PHENOTYPIC METHODS OF Bacterial identification- conventional & automated.pptx

Presented by : Dr. ReshmaVP
Moderator : Dr. Rashmi M.S

Importance of bacterial identification
• Determining the clinical significance of a particular pathogen ( a pathogen, a contaminant, or normal
microbiota)
• Guiding physician care of the patient through presumptive and final identification methods
• Determining whether laboratory testing for detection of antimicrobial resistance is warranted
• Determining the type of antimicrobial therapy that is appropriate
• Determining whether the infecting organism is a risk for other patients in the hospital, the public, or
laboratory workers
• Collecting epidemiologic data to monitor the control and transmission of organisms

BACTERIAL IDENTIFICATION
PHENOTYPIC METHODS
• Based on observable physical or metabolic
characteristics of bacteria
• Identification is through analysis of gene products
rather than through the genes themselves.
• Microscopic morphology and staining
characteristics
• Macroscopic (colony) morphology, including
odor and pigmentation
• Environmental requirements for growth
• Resistance or susceptibility to antimicrobial
agents
• Nutritional requirements and metabolic
capabilities
• Biochemical reactions including enzymatic
reactions or chemical profiles
GENOTYPIC METHODS
• Detecting the presence of a gene, or a part
thereof, or an RNA product that is specific
for a particular organism.
• In principle, the presence of a specific
gene or a particular nucleic acid sequence
unique to the organism is interpreted as a
definitive identification of the organism.
• Highly specific and often very sensitive.

• Conventional methods
• Automated methods

CONVENTIONAL METHODS
• Direct Microscopy
• Culture
• Morphology of Colony
• Culture Smear and Motility Testing
• Biochemical Reactions

DIRECT MICROSCOPY
• Microscopy is defined as the use of a microscope to magnify objects too small to be
visualized with the naked eye so that their characteristics are readily observable.
• Bright-field (light) microscopy
• Phase contrast microscopy
• Fluorescent microscopy
• Darkfield microscopy.

DIRECT MICROSCOPY (contd.,)
Direct examination of unstained specimens
• Examination of wet preparation/ saline mount:
• Bacterial morphology, arrangement
• Presence of yeast cells
• Presence of inflammatory cells
• Presence of epithelial cells
• Motility
• Serologic reactivity in specific antisera (quellung reaction)

Direct examination of unstained specimens (contd.,)
• Hanging drop procedure: for motility
• India ink/ Nigrosin preparation : for capsules
• Dark field examination : to visualize delicate organisms , invisible by bright field
optics- Treponema pallidum
• KOH mount, Iodine mount

APPLICATIONS OF MICROSCOPY IN DIAGNOSTIC MICROBIOLOGY:
• Rapid preliminary organism identification by direct visualization in patient specimens
• Detection of different organisms present in the same specimen
• Detection of organisms not easily cultivated in the laboratory
• Evaluation of patient specimens for the presence of cells indicative of inflammation or
contamination
• Determination of an organism’s clinical significance
• Preculture information about which organisms might be expected to grow so that appropriate
cultivation techniques are used
• Determination of which tests and methods should be used for identification and
characterization of cultivated organisms

All appropriate specimens should have a direct microscopic examination (smear of the primary
specimen).
Purposes:
Quality of the specimen can be assessed
Early indication of what may be wrong with the patient
Workup of the specimen can be guided by comparing what grows in culture to what was seen on
the original smear.

DIRECT MIICROSCOPY(contd.,)
• PREPARATION:
• Specimen samples are placed on the slide using a swab
or by using a pipette into which liquid specimen has
been aspirated
• Material to be stained is dropped (if liquid) or rolled (if
on a swab) onto the surface of a clean, dry, glass slide.

• Squash or crush prep : tissue, bone marrow aspirate, or other aspirated sample.
• The aspirate may be placed in the anticoagulant EDTA tube and inverted several times to mix the
contents.
• To prepare the slide, a drop of the aspirate is placed on a slide and a second slide is gently placed on
top; the two slides are pressed together, crushing or squashing any particulate matter.
• The two slides are then gently slid or pulled apart using a horizontal motion and air-dried before
staining.
• Cytocentrifugation or concentration of a sterile body fluid such as CSF
• In a cytocentrifuge, the hydraulic forces of the liquid cause the fluid to move away from the
sediment, which is then collected on an absorbent material, leaving the particulate matter and
cellular debris in the center of the microscope slide.
• The slide may then be stained for microscopy.

Staining methods:
1. Gram stain:
Principal stain used for microscopic examination of bacteria
Rapid presumptive identification of pathogens,
Quality of a specimen
To know whether bacterial pathogens from a specific body site are considered normal microbiota colonizing
the site or the actual cause of infection.
Nearly all clinically important bacteria can be detected using this method- EXCEPTIONS :
Organisms that exist almost exclusively within host cells (e.g., chlamydia)
Lack cell wall (e.g., mycoplasma and ureaplasma)
Insufficient dimension to be resolved by light microscopy (e.g., spirochetes).
Differential stain
 Procedure

Staining methods (contd.,):
Gram Stain Examination (Direct Smear):
• 400X magnification : WBCs , epithelial cells, debris, and larger
organisms such as fungi or parasites.
• Oil immersion:
• Presence of bacterial cells as well as the Gram reactions,
morphologies (e.g., cocci or bacilli), and arrangements (e.g., chains,
pairs, clusters) of the cells seen
• Presence of inflammatory cells (e.g., phagocytes) that are key
indicators of an infectious process.
• Presence of other host cells, such as squamous epithelial cells in
respiratory specimens
• Background tissue debris and proteinaceous material- indicates that
specimen material was adequately affixed to the slide.

Staining methods (contd.,):
CLINICAL APPLICATIONS OF GRAM STAINING:
• To differentiate Gram positive and Gram negative organisms.
• To know the morphology of bacteria.
• Direct diagnosis of emergency conditions like – Meningococcal meningitis, Pneumococcal meningitis.
• Diagnosis of – Candidiasis, Gonococcal urethritis etc.,
• Choose antibiotics for antibiotic sensitivity testing.
• Choose antibiotics for presumptive treatment in emergency condition

Staining methods:
2. Ziehl Neelsen staining:
• Acid fast and non acid fast bacteria
• Mycobacterium tuberculosis
• Nocardia spp., coccidian parasites, such as Cryptosporidium spp
• To detect acid-fast bacteria (e.g., mycobacteria) directly in clinical specimens
• Only performed on specimens from patients highly suspected of having a mycobacterial
infection.
• Procedure:
• Grading:

Staining methods:
• Albert’s staining
Procedure
 Albert’s stain – 3-5 min
 Albert’s iodine – 1 min
 Wash & blot dry
• Granules- Bluish black
• Protoplasm – green
• Other organisms –Light green

Staining methods:
Silver Impregnation stains
• Spirochaetes, Bartonella henselae
• Too slender to be visualized by bright field microscopy
• Warthin Starry, Dieterle, Steiner silver impregnation stains

Staining of spores:
 Unstained wet films under phase contrast microscope: large, refractile, oval or spherical
bodies within bacterial mother cells or else free from the bacteria.
 Ordinary dyes/ Gram’s stain:
 body of the bacillus –deeply coloured
 Spore unstained and appear as clear area
 Modified ZN stain : 0.25% sulphuric acid as decolourizer
 Red spores in blue stained bacteria
• MALACHITE GREEN STAIN FOR SPORES
• Colours the spores green & the vegetative bacilli red

Capsule Staining methods:
• India Ink method
• Nigrosin

PHASE CONTRAST MICROSCOPY
• Wet mount – non viscous liquid- urine
• Sample suspended in sterile saline – vaginal sample
• Observation of viable microorganisms

FLOURESCENT MICROSCOPY
• Fluorochroming and immunofluorescence
• Fluorochroming : direct chemical interaction between fluorescent dye & component of bacterial cell
• Min concentration of 104 /mL organisms
• Acridine orange: binds to nucleic acid - bright orange fluorescence
• to confirm the presence of bacteria in blood cultures when Gram stain results are difficult to interpret
• To detect bacteria , when the presence is highly suspected but none are detected using light microscopy.
• Nonspecific.
• Does not discriminate between gram-negative and gram-positive bacteria.
• For detection of cell wall–deficient bacteria (e.g., mycoplasmas) grown in culture that are incapable of retaining the
dyes used in the Gram stain
• Auramine-rhodamine: binds to mycolic acid- all mycobacteria
• Bright yellow/orange against a greenish background
• Calcofluor white : for fungus

DARK-FIELD MICROSCOPY
• For detecting certain bacteria directly in patient specimens
• Cannot be seen by light microscopy - because of their thin dimensions
• Difficult to grow in culture- because of their physiology,
• To detect spirochetes- Treponema pallidum- appear extremely bright against a black field.

RECENT ADVANCES:
DIGITAL AUTOMATED MICROSCOPY
• Automation in digital microscopy- using sophisticated software and unique technology
• To acquire microscopic digital images of Gram stains using a web-based interface.
• This interface allows images to be viewed on a single screen, using a fully automated
microscope
• Allows the viewer to track the slide on the x and y axis, very much like using a standard
microscope.
• A number of technologies, including Leica and PathXL, currently provide mobile device
viewers for virtual microscopy.

RECENT ADVANCES:
DIGITAL HOLOGRAPHIC MICROSCOPY
• To visualize bacteria in aqueous environments without the loss of resolution in samples 1mm
thick.
• 100 fold greater resolution than most bright field microscopes
• Has the potential to improve the identification of low concentrations of organisms in clinical
samples.

CULTURE
• Purposes:
• To grow and isolate all bacteria present in a clinical specimen
• To determine which of the bacteria that grow are most likely causing infection and which are likely
contaminants or colonizers
• To obtain sufficient growth of clinically relevant bacteria to allow identification, characterization,
and susceptibility testing
• Cultivation is the process of growing microorganisms in culture by taking bacteria from the infection
site (i.e., the in vivo environment) by some means of specimen collection and growing them in the
artificial environment of the laboratory (i.e., the in vitro environment).
• The successful transition from the in vivo to the in vitro environment requires that the nutritional and
environmental growth requirements of bacterial pathogens be met.
• EXCEPTIONS : Treponema pallidum, Mycobacterium leprae

CULTURE MEDIA (contd.,)
• Broth (liquid)/ solid(agar) / biphasic
• Broth media- nutrients are dissolved in water
• Bacterial growth is indicated by a change in the broth’s appearance from clear to turbid
• At least 106
bacteria per milliliter of broth are needed for turbidity to be detected with the
unaided eye.
• Some broths contain a pH indicator, such as phenol red

Thioglycollate broth, which contains a small amount of agar (making it a semisolid medium)
• Provides an indication of the type of organism present based on oxygen requirements.
• Strict anaerobes will grow at the bottom of the broth tube
• Aerobes will grow near the surface.
• Microaerophilic organisms will grow slightly below the surface where oxygen concentrations are lower
than atmospheric concentrations.
• Facultative anaerobes and aerotolerant organisms will grow throughout the medium, because they are
unaffected by the variation in oxygen content

• Solid medium : solidifying agent added to the nutrients and water.
• Agar- melting at high temperatures (≥95°C) but resolidifying after the temperature falls below 50°C.
• Agar plate.
• Agar deep
• Agar slant
• Different agar media usually are identified according to the major nutritive components of the medium
• The resulting bacterial population is considered to be derived from a single bacterial cell and is known
as a pure colony.
• All bacterial cells within a single colony are the same genus and species, having identical genetic and
phenotypic characteristics (i.e., are derived from a single clone).
• Pure cultures are required for subsequent procedures used to identify and characterize bacteria.

Primary culture media: Nutritive media, selective media, differential media, enrichment media .
Nutritive media: tryptic soy agar, nutrient agar
• Support the growth of a wide range of microorganisms
• Nonselective the growth of most organisms is supported.
Selective media support the growth of one group of organisms but not another, by adding antimicrobials,
dyes, or alcohol to a particular medium.
• MacConkey agar, Columbia agar with colistin and nalidixic acid.
• Selective media can also be differential media

• Enrichment media:
• Contain specific nutrients for the growth of particular bacterial
pathogens that may be present alone or with other bacterial species
in the specimen
• Buffered Charcoal –Yeast Extract agar (BCYE)- Legionella
pneumophilia (L-cysteine)
• Differential media : employ some factors that allows bacterial colonies
of one type to exhibit certain metabolic or culture characteristic to
distinguish from other bacteria.
• MacConkey agar, blood agar

CULTURE (contd.,)
• BACK UP BROTH:
• In some cases (sterile body fluids, tissues, or deep abscesses in a patient receiving antimicrobial
therapy)
• Along with primary solid (agar) media, backup broth (also called supplemental or enrichment
broth) medium is inoculated, so small numbers of organisms present may be detected.
• Allows detection of anaerobes in aerobic cultures and organisms that may be damaged by either
previous or concurrent antimicrobial therapy.
• Thioglycollate broth, brain-heart infusion broth (BHIB), and tryptic soy broth (TSB)

CULTURE (contd.,)
Selection of media - usually based on the organisms most likely to be involved in the disease
process.
• Example: for a CSF specimen- blood /chocolate agar at a minimum
• Example : if a specimen is collected from a source likely to be contaminated with normal
microbiota – selective media

CULTURE (contd.,)
Sheep Blood Agar :
• Supports growth for all but the most fastidious clinically significant bacteria.
• The medium consists of a base containing a protein source (e.g., tryptones), soybean protein
digest (containing a slight amount of natural carbohydrate), sodium chloride, agar, and 5% sheep
blood.
• Certain bacteria produce extracellular enzymes that lyse red blood cells in the agar (hemolysis).
• Beta -hemolysis
• Alpha -hemolysis
• Gamma - hemolysis or nonhemolytic.
• Transmitted light

CULTURE (contd.,)
Chocolate agar:
• During preparation the red blood cells are lysed when added to molten agar base.
• Release of intracellular nutrients such as hemoglobin, hemin (“X” factor), and the coenzyme
nicotinamide adenine dinucleotide (“V” factor)
• Fastidious bacteria :Neisseria gonorrhoeae, Haemophilus spp.

CULTURE (contd.,)
Columbia CNA with Blood
• Three peptone sources and 5% defibrinated (whole blood with fibrin removed to
prevent clotting) sheep blood.
• Differentiate bacterial colonies based on the hemolytic reactions they produce.
• CNA refers to the antibiotics colistin (C) and nalidixic acid (NA)
• Colistin disrupts the cell membranes of gram-negative organisms
• Nalidixic acid blocks DNA replication in susceptible organisms

CULTURE (contd.,)
MacConkey agar
• Selective and differential agar.
• Crystal violet dye - inhibit the growth of gram-positive bacteria and fungi and allows many types of
gram-negative bacilli to grow.
• Neutral red - pH indicator
• Fermentation of lactose results in acid production, which decreases the pH of the medium and causes
the neutral red indicator to give bacterial colonies a pink to red color.
• Non–lactose-fermenters, such as Shigella spp., remain colorless and translucent
• Slow fermenters and may not demonstrate a positive fermentation reaction in the first 24 hours of
growth.

CULTURE (contd.,)
CLED agar (Cysteine Lactose Electrolyte Deficient agar)
• Differential, non selective culture medium
• Used for the isolation, enumeration and differentiation of urinary microorganisms.
• It promotes the growth of urinary pathogens, but prevents excessive swarming
of Proteus species due to its lack of electrolytes.
• The medium allows quantitative determination of urinary pathogens when
calibrated loops are used for inoculation.
• Cysteine- promotes coliforms
• Lactose – energy source
• Bromothymol blue –indicator – yellow(acid), dark blue (alkalinization)

CULTURE (contd.,)
Gram-Negative Broth
• A selective broth for the cultivation of gastrointestinal pathogens (i.e., Salmonella spp. and Shigella spp.)
from stool specimens and rectal swabs.
• The broth contains several active ingredients, including sodium citrate and sodium deoxycholate- inhibit
gram-positive organisms and the early multiplication of gram-negative, non enteric pathogens.
• Mannitol - primary carbon source.
• GN broth should be subcultured 6 to 8 hours after initial inoculation and incubation.

CULTURE (contd.,)
Hektoen Enteric Agar
• Selective medium : Bile salts and dyes (bromthymol blue & acid fuchsin)
• Slow the growth of most nonpathogenic gram-negative bacilli found in the GIT
• Allow Salmonella spp. and Shigella spp. to grow.
• Differential: many non enteric pathogens that do grow will appear as orange to salmon-colored colonies.
• Fermentation of lactose, sucrose or salicin in the medium, resulting in the production of acid, which
lowers the medium’s pH and causes a color change in the pH indicator bromthymol blue.
• Salmonella spp. and Shigella spp.- do not ferment these carbon compounds - no color change occurs and
their colonies maintain the original blue-green color of the medium.
• Contains ferric ammonium citrate - 𝐻2S -producing organisms, such as Salmonella spp., can be
visualized as colonies exhibiting a black precipitate

CULTURE (contd.,)
Phenylethyl alcohol (PEA) agar:
• Sheep blood agar that is supplemented with phenylethyl alcohol to inhibit the growth of gram-
negative bacteria.
• The 5% sheep blood in PEA provides nutrients for common gram-positive cocci such as
enterococci, streptococci, and staphylococci .
• Although it contains sheep blood, PEA agar should not be used in the interpretation of hemolytic
reactions

CULTURE (contd.,)
Modified Thayer-Martin (MTM) agar
• Enrichment and selective medium
• For the isolation of Neisseria gonorrhoeae and Neisseria meningitidis
• Enrichment :basal components +chocolatized blood
• Selective capacity : Antibiotics
• Colistin - to inhibit other gram-negative bacteria
• Vancomycin- to inhibit gram-positive bacteria
• Nystatin - to inhibit yeast.
• Trimethoprim - to inhibit Proteus spp.
• Martin-Lewis agar, substitutes ansamycin for nystatin and has a higher concentration of
vancomycin

CULTURE (contd.,)
Xylose-lysine-deoxycholate (XLD) agar
• Selective and differential for Shigella spp. and Salmonella spp.
• Sodium deoxycholate- inhibits many gram-negative bacilli that are not enteric pathogens and inhibits gram
positive organisms.
• Phenol red indicator - detects increased acidity from carbohydrate (i.e., lactose, xylose, and sucrose)
fermentation.
• Enteric pathogens, such as Shigella spp. - do not ferment these carbohydrates, so their colonies remain
colorless (i.e., the same approximate pink to red color of the uninoculated medium).
• Salmonella spp. - Even though they ferment xylose- colorless colonies on XLD because of the
decarboxylation of lysine, which results in a pH increase that causes the pH indicator to turn red.
• These colonies often exhibit a black center that results from Salmonella spp. producing 𝐻2S
• Several of the nonpathogenic organisms ferment one or more of the sugars and produce yellow colonies

CULTURE (contd.,)
• Combination of Blood agar & MacConkey agar
• Chocolate agar : respiratory and sterile body fluids
• Stool specimen :
• Mildly selective media : MacConkey agar
• Highly selective media : DCA, XLD, TCBS agar
• Blood- blood culture bottles
• CLED agar – urine specimen

ANAEROBIC CULTURE MEDIA
• Robertson’s cooked meat (RCM) broth:
• It contains chopped meat particles (beef heart), which provide glutathione (a sulfhydryl
group containing reducing substance) and unsaturated fatty acids.
• It is the most widely used anaerobic culture medium .
• It is also used for maintenance of stock cultures.
• Thioglycollate broth
• Anaerobic blood agar
• Egg yolk agar
• Phenyl ethyl agar

CONVENTIONAL BLOOD CULTURE MEDIA
• BHI broth
• Biphasic medium

Other culture media
• Mannitol salt agar: yellow colonies of Staphylococcus aureus
• Milk agar
• Loeffler’s serum slope
• Tellurite blood agar
• TCBS agar

• Carrom coin appearance – Pneumococcus
• Medusa head appearance of colony on NA

CULTURE MEDIA- INOCULATION
• Specimen inoculation onto solid media can be done:
• Quantitatively by a dilution procedure or by means of a quantitative loop
• Semiquantitatively using an ordinary inoculating loop.
• Semiquantitation is referred to as streaking for isolation
• The microorganisms present in the specimen are successively diluted out as each quadrant is
streaked until finally each morphotype is present as a single colony.
• Numbers of organisms present can subsequently be graded as:
• 4+ (many, heavy growth) if growth is out to the fourth quadrant,
• 3+ (moderate growth) if growth is out to the third quadrant
• 2+(few or light growth) if growth is in the second quadrant
• 1+ (rare) if growth is in the first quadrant.

INOCULATED MEDIA - INCUBATION
The four most critical environmental factors :
• Oxygen and carbon dioxide (CO2 ) availability
• Temperature
• pH
• Moisture content of the medium

INOCULATED MEDIA- INCUBATION (contd.,)
• Aerobes grow in ambient air, which contains 21% O2 and a small amount (0.03%) of CO2 .
• Anaerobes usually cannot grow in the presence of O2
• Anaerobe jars, bags, or chambers - 5% to 10% H2, 5% to 10% CO2 , 80% to 90% N2 , 0% O2 .
• Capnophiles- increased concentrations of CO2 (5% to 10%) and approximately 15% O2 .
• Candle jar (3% CO2 ) or a CO2 incubator, chamber jar, or bag.
• Microaerophiles grow under reduced O2 (5% to 10%) and increased CO2 (8% to 10%).
• Specially designed chamber jars or bags.
• Automated microprocessor-controlled system, the Advanced Axonomat:
• to create the desired atmospheric balance of gases required for specific organismal growth
• Both anaerobic and microaerophilic environments may be produced

TEMPERATURE :
• Most medically relevant bacteria uses incubators with temperatures maintained in the 35°C to 37°C
• For others, an incubation temperature of 30°C (i.e., the approximate temperature of the body’s
surface) may be preferable
• Recovery of certain organisms can be enhanced by incubation at other temperatures.
• Campylobacter jejuni is able to grow at 42°C - Incubation at this temperature can be used as
temperature enrichment procedure.
• Listeria monocytogenes and Yersinia enterocolitica, can grow at 4°C to 43°C but grow best at
temperatures between 20° and 40°C - Cold enrichment

pH
• Most clinically relevant bacteria prefer a near-neutral pH range, from 6.5 to 7.5.
Moisture
• Water is provided as a major constituent of both agar and broth media.
• When media are incubated -> a large portion of water content can be lost by evaporation.
• Loss of water from media can be deleterious to bacterial growth in two ways:
• Less water is available for essential bacterial metabolic pathways
• Relative increase in the solute concentration of the media.
• An increased solute concentration can osmotically shock the bacterial cell and cause lysis.
• In addition, increased atmospheric humidity enhances the growth of certain bacterial species.
• Measures such as sealing agar plates or using humidified incubators are used to ensure appropriate
moisture levels are maintained throughout the incubation period

MORPHOLOGY OF BACTERIAL COLONY
• Size
• Shape
• Surface
• Edge
• Elevation
• Consistency
• Density
• Hemolysis
• Pigmentation

HEMOLYSIS ON BLOOD AGAR
• ALPHA
• BETA
• GAMMA

PIGMENT PRODUCTION
• DIFFUSIBLE PIGMENTS : P. aeruginosa
• NON-DIFFUSIBLE PIGMENTS: S. aureus

CELL CULTURES
• Obligate intracellular parasites - require viable host cells for propagation.
• Chlamydiae, rickettsiae, and rickettsiae-like organisms are bacterial pathogens
• Comprise layers of living cells growing on the surface of a solid matrix such as the inside of a glass tube
or the bottom of a plastic flask.
• The presence of bacterial pathogens within the cultured cells is detected by specific changes in the
cells’ morphology.
• Specific stains, composed of antibody conjugates, may be used to detect bacterial antigens within the
cells.
• Cell cultures may also detect certain bacterial toxins (e.g., Clostridium difficile cytotoxin).
• Cell cultures are most commonly used in diagnostic virology.

CULTURE SMEAR
• Indirect smear : contains organisms obtained after growth in artificial media.
• Prepared from solid / semisolid media / broth.
• Occasionally an organism may grow in culture that was not seen in the direct smear.
• Liquid broth culture smears - more clearly and accurately represent the native cellular morphology
and arrangement compared with smears from solid media.
• Preparation :
• Solid medium : using a sterile loop or needle - Emulsified in a drop of sterile water or saline on the
slide.
• For small amounts of growth - a sterile wooden applicator stick
• Allowed to air-dry and is affixed to the slide by placing it on a slide warmer (60° C) for at least 10
minutes or by flooding it with 95% methanol for 1 minute.
• Liquid medium : an aspirated sample of the broth culture is applied to the slide, air-dried, and fixed
before staining

MOTILITYTESTING
• Tumbling motility – Listeria
• Gliding motility – Mycoplasma
• Stately motility – Clostridium
• Darting Motility – Vibrio cholerae, Campylobacter
• Swarming on agar plate – Proteus, Clostridium tetani
• Corkscrew, lashing, flexion extension- Spirochete

BIOCHEMICAL REACTIONS
• Catalase test
• Oxidase test
• ICUT
• Sugar fermentation test
• MR test
• VP test
• OF test
• Nitrate reduction test
• Decarboxylase test
• PPA test
• Coagulase test
• DNase test
• CAMP test
• Bile esculin hydrolysis test
• Heat tolerance test
• Sugar fermentation test
• PYR test
• Bile solubility test
• Novobiocin susceptibility test
• Optochin susceptibility test
• Bacitracin susceptibility test

ENZYME BASEDTESTS
• Enzymatic content of an organism is a direct reflection of the organism’s genetic makeup -> is specific
for individual bacterial species.
• Enzyme-based tests are designed to measure the presence of a specific enzyme or a complete
metabolic pathway that may contain several different enzymes.
• Single Enzyme Tests :to determine the presence of a single enzyme.
• Usually provide rapid results because they can be performed on organisms already grown in
culture. Play a key role in the identification scheme.
• Used extensively to determine which subsequent identification steps should be followed.
• Catalase test, oxidase test, indole test, urease test, PYR test

Tests for the Presence of Metabolic Pathways
• Based on determining what metabolic pathways an organism uses and the substrates
processed by these pathways.
• These pathways may involve several interactive enzymes.
• The presence of an end product resulting from these interactions is measured in the
testing system.
• Three general categories:
• Carbohydrate oxidation and fermentation
• Amino acid degradation
• Single substrate utilizations

1. Oxidation and Fermentation Tests
• Oxidative processes require oxygen; fermentative ones do not.
• Observe acid byproducts are produced in the presence or absence of oxygen (fermentation).
• Presence of acid byproducts is detected by a change in the pH indicator incorporated into the medium.
• Usually accomplished using a special semisolid medium (oxidative fermentative [O-F] medium) that
contains low concentrations of peptone and a single carbohydrate substrate such as glucose.
• The organism to be identified is inoculated into two glucose O-F tubes, one of which is then overlaid
with mineral oil as a barrier to oxygen.
• Common pH indicators :
• Bromocresol purple : purple to yellow
• Andrade’s acid fuchsin indicator: pale yellow to pink
• Phenol red - red to yellow
• Bromothymol blue: green to yellow.

2. Amino acid degradation
• Determining the ability of bacteria to produce enzymes that either deaminate, or decarboxylate certain
amino acids
• Lysine, tyrosine, ornithine, arginine, and phenylalanine
• Decarboxylases cleave the carboxyl group from amino acids so that amino acids are converted into
amines; lysine is converted to cadaverine, and ornithine is converted to putrescine.
• Because amines increase medium pH, they are readily detected by color changes in a pH indicator.
• Decarboxylation is an anaerobic process that requires an acid environment for activation.
• The most common medium used for this test is Moeller decarboxylase base that contains glucose, the
amino acid substrate of interest and a pH indicator.
• Deamination, the cleavage of the amine group from an amino acid, occurs in air.
• Lysine iron agar medium is a combination medium used for the identification of decarboxylation and
deamination in a single tube.

3. Single Substrate Utilization
• Whether an organism can grow in the presence of a single nutrient or carbon source
• Such tests entail inoculating organisms to a medium that contains a single source of nutrition (e.g.,
citrate, malonate, or acetate) and, after incubation, observing the medium for growth.
• Growth is determined by observing the presence of bacterial colonies or by using a pH indicator to
detect end products of metabolic activity

Establishing Inhibitor Profiles
• The ability of a bacterial isolate to grow in the presence of one or more inhibitory substances
• Growth in the presence of various NaCl concentrations (identification of Enterococci spp.
and Vibrio spp.)
• Susceptibility to optochin and solubility in bile (identification of Streptococcus
pneumoniae)
• Ability to hydrolyze esculin in the presence of bile (identification of Enterococci spp. in
combination with NaCl)
• Ethanol survival (identification of Bacillus spp.)

1. CATALASE TEST
• Principle :
• Aerobic & facultative anaerobic organisms produce 𝐻2𝑂2& 𝑂2
−
.
• Catalase enzyme
• 2𝐻2𝑂2 → 2𝐻2𝑂 + 𝑂2
• 3% 𝐻2𝑂2
• METHODS :
• Slide method
• Tube method
• 1 mL 𝐻2𝑂2 is poured over a 24 hr nutrient agar slope culture

1. CATALASE TEST (contd.,)
• Expected results:
• Positive : rapid production of bubbles
• Negative : no or few bubbles
• LIMITATIONS :
• Some organisms produce a peroxidase that slowly catalyzes breakdown of 𝐻2𝑂2
(Enterococci)
• Test performed from colonies grown in blood agar.
• CAUTION :
• Possibility of airborne transmission of pathogen

1. CATALASE TEST (contd.,)
• QUALITY CONTROL :
• Positive : S. aureus ATCC 25923
• Negative : S. pyogenes ATCC 19615
CATALASE POSITIVE CATALASE NEGATIVE
Staphylococci Streptococci
Listeria monocytogenes Enterococci
Corynebacteria Gram positive spore forming bacilli
Bacillus Lactobacillus
Micrococcus , Macrococcus, Planococcus Gardnerella vaginalis
Gonococci Eikenella corrodens
Alloiococcus Kingella kingae
Enterobacteriaceae Erysipelothrix rhusiopathiae
Actinomycetes Acranobacterium hemolyticum

2. OXIDASE TEST
• PRINCIPLE :
• To determine the presence of cytochrome oxidase using the oxidation of the
substrate tetra methyl –p- phenylene diamino dihydrochloride.
• METHOD :
• Kovac’s method
• Plate method
• Dry filter paper method
• Wet filter paper method

2. OXIDASE TEST (contd.,)
• CAUTION/ LIMITATION:
• Freshly prepared reagent
• Platinum loop/ clean glass rod
• Avoid iron/ chromium/ nichrome loops
• Avoid picking colonies from media containing fermentable sugars
• Positive : P. aeruginosa ATCC 27853
• Negative : E. coli ATCC 25922

2. OXIDASE TEST (contd.,)
OXIDASE POSITIVE OXIDASE NEGATIVE
Neisseria Enterobacterales
Vibrio Staphylococcus
Campylobacter Acinetobacter spp.
Pseudomonas Stenotrophomonas maltoplilia
Aeromonas, Plesiomonas Bordetella
Micrococcus
Alcaligenes
Chromobacterium
Chryseobacterium

3. INDOLE TEST
• PRINCIPLE :
• To determine an organism’s ability to hydrolyze tryptophan to form indole
• Tryptophan Indole + Pyruvic acid + Ammonia
• Indole + p- di methyl amino benzaldehyde RED coloured product
Tryptophanase
KOVAC’S REAGENT EHRLICH’S REAGENT
Amyl / iso amyl alcohol Absolute ethyl alcohol
p- dimethylamino benzaldehyde p- dimethylamino benzaldehyde
HCl (conc.) HCl (conc.)

3. INDOLE TEST (contd.,)
• Medium rich in tryptophan must be used.
• METHOD
• EXPECTED RESULTS:
• Positive : pink to wine coloured ring
• Negative : no colour change/ yellow ring
Kovac’s method Ehrlich’s method
Positive E. coliATCC 25922 H. influenzae ATCC 49766
Negative K. pneumoniae ATCC 13883 H. parainfluenzae ATCC 76901

3. INDOLETEST (contd.,)
INDOLE POSITIVE INDOLE NEGATIVE
E. coli K. pneumoniae
K. oxytoca Shigella sonnei
Citrobacter koseri Salmonella genus
Edwardsiella tarda Citrobacter freundii
Proteus vulgaris Enterobacter aerogenes
Morganella morganii Enterobacter cloacae
Providencia Hafnia
Serratia marcescens
Proteus mirabilis
Pseudomonas

4. CITRATE UTILIZATION TEST
• PRINCIPLE :
• To identify organisms capable of using sodium citrate as the sole carbon source & inorganic ammonium
salts as the sole nitrogen source
• Citrate Oxaloacetic acid + acetate
• Pyruvic acid + CO2
CITRASE

4. CITRATE UTILIZATION TEST (contd.,)
KOSER’S MEDIUM SIMMON’S CITRATE MEDIUM
NaCl (5 g) Koser’s medium 1L
MgSO4 (0.2 g) Agar (20 g)
NH4PO4 (1 g) Bromothymol blue (0.2%) – 40 mL
KH2PO4 (1 g)
Sodium citrate (5 g)
Distilled water ( 1L)

• METHOD :
• EXPECTED RESULTS :
KOSER’S CITRATE MEDIUM SIMMON’S CITRATE MEDIUM
POSITIVE Turbidity Growth on the medium with/ without
change in colour of indicator
NEGATIVE No turbidity No growth and no colour change

• QUALITY CONTROL
• Positive : K. aerogenes ATCC 13048
• EXAMPLES CITRATE POSITIVE CITRATE NEGATIVE
Salmonella E. coli
Citrobacter Shigella
Klebsiella Edwardsiella
Enterobacter Hafnia
Serratia Yersinia
Providencia
Vibrio vulnificans

5. UREA HYDROLYSIS TEST
• PRINCIPLE :

5. UREA HYDROLYSIS TEST (contd.,)
CHRISTENSEN’S MEDIUM STUART’S UREA BROTH
Components Peptone 1g
NaCl 5 g
K2PO4 2 g
Phenol red 6 ml
Agar 20g
Distilled water 1 L
Glucose ( 10%) 10mL
Urea 20% - 100 mL
Yeast extract 0.1 g
Monopotassium phosphate 9.1 g
Disodium phosphate 9.5 g
Urea 20 g
Phenol red 0.01 g
Distilled water 1 L
Final pH 6.8 6.8
Less buffered Heavily buffered with phosphate salts
POSITIVE Change in colour of slant from light
orange to magenta
Red colour throughout the medium
Negative No colour change No colour change

5. UREA HYDROLYSIS TEST (contd.,)
• Positive :Proteus vulgaris ATCC 13315
• Weak positive : K. pneumoniae ATCC 13883
Strong / most rapid urease producers Brucella , H. pylori
Rapid urease producers Proteus, Morganella
Slow urease producers Klebsiella , Enterobacter
Urease producing fungi Cryptococcus neoformans
Trichophyton mentagrophytes

6. TRIPLE SUGAR IRON (TSI) AGAR TEST
• PURPOSE : To determine whether a Gram Negative Bacilli ferments glucose and lactose/ sucrose and
forms gas/ H2S
• TSI Composition :
• 10 parts lactose : 10 parts sucrose : 1 part glucose & peptone
• Phenol red
• Ferrous sulfate
• After 8-12 hrs of incubation, glucose fermenting bacteria ferments glucose -> produces acid ->
turns entire medium acidic (yellow)
• 18-24 hrs –in the butt- glucose fermentation under anaerobic conditions -> organic acids formed ->
acidic (yellow)

6. TRIPLE SUGAR IRON (TSI) AGAR TEST (contd.,)
• 18- 24 hrs – in the slant – oxidation of fermented products under aerobic conditions ->
alkaline amines are formed -> alkaline slant (red)
• 18- 24 hrs - in addition to glucose, lactose and/ or sucrose are also fermented -> large
amount of fermentation products formed on slant neutralizes alkaline amines and renders
the slant acidic -> acidic slant (yellow)
• CO2 and H2 formation -> bubbles/ cracks in agar/ separation of agar from bottom/ sides of
tube
• H2S production -> blackening of agar

TSI AGAR KLIGER’S IRON AGAR
Beef extract 3g
Yeast extract 3 g
Peptone 20 g
Glucose 1 g
Lactose 10 g
Sucrose 10 g
Ferric citrate 0.3 g
NaCl 5 g
Agar 12 g
Phenol red (0.2%) 12 mL
Distilled water 1 L
Sodium thiosulfate 0.3 g
Beef extract 3 g
Yeast extract 3 g
Peptone 15 g
protease peptone 5 g
Lactose 10 g
Glucose 1 g
Ferrous sulfate 0.2 g
NaCl 5 g
Sodium thiosulfate 0.3 g
Agar 12 g
Phenol red 0.024 g
Distilled water 1L
Final pH: 7.3 Final pH: 7.4

6. TRIPLE SUGAR IRON (TSI)
AGAR TEST (contd.,)
• METHOD :
• Non fermenter (K/K)
• Non Lactose( and sucrose) fermenter : only ferments glucose (K/A)
• Lactose and/ or sucrose fermenter : A/A
• H2S production
• CO2 production

• A/A + gas production : E.coli ATCC 25922
• K/A +/- gas production, H2S + : Salmonella enterica subsp.enterica serovar typhimurium ATCC 14028
• K/K : P. aeruginosa ATCC 27853
• K/A, H2S + : Proteus mirabilis ATCC 12453
• K/A : Shigella flexneri ATCC 12022

REACTION EXAMPLES
K/K P. aeruginosa
K/A Citrobacter koseri, Hafnia, Serratia, Providencia, Morganella
K/A, 𝐻2S + Salmonella, C. freundii, Proteus, Edwardsiella tarda
A/A E.coli, Klebsiella, Enterobacter
A/A with more gas Klebsiella, Enterobacter

7. SUGAR FERMENTATION TEST
• Carbohydrate fermentation is the process microorganisms use to produce energy.
• Most microorganisms convert glucose to pyruvate during glycolysis; however, some organisms use
alternate pathways.
• A fermentation medium consists of a basal medium containing a single carbohydrate (glucose, lactose,
or sucrose) for fermentation.
• Medium may contain various colour indicators, such as Andrade’s indicator, bromocresol, or others.
• A Durham tube is placed in each tube to capture gas produced by metabolism.
Basal media:
• Pancreatic digest of casein (10 g), beef extract (3 g), NaCl (5 g), carbohydrate (10 g)
• Specific indicator (Andrade’s indicator [10 mL, pH 7.4] or bromocresol purple [0.02 g, pH 6.8]).

7. SUGAR FERMENTATION TEST (contd.,)
PROCEDURE:
Peptone Medium with Andrade’s Indicator (for Enterics and Coryneforms)
• Inoculate each tube with one drop of an 18- to 24-hour brain-heart infusion broth culture.
• Incubate at 35°C to 37°C for up to 7 days in ambient air.
• Examine the tubes for acid (indicated by a pink color) and gas production.
• Tubes must show growth for the test to be valid.
• If no growth in the fermentation tubes or control is seen after 24 hours of incubation, add one to two
drops of sterile rabbit serum per 5 mL of fermentation broth to each tube.
Expected Results
• Positive: Indicator change to pink with or without gas formation in Durham tube
• Negative: Growth, but no change in color. Medium remains clear to straw colored

Broth (Brain-Heart Infusion Broth May Be Substituted) with Bromocresol Purple Indicator (for
Streptococci and Enterococci)
• Inoculate each tube with two drops of an 18- to 24-hour brain-heart infusion broth culture.
• Incubate 4 days at 35°C to 37°C in ambient air.
• Observe daily for a change of the bromocresol purple indicator from purple to yellow (acid).
Expected Results
• Positive: Indicator change to yellow
• Negative: Growth, but no change in color. Medium remains purple

Quality Control
• Peptone Medium with Andrade’s Indicator
• Dextrose: Positive, with gas: Escherichia coli (ATCC25922)
• Positive, no gas: Shigella flexneri (ATCC12022)
• Brain-Heart Infusion Broth with Bromocresol Purple Indicator
• Dextrose: Positive, with gas: Escherichia coli (ATCC25922)
• Negative, no gas: Moraxella osloensis (ATCC10973)

8. DECARBOXYLASE TEST (Moeller’s Method)
• Decarboxylases - group of substrate-specific enzymes that are capable of reacting with the carboxyl
(COOH) portion of amino acids, forming alkaline-reacting amines.
• Decarboxylation - forms carbon dioxide as a second product.
• Each decarboxylase enzyme is specific for an amino acid.
• Lysine, ornithine, and arginine - amino acids routinely tested in the identification of the
Enterobacteriaceae.
• Lysine → Cadaverine
• Ornithine → Putrescine
• Arginine → Citrulline
• The conversion of arginine to citrulline is a dihydrolase, rather than a decarboxylase reaction
• An NH2 group is removed from arginine
• Citrulline is next converted to ornithine, which then undergoes decarboxylation to form
putrescine.

8. DECARBOXYLASE TEST (contd.,)
• Moller decarboxylase medium.
• The amino acid to be tested is added to the decarboxylase base before inoculation with the
test organism.
• A control tube, consisting of only the base without the amino acid, must also be set up in
parallel.
• Both tubes are anaerobically incubated by overlaying with mineral oil.
• During the initial stages of incubation, both tubes turn yellow - fermentation of the small
amount of glucose in the medium.
• If the amino acid is decarboxylated, alkaline amines are formed and the medium reverts to
its original purple color.

• Moller Decarboxylase Broth Base
• Peptone 5 g
• Beef extract 5 g
• Bromcresol purple 0.01 g
• Cresol red 0.005 g
• Glucose 0.5 g
• Pyridoxal 0.005 g
• Distilled water 1 L
• Final pH 6.0
• Amino Acid
• Add 10 g (final concentration = 1%) of the L (levo) -form of the amino acid (lysine,
ornithine, or arginine).
• Double this amount if the D (dextro) -form is to be used, because only the L-form is active.

• PROCEDURE :
• From a well-isolated colony of the test organism previously recovered on primary isolation agar,
inoculate two tubes of Moeller decarboxylase medium
• TEST : containing the amino acid to be tested
• CONTROL: devoid of amino acid.
• Overlay both tubes with sterile mineral oil to cover about 1 cm of the surface
• Incubate at 35°C for 18–24 hours.
:

• Positive:
• Lysine—Klebsiella pneumoniae (ATCC33495)—yellow to purple
• Ornithine—Enterobacter aerogenes (ATCC13048)—yellow to purple
• Arginine—Pseudomonas aeruginosa (ATCC27853)—yellow to purple
• Base Control:
• Positive Glucose Fermenters: Klebsiella pneumoniae (ATCC27736)—yellow
Enterobacter aerogenes (ATCC13048)—yellow
• Negative:
• Lysine—Citrobacter freundii (ATCC331218)—yellow
• Ornithine—Proteus vulgaris (ATCC6380)—yellow
• Arginine—Escherichia coli (ATCC25922)—yellow

• INTERPRETATION :
• Conversion of the control tube to a yellow color indicates that
• The organism is viable and
• The pH of the medium has been lowered sufficiently to activate the decarboxylase
enzymes.
• POSITIVE : Reversion of the tube containing the amino acid to a blue-purple color ( formation of
amines from the decarboxylation reaction)< Alkaline (purple) color change compared with the
control tube >
• Negative: No color change or acid (yellow) color in test and control tube. Growth in the control
tube.

9. METHYL RED TEST
• PRINCIPLE :
• Quantitative test for acid production
• Requires positive organisms to produce strong acids (lactic acid, acetic acid, formic acid)
from glucose through mixed acid fermentation pathway
• Organisms that can maintain the low pH after prolonged incubation (48- 72 hrs )
overcoming the pH buffering system of the medium can be called methyl red positive.
• Methyl red : pH 6.0- yellow; pH 4.4 – red

METHYL RED TEST (contd.,)
• COMPONENTS
• METHYL RED TEST BROTH- GLUCOSE PHOSPHATE PEPTONE WATER
• Peptone : 5 g
• K2HPO4 : 5 G
• Water : 5 L
• Glucose (10%) : 50 mL
• Methyl red indicator solution :
• Methyl red : 0.1 g
• Ethanol : 300 ml
• Distilled water : 200 ml

• METHOD :
• Inoculate the medium lightly and incubate at 37C for 48 hrs
• Add five drops of methyl red reagent
• Mix and read immediately
• Positive : bright red
• Negative : yellow

• Positive : E.coli
• Negative : E. aerogenes
MR POSITIVE MR NEGATIVE
E. coli
K. ozaenae
K. rhinoscleromatis
K. ornitholytica
Edwardsielleae
Salmonellae
Proteae
Citrobacter
Yersinia
K. pneumoniae
Enterobacter spp.

10. VOGES – PROSKAUER TEST (acetoin production)
• Many bacteria ferment carbohydrates with the production of acetyl methyl
carbinol or its reduction product 2,3 butylene glycol
• These substances can be tested by a colorimetric reaction between diacetyl
(formed by oxidation ) and a guanidino group under alkaline conditions

VOGES – PROSKAUER TEST (contd.,)
• Medium : glucose phosphate peptone water
• Reagent 1 : α-Naphthol, 5% color intensifier
• α-Naphthol 5 g
• Absolute ethyl alcohol 100 mL
• Reagent 2. Potassium hydroxide, 40%, oxidizing agent
• Potassium hydroxide 40 g
• Distilled water 100 mL

• METHOD
• Inoculate a tube of MR/VP broth with a pure culture of the test organism.
• Incubate for 24 hours at 35°C.
• At the end of this time, aliquot 1 mL of broth to a clean test tube.
• Add 0.6 mL of 5% αnaphthol, C-13 followed by 0.2 mL of 40% KOH.
• It is essential that the reagents be added in this order.
• Shake the tube gently to expose the medium to atmospheric oxygen and allow the
tube to remain undisturbed for 10–15 minutes.

• Positive test : development of a red color 15 minutes
or more after addition of the reagents, indicating the
presence of diacetyl.
• QUALITY CONTROL
• Positive : E. aerogenes
• Negative : E.coli
VP POSITIVE VP NEGATIVE
K. Pneumoniae
E. Cloacae
Serratia
Ewingella americana
Aeromonas sobria
Vibrio cholerae
Chryseomonas
E. Coli
Edwardsiella tarda
Salmonellae
Proteae
Yersinieae

11. OXIDATION – FERMENTATION TEST
• Saccharolytic microorganisms degrade glucose either fermentatively or
oxidatively
• The end products of fermentation are relatively strong mixed acids that can be
detected in a conventional fermentation test medium.
• The acids formed in oxidative degradation of glucose are extremely weak
• More sensitive oxidation-fermentation medium of Hugh and Leifson (OF
medium) is required for their detection.

O/F TEST (contd.,)
• The OF medium of Hugh and Leifson differs from carbohydrate fermentation media as follows:
• The concentration of peptone is decreased from 1% to 0.2%.
• The concentration of carbohydrate is increased from 0.5% to 1.0%.
• The concentration of agar is decreased from 1.5% to 0.3% -> semisolid
• The lower protein/carbohydrate ratio reduces the formation of alkaline amines that can
neutralize the small quantities of weak acids that may form from oxidative metabolism.
• The relatively larger amount of carbohydrate serves to increase the amount of acid that can
potentially be formed.
• The semisolid consistency of the agar permits acids that form on the surface of the agar to
permeate throughout the medium, making interpretation of the pH shift of the indicator easier
to visualize.
• Motility can also be observed in this medium.

O/F TEST (contd.,)
• MEDIA &REAGENTS :
• pH adjusted to 7.1 before adding bromothymol blue
• Medium autoclaved in a flask at 121C for 15 min
• Carbohydrate (glucose) to be added is sterilized separately and added to give a final concentration
of 1%.
• OF medium should be poured without a slant into tubes with an inner diameter of 15–20 mm to
increase surface area.
• Depth 4 cm.

O/F TEST (contd.,)
• PROCEDURE:
• Two tubes are required for the OF test, each inoculated with the unknown organism, using a
straight needle, stabbing the medium three to four times halfway to the bottom of the tube.
• One tube of each pair is covered with a 1-cm layer of sterile mineral oil or melted paraffin
• Other tube left open to the air.
• Incubate both tubes at 35°C
• Examine daily for several days (upto 30 days)

O/F TEST (contd.,)
• QUALITY CONTROL:
• Glucose fermenter: Escherichia coli
• Glucose oxidizer: Pseudomonas aeruginosa
• Nonsaccharolytic: Moraxella species
• INTERPRETATION:

12. NITRATE REDUCTION TEST
• Organisms demonstrating nitrate reduction have the capability of extracting oxygen
from nitrates to form nitrites and other reduction products
• The presence of nitrites in the test medium is detected by the addition of α-
naphthylamine and sulfanilic acid, with the formation of a red diazonium dye, p-
sulfobenzeneazo-αnaphthylamine

12. NITRATE REDUCTION TEST (contd.,)
Nitrate Broth or Nitrate Agar (Slant)
• Peptone 5 g
• Potassium nitrate (KNO3 ) 1 g
• Agar (nitrite-free) 12 g
• Reagent A
• α-Naphthylamine 5 g
• Acetic acid (5 N), 30% 1 L
• Reagent B
• Sulfanilic acid 8 g
• Acetic acid (5 N), 30% 1 L

• METHOD :
• Inoculate the nitrate medium with a loopful of the test organism isolated in pure
culture on agar medium, and incubate at 35°C for 18–24 hours.
• At the end of incubation, add 1 mLeach of reagents A and B to the test medium, in
that order.

• EXPECTED RESULTS:
• POSITIVE : The development of a red color within 30 seconds after adding the test reagents
indicates the presence of nitrites
• If no color develops after adding the test reagents:
• True negative :nitrates have not been reduced
• False negative :Nitrates have been reduced to products other than nitrites, such as ammonia,
molecular nitrogen (denitrification), nitric oxide (NO) or nitrous oxide (N2O), and
hydroxylamine.
• It is necessary to add a small quantity of zinc dust to all negative reactions.
• Zinc ions reduce nitrates to nitrites, and the development of a red color after adding zinc dust
indicates the presence of residual nitrates and confirms a true negative reaction.

QUALITY CONTROL :
• Positive control: Escherichia coli
• Negative control: Acinetobacter baumannii

13. PHENYL PYRUVIC ACID TEST
• Phenylalanine on deamination forms a keto acid, phenylpyruvic acid.
• Of the Enterobacteriaceae, only members of the Proteus, Morganella, and Providencia genera possess
the deaminase enzyme necessary for this conversion.
• The phenylalanine test depends on the detection of phenylpyruvic acid in the test medium after
growth of the test organism.
• The test is positive if a visible green color develops on addition of a solution of 10% ferric chloride.

PHENYL PYRUVIC ACID TEST (contd.,)
• MEDIA & REAGENTS:
• Phenylalanine agar is poured as a slant into a tube.
• Meat extracts or protein hydrolysates cannot be used because of their varying
natural content of phenylalanine.
• Yeast extract serves as the carbon and nitrogen source.

PHENYL PYRUVIC ACID TEST (contd.,)
• Positive: Proteus mirabilis (ATCC12453)
• Negative: Escherichia coli (ATCC25922)
• PROCEDURE:
• The agar slant of the medium is inoculated with a single colony of the test
organism isolated in pure culture of primary plating agar.
• After incubation at 35°C for 18–24 hours, 4 or 5 drops of the ferric chloride
reagent are added directly to the surface of the agar.
• As the reagent is added, the tube is rotated to dislodge the surface colonies.

PHENYL PYRUVIC ACID TEST
(contd.,)
• Interpretation
• POSITIVE : The immediate appearance of an intense green color indicates the
presence of phenylpyruvic acid
• Negative: Slant remains original color after the addition of ferric chloride

14. COAGULASE TEST
• S. aureus produces 2 forms of coagulase.
BOUND COAGULASE FREE COAGULASE
Clumping factor Thrombin like substance
Bound to bacterial cell wall Secreted extracellularly
Reacts directly with fibrinogen in plasma Causes clot formation when S. aureus colonies are
incubated with plasma
Precipitation of fibrinogen on staphylococcal cell ->
rapid agglutination/ clumping
Free coagulase + Coagulase reacting factor in
plasma -> Coagulase- CRF complex -> react with
fibrinogen -> clot formation
Not present in culture filtrates Present in culture filtrates

SLIDE COAGULASETEST TUBE COAGULASETEST
For bound coagulase For free coagulase
Rabbit plasma with EDTA Method 1:
1 in 6 dilution of plasma in 0.85% NaCl
Place 1 mLof diluted plasma in a tube
Emulsify Staphylococcus colony into it
Incubate at 37C for 4 hrs
Method 2: using undiluted plasma (0.5ml) and 0.1 ml
of culture
Positive :clumping within 10 sec Positive : any degree of clot formation
Negative : no clumping Negative : no clot
Kit slide tests are also available

COAGULASE TEST (contd.,)
• Positive : S. aureus ATCC 25923
• Negative : S. epidermidis ATCC 12228
• EXAMPLES
• Coagulase positive : S. aureus
• Coagulase negative : CoNS

15. Deoxyribonucleic Acid Hydrolysis (DNase Test Agar)
• To determine the ability of an organism to hydrolyze DNA.
• The medium is pale green because of the DNA–methyl green complex.
• If the organism growing on the medium hydrolyzes DNA, the green color fades and the colony is
surrounded by a colorless zone.
• Media:
• Pancreatic digest of casein (10 g),
• yeast extract (10 g),
• deoxyribonucleic acid (2 g),
• NaCl (5 g), agar (15 g),
• methyl green (0.5 g),
• pH 7.5

Method
• Inoculate the DNase agar with the organism to be tested and streak for isolation.
• Incubate aerobically at 35°C to 37°C for 13 to 24 hours.
Expected Results
• Positive: When DNA is hydrolyzed, methyl green is released and combines with highly polymerized
DNA at a pH of 7.5, turning the medium colorless around the test organism
• Negative: If no degradation of DNA occurs, the medium remains green

Quality Control
• Positive: Staphylococcus aureus (ATCC25923)
• Negative: Escherichia coli (ATCC25922)

16. CAMP test
• Presumptive identification of group B β-hemolytic streptococci
• First described in 1944 by Christie, Atkins, and Munch–Petersen
• The hemolytic activity of the β-hemolysin produced by most strains of Staphylococcus aureus is
enhanced by an extracellular hemolytic protein (CAMP factor) produced by group B streptococci.
• Interaction of the β-hemolysin with this factor causes “synergistic hemolysis,” which is easily observed
on a blood agar plate.
• This phenomenon is seen with both hemolytic and nonhemolytic isolates of group B streptococci.
• Other bacteria, such as Listeria monocytogenes, Rhodococcus equi, and certain strains of Vibrio cholera,
are also CAMP-positive.

CAMP test (contd.,)
• MATERIALS:
• β-hemolysin-producing strain of Staphylococcus aureus
• Sheep blood agar plate
• Positive: Streptococcus agalactiae (ATCC13813)—enhanced arrowhead hemolysis
• Negative: Streptococcus pyogenes (ATCC19615)—beta-hemolysis without enhanced
arrowhead formation

CAMP test (contd.,)
• PROCEDURE:
• Down the center of a blood agar plate, and make a single straight line streak of
βhemolysin-producing S. aureus.
• Taking care not to intersect the staphylococcal streak, inoculate a streak of the
β-hemolytic streptococcus to be identified perpendicular to the staphylococcal
streak.
• Make these streaks so that, after incubation, the growth of the two organisms
will not be touching.
• The streptococcal streak should be 3–4 cm long.
• Known group A and B streptococcal strains should be similarly inoculated on
the same plate as negative and positive controls, respectively.
• Incubate the plate at 35°C in ambient air for 18–24 hours.

CAMP test (contd.,)
• INTERPRETATION:
• Positive: Enhanced hemolysis is indicated by an arrowheadshaped zone of beta-
hemolysis at the juncture of the two organisms
• Negative: No enhancement of hemolysis
• The area of increased hemolysis occurs where the β-hemolysin secreted by the
Staphylococcus and the CAMP factor secreted by the group B Streptococcus
intersect.
• Any bacitracin-resistant, trimethoprim-sulfamethoxazole–resistant, CAMP test-
positive, β-hemolytic Streptococcus can be reported as “β-hemolytic
Streptococcus, presumptive group B by CAMP test.”

17. ESCULIN HYDROLYSIS TEST
• Esculin medium without bile is useful in differentiating several species on nonfermenting
bacilli.
• Esculin is a substituted glucoside that can be hydrolyzed by certain bacteria to yield glucose
and esculetin.
• Esculin is a fluorescent compound that fluoresces under long-wave UV light at 360 nm.
• When esculin is hydrolyzed, fluorescence is lost, and the medium turns black due to the
reaction of esculetin with the ferric ions in the medium.

ESCULIN HYDROLYSIS test (contd.,)
• MEDIA & REAGENTS:

• Quality Control
• Positive control: Aeromonas hydrophila ATCC 7965
• Negative control: P. aeruginosa ATCC 27853
• Procedure :
• After touching the center of one well-isolated colony with a sterile inoculation loop or
wooden stick, inoculate the organism onto the surface of the agar slant or into the broth.
• Incubate for 18– 24 hours at 35°C

• Interpretation:
• POSITIVE : The development of a black color or the loss of fluorescence
under UV light (360 nm)
• NEGATIVE : Fluorescence or lack of a black color

• EXAMPLES:
• ESCULIN POSITIVE : Several species of Chryseobacterium, Sphingobacterium,
as well as C. luteola, B. vesicularis, S. paucimobilis, S. maltophilia, R. radiobacter,
and some species of B. cepacia, B. pseudomallei, and O. anthropic
• ESCULIN NEGATIVE :

18 . PYR TEST (l-Pyrrolidonyl Arylamidase)
• The enzyme l-pyrrolidonyl arylamidase hydrolyzes the l-pyrrolidonyl-b-naphthylamide substrate to
produce a b-naphthylamine.
• The b-naphthylamine can be detected in the presence of N,N-methylamino-cinnamaldehyde reagent by
the production of a bright red precipitate.
• Method
• Before inoculation, moisten the disk slightly with reagentgrade water. Do not flood the disk.
• Using a wooden applicator stick, rub a small amount of several colonies of an 18- to 24-hour pure
culture onto a small area of the PYR disk.
• Incubate at room temperature for 2 minutes.
• Add a drop of detector reagent, N,N-dimethylaminocinnamaldehyde, and observe for a red color within
1 minute.

PYR Test (contd.,)
Expected Results
• Positive: Bright red colour within 5 minutes
• Negative: No colour change or an orange colour
Quality Control
• Positive: Enterococcus faecalis (ATCC29212)
Streptococcus pyogenes (ATCC19615)
• Negative: Streptococcus agalactiae (ATCC10386)

19. BILE SOLUBILITY TEST
• Bile solubility reagent (10% sodium deoxycholate) - presumptive identification and differentiation of S.
pneumoniae from other α-hemolytic streptococci.
• S. pneumoniae cells visibly lyse when 10% sodium deoxycholate is applied, while other αhemolytic
streptococci do not.
• Bile or a solution of a bile salt (e.g., sodium deoxycholate) rapidly lyses pneumococcal colonies.
• Lysis depends on the presence of an intracellular autolytic enzyme, amidase.
• Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus
accelerating the organism’s natural autolytic process

Reagents
• Sodium desoxycholate, 10% aqueous solution
• Blood agar plate with growth of an α-hemolytic Streptococcus
Quality Control
• Bile (deoxycholate) soluble: Streptococcus pneumoniae (ATCC49619)
• Bile (deoxycholate) insoluble: Enterococcus faecalis (ATCC29212)

19. BILE SOLUBILITY TEST (contd.,)
Tube Test
• Prepare a saline suspension of the test isolate
from an 18- to 24-hour, pure culture.
• Adjust turbidity to that of a 0.5 to 1.0 McFarland
standard or equivalent.
• Aliquot 0.5 mL of the suspension into each of
two tubes. Label one tube as Test, the other as
Control.
• Add 0.5 mL of 10% bile solubility reagent to the
tube marked Test, and 0.5 mL of saline (pH 7.0)
to the tube marked Control.
• Gently agitate tubes to suspend bacteria.
• Incubate tubes at 35°C to 37°C and examine
periodically for up to 3 hours.
• Observe for clearing in the Test suspension. The
Control suspension should remain turbid.
Plate Spot Test
• Place one drop of 10% bile solubility reagent
near suspected 18- to 24-hour-old colonies
growing on sheep blood agar.
• Gently roll the drop over several representative
colonies by tilting the plate. Take care not to
dislodge the colonies.
• Incubate the plate aerobically in an upright
position at 35°C to 37°C, and examine
periodically for up to 30 minutes.
• Leave lid slightly ajar to enhance evaporation of
the reagent.
• Observe the colony for disintegration or
solubility.

(contd.,)
Tube Test
• Positive test — Clearing or loss of
turbidity of the test suspension
within 3 hours. The Control
suspension remains turbid.
• Negative test — Test and Control
suspensions remain turbid after 3
hours.
Spot Test
• Positive test—Disintegration of
colonies and/or the appearance of
an α-hemolytic zone on the plate
where the colony was located
within 30 minutes.
• Negative test—Colonies on the plate
remain intact with no change in
colony integrity within 30 minutes.

20. BILE ESCULIN TEST
• The bile esculin test is based on the ability of certain bacteria (group D streptococci and Enterococcus
species ) to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile).
• Esculin is a glycosidic coumarin derivative (6-β-glucoside-7-hydroxy-coumarin).
• The two moieties of the molecule (glucose and C-29 7-hydroxycoumarin) are linked together by an
ester bond through oxygen.
• For this test, esculin is incorporated into a medium containing 4% bile salts.
• Bacteria that are bile esculin–positive are able to grow in the presence of bile salts
• Subsequent hydrolysis of the esculin in the medium results in the formation of glucose and a
compound called esculetin.
• Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate)
to form a black diffusible complex.

20. BILE ESCULIN TEST (contd.,)

Medium: Bile esculin agar medium - agar slants or plates.
• Peptone 5 g
• Oxgall (bile) 40 g
• Esculin 1 g
• Ferric citrate 0.5 g
• Agar 15 g
• pH 7.0

Quality Controls
• Positive: Enterococcus faecalis (ATCC19433)—growth; black precipitate
• Negative: Escherichia coli (ATCC25922)—growth; no color change
Streptococcus pyogenes (ATCC19615)—no growth; no color change
Procedure
• With an inoculating wire or loop, touch two or three morphologically similar streptococcal colonies
and inoculate the slant of the bile esculin medium with an S-shaped motion, or streak the surface of a
bile esculin plate for isolation.
• Incubate the tube or plate at 35°C for 24–48 hours in an ambient air incubator.

Interpretation :
• On plates, black haloes will be observed around isolated colonies and any blackening is
considered positive.
• Positive: Growth and blackening of the agar slant
• Negative: Growth and no blackening of medium
No growth
• All group D streptococci will be bile esculin–positive within 48 hours.

21. NOVOBIOCIN SUSCEPTIBILITY TEST
• CoNS can be divided into novobiocin-susceptible and novobiocin resistant species.
• Among the novobiocin-resistant species, S. saprophyticus is the one commonly recovered from
humans as a cause of urinary tract infections.
• Therefore, screening coagulase-negative staphylococci isolated from quantitative urine cultures for
susceptibility to novobiocin provides a reliable presumptive identification of this species.
• Reagents
• Novobiocin disks, 5 mg

21. NOVOBIOCIN SUSCEPTIBILITY TEST (contd.,)
• Quality Control
• A known S. saprophyticus strain and an S. epidermidis strain
• Procedure
• Prepare a suspension of the organism to be identified in sterile distilled water or broth.
• The suspension should be equivalent in turbidity to a 0.5 McFarland standard.
• With a sterile swab, spread some of the suspension over half of a blood agar plate.
• Aseptically place a novobiocin disk on the inoculated area.
• Susceptibility to furazolidone may be assessed on the same plate by placing the disks about 4 cm
apart on the inoculated area.
• Gently tap the disk(s) with sterile forceps to assure contact with the agar surface.
• Incubate the plate aerobically for 18–24 hours at 35°C.

21. NOVOBIOCIN SUSCEPTIBILITY TEST (contd.,)
Interpretation :
• S. saprophyticus are novobiocin-resistant and will show zones of inhibition of
6 mm (no zone)–12 mm.
• Other Coagulase-Negative Staphylococci and S. aureus are novobiocin-
susceptible and will show zones of 16 mm or larger.

22. OPTOCHIN SUSCEPTIBILITY TEST
• Ethylhydrocupreine hydrochloride (optochin), a quinine derivative, selectively inhibits the growth of
Streptococcus pneumoniae at very low concentrations (5 mg/mLor less).
• Optochin may also inhibit other Viridans Streptococci, but only at much higher concentrations.
• The test has a sensitivity of more than 95%, is simple to perform, and is inexpensive.
• Optochin is water-soluble and diffuses readily into agar medium.
• Filter paper disks impregnated with optochin - disk diffusion test - to determine susceptibility of
suspected pneumococci
• S. pneumoniae cells surrounding the disk are lysed owing to changes in the surface tension, and a zone
of inhibition is produced.

Media and Reagents
• Well-isolated colonies of the organism to be tested on sheep blood agar
• Optochin disks (5 μg)
Quality Control
• Positive control: Streptococcus pneumoniae
• Negative control: Viridans Streptococcus or Enterococcus faecalis

Procedure
• Using an inoculating loop or wire, select three to four well-isolated colonies of the organism to be
tested and streak onto one-half to a one-third of a blood agar plate.
• The inoculated area should be about 3 cm2 .
• Place an optochin disk in the upper third of the streaked area.
• Tap down the disk with flamed or otherwise sterilized forceps so that the disk adheres firmly to the
agar surface.
• Incubate the plate at 35°C for 18–24 hours in a candle jar or in 5%–7% CO2 .

Interpretation
• A Viridans Streptococcus can be presumptively identified as S. pneumoniae if it
shows a zone of inhibition of 14 mm or more around a 6-mm or a zone of 16
mm or more around a 10-mm disk
• Organisms showing zones smaller than these should be tested for bile
solubility.

23. BACITRACIN & SXT SUSCEPTIBILITY TEST
• Susceptibility to low concentrations of the polypeptide antibiotic bacitracin and to the
combination sulfonamide–trimethoprim–sulfamethoxazole (SXT) - presumptive
identification of both group A and group B β-hemolytic streptococci.
• Group A Streptococci are susceptible to relatively low concentrations of bacitracin and are
resistant to SXT.
• Group B Streptococci are resistant to both antibiotics.
• Other β-hemolytic Streptococci show varying susceptibility to bacitracin, but these organisms
are usually susceptible to SXT.
• The performance of the SXT test along with the bacitracin test increases the sensitivity and
predictive value of the bacitracin test.
• Bacitracin is also used to distinguish Staphylococci species (resistant) from Micrococci
(susceptible).

Reagents
• Bacitracin disks (0.04 units/disk)
• SXT disks (trimethoprim–sulfamethoxazole, 1.25 μg/23.75 μg)
Quality Control
Positive:
Streptococcus pyogenes (ATCC19615)—susceptible
Micrococcus luteus (ATCC10240)—susceptible
Negative:
Streptococcus agalactiae (ATCC27956)—resistant
Staphylococcus aureus (ATCC25923)—resistant

Procedure
• Pick three to four isolated colonies of the β-hemolytic Streptococcus, and streak the
inoculum down the center of half of a blood agar plate.
• Using a sterile swab or a bacteriologic loop, spread the inoculum as a lawn over the entire
half of the plate.
• Aseptically place a bacitracin disk and an SXT disk on the inoculated area. Make sure that
the disks are spaced evenly. Using flamed forceps, gently tap down the disks so that they
adhere to the agar surface.
• Incubate the plate in ambient air at 35°C.

• Interpretation :
• Susceptible (S): Any zone around either of the disks
• Resistant (R): Growth up to the edge of the disk

AUTOMATED CULTURETECHNIQUES
Automated blood culture
techniques
• BacT/ALERT 3D
• Bact/ALERT VIRTUO
• BACTEC
• VersaTREK
Automated systems for bacterial
identification
• MALDI TOF
• VITEK
• Phoenix
• MicroScanWalkAway system

CONVENTIONAL BLOOD
CULTURE METHODS
• Often yield poor results
• Low bacterial load
• Increased chance of contamination
• Less sensitive
• Takes more time
• More labour intensive
AUTOMATED BLOOD CULTURE
METHODS
• Continuous automated monitoring
• More sensitive
• Rapid
• Less labour intensive
• High cost of instrument & culture bottles
• Inability to observe colony morphology as
liquid medium is used.

BacT/ALERT 3D
• Principle : colorimetric detection of bacterial growth
• BacT/ALERT bottles –
• Tryptic soy broth
• Brain heart infusion broth
• Adsorbent polymeric beads
• Liquid emulsion sensor – bottom of each bottle; differentially permeable membrane
• CO2 produced by growing microorganisms – diffuse across the sensor – reacts with
water- generates hydrogen ions
• H+ ions -> pH -> blue green sensor becomes yellow
• The algorithm of detection of growth is based on the analysis of the rate of change of
CO2 concentration occurring in each individual bottle.

BacT/ALERT VIRTUO
• Automatic loading & unloading of bottles
• Faster detection of growth
• Can determine the volume of blood present in the bottle

BACTEC
• Flourometric detection
• BACTEC bottle
• Soybean- casein digest broth
• Polymeric resin beads
• Oxygen sensitive fluorescent compound dissolved in the broth
• Uninoculated broth : large amount of dissolved oxygen quenches the fluorescence dye
• Actively dividing microorganisms consume oxygen, remove the quenching effect, and allow
the fluorescence to be detected.

VersaTREK
• PRINCIPLE : CO2 liberated from bacteria, causes a pressure change which is then detected by
manometry.
• Differs from the BacT/ALERT3D and the BD BACTEC systems in the following ways:
• The production of CO2 is monitored manometrically
• Both gas consumption and production are monitored
• Changes in the concentrations of H2 and O2 in addition to CO2 are detected.

VersaTREK (contd.,)
• The data unit is also a cabinet that serves as a self-contained incubator, agitator and detector.
• Capacity : 96-to-240 or up to 528 bottles are currently available.
• After inoculation of up to 10 mL of venous blood, each bottle is fitted with a disposable connector,
which includes a recessed needle that penetrates the septum of the blood culture bottle.
• Each bottle is then placed in a defined position on a carrying rack that is aligned such that the
connector attaches directly to a sensing probe located at the top of each position.
• Once the bottle is properly aligned, the pressure of the head–gas is continuously monitored.
• A reading is taken every 12 minutes

Versa TREK ( contd.,)
• When the change in reading exceeds a delta value, lights are illuminated that indicate the position
of any positive bottle.
• A reading may occur during a phase of consumption of 𝐻2 and 𝑂2 .
• Oxygen consumption is accelerated at the time replicating organisms enter the log phase of
growth.
• A reading may be possible, therefore, early in the incubation period before a detectable amount of
CO2 is produced.
• Testing multiple gases is a theoretical advantage for the electrostatic precipitator (ESP) system,
especially for the detection of a saccharolytic microorganisms that may not produce sufficient
CO2 for detection by the indicator.

CONTINUOUS MONITORING BLOOD CULTURE SYSTEMS
ADVANTAGES
• Decrease in laboratory workload
• Decrease in the number of false-positive
results and pseudobacteremia (because
of decreased handling and sampling of
the bottles)
• Significant increase in the speed of
detection and in the rate of microbial
recovery..
DISADVANTAGES
• Limited selection of media
• Large size of the instruments
• Expensive

AUTOMATED SYSTEMS FOR BACTERIAL IDENTIFICATION
• MALDITOF
• VITEK 2
• Phoenix
• MicroScanWalkAway system

MALDITOF
• It can identify bacteria, fungi, and mycobacteria with a turnaround time of few
minutes and with absolute accuracy
• Two systems are commercially available: VITEK MS (bioMérieux) and Biotyper
system (Bruker).
• MALDI-TOF examines the pattern of ribosomal proteins present in the organism.
Sample preparation:
• The colony of an organism is smeared onto a well of the slide and one drop of
matrix solution (composed of cyano-hydroxy-cinnamic acid) is added to the same
well and mixed
• Slide is loaded in the system.

Steps after loading:
• Ionization chamber:
• Wells are irradiated with the laser beam.
• The matrix absorbs the laser light causing desorption and ionization of bacterial ribosomal proteins,
generating singly protonated ions
• Analyzer:
• These ions are then accelerated into an electric field which directs them to the analyzer chamber.
• The analyzer (mass spectrometer) separates them according to their time-of-flight (TOF) in the flight
tube
• . The smaller molecules travel faster, followed by the bigger, according to the mass to charge (m/z)
ratio
• Detector:
• It converts the received ion into an electrical current which is then amplified and digitized to
generate a characteristic spectrum that is unique to a species due to its conserved ribosomal
proteins.
• The test isolate is identified by comparing its spectrum with a known database.

• A summation of the time of flight for all molecules present will produce a spectrum
• This spectrum is then electronically compared with all the spectra in the library to
determine the best match
• Subsequently the identification of the microbe

VITEK 2 AUTOMATED SYSTEM
• Perform both identification and antimicrobial susceptibility testing (AST) of bacteria and yeast.
• It 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
• Substrates in the well measure various metabolic activities such as acidification, alkalinization,
enzyme hydrolysis, etc. which helps in identification of the organism

• The reaction pattern obtained from the test organism is compared with the database and the
identification is reported with a confidence level of matching
• The cards are incubated in the system at 35.5 ± 1°C.
• The reading is taken once every 15 minutes by the optical system of the equipment, which
measures the presence of any colored products of substrate metabolism (by advanced colorimetry
method)
• The result of identification is usually available within 4–6 hours

REFERENCES
• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology – 7th edition
• Bailey & Scott’s Diagnostic Microbiology – 15th edition
• Mackie & McCartney Practical Medical Microbiology – 14th edition
• Essentials of Medical Microbiology – 3rd edition -Apurba S Sastry, Sandhya Bhat

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