microbiology

1. CONVENTIONAL
METHODS
OF
BACTERIAL
IDENTIFICATION
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1:00:29 AM
1

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HISTORY
The approach to diagnostic microbiology outlined
should be conservative ,therefore microbiologists must maintain
quality service despite increasing stringent cost-containment
policies.
McCUBE AND STOTTMEIER noted that various “ special
interests” have adversely affected functions in hospitals and
laboratory .
The decision of how far to process the individual specimen
cultures must be based on a thorough knowledge of host – parasite
relations that may apply in any given case.
BARTLETT has recommended “ processing control” that is
“restricting the processing and reporting of culture specimens to the
production of prediction useful information” and this principle is
followed from the last three decades.

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PROCESSING OF SPECIMEN FOR IDENTIFICATION STARTS WITH
DIRECT MOUNTS OR STAINED SMEARS TO ESTABLISH A
PRESUMPTIVE DIAGNOSIS
Advantages of microscopic examination :
1. The number of segmented neutrophils represent the magnitude
of inflammation
2. The quality of specimens can be validated and observation of
bacteria,fungi,parasite structures etc provide information to
render an immediate presumptive diagnosis and help in
selecting the media to be used and therapy.
3. It also gives evidence that anaerobic bacteria are present in the
sample.

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4
specimens
General Procedure of Bacteriological Diagnosis
Isolation, identification
Biochemical tests
EIA, ELISA, IF test, agglutination test
Antigen detection
Molecular Biological Diagnosis(hybridization, PCR, RT-PCR,etc)
Serological diagnosis (Ab titer)
convalescent phase / acute phase≥4
Morphologic identification
microscopy & staining

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TECHNIQUES OF DIRECT EXAMINATION OF
UNSTAINED SPECIMENS
1. SALINE MOUNT : MOTILITY OR
REACTIONS TO CHEMICALS OR SPECIFIC ANTISERA
2. HANGING DROP : MOTILITY
3.IODINE MOUNT : IODINE STAINS THE NUCLEI AND
INTRACYTOPLASMIC ORGANELLES SO THEY ARE
EASILY SEEN
USED PARALLEL WITH SALINE MOUNT FOR FECES
IT CANNOT BE EXCLUSIVELY USED FOR FECES
WITHOUT SALINE MOUNT AS IT PARALYSES
MOTILITY OF BACTERIA AND TROPHOZOITES

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4.POTASSIUM HYDROXIDE : KOH dissolves background
MOUNT keratin ,unmasking the
fungal elements
5. INDIAN INK : Indian ink or nigrosin preparation
PREPARATION to visualize capsules
6. DARKFIELD : used for delicate microorganisms
EXAMINATION that are invisible by bright – field
optics like spirochetes,leptospira etc
7.NEUFELDS QUELLUNG : when capsulated bacteia comes contact
REACTION with serum having anticapsular antibody,
Capsules undergo change in refractive
index to be seen visibly by swelling .
Mainly used for streptococcus pneumoniae
and hemophilus infuenzae

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COMMON BIOLOGIC STAINS USED IN
BACTERIOLOGY
1. GRAMS STAIN : differential stain for staining properties of all
bacterias
2. LOFFLER'S METHYLENE BLUE : simple direct stain , used
specifically to detect bacteria in spinal fluid
3. ZIEHL NEELSEN ACID FAST STAIN : apart from mycobacteria
also stain oocysts of crptosporidium and isospora belli
KINYOUN MODIFICATION OF ACID FAST STAIN is called “cold
method” because a surface active agent is used rather than
heat treatment .
4. SILVER STAINS: mainly used to examine the tissue sections of
lymes disease
The WARTHIN STARRY AND STEINER-STEINER silver
impregnation stains have been used for years to demontrates
spirochetes in formalin fixed tissue sections.

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WRIGHT GIEMSA : used for staining cellular elements of the peripheral
blood smear
used mainly for intracellular organisms such as
histoplasma capsulatum and leishmania species
Also used for intracellular inclusions like corneal
scrapings of trachoma.
LACTOPHENOL COTTON BLUE : because of its sulfonic groups, the dye is
strongly acidic therefore it used to
counter stain the unfixed tissues
Presently it is mainly used for direct
staining of fungus
TOLUEDENE BLUE : used for lung biopsy or respiratory secretions to
detect pneumocystis carinii.
METHYLENE BLUE : used adjunct with grams stain for CSF for
hemophilus influenzae and neiserria
meningitidis

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1. FLUOROCHROME
AURAMINE RHODAMINE : stains selectively mycobacteria by
binding to the mycolic acid
ACRIDINE ORANGE : at acidic ph bacteria and yeast stain brilliant
orange against a black ,light green or yellow
background .
used for bacteria demonstration in blood
cullture broth ,CSF and other exudates where
very low colony forming units are present .

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FLUORESCIN ISOTHIOCYANATE (FITC) AND TETRAMEHLY RHODAMINE
ISOTHIONATE (TMRI ) are commonly used florochromes that , open
exciting or short wavelength visible light emit light waves in visible range
with absorption maximum of 490 nm and 550nm respectively.
This FLOROCHROMES bind chemically with a variety of proteins,including
antigens and antibody,providing a label or tag by which immunologic
reaction can be visualised in direct smears of biologic fields or secretions
and in tissue sections.
FLOROCHROMES /protein ratio vary with different reagents to produce
optimal stain of desired objects with a minimum of non specific
background interference .the current development of monoclonal
antibodies , which are monospecific to there respective antigens ,has lead
to the preparation of FLOROCHROMES reagents for direct and indirect
detection of several antigens of human and animal specimens chlamydia
trachomatis,legionella, treponeam,toxoplasma and virus,

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PRINCIPLES OF BACTERIAL CULTIVATION :
Cultivation is a process of growing microorganisms in culture by taking
from the infection site (i.e. invivo environment ) by some means of
specimen collection and growing them in artificial environment of the
laboratory (i.e. invitro environment )
Cultivation has three main purposes :
1. To grow and isolate all bacteria present in the clinical specimen
2. Identify which bacteria are causing infection and which are
contaminants
3. to obtain sufficient growth of clinically relevant bacteria to allow
identification and characterization.

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Requirements for bacterial growth
• Temperature
 Nutrients
H2O, C-source, N-source,
Inorganic salts, Growth factors
 pH
 Gas
incubator  Temperature, gas
culture medium  Nutrients, pH
The successful transition from the in vivo to invitro environment
requires that the nutritional and environmental growth
requirement of bacterial pathogens are met.

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The nutritional requirements of a bacterial species can
be exploited to prepare an elective medium which will
preferentially encourage growth of that organism.
The efficacy of such media can be enhanced further by
the incorporation of selectively inhibitory compounds which
are substantially less toxic to the desire isolate than to its
normal competitors.
The inhibitors chosen for this purpose are many and
varied e.g. tellurite in media designed to isolate C. diphtheriae
from throat swab culture, or bismuth sulphite for isolation of
salmonella, including S. typhi from fecal material.

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A selective and diagnostic medium may combine
the effect of several agents. For example, MacConkey
medium which besides nutrients (peptones and lactose)
contain sodium chloride, bile salts and neutral red favours
the growth of lactose positive, gram-negative bacteria,
especially enterobacteria, while discouraging that of many
gram-positive bacteria which are more sensitive to the
combined toxic action of detergent and hydrophilic
bactericidal dyes.
Similar use can be made of combinations of
antibiotics for repressing the growth of sensitive bacteria
but not of resistant ones.

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SELECTION OF PRIMARY CULTURE MEDIAS
 Selection of medias is dependent on knowing the normal flora
present in the specimen being examined and the spectrum of
pathogenic organisms that may be found.
 Agar plates are commonly used for culture medias.
 Inoculation into broth media for primary recovery of specimens
such as body fluids ,needle biopsy and deep body aspirations in
which the recovery of only a few organisms in low concentrations
may be significant .

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CULTURES FROM VARIOUS ANATOMIC SITES
1.THROAT
Oral Cavity (Mouth And Teeth): Microbes normally found on the
mouth and teeth include
staphylococci,
streptococci (particularly Streptococcus mutans),
Lactobacillus acidophilus,
Actinomyces odontolyticus,
anaerobic spirochetes

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Upper Respiratory Tract (Pharynx and Trachea): Microbes normally
found in the upper respiratory
tract include
staphylococci, S. aureus
S.epidermidis
streptococci (such as Streptococcus agalactiae),
diphtheroid bacilli (such as Corynebacterium diphtheria),
spirochetes,
Neisseria,
Haemophilus, and
Branhamella.

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POTENTIAL PATHOGENS RECOMMENDED ISOLATION
1.Group A B hemolytic streptococci
Streptococcus pneumoniae
2.Klebsiella pneumoniae
And other enterobacteriaceae
3.Pseudomonas aeroginosa
4.Neisseria gonorrhoea and N.
meningitidis
5.Staphylococcus aureus
5% sheep blood agar with or
without SXT
5% sheep blood agar and
MacConkey ( or EMB ) agar
Modified thayer martin medium
Horse blood with colistin and
nalidixic acid (CNA)

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horse blood agar ,
chocolate agar,
staphylococcus streak technique
Loefflers serum glucose ( or
modified pai medium ) , a cysteine
tellurite agar plate ( ex: tinsdale
agar ) and 5% sheep blood agar
Bordet gengou potato infusion agar
or regan and lowe medium (
charcoal agar with horse blood and
cephalexin )
Lowenstein jensen egg medium
Buffered Charcoal yeast extract
6.Haemophilus influenzae type b
Non typable Haemophilus
influenzae
7. C. Diptheriae
8. B. Pertusis
9. Mycobacterium species
10. Legionella pneumophila and
other Legionella species

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In summary , a single 5% sheep blood agar plate streaked for
isolation with two or three stab marks in the blood agar ( which
allows a anaerobic environment allowing expression of full activity of
both oxygen labile streptolysin O and oxygen stable streptolysin S. ) is
suficient for most throat cultures .
By special request selective media for the recovery of unusual
pathogens may be required .

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2. SAMPLES FROM LOWER RESPIRATORY TRACT
The tracheobronchial tree below the larynx is normally sterile .
To avoid contamination with the oral flora : translaryngeal
aspirations and bronchoscopic brushings or lavages are preferred
clinical samples
As both gram positive and negetive infections are common so
Gram -ve selective media : MacConkey or EMB agar ; and
Gram +ve selective media : phenylethyl alchohol (PEA) and colistin
nalidixic acid (CNA) blood agar are used .
And special media for the recovery of organisms having specific
growth requirements must be used for all lower respiratory
infections .

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3. URINE
 STAPHYLOCOCCI COAGULASE NEGETIVE
 DIPTHEROIDS
 E.COLI AND OTHER COLIFORMS
 LACTOBACILLUS SPECIES
 ALPHA HEMOLYTIC STREPTOCOCCI
 BACILLUS SPECIES
COMMENSAL FLORA ( CONTAMINANTS) IN
PERIURETHRAL AREA
Urine is normally sterile but is easily contaminated in the
periurethral area

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E.COLI
KLEBSIELLA-
ENTEROBACTOR- SERRATIA
SPECIES
PROTEUS MIRABILIS AND
OTHER PROTEUS SPECIES
GROUP D ENTEROCOCCI
S. AUREUS AND
S.SAPROPHYTICUS
ACINETOBACTOR SPECIES
PSEUDOMENAS SPECIES
N. GONNORHOEAE
MacConkey ( or EMB ) agar
5% sheep blood agar
Modified thayer martin medium
Chocolate agar
POTENTIAL PATHOGENS RECOMMENDED ISOLATION

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In general ,
UTI can be caused by both gram positive and negetive
bacteria so a mixture of 5% sheep blood agar and macconkey
( or emb ) agar is sufficient to isolate most of pathogens.
CLED MEDIUM

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GENITAL TRACT
E.COLI AND OTHER COLIFORMS
GROUP D ENTEROCOCCI
STAPHYLOCOCCI COAGULASE NEGETIVE
DIPTHEROIDS
LACTOBACILLUS SPECIES
MANY SPECIES OF ANAEROBES
COMMENSAL FLORA ( CONTAMINANTS)

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N. GONNORHOEAE
Group B streptococci
Group D streptococci
Gardenella vaginalis
Mobiluncus species
E.Coli and other
enterobacteriaceae
Certain anaerobes
Virtually any
organism in high
concentration and in
pure culture
Modified thayer martin medium
5% sheep blood agar
Clue cells in smear
Columbia CNA agar with 5% packed red
blood cells
MacConkey ( or EMB ) agar
Prereduced anaerobic blood agar plates
with and without selective agents
5% sheep blood agar, MacConkey agar
or other special medium depending on
the clinical diagnosis
POTENTIAL PATHOGENS RECOMMENDED ISOLATION

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Most samples of genital tract has infection by N.
GONORRHOEAE & CHLAMYDIA TRACHOMATIS.
The recovery of latter requires tissue cultures , whereas the
former requires Modified thayer martin medium whereas routinely 5%
sheep blood agar and MacConkey ( or EMB ) agar is used.

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GASTROINTESTINAL TRACT CLINICAL SAMPLE
Microbes normally found in the upper intestine Include
lactobacilli and
enterococci (such as Enterococcus faecalis).
Microbes of the lower intestine and colon include
mostly anaerobes where >90% are anaerobes (such as
Bacteroides &Clostridium.),
enterobacteriaceae (Escherichia coli and others),
Pseudomonas, and
Candida species.

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SALMONELLA SPECIES
SHIGELLA SPECIES
E.COLI ( ETEC & EPEC)
S.AUREUS
CAMPYLOBACTER SPECIES
VIBRIO AND HALOPHILIC VIBRIO
SPECIES
YERSINA ENTEROCOLITICA
C.DIFFICILE
MacConkey ( or EMB ) AGAR
HEKTOEN ( OR XLD ) AGAR
SS AGAR
GN OR SELENITE ENRICHMENT BROTH
PHENYL ETHYL ALCHOHOL (PNA ) AGAR
OR CNA BLOOD AGAR
SELECTIVE CAMPY BLOOD AGAR
THIOSULPHATE CITRATE BILE SUCROSE AGAR
(TCBS)
CIN (CEFSULODIN –IRGASAN – NOVOBIOCIN )
AGAR
CYCLOSERINE – CEFOXITIN – EGGE YOLK –
FRUCTOSE AGAR ( MUST DO CYTOTOXIN ASSAY
ON STOOL SPECIMEN )
POTENTIAL PATHOGENS RECOMMENDED ISOLATION

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CSF

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N.MENINGITIDIS
H.INFLUENZAE GROUP B
S.PNEUMONIAE
GROUP B STREPTOCOCCI
L. MONOCYTOGENES
E.COLI
MYCOBACTERIUM
TUBERCULOSIS
CRYPTOCOCCUS
CHOCHOLATE AGAR (MTM )
CHOCOLATE AGAR,
STAPHYLOCOCCUS STREAK TECHNIQUE
CNA AGAR
5% SHEEP BLOOD AGAR
5% SHEEP BLOOD AGAR
MACCONKEY ( OR EMB ) AGAR
L-J MEDIUM
INDIAN INK SMEAR
POTENTIAL PATHOGENS RECOMMENDED ISOLATION

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SPECIMEN FROM WOUND OR ABSCESS
CORYNEBACTERIUM SPECIES
STREPTOCOCCI ALPHA HEMOLYTIC
STAPHYLOCOCCI COAGULASE NEGETIVE
PRPIONIBACTERIUM SPECIES
BACILLUS SPECIES
COMMENSAL FLORA ( CONTAMINANTS)

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S. AUREUS
STREPTOCOCCI ALPHA
HEMOLYTIC
ENTEROCOCCI
E.COLI
PROTEUS SPECIES
P.AERUGINOSA
ANAEROBES
PEPTOSTEPTOCOCCI
BACTEROIDS SPECIES
CLOSTRIDIUM PERFRINGES
ACTINOMYCES ISRAELII
MYCOBACTERIUM MARINUM
5% sheep blood agar
5% sheep blood agar
MacConkey ( or EMB ) agar
PREREDUCED & VIT. K1 SUPPLEMENTED
ANAEROBIC BLOOD AGAR PLATES
LJ MEDIUM
POTENTIAL PATHOGENS RECOMMENDED ISOLATION

34. Isolation Technique
 In nature microbial cultures are mixed
 Identification relies upon isolating individual
colonies
 Testing requires pure cultures
 As a result isolation technique provides an
essential microbiological tool

35. Mixed Culture from Raw Poultry

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METHODS OF ISOLATING PURE CULTURE
1. STREAK PLATE ISOLATION TECHNIQUE
2. PRETREATMENT OF SPECIMEN :
Suitable bactericidal substances are used for pretreatment of
specimens to isolate a particular bacteria . ex: concentraction
and decontamination of sputum before culture of M.
Tuberculosis.
3. BY HEATING LIQUID MEDIUM
specimen heated to 800C to destroy vegetative forms of
bacteria but spore bearers survive . Ex: isolation of tetanus
from dust and similar sources

37. Streak Plate Isolation Principle
 An original inoculum containing a mixture of bacteria
is spread into 4 quadrants on solid media.
 The goal is to reduce the number of bacteria in each
subsequent quadrant.
 Colonies are masses of offspring from an individual
cell therefore streaking attempts to separate
individual cells.
 Discrete colonies form as the individual cells are
separated and then multiply to form isolated colonies
in the later quadrants.

38. Streaking the Quadrants
Flame between each quadrant.
Q
Q
Qu
u
ua
a
ad
d
dr
r
ra
a
an
n
nt
t
t 1
1
1
Q
Q
Q 4
4
4 Q
Q
Q 2
2
2
Q
Q
Q 3
3
3

39. Quadrant 1- Streak with broad narrow strokes in the
upper half of the first quarter of the plate.

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41. Incinerate and cool the loop between
the quadrants

42. Quadrant 2 – Rotate the plate, enter the previous streak mark
one or two times and then streak the upper portion of the second quarter of
the plate with broad strokes.

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44. Incinerate and cool the loop
between the quadrants

45. Quadrant 3 – Rotate the plate, enter quadrant 2 one or
two times and then streak with shorter more separated strokes
from the top of the quadrant to the center.

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47. Incinerate and cool the loop
between the quadrants

48. Quadrant 4 – Enter quadrant 3 and then streak with broad
S-shaped motions through the center of the plate.

49. Streaking the Quadrants
Flame between each quadrant.
Q
Q
Qu
u
ua
a
ad
d
dr
r
ra
a
an
n
nt
t
t 1
1
1
Q
Q
Q 4
4
4 Q
Q
Q 2
2
2
Q
Q
Q 3
3
3

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GROWTH IN FIRST QUADRANT

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GROWTH IN FIRST AND SECOND QUADRANT

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GROWTH IN FIRST ,SECOND AND THIRD QUADRANT

53. Isolated Colonies

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EVALUATION OF COLONY MORPHOLOGY
1. COLONY CHARACTERISTICS
1. Colony size - pin point / small / medium /large
2. Colony Pigmentation
3. Colony shape : form,elevations
4. Colony surface appearance - glistening / opaque
/dull / transparent
5. Changes in agar media : hemolysis , color
6. Consistency : mucoid , friable , firm , butyrous
7. Odor
8. Edge : entire / crenated / lobate / undulate
/ciliate

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2. TYPE OF MEDIA SUPPORTING GROWTH
Like growth in MacConkey media indicates gram negative growth and
the incubator condition that growth occured indicates whether the
bacteria is aerobic or anaerobic .
3. RELATIVE QUANTITIES OF EACH COLONY TYPE
Predominance of a colony helps in establishing the
organisms clinical significance

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GROWTH IN LIQUID MEDIA
1. Uniform turbidity : mainly gram negative
2. Deposit at bottom : in case of steptococci, as chain become heavier
settle down
3. Surface pellicle formation : aerobe mainly grow at surface due to
more oxygen ex: pseudomonas

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MICROSCOPIC MORPHOLOGY AND STAINING CHARACTERISTICS
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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Gram stain reactions when observed in conjunction with types and
arrrangements can help in a presumtive diagnosis.
ex: gram positive cocci in cluster – staphylococcus
lancet shaped diplococci – S. pneumoniae
large gram positive bacilli – clostridium species

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ORGANISM IDENTIFICATION BY RESISTANCE OR SUSCEPTIBLE
TO INHIBITORY SUBSTANCES
Accomplished by adding inhibitory substances or antibiotics to
agar media or measuring sensitivity during antimicrobial
susceptibility for therapy.
Growth in presence of various NaCl concentrations indicate vibrio
species or enterococci
Susceptibility to optochin and solubility in bile indicate S.
Pneumoniae
Ability o hydrolyse esculin indicate enterococci
Ethanol survival indicate: bacillus species

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Motility test:
. • The test is based on
the flagellum of the
b a c t e r i a .
• If the bacteria have
f l a g e l l u m , t h e
semisolid medium
will be clouding and
t h e t e s t w i l l b e
positive. .
Semisolid medium

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Motility Agars
 Sulfide-indole-motility (SIM) is a semisolid motility agar
that contains peptonized iron for detection of H2S and
tryptophan for indole production.
 Pure motility agar lacks an H2S indicator and tryptophan
for indole production, and contains tetrazolium salts that
are reduced to red formazan complexes to enhance visual
assessment of motility.

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IDENTIFICATION ACCORDING TO NUTRITIONAL
REQUIREMENTS AND METABOLISM CHARACTERS
ESTABLISHING ENZYMEATIC CAPABILITIES
ENZYME BASED TESTS ARE OF TWO TYPES :
1. PRESENCE OF A SPECIFIC ENZYME
2. COMPLETE SET OF ENZYMES FOR A COMPLETE
METABOLIC PATHWAY

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SINGLE ENZYME TESTS
Oxidase test –
 Used to distinguish fermenters (Oxidase negative) from non-
fermenters(Oxidase positive).
 Testing for the presence of the enzyme indophenol oxidase.
 The reagent (Tetramethyl-para-phenylenediamine) will be
oxidized in the presence of oxygen by the
 enzyme indophenol oxidase producing
 a dark-purple product called indophenol.
 Production of a dark purple product
 (within 10 seconds) is a positive
( + ) test for oxidase.

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Oxidase positive Oxidase negative
Alcaligenes spp. Acinetobacter spp.
Bordatella pertussis Bordatella parapertussis
Campylobacter spp. Francisella tularensis
Brucella spp. Brucella canis
Kingella kingae Flavimonas oryzihabitans
Moraxella spp. Stenotrophomonas maltophilia
Neisseria spp. Enterobacteriaceae
Pseudomonas spp.

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CATALASE TEST
The presence of catalase enzyme in the test isolate is detected
using hydrogen peroxide. If the bacteria possess catalase (i.e., are
catalase-positive), when a small amount of bacterial isolate is added to
hydrogen peroxide, bubbles of oxygen are observed.
The test is done by placing a drop of hydrogen peroxide on a microscope
slide. Using an applicator stick, a scientist touches the colony and then
smears a sample into the hydrogen peroxide drop.
If bubbles or froth forms, the organism is said to be
catalase-positive.
Staphylococci[27] and Micrococci[28] are catalase-positive.
If not, the organism is
catalase-negative.
Streptococci[29] and Enterococci are catalase-negative.

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CATALASE-POSITIVE.
CATALASE-NEGATIVE.

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Urease test –
 Testing for the presence of the enzyme
urease.
 This enzyme will break down urea to
ammonia and carbon dioxide.
CO(NH2)2  NH3 + CO2
 The ammonia produced increases the pH
of the media (becomes more alkaline)
and causes the indicator present in the
media (Phenol red) to change from a
yellow-orange color to a hot pink color.
 A hot-pink color is an indication of a
positive test for Urease.

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Urease-Producing Enterobacteriaceae
 Proteus
 Morganella
 Providencia rettgeri
 Klebsiella pneumoniae
 Klebsiella oxytoca
 Enterobacter cloacae
 Yersinia enterocolitica

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Triple Sugar Iron (TSI) Test
Test conditions:
Provides information about sugar fermentation and the possible by-
products produced by the organisms tested.
Three sugars, glucose, lactose, and sucrose are present in the media at
different concentrations (Glucose at 0.1%, Lactose at 1.0%, and Sucrose at
1.0%).
Fermentation is an anaerobic process.
When sugar is fermented there may be an acid end-product produced.
Phenol red is again used in the media as a pH indicator and will change
from red to yellow under acidic conditions.
Some organisms also may produce hydrogen gas (H2) as a by-product of
fermentation.
Also present in the media is ferrous sulfate, which will react with any
hydrogen sulfide (H2S) produced as a another possible by-product to form a
black precipitate (ferrous sulfide). H2S production is an anaerobic process
so the black precipitate produced will only appear in the butt of the tube.

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Procedure:
Work in pairs
will be testing three organisms:
1.Pseudomonas aeruginosa
2.Escherichia coli
3.Proteus vulgaris
need one TSI agar slant for each organism/group (3 tubes).
The tests require anaerobic conditions, therefore the organisms must
be stabbed into the agar media. For this reason you will use sterile
inoculating needles (not loops) for inoculating the tubes.
The best method is to obtain one of the organisms, preferably on a
nutrient agar plate, and roll the tip of a sterile inoculating needle in the
organism.
Next stab the agar slant nearly to the bottom of the agar tube and
when removing the inoculating needle, lightly streak the surface of the
slant.
Follow the same procedure for each organism tested.
The tubes are placed in the incubator for 18 - 24 hours at 37 C.

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Interpretation of Results:
Many of the enteric organisms will ferment glucose with the production of
acids which will change the color of the medium in the butt and along the
slant from red to yellow because of a reduction in the pH (within the first
few hours).
However, since the glucose is present in small amounts (0.1%), the supply
is soon exhausted and the organisms growing on the surface of the slant in
the presence of oxygen are forced to catabolize peptones and amino acids
in the media for their energy supply. Alkaline end-products ( NH4OH) are
produced from these substances which revert the pH of the slant to an
alkaline pH and thus change the color of the agar slant back to red (after
18-24 hours).
Organisms such as Salmonella spp. or Shigella spp. and other organisms
which attack glucose but do not ferment lactose or sucrose will produce an
alkaline slant and acid butt in TSI slants in 18 to 24 hours. Since
metabolism is progressing at a slower rate in the butt, this reversion does
not usually take place in the butt until 48 hours or longer.

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If the glucose is metabolized to CO2, the gas will be seen
as bubbles or cracks in the agar butt. If hydrogen sulfide is
formed during growth, a gray or black streak of iron sulfide
is seen originating where the inoculating needle entered and
throughout the agar butt.
Organisms which attack lactose and/or sucrose, such as
Escherichia, will produce acid slants and acid butts usually
with the formation of gas. In these cases, the acid slants do
not revert to an alkaline status because lactose (1%) and
sucrose (1%) are being fermented and are present in
concentrations ten times that of glucose.
Some organisms (e.g., Pseudomonas, Acinetobacter) fail to
ferment even glucose, and because they are strictly aerobic,
they fail to grow in the butt of the tube. In these cases, the
butt will be unchanged in color, and the slant either alkaline
or unchanged.

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TSI Test Results
Pseudomonas aeruginosa
Shigella
Salmonella enteritidis
Eschericihia
Klebsiella
Enterobacter
Proteus vulgaris
Control

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K = alkaline = Red; A = acid = Yellow; NC = No change; G = gas produced; H2S = hydrogen sulfide produced
Acid or alkaline results in the slant are reported first, followed by the butt results (e.g., K/A would be read as "K over A"
or "alkaline over acid" and refers to an alkaline slant and acid butt).
Slant result / Butt result
K/A Only Glucose fermented; peptone utilized
A/A  Glucose and Lactose/Sucrose fermented
K/K  No sugars fermented, only peptone utilized
K/NC  No sugars fermented, Peptone used aerobically only
NC/NC  No growth, neither sugars or peptone used
A/AG  Glucose and Lactose/Sucrose fermented, gas produced
A/A + H2S  Glucose and Lactose/Sucrose fermented, H2S produced
A/AG + H2S  All sugars fermented and both H2S and gas produced
K/AG  Only Glucose fermented; peptone utilized, gas produced
K/A + H2S  Only Glucose fermented; peptone utilized, H2S produced
K/AG + H2S  Only glucose fermented; Peptone utilized, both gas and H2S
produced
Recording the Results of the TSI Tests

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Identification of GNB – IMViC Tests
Mnemonic for the four Biochemical tests performed on the Gram
Negative Bacilli being studied
1. Indole test
2. Methyl Red test
3. Voges-Proskauer test
4. Citrate utilization test
These tests divide the Enterobacteriaceae into two major groups:
1. Escherichia coli group
2. Enterobacter-Klebsiella group

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Indole test:
Tests for the production and secretion of the
enzyme tryptophanase.
If the organism can produce tryptophanase
and break down tryptophan to Indole the
Indole will react with Kovac’s reagent
(p-dimethylaminobenzaldehyde) forming
a pink-colored chemical complex.
Indole positive :
Escherichia coli
Enterobacter aerogenes
Proteus Vulgaris
Pseudomonas aeruginosa

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Some organisms produce acid from the metabolism of
glucose (fermentation) in a sufficient quantity to alter the pH of the
media to about 4.4. These are stable acids and are not further
metabolized.
Methyl red indicator is used to detect the presence of these
acids in the MRVP broth medium. Methyl red indicator at this pH (~
4.4) changes to a bright cherry red color.
Methyl Red Test
MR +VE
Escherichia coli,
Enterobacter aerogenes
Proteus Vulgaris
Pseudomonas aeruginosa

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The Methyl Red Test: Left to Right:
positive, positive, negative, control.

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Voges-ProskauerTest
 Some microorganisms produce organic acids from glucose
metabolism, but further metabolize the acid produced to various
neutral end products, like acetoin, and 2,3-butanediol.
 There is an initial pH drop in the MRVP broth, but the neutral end
product raise the pH so that the methyl red test will be negative.
 The presence of acetoin, and 2,3-butanediol is tested for using a-
naphthol which reacts with these two neutral products to produce a
mahogany red color.
Escherichia coli,
Enterobacter aerogenes
Proteus Vulgaris
Pseudomonas aeruginosa
Voges-Proskauer positive

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Voges-Proskauer Test
Left: uninoculated control
Right: negative (copper color)
Left: uninoculated control
Right: positive (red color)

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Citrate Test
 Citrate may be used as a carbon source by some microorganisms.
 The Citrate test uses an agar medium with citrate and the pH
indicator Bromothymol blue present. At the pH of the un-inoculated
medium the color is Blue-green.
If the organism can utilize Citrate as a
carbon source the breakdown of citrate
releases bicarbonate ions (HCO3
-) into the
medium. The bicarbonate ions raise the pH
of the medium above 7.4 pH. This causes
the Bromothymol blue indicator to turn
dark blue in color.

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CITRATE POSITIVE
Escherichia coli,
Enterobacter aerogenes
Proteus Vulgaris
Pseudomonas aeruginosa
(citrase)
Citrate CO2 + Na HCO + H2O
(blue color change)

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Phenylalanine Deaminase Reaction
 Enterobacteriaceae utilize amino acids in a variety of ways
including deamination.
 Phenylalanine is an amino acid that forms the keto acid
phenylpyruvic acid when deaminated. Phenylpyruvic acid
is detected by addition of ferric chloride that forms an
intensely dark olive-green colored complex when binding
to phenylpyruvic acid.
 The deamination of phenylalanine is an important
biochemical property of Proteus, Morganella, and
Providencia.

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IMViC Reactions
I M Vi C
Escherichia coli + + – –
Edwardsiella tarda + + – –
Proteus vulgaris + + – –
Klebsiella pneumoniae – – + +
Klebsiella oxytoca + – + +
Enterobacter spp. – – + +
Serratia marcescens – – + +
Citrobacter freundii – + – +
Citrobacter koseri + + – +

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IPViC Reactions
I P Vi C
Eschericia + – – –
Shigella +/– – – –
Yersinia +/– – – –
Edwardsiella + – – –
Salmonella – – – +
Citrobacter – – – +
Klebsiella +/– – + +
Enterobacter – – + +
Serratia – – + +
Proteus +/– + +/– +
Morganella + + – +
Providencia – + – +

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Gelatin Hydrolysis
 Gelatin is another protein
 Gelatinase (enzyme) breaks down gelatin
into amino acids to be taken up by
bacteria
 Gelatin liquifies when it’s broken down

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MACROMOLECULES HYDROLYSIS
•Exoenzymes
diastase（amylase）
protease、
lipase
• endoenzymes
• Starch hydrolysis
Some bacteria are capable of using starch as a source of carbohydrate
but in order to do this, they must first hydrolyze or break down the
starch so it may enter the cell. The bacterium secretes an exoenzyme
which hydrolyzes the starch by breaking the bonds between the glucose
molecules. This enzyme is called a diastase（amylase）.

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Starch hydrolysis

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2、fermentation of carbohydrates
Facultative anaerobic and anaerobic bacteria are capable of fermentation, an
anaerobic process during which carbohydrates are broken down for energy
production. A wide variety of carbohydrates may be fermented by various
bacteria in order to obtain energy and the types of carbohydrates which are
fermented by a specific organism can serve as a diagnostic tool for the
identification of that organism.
We can detect whether a specific carbohydrate is fermented by looking for
common end products of fermentation. When carbohydrates are fermented as a
result of bacterial enzymes, the following fermentation end products may be
produced:
• acid end products.
• acid and gas end products.

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In order to test for these fermentation products, you inoculate and
incubate tubes of media containing a single carbohydrate (such as
lactose or maltose), a pH indicator (such as phenol red) and a durham
tube (a small inverted tube to detect gas production). If the particular
carbohydrate is fermented by the bacterium, acid end products will be
produced which lowers the pH, causing the pH indicator to change color
(phenol red turns yellow) .If gas is produced along with the acid, it
collects in the durham tube as a gas bubble.

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Amino Acid Decarboxylation
 If an Enterobacteriaceae contains amino acid
decarboxylase, amines produced by decarboxylase action
cause an alkaline pH, and bromcresol purple turns
purple.
 Lysine, ornithine, and arginine are utilized. A base broth
without amino acid is included in which glucose
fermentation acidifies the broth, turning the bromcresol
purple yellow.

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Amino Acid Decarboxylation1
Lysine → Cadaverine
Ornithine → Putrescine
Arginine → Citrulline → Ornithine → Putrescine
1Conversion of arginine to citrulline is a dihydrolase reaction

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Amino Acid Decarboxylation
Tube Amino Acid Color Interpretation
Base None Yellow Broth acidified1
1 Lysine Purple Positive
2 Ornithine Yellow Negative
3 Arginine Yellow Negative
1Indicates organism is a viable glucose fermenter, and pH of broth
medium sufficiently acidified to activate decarboxylase
enzymes.

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Amino Acid Decarboxylation
 Decarboxylation patterns are essential for the genus
identification of Klebsiella, Enterobacter, Escherichia, and
Salmonella.
 Decarboxylation patterns are also essential for the
species identification of Enterobacter aerogenes,
Enterobacter cloacae, Proteus mirabilis, and Shigella
sonnei.

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Culture Media and Isolation
Methods FOR MYCOBACTERIUM
TUBERCULOSIS
• Mycobacterium are strictly aerobic
• Slow growers; cultures held for 6 weeks before calling
negative
• Media
– Lowenstein-Jensen (LJ) media – egg based
– Middlebrook 7H10 and 7H11 agar – serum based
– Middlebrook 7H9 broth
– BACTEC broth contains 14C-labeled substrate
– When organisms grow, 14C in the form of 14CO2 is
released and detected radiometrically

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Biochemical Identification
Niacin accumulation
Nitrate reduction
Catalase
Hydrolysis of Tween 80
Iron uptake
Arylsulfatase
Pyrainamidase
Urease
Inhibitory tests
NAP
TCH
Growth in 6.5% NaCl
Tellurite reduction
Growth on MacConkey

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101. THANK YOU
REFERENCES
7/28/2022 1:00:29 AM
10
1
1. BAILEY & SCOTT’S : DIAGNOSTIC MICROBIOLOGY
2. KONEMAN : DIAGNOSTIC MICROBIOLOGY
3. MACKIE & MCCARTNEY : PRACTICAL MEDICAL
MICROBIOLOGY
4. PROF. C.P. BAVEJA : TEXTBOOK OF MICROBIOLOGY
5. JAWETZ, MELNICK & ADELBERG’S : MEDICAL
MICROBIOLOGY