Chapter 5 Identification of Bacteria.pptx

Essentials of Medical Microbiology
Learning objectives
At the end of the session, the students will be able to
• To know the conventional culture methods
• Automated culture techniques
• Molecular methods

Representation of microbial identification

Type of infections and various specimens collected

CONVENTIONAL METHOD
• Direct Microscopy-
o Direct smear examination- Specimens are subjected to
the following staining techniques.
o Gram staining
o Albert's staining
o Ziehl-Neelsen (ZN) acid fast staining

Culture
• Depending on the type of specimen, various culture
media are used.
• Combination of blood agar and MacConkey agar is most
commonly employed for most specimens.

Culture (cont..)
• Combination of blood agar and MacConkey agar - pus, wound swab &other
exudate specimens, sterile body fluids, urine, sputum and other respiratory
specimens.
• Chocolate agar - Respiratory and sterile body fluid specimens.
• Stool specimen should be inoculated on to selective media such as-
o Mildly selective media-MacConkey agar and
o Highly selective media-DCA, XLD and TCBS
• Blood specimen should be directly inoculated into blood culture bottles without
performing direct microscopy methods.
• CLED agar - urine specimen

Morphology of Bacterial Colony
• Size-in millimetres e.g. pin head size is characteristic of
staphylococcal colony & pin point size is characteristic of streptococci
colony
• Shape – Circular or irregular
• Surface –glistening or dull
• Edge-Entire, crenated, lobate, undulated or filamentous
• Elevation-flat, raised, convex, umbonate, or pulvinate
• Consistency - Mucoid, friable, firm, butyrous
• Density–opaque, translucent or transparent

Morphology of Bacterial Colony
• Colour of the colony- Colonies may be colored due to
properties of the media used or due to pigment
production.
• Pigment produced by certain bacteria may also color the
colony. Pigments are of two types.
o Diffusible pigments
o Non-diffusible pigments

Haemolysis on blood agar
• Partial or α hemolysis
• Complete or β hemolysis
• No hemolysis (γ hemolysis, a
misnomer)
• α prime hemolysis

Culture smear and motility testing
• Colonies grown on the culture media should be
subjected to Gram staining and motility testing by
hanging drop method.

Biochemical reactions
• Based on the type of organisms detected in culture smear, the appropriate
biochemical tests are employed.
• Initially, catalase and oxidase tests are done on all types of colonies grown on
the media.
• For Gram negative bacilli- Common biochemical tests done routinely are-
o Indole test
o Citrate utilization test
o Urea hydrolysis test
o Triple sugar iron test (TSI)

• If there is any doubt in correct identification of bacteria, then
further biochemical tests are put such as-
o Sugar fermentation test
o MR (methyl red) test
o VP (Voges Proskauer) test
o OF test (oxidation –fermentation test)
o Nitrate reduction test
o Decarboxylase test
o PPA test (phenyl pyruvic acid test)
Biochemical reactions (cont)

For Gram positive cocci; certain useful biochemical tests are
• Coagulase test (for Staphylococcus aureus)
• DNase test (for Staphylococcus aureus)
• CAMP (Christie Atkins Munch-Petersen) test for Group B Streptococcus.
• Bile esculin hydrolysis test (for Enterococcus)
• Heat tolerance test (for Enterococcus)
• Sugar fermentation test
• PYR test (for Streptococcus pyogenes and Enterococcus)
• Bile solubility test (for pneumococcus)

Antimicrobial susceptibility tests done for bacterial identification
1. Novobiocin susceptibility test- done for
Staphylococcus saprophyticus
2. Optochin susceptibility test (for pneumococcus)
3. Bacitracin susceptibility test-done to differentiate
group A and group B Streptococcus)

Catalase test
• When a drop of hydrogen peroxide (3% H2O2) is added to a
colony of any catalase producing bacteria, effervescence
or bubbles appear due to breakdown of H2O2 by catalase
to produce oxygen.

Oxidase tests
• Detects the presence of cytochrome oxidase enzyme in bacteria
which catalyses the oxidation of reduced cytochrome by
atmospheric oxygen.
• Oxidase positive (deep purple)- Examples include Pseudomonas ,
Vibrio, Neisseria, Bacillus etc.
• Oxidase negative (no colour change)-Examples include;
members of family Enterobacteriaceae, Stenotrophomonas, etc.

Indole test
• Detects the ability of certain bacteria to produce
enzyme tryptophanase that breaks down amino acid
tryptophan present in the medium into indole.
• Indole positive Escherichia coli, Proteus vulgaris, Vibrio
cholerae etc.
• Indole negative - Examples include- Klebsiella
pneumoniae, Proteus mirabilis, Pseudomonas, Shigella,
Salmonella, etc.

Citrate utilization test
• Dtects the ability of a few bacteria to utilize
citrate as the sole source of carbon for its growth,
with production of alkaline metabolic products.
• Citrate test is positive for Klebsiella pneumoniae,
Citrobacter, Enterobacter etc.
• Test is negative for Escherichia coli, Shigella,
Salmonella Typhi, etc.

Urea hydrolysis test
• Urease producing bacteria can split urea present in
the medium to produce ammonia that makes the
medium alkaline.
• Urease test is positive for-Klebsiella pneumoniae,
Proteus species, Helicobacter pylori, Brucella, etc.
• Urease negative - Escherichia coli, Shigella,
Salmonella, etc.

Triple sugar iron agar test (TSI)
• Composition- It is a composite solid agar medium in tube
having a butt & a slant. Its constituents include-
• Three sugars- glucose, sucrose and lactose in the ratio of
1:10:10 parts
• Phenol red as an indicator of acid production
• Ferric salts as an indicator of hydrogen sulphide (H2S).

TSI (cont..)
Interpretation
• Ability to ferment sugars to produce acid
• Ability to produce gas
• Ability to produce H2S

TSI

Various reactions in TSI
Reactions in TSI Examples
Acidic slant/acidic butt ≥ 2 sugars fermented
• Glucose fermented &
• Lactose and/or sucrose fermented
A/A, gas produced, no H2S
(fig 6B)
Escherichia coli
Klebsiella pneumoniae
Alkaline slant/acidic butt Only glucose fermenter group
K/A, no gas, no H2S
(fig 6C)
Shigella
K/A, no gas, H2Sproduced (small amount), fig
6D
S.Typhi
K/A, no gas, H2S produced (abundant), fig 6E Proteus vulgaris
K/A, gas produced, H2Sproduced (abundant) S.Paratyphi B
K/A, gas produced, no H2S S.Paratyphi A
Alkaline slant/alkaline butt Non fermenters group
K/K, no gas, no H2S
(fig 6F)
Pseudomonas
Acinetobacter

Sugar fermentation test
• Detects the ability of an organism to ferment
• a specific carbohydrate (sugar) incorporated in a medium
producing acid with/without gas. Glucose, lactose, sucrose and
mannitol are widely used for sugar fermentation.
• Acid production is detected by using indicators such as-
o Andrade's indicator
o Phenol red indicator
o Gas production is detected by using an inverted Durham’s tube

Biochemical tests

Decarboxylase test
• Detects the presence of substrate specific decarboxylase
enzyme in the bacteria that break down amino acids such
as lysine, arginine and ornithine to produce alkaline by-
products.

MR (methyl red) test
• In glucose phosphate broth, certain bacteria ferment
glucose to produce stronger acids that maintain the pH
below 4.4 which turns methyl red indicator from yellow
to red color
o MR Positive (red color)-Escherichia coli
o MR negative(yellow, i.e. no change in color)-
Klebsiella pneumoniae

VP (Voges Proskauer) test
• In the presence of alkali and atmospheric oxygen,
acetoin is oxidised to diacetyl which reacts with α-
naphthol to give red colour
• VP positive- Klebsiella pneumoniae, Enterobacter, El
Tor vibrios, Staphylococcus, etc.
• VP negative - Escherichia coli, Shigella, Salmonella,
etc.

OF test (oxidation –fermentation test or Hugh and Leifson test)
Open tube Covered tube Metabolism
Acid (yellow) Acid (yellow) Fermentative bacteria
Acid (yellow) Alkaline (green) Oxidative bacteria (i.e.
non fermenters)
Alkaline
(green)
Alkaline (green) Asaccharolytic bacteria

Nitrate reduction test
• Detects the presence of an enzyme nitrate reductase in
the organism, which reduces nitrate present in the
medium (nitrate broth) to nitrite or free nitrogen gas.
• Nitrate test positive- All members of family
Enterobacteriaceae.

PPA test (phenyl pyruvic acid test)
• Specific test done for members of tribe Proteeae; which
includes Proteus, Morganella and Providencia.
• They possess a specific enzyme that deaminates
phenylalanine present in the medium to phenyl pyruvic
acid (PPA).
• PPA reacts with few drops of 10% ferric chloride solution
to produce green color.

Automated blood culture techniques
Advantages:
• Continuous automated monitoring
• More sensitive- increase in the yield of positive cultures
from clinical specimens.
• Rapid- takes much less time than conventional methods.
• Less labor intensive- saves man power

Automated blood culture techniques
Disadvantages
i) high cost of the instrument and culture bottles
ii) inability to observe the colony morphology as liquid
medium is used
iii) no separate detection in mixed cultures
iv) ↑overgrowth by contaminants
v) v) for techniques based on radiometric detection- there is
need for disposal of radioactive materials.

Common automated blood culture systems-
BacT/ALERT automated blood culture system
• Principle of BacT/ALERT is
based on colorimetric
detection of growth in
contrast to fluorometric
detection by BACTEC.

Common automated blood culture systems-
BacT/ALERT VIRTUO automated blood culture
BacT/ALERT VIRTUO (bioMieux)
• Advanced form of BacT/ALERT which
offers several advantages such as:
(i) automatic loading and unloading of
bottles,
(ii) faster detection of growth
(iii) can determine the volume of blood
present
• in bottle

Automated Systems for Bacterial Identification

VITEK 2 automated system
The VITEK 2 is an automated system used for
identification and antimicrobial susceptibility
testing (AST) of bacteria and yeast.
•
Uses colorimetric reagent card containing 64
wells; each well contains an individual test
substrate.
• Separate cards are available for gram-
negative, gram-positive bacteria, fastidious
bacteria and yeasts

MOLECULAR METHODS
A. Thermocycler machine (Eppendorf ); B. Gel electrophoresis of
amplified product; C. Visualization of amplified DNA under UV light

Polymerase chain reaction cycle

Biofire FilmArray
• Multiplex nested PCR system
• Four panels are available such
as respiratory, gastrointestinal,
meningitis-encephalitis and
blood culture identification
panels

Real-time PCR (rt-PCR)
• PCR technology, which is
used to amplify and
simultaneously detect or
quantify a targeted DNA
molecule on real-time
basis.

Loop Mediated Isothermal Amplification (LAMP)
• LAMP is an isothermal nucleic acid amplification
technique

MICROBIAL TYPING
• Microbial typing refers to characterization of an organism
beyond its species level.

Chapter 5 Identification of Bacteria.pptx

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Chapter 5 Identification of Bacteria.pptx