Microbial growth dependent identification

1. Growth Dependent
Identification

2. Content
 Introduction
 Identification Method
 Isolation in PureForm
 Staining Reaction
 Morphology of Bacterial Colony
 Cultural Characteristics
 Metabolism
 Biochemical Properties
 Challenges in Bacterial Identification
 References

3. Introduction
The most important task of a bacteriology is to
identify the pathogens from the clinical sample.
While depends on some growth related
characteristics to easily identify the pathogens
either it is growing or not, can be said as growth
dependent identification.

4. Identification Method
Isolation in pure form
There are several
methods to identified
the different
type of bacteria.
Staining reaction
Morphology of bacterial colony
Cultural characteristics
Metabolism
Biochemical properties

5. 1. 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:
1. 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.
2. 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.
←

6. 2. Staining Reaction
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7. 3. Morphology of the Bacterial
Colony
Shape: circular, irregular orrhizoid.
Size: diameter inmm

8. Elevation: flat, raised, low convex, domeshaped
Surface: smooth, wavy, rough, granular, papillate, glistening etc.
Margin: Entire, wavy, lobate, filiform
←

9. 4. Cultural Characteristics
Microorganisms may show distinguishing gross
morphologies when cultured on different media.
This macroscopic appearance of bacteria is often
used in their identification. Selected strains of
bacteria will be inoculated on several types of
media for the purpose of observing and comparing
their colonial growth characteristics.
These provide additional information for the
identification of a bacterium.
←

10. On solid medium the following
characters are observed
i. Shape: circular, irregular, radiate orrhizoid.
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.

11. Mac Conkey Agar
Enterobacter cloacae on
MacConkeyAgar:
growth with pinkcolonies
Eschericia coli on MacConkeyAgar:
growth with pinkcolonies

12. In Fluid medium following characters
are observed
i. Degree of growth‐ Absence, scanty,
moderate, abundant etc.
ii. Present of turbidity and itsnature.
iii. Presence of deposit and itscharacter.
iv. Nature of surfacegrowth.
v. Ease and disintegration andodor.

13. 5. Metabolism
Toclassify the differentiate species following aspects are
studied-
←
Requirement of
oxygen
The need of
CO2
Capacity to
form pigments
Power of
hemolysis

15. Tests to know
 Case Study Tests
o Indole
o Methyl Red
o Motility
o Lactose fermentation
o Sucrose fermentation
 Staphylococcus identification tests
o MSA

16. 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 acidtryptophan.
Bacteria that possess the enzyme tryptophanase are capable of
hydrolyzing and deaminating tryptophan with the production of
Indole, Pyruvic acid and ammonia.
Property it testsfor:
● This test is performed to help differentiate species of the family
Enterobacteriaceae.
Media and Reagents Used:
● Tryptone broth containstryptophan.
● Kovac’s reagent—contains hydrochloric acid,
dimethylaminobenzaldehyde, and amyl alcohol—yellow incolor.

17. Contd…
• Procedure:
‐Inoculate Tryptone broth with the
test organism and incubate for 18
to 24 hrs at 37 oC
‐Add 15 drops of Kovac’s reagent
down the inner wall of thetube
• 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

18. Methyl Red (MR)
Properties these test for: Both tests are used to
differentiate species of the familyEnterobacteriaceae.
Media and ReagentsUsed:
● Glucose Broth
● Methyl Red indicator for MRtest
● 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 thesystem
This is a qualitative test for acid production.

19. (Contd…)
Procedure:
‐ Inoculate the MR/VP broth with a pure culture of the test organism and
incubate at 35°C for 48 to 72hrs.
Add 5 drops of MR reagent to thebroth
Result interpretation:
‐ Positive result is red (indicating pH below6)
‐ Negative result is yellow (indicating noacid production)
MR Positive:
E. coli
MR Negative:
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella spp.
negative positive

20. 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.

21. + – +
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.
Contd…

22. Lactose Fermentation
Property it tests for: This tests for the
bacteria’s ability to ferment lactose.
Media and Reagents Used: Lactose broth
contains beef extract, gelatin peptone, and
lactose. A phenol red indicator is added to
indicate acid production from fermentation.
How to Perform Test: Inoculate lactose broth
with inoculatingloop.
Results
● A positive result is yellow after indicator is
added (indicating lactose fermentation)
● A negative result will have no color change
or will be reddish. +–

23. Sucrose Fermentation
Property it tests for: This test is done to help
differentiate species of the family
Enterobacteriaceae. This tests the bacteria’s
ability to ferment sucrose and production of
acidend‐product
Media and Reagents Used: Sucrose broth
contains beef extract, gelatin peptone, and
sucrose. Phenol red indicator is added to
indicate an acid end‐product.
How to Perform Test: Inoculate sucrose broth
withinoculating loop.
Results
● A positive result is yellow after
indicator is added (indicating
sucrose fermentation)
● A negative result has no color change or is
reddish.
+ –

24. Mannitol Salt Agar (MSA)
Property it tests for: This tests for the bacteria’s ability
to tolerate 7% salt concentration and ferment
mannitol. The media is selective because it selects for
salt tolerant bacteria.
Media and Reagents: MSA media contains nutrient
agar, mannitol, 7% sodium chloride and phenol red
indicator.
How to Perform Test: Inoculate an MSA plate using
streak plate method and incubate 24‐48 hours.

25. MSA Results
Reading Results:
● If the organism is tolerant to salt it will grow.
● If the organism is not tolerant to salt it will not grow.
● If the salt tolerant organism can ferment mannitol then there will be
yellow zones around thecolonies.
● If the salt tolerant organism cannot ferment mannitol then the media will
remain pink.
Growth with no mannitolfermentation. Growth with + mannitol fermentation.

26. Test for Enzymes
Urease Test
Nitrate Reduction
Coagulase Test

27. 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 redindicator.
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 withinoculating loop.

28. 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 turnsa
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.

29. Coagulase test
This test is used to differentiate Staphylococcus aureus (positive) from
coagulase negative Staphylococci
When a bacterial suspension is mixed with plasma, this enzyme causes
alteration in fibrinogen.
leading to precipitation on the staphylococcal cells, causing the cells to
clump.
Slide test: Positive when there is Macroscopic clumping in 10 seconds or
less in a plasma drop and no clumping in a saline or water drop.
Tube test: Positive when there is a Clot of any size

30. Coagulase Results
Tube test:
-Positive: Clot of any size (a)
-Negative: No clot (b)
Coagulase Positive : Staphylococcus aureus
Coagulase negative: Staphylococcus epidermidis
(a) (b)

31. Nitrate reduction test

32. Challenges in Bacterial Identification
 Traditional methods of bacterial identification
rely on phenotypic identification of the
causative organism
 These methods of bacterial identification suffer
from two major drawbacks.
 Firstly, they can be used only for organisms that
can be cultivated in vitro.
 Secondly, some strains exhibit unique
biochemical characteristics that do not fit into
patterns that have been used as a characteristic
of any known genus and species.

33. References
 Biology of Microorganisms (7th edn.), Brock
T.D., Smith D.W. and Madigan M.T.
 Microbiology (6/E), Prescott L.M., John P.H.
Donald A.K.
Web Resources.

34. THANKs….