.T.V.Rao MD
Dr.T.V.Rao MD 1

2. Q: What was that clue to help us
remember the hierarchy of
biological classification?
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Dr.T.V.Rao MD 2

3. Classification
Family: a group of related genera.
Genus: a group of related species.
Species: a group of related strains.
Type: sets of strain within a species (e.g.
biotypes, serotypes).
Strain: one line or a single isolate of a
particular species.
Dr.T.V.Rao MD 3

4. 4
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
Dr.T.V.Rao MD

5. Methods in bacterial identification
1. Phage Typing – which phage infects the
bacterium
2. Serological analysis – what antibodies are
produced against the bacterium
3. Pathogenicity – what diseases does the
bacterium cause.
4. Genetic & molecular analysis
• G + C base composition
• DNA analysis using genetic probes
• Nucleic acid sequencing & rRNA analysis
Dr.T.V.Rao MD 5

6. Steps in diagnostic isolation and
identification of bacteria
• Samples of body fluids are streaked on culture
plates and isolated colonies of bacteria appear
after incubation.
Observation of these colonies for size, texture,
color, and (if grown on blood agar) hemolysis
reactions, is highly important as a first step in
bacteria identification.
Whether the organism requires oxygen for
growth is another important differentiating
characteristic.
Dr.T.V.Rao MD 6

7. Identification of Bacteria after
isolation in pure forms
• The bacteria
is obtained in
pure culture
it has to be
identified
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8. Bacterial cells

9. Morphology
• Depends on
number of factors
such as strain
studied nature of
the culture
medium
• Temperature and
time of incubation
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10. Shape of the colony
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11. Shape of Bacteria
 Bacteria display three basic
shapes:
i. round- cocci, (from the
Greek kokkos - a berry),
ii. rod shaped – bacilli (from the
Latin bacillus - a stick or rod),
iii.spiral (quelled).
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12. Gram stain
a. Gram stain divides the bacteria into Gram positive
& Gram negative.
The basic procedure :
i. Take a heat fixed bacterial smear.
ii. Flood the smear with CRYSTAL VIOLET or Methyl violet for 1
minute, then wash with water. [PRIMARY STAIN]
iii. Flood the smear with IODINE for 1 minute, then wash with water.
iv. Flood the smear with ETHANOL-ACETONE, quickly, then wash with
water. [DECOLORI
v. Flood the smear with SAFRANIN for 1 minute, then wash with
water. [COUNTERSTAIN]
vi. Blot the smear, air dry and observe.
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13. Crystal
violet
Gram's
iodine
Decolorise with
acetone
Counterstain with
e.g. methyl red
Gram-positives
appear purple
Gram-negatives
appear pink
The Gram Stain

14. Dr.T.V.Rao MD 14

15. Gram-positive rods
Gram-negative rods
Gram-positive cocci
Gram-negative cocci

16. Coccus
Staphylococcus species
Streptococcus species
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17. Gram stain morphology
• Shape
– cocci (round)
– bacilli (rods)
– spiral or curved (e.g. spirochetes)
• Single or multiple cells
– clusters (e.g. streptococci)
– chains (e.g. streptococci)
• Gram positive or negative
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18. Gram-positive Cocci
• Staphylococci
– Catalase-positive
– Gram-positive cocci in
clusters
• Staphylococcus aureus
– coagulase-positive
• Staph. epidermidis
– and other coagulase
negative staphylococci

19. Gram-Positive Cocci
• Streptococci
– Catalase-negative
– Gram-positive cocci in
chains or pairs
• Strep. pyogenes
• Strep. pneumoniae
• Viridans-type streps
• Enterococcus faecalis

20. Gram-Negative Cocci
• Neisseria gonorrhoea
– The Gonococcus
• Neisseria meningitides
– The Meningococcus
• Both Gram-negative
intracellular diplococci

21. Gram-Negative Bacilli
• Enteric Bacteria
– E. coli
– Salmonella
– Shigella
– Yersinia
– Pseudomonas
– Proteus
– Vibrio cholerae
– Klebsiella pneumoniae

22. Classifying Bacteria: Gram-Negative & Gram-Positive
Gram __________
• Peptidoglycan is the thick, outermost
layer of the cell wall.
• About 90% of cell wall is made of
peptidoglycan.
Gram __________
• Cell wall is more chemically complex, thinner
and less compact.
• Peptidoglycan only 5 – 20% of the cell wall.
• Peptidoglycan is not the outermost layer,
but between the plasma membrane and the
outer membrane.
• Not accessible to the action of antibiotics.
• Outer membrane is similar to the plasma
membrane, but is less permeable and
contains lipopolysaccharides (LPS).
• LPS is a harmful substance classified as an
endotoxin.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Dr.T.V.Rao MD 22

23. Spirochetes
• Thin spiral bacteria
• Viewable by phase-
contrast
microscopy or
silver stain
– Trepanoma
palladium
– Borrelia burgdorferi
– Leptospira

24. Classification: Dichotomous Key
Simple Stain
Cocci
Bacilli
Gram Stain
Gram
negative cocci
Gram
positive
cocci
Mannitol Salt
yellow pink
Staphylococcus
aureus
Staphylococcus
epidermis
Gram Stain
Gram
negative
bacilli
Gram positive bacilli
MacConkey’s
No
color
change
Salmonella
pullorum
Pink
colonies
E. coli
Enterobacter
aerogenes
Acid Fast stain
Acid Fast
Mycobacterium
tuberculosis
Not
acid
fast
Endospore stain
Forms
endospores
Bacillus subtilus
smegmatis
Since we will be
working with a
limited number
of bacterial
species and
identification
techniques, we
will be using a
limited
dichotemous key
in lab.
Dr.T.V.Rao MD 24

25. GRAM-POSITIVE
Facultative anaerobe, cocci
 Thick cell wall, ~50% of cell’s mass.
(When you Gram stain it, the cells are
intensely purple.)
 Found in many places throughout the
environment human skin, animals, water,
dust, and soil.
 M. luteus on human skin transforms
chemicals in sweat into body odor.
 Grow well even with little water or high
salt concentrations. (You may find it growing
on your Mannitol Salt nasal sample.)
 Normal flora that can become
opportunistic in immune compromised.
Bacterial Genus: _______________
This is your lab
friend Micrococcus
luteus.
Images: M. luteus colonies, T.Port;
M. luteus, Janice Carr, PHIL #9761
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26. Isolation in pure form
 Studies on the biochemical, antigenic and other
characters of bacteria can be done only if the organism
available in the pure form.
Technique:
a. Plating on solid culture media- clinical sample is
streaked onto a solid medium (like: MacConkey agar,
nutrient agar or blood agar) in such a way so as to
ensure isolated discrete colonies.
b. Use of selective growth condition-most important
example of this is the growth of anaerobic bacteria
which will not take place in an environment having
oxygen.
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27. Isolation of Bacteria on Culture
plates
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28. Dr.T.V.Rao MD
Flagella
• Motility - movement
• Swarming occurs with some bacteria
– Spread across Petri Dish
– Proteus species most evident
• Arrangement basis for classification
– Monotrichous; 1 flagella
– Lophotrichous; tuft at one end
– Amphitrichous; both ends
– Peritrichous; all around bacteria.
28

29. Flagella
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30. India ink (capsule stain)
• The capsule stain employs
an acidic stain and a basic
stain to detect capsule
production.
• Capsules are formed by
organisms such
as Klebsiella
pneumoniae . Most
capsules are composed of
polysaccharides, but
some are composed of
polypeptides.
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31. Bacterial Spores
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32. Different types of Haemolysis
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33. Different types of Haemolysis
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34. On solid medium the following characters
are observed
i. Shape: circular, irregular, radiate or rhizoid.
ii. Size: The size of the colony can be a useful
characteristic for identification. The diameter of a
representative colony may be measured.
iii. Elevation:
iv. Margin: Entire, wavy, lobate, filiform
v. Surface: smooth, wavy, rough, granular, papillate,
glistening etc.
vi. Size in mm
vii. Texture : dry, moist, mucoid, brittle, viscous,
butyrous (buttery).
viii. Color : colorless, pink, black, red, bluish-green.
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35. contd
Bacillus subtilis Proteus spp.
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36. Lactose Fermentation
MacConkey’s agar helps us
identify different types of
Gram negative bacteria.
There is lactose in the
medium that the bacteria
can eat, and a pH
sensitive dye that
turns pink, if they
“poop out” waste
from breaking
down lactose.
Lac+ genera
include E. coli,
Enterobacter,
and Klebsiella.
Lac- genera
include
Salmonella,
Shigella, &
Proteus.
Image: MacConkey’s agar, T. Port
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37. Tests To Know
• Case Study Tests
– Indole
– Methyl Red/Voges Proskauer
– Citrate
– H2S production in SIM
– Motility
– Lactose fermentation
– Sucrose fermentation
– Glucose fermentation & gas production
– Triple Sugar Iron Agar (TSI) test
• Staphylococcus identification tests
– MSA
Dr.T.V.Rao MD 37

38. PHENOTYPIC CHARACTERISTICS
Metabolic differences
• Biochemical tests
–Sugar fermentation
• e.g., Lactose, sucrose,
glucose, etc.
• Fermentation results in
acid production
– pH indicator changes color
– Pink  yellow
• Inverted tube (Durham tube) collects any gas
produced

39. Key identification characteristics for
Enterobacteriaceae
GENUS/SPECIES Fermentation of Gas MR VP Indole Citrate Urease H2S M
G L S M
Escherichia coli (+) (+) (+) (+) (+) (+) (-) (+) (-) (-) (-)
Shiegella (+) (-) (-) (+) (-) (+) (-) (-/+) (-) (-) (-)
Shiegella sonnei (+) (+) (-) (+) (-) (+) (-) (-) (-) (-) (-)
Salmonella (+) (-) (-) (+) (+) (+) (-) (-) (+) (-) (+)
Klebsiella Pneumo. (+) (+) (-) (+) (+) (-) (+) (-) (+) (+) (-)
Enterobacter (+) (-) (+) (+) (-) (+) (-) (+) (-) (+) (+)
Serratia (+) (+) (-) (+) (+) (-/+) (+) (-) (+) (-) (-)
Proteus (+) (-) (-) (+) (-/+) (+) (-) (+) (-/+) (+) (+)
morganella (+) (-) (-) (+) (+) (+) (-) (+) (-) (+) (+)
Yersinia (+) (-) (-) (+) (-) (+) (-) (-/+) (-) (-/+) (-)
G: Glucose, L:Lactose, S:Sucrose, M: Manitol, MR: Methyl Red, VP: Voges Proskauer
Dr.T.V.Rao MD 39

40. Indole Test
Principle:
Indole test is performed to determine the ability
of the organism to split tryptophan molecule into
Indole. Indole is one of the metabolic degradation
product of the amino acid tryptophan
Bacteria that possess the enzyme tryptophanase
are capable of hydrolyzing and deaminating
tryptophan with the production of Indole, Pyruvic
acid and ammonia.
Dr.T.V.Rao MD 40

41. Indole Test
Property it tests for:
• This test is performed to help differentiate
species of the family Enterobacteriaceae.
Media and Reagents Used:
• Tryptone broth contains tryptophan.
• Kovac’s reagent—contains hydrochloric acid,
dimethylaminobenzaldehyde, and amyl
alcohol—yellow in color.
Dr.T.V.Rao MD 41

42. Indole test
• Procedure:
-Inoculate Tryptone broth with
the test organism and incubate
for 18 to 24 hrs at 37°c
-Add 15 drops of Kovac’s reagent
down the inner wall of the tube
• Interpretation:
-Development of bright red color
at the interface of the reagent
and the broth within seconds
after adding the reagent is
indicative of the presence of
Indole and is a positive test
Indole Positive:
E.coli
Proteus vulgaris
Indole Negative:
Salmonella spp.
Klebsiella spp.
Enterobacter aerogens
Dr.T.V.Rao MD 42

43. Oxidase Test
Principle
Oxidase test is used to determine the
presence of bacterial cytochrome oxidase
enzyme using the oxidization of the
substrate “tetramethyl-p-
phenylenediamine dihydrochloride” to
indophenol a dark purple colored end
product.as positive test. No colour
development indicates a negative test and
the absence of the enzyme.
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44. Oxidase Test….
Methods
Quality controls
Positive control- Pseudomonas spp
Negative control – E. coli
1. Moist filter paper
method
2. Direct plate method
.
 The reagent acts as an artificial electron acceptor substituting the oxygen. In the
reduced stage dye is colorless , but in the presence of enzyme cytochrome
oxidase dye is oxidised to indophenol blue
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45. Oxidase Test…..
Positive
• Pseudomonas spp.
• Aeromonas spp.
• Vibrio spp.
• Alcaligenes spp.
• Neisseria spp.
• Haemophilus sps
Negative
• Enterobacteriaceae
• Acenitobacter spp.
Dr.T.V.Rao MD 45

46. Methyl Red/Voges- Proskauer (MR/VP)
• Properties these test for: Both tests are used to differentiate
species of the family Enterobacteriaceae.
• Media and Reagents Used:
– Glucose Broth
– Methyl Red indicator for MR test
– Voges Proskauer reagents- A: 5% Alpha-Naphthol &
ethanol, B: Potassium Hydroxide; (3:1 ratio) & Deionized
Water.
Principle of MR test:
To test the ability of the organism to produce and maintain stable acid
end products from glucose fermentation and to overcome the buffering
capacity of the system
This is a qualitative test for acid production.
Dr.T.V.Rao MD 46

47. Principle of MR test:
To test the ability of the organism to produce
and maintain stable acid end products from
glucose fermentation and to overcome the
buffering capacity of the system
This is a qualitative test for acid production.
Dr.T.V.Rao MD 47

48. MR test (contd…)
Procedure:
- Inoculate the MR/VP broth with a pure culture of the test organism and
incubate at 35° for 48 to 72 hrs.
Add 5 drops of MR reagent to the broth
Result interpretation:
- Positive result is red (indicating pH below 6)
- Negative result is yellow (indicating no acid production)
Left: negative/Right: positive
MR Positive: E.
coli
MR Negative:
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella spp.
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49. Nitrate reduction….
• To distinguish between these two reactions, zinc
dust must be added. Zinc reduces nitrate to
nitrite. If the test organism did not reduce the
nitrate to nitrite, the zinc will change the nitrate
to nitrite. The tube will turn red because alpha-
naphthylamine and sulfanilic acid are already
present in the tube
• Thus a red color after the zinc is added indicates
the negative nitrate reduction test.
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50. Negative
Negative
Posiitive Positive
Nitrate reduced to
NH3 or N2 gas,
nitrite absent
Nitrate reduced to
nitrite
Nitrate not
reduced
Nitrate not
reduced
Nitrate reduction test….
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51. Addition of Zn dust or
Nitrate reduction test….
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52. MR test (contd…)
Procedure:
- Inoculate the MR/VP broth with a pure culture of the test organism and
incubate at 35° for 48 to 72 hrs.
Add 5 drops of MR reagent to the broth
Result interpretation:
- Positive result is red (indicating pH below 6)
- Negative result is yellow (indicating no acid production)
Left: negative/Right: positive
MR Positive: E.
coli
MR Negative:
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella spp.
Dr.T.V.Rao MD 52

53. Motility Test
• Property it tests for: This test is done to help
differentiate species of bacteria that are motile from
non-motile.
• Media and Reagents Used: Motility media contains
tryptose, sodium chloride, agar, and a color indicator.
• How to Perform Test: Stab motility media with
inoculating needle.
• Reading Results: If bacteria is motile, there
will be growth going out away from the stab
line, and test is positive. If bacteria is not
motile, there will only be growth along the
stab line. A colored indicator can be used to
make the results easier to see.
Dr.T.V.Rao MD 53

54. Urea Hydrolysis (Urease test)
• Property it tests for: This test is done to determine a bacteria’s
ability to hydrolyze urea to make ammonia using the enzyme
urease.
• Media and Reagents Used: Stuarts Urea broth (pH 6.8) contains a
yeast extract, monopotassium phosphate, disodium phosphate,
urea, and phenol red indicator.
• Principle
To determine the ability of the organism to split urea
forming 2 molecules of ammonia by the action of the
enzyme Urease with resulting alkalinity
• How to Perform Test: Inoculate Urea broth with
inoculating loop.
Dr.T.V.Rao MD 54

55. Reading Results:
• Urea broth is a yellow-orange
color. The enzyme urease will
be used to hydrolyze urea to
make ammonia. If ammonia is
made, the broth turns a bright
pink color, and is positive. If
test is negative, broth has no
color change and no ammonia
is made.
• Figure in the right shows negative (left) and
Positive (right) results. Dr.T.V.Rao MD 55

56. Coagulase test
Principle:
- This test is used to differentiate
Staphylococcus aureus (positive) from
coagulase negative Staphylococci. S. aureus
produces two forms of coagulase: bound and
free.
- Bound coagulase or clumping factor, is
bound to the bacterial cell wall and reacts
directly with fibrinogen. When a bacterial
suspension is mixed with plasma, this enzyme
causes alteration in fibrinogen of the plasma to
precipitate on the staphylococcal cells, causing
the cells to clump. Dr.T.V.Rao MD 56

57. Coagulase test
• Free coagulase is produced extra-
cellularly by the bacteria that
causes the formation of a clot
when S. aureus colonies are
incubated with plasma
Dr.T.V.Rao MD 57

58. Coagulase Results
 Reading Results:
A. Slide test:
- Positive: Macroscopic clumping in 10 seconds or
less in coagulated plasma drop and no clumping
in saline or water drop.
- Negative: No clumping in either drop.
- Note: All negative slide tests must be confirmed
using the tube test.
B. Tube test:
- Positive: Clot of any size (a)
- Negative: No clot (b)
a b
Coagulase Positive : Staphylococcus aureus
Coagulase negative: Staphylococcus epidermidis
Dr.T.V.Rao MD 58

59. Molecular analysis
It would be ideal to compare sequences of entire
bacterial chromosomal DNA.
Alternatively, genomic similarity has been assessed
by the guanine (G)+ cytosine (C) content (% GC).
This has been replaced by two alternatives:
1. Hybridization
2. Sequencing specific genes
Dr.T.V.Rao MD 59

60. DNA-DNA homology
1. How well two strands of DNA from different
bacteria bind (hybridize) together.
This technique is employed to compare the
genetic relatedness of bacterial strains/species.
2. If the DNA from two bacterial strains display a
high degree of homology (i.e. they bind well) the
strains are considered to be members of the same
species.
Dr.T.V.Rao MD 60

61. • Program created by Dr.T.V.Rao MD for
benefit of Medical Microbiologists
• Email
• doctortvrao@gmail.com
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