identification of bacteria

2. METHODS OF
IDENTIFICATION
 PHENOTYPIC
-MANUAL
-AUTOMATED
Traditional methods of bacterial identification rely on
phenotypic identification of the causative organism using
gram staining, culture and biochemical methods.
Two major drawbacks:
First, they can be used only for organisms that can be
cultivated in vitro.
Second, 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.
 GENOTYPIC

3. Gram Stain
 •Based on cell wall composition and
peptidoglycan thickness
 •Gram positive cell wall
 •Gram negative cell wall

5. Morphological Characteristics Colony
Isolation & Gram Stain

6. Biochemical tests
 Enzyme and Growth characteristics
Identification of bacteria involves testing
the pure culture.
 Incubating is many relevant indicating
media and collecting the data regarding
characteristics like growth in solid and
broth media, enzymic reactions,
morphology and its physical, chemical
and biological requirements for growth.
 Strict quality control required

7. Serology
 Serology In addition to enzyme
reactions and growth features a
bacterium can be tested to see what
anti-gens it carries on its cell and extra-
cellular material. This is done by testing
the cells with a range of anti-bodies to
produce a sero-type

8. Latex Agglutination – E. coli O157

9. Miniaturized Kits
API 20E Enterobacteriaceae +
18-24 h
Generate profile #
Biochemical reactions:

11. Interpretation
 All these phenotypic characteristics
need to be interpreted in a method that
allows for the probability of certain
features not occurring.
 Computer programs are available to
calculate these probabilities.
 Identification can be expressed as a
certain % probability of being one
species and another % of being
something else.

12. Automated ID and Sens
 Automated ID testing available since 1977
 Biochemical substrates miniaturised and read
by colourmetric or fluormetric means
 Available in many bigger labs
 Multiple Antibiotics in different dilutions
available on Cards or panels to ascertain MIC
– Expert Functions
 Suppliers: Microscan Walkaway (Dade
Behring) Vitek2 (BioMerieux) BD Phoenix (BD)
 Cost neutral on ID part but not on
susceptibility testing. What will happen to
these systems with introduction of Maldi-
tof?

14. MALDI-TOF
Will it replace traditional
diagnostic and Biochemical
tests?
 Matrix-assisted laser desorption ionisation –
time of flight mass spectroscopy
 uses 16s ribosomal proteins
 Compares the mass peaks achieved by test
strains to those of approx 3,500 known strains
in the MALDI Biotyper Library
 Organism identification within 20 minutes of
starting the process
 The resultant identification is meant to be
robust, as it relies on high abundance proteins.

15. Principle
The sample for MALDI is uniformly mixed in a large quantity
of matrix.
The matrix absorbs the ultraviolet light (nitrogen laser
light, wavelength 337 nm) and converts it to heat energy.
A small part of the matrix (down to 100 nm from the top
outer surface of the Analyte in the diagram) heats rapidly
(in several nano seconds) and is vaporized, together with
the sample.
Charged ions of various sizes are generated on the sample
slide. A potential difference V0 between the sample slide
and ground attracts the ions . Ions with smaller m/z value
(lighter ions) and more highly charged ions move faster
through the drift space until they reach the detector.
Consequently, the time of ion flight differs according to the
mass-to-charge ratio (m/z) value of the ion.

16. MALDI-TOF
 Protein based spectral identification of
bacteria
 Identifications available in literally minutes
– not hours
 Tiny amount of bacterial growth needed –
not affected by media or incubation
conditions
 Minimal cost per test, virtually no
consumables
 Suppliers : BD/Bruker, BioMerieux

19. Genotype
 It is possible that not all genes are
expressed and two different bacteria can
still appear the same with all the above
phenotypic information.
 An examination of the genetic material is
possible were the DNA is examined
directly

20. Real Time PCR Based
Bacterial Identification
 Using a DNA based assay, one can easily detect bacterial
strains directly from clinical samples or from small amounts
of cultured bacterial cells, thus improving the sensitivity and
decreasing the time required for bacterial identification. PCR
has been particularly useful in this regard, which relies on
primer sequences designed to facilitate bacterial
identification at any level of specificity: strain, species or
genus.
 Real-time PCR is a promising tool for distinguishing specific
sequences from a complex mixture of DNA and therefore is
useful for determining the presence and quantity of
pathogen-specific or other unique sequences within a
sample. Real-time PCR facilitates a rapid detection of low
amounts of bacterial DNA accelerating therapeutic decisions
and enabling an earlier adequate antibiotic treatment.

21. Microarray Based Bacterial
Identification
 Microarrays combines the potential of
simultaneous bacterial identification and
speciation. This method is versatile and makes
it possible to detect and discriminate different
bacterial samples on a single slide. The rapid
identification of the bacteria in clinical samples
is important for patient management and
antimicrobial therapy. DNA microarray-based
approach is used for the quick detection and
identification of bacteria using species-specific
oligonucleotide probes designed for specific
regions of various targeted genes.

22. Biochemical tests for
identification of bacteria

23. Test for metabolism of carbohydrates &
related compounds
 Bacteria differ widely in their ability of metabolize carbohydrates &
related compounds.
 Carbohydrates may be altered when exposed to heat, for these
reasons, it is recommended that it should be sterilized before
carbohydrate is added.
 For purposes of identification these differences can be
demonstrated by four varieties of test:
(1) Tests to distinguish between aerobic &
anaerobic breakdown of sugars.
(2) Tests to show the range of carbohydrates &
related compounds.
(3) Tests for specific breakdown products.
(4) Tests to show ability to utilize a particular
substrate.

24. Control of tests
 Sterility of each batch of test medium should be confirmed by
incubating one or two uninoculated tubes of each batch
along with test.
 If it show evidence of bacterial growth, the test and
remaining of that batch of medium should be discarded.
 Control test are also done to confirm that test media have
been made up correctly & that they are used and observed
under proper condition.
 One tube of each batch of test medium is inoculated with a
stock culture of a bacteria known to give a positive reaction
& another tube with a stock culture known to give a negative
reaction.
 Positive & negative controls are incubated and examined
along with the tests.

25. Test to show metabolism of different
carbohydrates
 Sterilization of solution of test compound by filtration is
recommended.
 Tyndallization may be used for sterilization of a sugar
solution incorporated in nutrient media, eg:-peptone water.
Look for production of acid & gas or acid alone when a pure
culture grows in the presence of the test compound.
Constituents of fermentation test media:
(a) A suitable nutrient medium as a base to allow growth of
organism under test
 Nature of this medium depends upon the nutritional
requirement of the organism.
 eg:- Peptone water, serum peptone water & serum agar.

26. Contd.
(b) Carbohydrate or related compound under test.
 A large variety are used & they are often referred to loosely
as ‘sugars’.
 Commonly used sugars includes the following:
 Monosaccharides:
Pentoses:- Arabinose, Xylose, Rhamnose.
Hexoses:- Glucose, fructose, mannose, sorbose,
galactose.
 Disaccharides:
Sucrose, Lactose, trehalose, cellobiose.
 Trisaccharides: Raffinose.

27. Contd.
 Polysaccharides:
Starch, inulin, dextrin, glycogen.
 Polyhydric alcohol:
Glycerol, erythritol, adonitol, mannitol, dulcitol, sorbitol,
inositol.
 Glycosides:
Salicin, coniferin, aesculin.
 Organic acids:
Tartrate( dextro, laevo & meso), citrate, gluconate, malonate.

28.  Sugar is usually added in the conc. of 0.5% to peptone water or
1% to serum bases.
 A suitable indicator that will change color only as a result of the
fermentation of acids during the fermentation is required.
 Variety of indicators with a pH between 6 to 8 have been used.
eg:
(A) Andrade indicator: Made by adding NaOH 1 mol/litre to a
0.5% solution of acid fuchsin until the color just become yellow.
It is used at a final conc. Of 0.005% in the medium & it turns
dark reddish pink at about pH 5.5.
(B) Bromocresol: Made up as a 0.2% solution.
2.5 ml of this solution is added to each 100 ml medium to give
a final conc. Of 0.005%.
Its yellow at pH 5.2 & violet purple at pH 6.8.
Adv:- Used in media that are likely to be stored for long time.

29. Contd.
(C) Phenol red: Solution of phenol sulphonthalein. Add 1.0
gm of it into 10 ml NaOH 0.1 mol/litre & add 20 ml distilled
water & dissolve it by gentle heat.
 Then add 10 ml HCl 0.1mol/litre & make up to 500 ml with
distilled water to give a 0.2% solution.
 For use 5 ml of it is added to each 100 ml of medium, giving
a final conc. of 0.01%.
 It is yellow at pH 6.8 & purple-pink at pH 8.4.
(D) Bromothymol blue: Made up as a 0.2% solut. By
dissolving 1 g in 25 ml NaOH 0.1 mol/litre mixed with 475 ml
distilled water.
 For use, 1.25 ml of 0.2% solut. is added to each 100 ml
medium giving a final conc. Of 0.0025%.
 It is yellow at pH 6.0 & blue at pH 7.6.
 It is alternative to phenol when pH change is small.

30. Serum agar fermentation media
 Recommended for organism such as Meningococci &
Gonococci that grow poorly in liquid media.
 Basal media: Consist of Peptone, NaCl, distilled water,
digest broth, agar, phenol red 0.2% & final pH should be 7.6.
 Complete medium: Basal medium ----100 ml
Sterile serum-------5 ml
Test compound, 10%---10 ml
 After incubation, examine the media for presence of a color
change, indicating acid.
 Slope of solid media are seeded over the whole surface.

31. Tests for specific breakdown products
 Methyl red test:
Principle: Ability of bacteria to ferment glucose & produce
mixed acid by product & maintain the acidic condition & to
overcome the buffering capacity of the system.
 Employed to detect the production of sufficient acid during
fermentation of glucose & maintenance of pH of culture is
sustained below 4.5, as shown by a color change of methyl
red indicator.
 MR result is determined after 48 hrs, or longer incubation as
all enteric organism give a positive reaction after 18 to 24
hrs.
 MR positive— E. coli ATCC 25922 ( Red To Magenta )
 MR negative- Enterobacter aerogenes ATCC 13048 ( Yellow
color)

32. e
Positive
Negative

33. Tests to Show Ability to Utilize a
Specific Substrate
 (A) Citrate Utilization Test
 (B) Malonate Utilization Test

34. (A) Citrate Utilization Test
Principle- Some bacteria can obtain energy in a manner
other than by CHO fermentation, by using citrate as a sole
source of carbon. Citrate utilization in the medium is
detected by the formation of alkaline by products (NH3).
 Used for identification of many members of
Enterobacteriaceae
 Medium- Used to detect Citrate utilization by test bacteria
must be devoid of protein and carbohydrate as a source of
carbon
 Media used- Simmons Citrate, Christensen’s Citrate sulphide
medium, Koser’s Citrate medium.

35. Citrate test
 Result interpretation
 Positive results-
Represented by development of
deep blue color within 24 – 48
hrs.
 Positive Control-
Enterobacter aerogenes
 Negative Control- E. coli Positive Control

36. H2S production test
Lead Acetate Strip
Showing H2S
production
Lead Acetate Strip
Showing lack of H2S
production

37. Indole test
 Principle:- It demonstrate the ability of certain bacteria to
decompose the amino acid tryptophan to indole which accumulates in
the medium.
 Indole is then tested for by a colorimetric reaction with
p-dimethylaminobenzaldehyde.
 Indole is a volatile & can be detected either by testing medium with p-
dimethylaminobenzaldehyde or by a paper impregnated with oxalic
acid held near the mouth of tube or container.
 Temp. of the incubation may affect the result.
 Uses:-Differentiate b/w E. coli (+) from Klebsiella & Enterobacter(-)
Citrobacter koseri (+) from Citr. freundii (-)
Proteus vulgaris (+) from other Proteus spp.(-)

38. Contd.
 Quality control:- Positive- E. coli
Negative- Klebsiella pneumoniae
Procedure:- Inoculate tryptophan broth (or other suitable
indole media) with test organism & incubate at 35° C for 24-
48 hrs. At the end add 15 drops of reagent ( Ehrlich’s
reagent= p-dimethylaminobenzaldehyde, Absolute ethyl
alcohol & Conc HCl or Kovac’s reagent= Pure amyl or
isoamyl alcohol, p- dimethyl aminobenzaldehyde & Conc
HCl) down the inner wall of the tube.
Interpretation:- A bright fuchsia red color at the interface of
the reagent & broth within seconds after adding reagent is
indicative of presence of indole & is a positive colour.

39. Contd.

40. Amino acid decarboxylase test
 Principle:- Decarboxylases are group of substrate-
specific enzymes that are capable of reacting with the
carboxyl (COOH) portion of amino acids, forming alkaline
reacting amines.
 After decarboxylation reaction CO2 is released as second
product.
 Each decarboxylase enzyme is specific for an amino acids.
 Lysine, ornithine & arginine are three amino acids routinely
tested in identification of the Enterobacteriaceae.
 The specific amine products are as follows:-
Lysine– Cadaverine
Ornithine- Putrescine
Arginine– Citrulline
 Arginine reaction is recognised now as a dihydrolase test.

41. Contd.
Media used:- (a) Moller’s decarboxylase base ( Peptone, beef
extract, bromcresol purple, cresol red, pyridoxal phosphate, glucose
& deionized water ).
(b) Falkow’s lysine decarboxylase broth ( Peptone, yeast
extract, glucose, L-lysine, bromocresol purple, deionized water ).
Quality control:-
Amino acid Positive control Negative control
Lysine Enterobacter aerogenes Enterobacter cloacae
Ornithine Enterobacter cloacae Klebsiella pneumoniae
Arginine Enterobacter cloacae Enterobacter aerogenes

42. Contd.
 Procedure: From a well isolated colony of the test organism,
inoculate two tubes of Moller decarboxylase base medium, one
containing amino acid to be tested, the other to be used as control
devoid of amino acid.
 Overlay both tubes with sterile mineral oil to cover about 1 cm of the
surface & incubate at 35-37° C for 24-48 hrs.
 Result interpretation:-Positive (+):- Turbid purple–blue color.
Negative (-):- Bright, clear yellow color ( ie.
Only glucose is fermented).
Control:- Yellow color indicates that organism
is viable.

43. Decarboxylation reaction
Control
Positive Negative

44. Phenylalanine deaminase test
 Principle: - To determine the ability of an organism to
deaminate phenylalanine to phenylpyruvic acid
enzymatically by Phenylalanine deaminase, with resulting
acidity.
 Uses:- It is characteristic of Morganella morganii biogp. 1 &
2, & all Proteus & Providencia spp.
 Media used:- Phenylalanine Agar ( DL-Phenylalanine,
yeast extract, sodium chloride, di sodium hydrogen
phosphate, agar, distilled water )
 Reagent:- 10% solution of ferric chloride.
 Quality control:- Positive control--- Proteus spp.
Negative control---E. coli

45. Contd.
 Procedure:- Inoculate with a fairly heavy inoculum
from a well isolated colony.
 Incubate for 4hrs to 18-24 hrs at 35-37° C.
 After incubation 4-5 drops of ferric chloride (10%) added
directly to the surface of agar.
 As the reagent is added, rotate the tube to dislodge the
surface colonies.
 Result interpretation:- The immediate appearance of an
intense green color in the fluid & at agar slope indicates the
presence of phenylpyruvic acid & a positive result.

46. PPA Test
Positive Negative

47. Tests for enzymes
(1) Catalase test: -
 Principle: - To test for the presence of the enzyme
catalase.
 Catalase is an hemoprotein enzyme that decomposes H2O2
into water & nascent oxygen.
 This enzyme is essential to the biological defense against
oxygen toxicity.
 H2O2 is an oxidative end product of the aerobic breakdown
product of sugars.
 Uses:- Most commonly used to differentiate members of
Micrococcaceae from members of the Streptococcaceae.
 Bacillus (+) from Clostridium(-).

48. Contd.
 Reagent: H2O2 3% stored in a brown bottle under
refrigeration.
 It must be tested with positive & negative control
organism.
 Quality control:- Positive control– Staph. aureus
Negative control– Strept. spp.
 Procedure:- (1) Slide method:- With an
inoculating needle or a wooden applicator stick,
transfer growth from the center of a colony to the
surface of a glass slide, then add one drop of 3% H2O2
& observe for brisk effervescence of nascent oxygen.
 (2) Tube method:- Add 1 ml of 3% H2O2 directly to
an 18-24 hrs heavily inoculated pure agar slant culture.
Observe for immediate bubbling.

49. Contd.
 Result interpretation:-
Rapid & sustained appearance of
bubble constitutes a positive test.
 Caution:- Catalase is also
present in RBCs, so care must be
taken to avoid carryover of RBCs
with colony material.

50. Oxidase test
 Principle:- To determine the presence of the intracellular
oxidase enzyme that will catalyze the transport of electrons b/w
electrons donors in the bacteria & a dye tetramethyl-p-
phenylene-diamine dihydrochloride is reduced to a deep purple
color.
 This oxidase reaction is due to a cytochrome oxidase system
that activate oxidation of reduced cytochrome by molecular
oxygen, which in turn acts as an electron acceptor in the
terminal stage of the ETS.
 Uses:- (1) Identify all Neisseria spp., Pseudomonadaceae(+).
(2) Most GP Bacteria are oxidase negative.
(3) To differentiate b/w Moraxella, Vibrio, Campylobacter
&
Neisseria (+) from Acinetobacter(-).
(4) To exclude family Enterobacteriaceae (-).

51. Contd.
 Reagents:- Kovac’s reagent:- 1% solution tetramethyl-
p- phenylene-diamine dihydrochloride.
 Quality control:-
Positive control–
Ps. aeruginosa
Negative control– E. coli
 Procedure:- (A) Dry filter paper method:- Oxidase
reagent is unstable & has to be freshly prepared for use.
 Strips of Whatman’s No. 1 filter paper are soaked in a freshly
prepared 1% solution of tetramethyl-p-phenylene-diamine
dihydrochloride. After draining for about 30 seconds the
strips are freeze dried & stored in a dark bottle tightly sealed
with a screw caps.

52. Contd.
 The colony to be tested is picked with a platinum loop & smeared
over the moist (distilled water) area.
 Result:-A positive reaction is indicated by an intense deep-purple
hue, appearing within 5-10 sec.
 ‘Delayed reaction’-- Deep-purple hue in10-60 sec.
 Negative reaction--- Absence of coloration or by coloration later
than 60 sec.
(B) Wet filter paper method:- Reagent must be freshly
prepared & the bacterial growth must be transferred to the test
paper with a clean loop & look for color.
When testing colony from Mac Conkey medium, a pink violet color
is due to carry-over from the medium & not a true oxidase reaction.

53. Oxidase test
Caution:-Do not attempt to
perform an oxidase test on any
colonies growing on medium
containing glucose because it’s
fermentation inhibits oxidase activity
which may result in false negative.

54. Urease test
 Principle:- To determine the ability of an organism to split urea in to
two molecules of ammonia by the action of the enzyme urease, with
resulting alkalinity(NH3)& is tested by means of a suitable pH
indicator .
 An alternative method is to test for the production of ammonia from
urea by means of Nessler’s reagent.
 Medium:- (1) Christensen’s medium (Peptone, NaCl, dipotassium
hydrogen phosphate, phenol red,agar,DW,glucose10%,urea 20%)
 (2) Rustigian &Stuart’s urea broth.
 (3) Urea R (rapid) broth.

55. Urease test
Quality control:-Positive- rapid and strong
Proteus mirabilis ATCC 29906
Proteus vulgaris ATCC 13315
Positive( weak)– Kleb. peumoniae ATCC 13883
Negative-- E. coli ATCC 11775
Procedure:- Inoculate heavily the over the entire slope surface &
incubate at 37°C for 18-24 hrs.
Result:-Positive- Intense pink red color on the slant .
Rapid (+):- 1-6 hrs. for all proteeae organism
Delayed(+): 24 hrs.-6 days some Kleb. Enterobacter,
Citrobacter spp.
Negative- no color change.
Caution :- Don't stab the butt because it serves as a color control.

56. UREASE TEST
Positive Negative

57. ONPG TEST
 PRINCIPLE:- To demonstrate the presence or absence of
enzyme β-galactosidase by use of organic compound o-
nitophenyl-β-D-galactopyranoside(ONPG).
 β-galactosidase (intracellular enzyme) which hydrolyses
lactose & a permease which regulate the uptake of lactose in
to the cell.
 Certain bacteria possess the β-galactosidase enzyme but
not the permease .
 These potential lactose fermenters may not produce acid in
peptone water media.

58. Contd.
Reagents :- (1) o-nitophenyl-β-D-galactopyranoside
(2) sodium phosphate buffer 0.01mol/l
Quality control:- Positive- E. coli ATCC 25922
Negative- Proteus mirabilis ATCC 29245
Neisseria meningitis ATCC 13077
Procedure & result:- Inoculate & incubate for 24 hrs. at 37°C
if the
test is positive a yellow color will develop in the
fluid .
 Late lactose fermenters give a positive result more quickly
in this medium.

59. ONPG Test
Positive Negative

60. Nitrate reduction test
 Principle :- To determine the ability of an organism to reduce
nitrate to nitrite or to free nitrogen gas.
 It test for the presence of the enzyme the nitrate reductase which
causes reduction nitrate in the presence of suitable electron donor
to nitrite which can be tested for by an appropriate colorimetric
reagent
 Uses :- Almost all Enterobacteria reduces nitrate
 Helpful in identifying members of Haemophilus, Neisseria,
Moraxella genera
 Media & reagent :-(1) nitrate broth or nitrate agar
 (2) reagent A- α naphthylamine (5 mol/l)
 (3)Reagent B- Sulfanilic acid 5 mol/l
 Immediately before use mix equal volumes of sol A & B to give test
reagent.

61. Contd.
Quality control:-Positive- E.coli ATCC
11775
Negative-Acinetobacter
lwoffii ATCC15309
 Procedure :-Inoculate the media with
loopful of the test organism & incubate for
12-24 hrs. rarely for 5 days at 35°C.
 At the end of incubation add 0.1 ml of the
test reagent to the culture.
 Result :- A red color developing with in a
few min. indicates the presence of nitrite.

62. TSI TEST
 PRINCIPLE :- To determine the ability of an organism to attack a specific
carbohydrate incorporated in a basal growth medium with or with out
production of gas a long with determination of H2S production
 USES :-(1) K/K or No change-No carbohydrate fermentation eg-
Ps. aeruginosa
 (2) K/A – Only glucose fermented, lactose or sucrose not fermented
eg- Shigella spp.
 (3) K/A/H2S- Only glucose fermented lactose not fermented
hydrogen sulfide produced eg- Salmonella spp., Citrobacter spp.&
Proteus spp.
 (4) A/A -Glucose and lactose (or sucrose) fermented it is
characteristic of lactose fermenting coliforms eg –E. coli, Kleb,
Enterobacter spp.

63. Contd.
 MEDIA:-(1) TRIPLE SUGAR IRON AGAR (glucose ,
lactose, sucrose -1:10:10, ferric citrate, sodium chloride, sodium
thiosulphate, agar, peptone yeast extract, phenol red 0.2% & Dw)
 (2) KLIGLER’S IRON AGAR:- glucose, lactose, 1:10, ferrous
sulphate ,sodium chloride, sodium thiosulphate, agar, peptone,
proteose peptone, beef extract yeast extract, phenol red 0.2% & Dw
 PROCEDURE:- Streak a heavy inoculum over the surface of the
slope & stab in to the butt incubate aerobically at 37°C for 24 hrs.
and result interpreted.
 RESULT:- (A) Fermentation of only glucose:-
Slant Butt
Alkaline reaction Acid reaction
Red color Yellow color

64. Contd.
 (B) Fermentation of glucose & lactose:-
Slant Butt
Acid reaction Acid reaction
Yellow color Yellow color
 (C) Neither glucose nor lactose fermented:-
Slant Butt
Growth only & no color Growth only & no color
 (D) Gas production:-
No Gas production Gas production
 (F) H2S production:-
Black color No black color

65. TRIPLE SUGAR IRON AGAR TEST

66. Coagulase Test
Principle- To test the ability of an organism to clot plasma by
the action of enzyme coagulase
Two types of Coagulase produced
 Free coagulase (thrombin like substance present in culture
filtrates)- Detected by Tube Method
 Bound Coagulase (Clumping Factor, attached to the
bacterial cell wall)- Detected by Slide Method
Purpose- To speciate among the species Staphylococcus
Reagents- Rabbit Plasma with EDTA. (Reconstituted plasma
should be refrigerated)
QC- Positive- S. aureus subsp aureus ATCC 12600
Negative– S. epidermidis ATCC 14990

67. Procedure- Coagulase Test
Slide Test
 Place two drops of NS on a clean glass slide,& gently emulsify colony.
 Put a drop of coagulase plasma in the suspension with a wooden
applicator stick and observe for agglutination.
 Positive Reaction is detected within 10-15 sec. by formation of white
precipitate and agglutination.
 All strains of negative slide test must be tested by tube coagulase test
Tube Test
 Emulsify a small colony growth in a tube containing 0.5 ml coagulase
plasma.
 Incubate the tube at 37°C for 4 hrs and observe for clot formation by gently
tilting the tube.
 If no clot is observed, reincubate the tube at room temperature and read
again after 18 hrs
 Considered positive if any degree of clotting is observed.

68. Slide Coag. Tube Coag.

69. DNase Test
Principle- To detect the ability of an organism to produce
DNase
capable of depolymerizing DNA.
Uses:- To detect Staph. aureus strains that may produce weak
or equivocal tube coagulase reaction.
Method:-
 To plate contain DNA, a spot inoculum of the test organism is
inoculated at 37° C for 18-24 hrs. Detection can be done by
two methods:-
 Flooding the plate with 3.6% HCl to precipitate the DNA,
leaving a clear zone of hydrolyzed DNA around the growth
(positive)
 Incorporating Toludiene Blue O dye to the media that gives a
pink color around the growth showing the hydrolyzed DNA
(positive)

70. Oxidation Fermentation Test
 Principle:- Saccharolytic micro-organisms degrade glucose
either fermentatively or oxidatively.
 The end products of fermentation are relatively strong mixed
acids that can be detected in conventional fermentative test
medium
 Acids formed in the oxidative pathway are weak and more
sensitive OF medium is required for their detection.
 Uses-
 To differentiate Enteric and Non Enteric gram negative aerobic to
facultative anaerobic bacilli from Enterobacteriaceae which are
fermenters.
 Determination of Motility and Gas Production.
 Medium- Hugh Leifson’s Medium (High CHO : Protein ratio)

71. Contd.
 QC
 Glucose Fermenter- S. aureus subspp aureus, E.coli
 Glucose Oxidizer- Micrococcus luteus, Ps. Aeruginosa
 Non- Saccharolytic- Moraxella spp.
 Procedure
 Two tubes of OF test inoculated with test organism using a
straight needle stabbing the medium 3-4 times half way to the
bottom of the tube.
 One tube of each pair is covered with 1 cm layer of sterile
mineral oil (closed tube) leaving the other tube open to air (open
tube).
 Incubate both tubes at 37°C and examine daily for 7 days.

72. O/F Test- Result Interpretation
OPEN TUBE CLOSED TUBE METABOLISM
Acid (yellow) Alkaline (Green) Oxidative
Acid (yellow) Acid (yellow) Fermentative
Alkaline (Green) Alkaline (Green) Non-Saccharolytic

73. O/F Test
Fermentative
Oxidative
Non Saccharolytic

74. CAMP/Bacitracin
Principle:-
 To determine an organisms ability to produce and elaborate
the CAMP factor which acts synergistically with Staph. β
hemolysin on Sheep or Bovine RBC’s to produce a lytic
phenomenon at the juncture of 2 organisms.
 The same phenomena of synergistic hemolysis with CAMP
factor and alpha toxin of Cl. perfringens.
Uses:-
 To differentiate Group B β H Streptococci (S. agalactiae)
from other Streptococcus spp.
 To identify hemolytic pathogenic spp of Listeria

75. Contd.
 Medium used:- Sheep blood
agar plate.
 Quality Control:-
 Positive Control-
Streptococcus agalactiae
 Negative Control-
Streptococcus pyogenes
 Reverse CAMP Test- Uses
the alpha toxin of Cl. perfringens
(Uses HBA)
 Clostridium spp replaces S.
aureus and a known Group B
Strept. is used.
Clostridium
Reverse Arrow
(+)
Reverse CAMP Test

76. CAMP Test

77. Growth in Presence of 6.5% NaCl Agar / Broth
Principle- To detect the ability of an organism to grow in the presence of
6.5% NaCl.
Use- To identify Enterococcus spp (positive) from Non Enterococcal group
D Streptococci (negative)
Bile Esculin Agar:-
Principle-To determine the ability of
an organism to hydrolyze the
glycoside esculin to esculetin and
glucose in presence of bile
Use- Identification and differentiation
of Non Enterococcal group D
Streptococci(+) and
Enterococcus spp(+)

78. -Pottasium Tellurite Agar
-Mannitol / Arabinose / Sorbitol
-NH3 production form Arginine
Uses- To speciate among the
Enterococcus spp.
PTA-
Principle- To determine the ability of
an organism to reduce tellurite to
tellurium
Positive- Black colored colony
Eg- E fecalis
Negative- Colorless colony
Eg-E faecium
Note- Mannitol / Arabinose / Sorbitol
fermentation and NH3 production
form Arginine are used to classify
Enterococcus spp.
Positive
Negative

79. THANK YOU