Transcript 2- Macroscopic (Colony)

Bacterial Identification
General
Function of
Clinical Microbiology Lab
• Participation in management of patients
with infectious diseases by processing of
clinical specimens:
1- Microscopy.
2- Cultivation & isolation of pathogens.
3- Identification of isolates: macroscopy,
microscopy, biochemical tests, serology.
4- Antimicrobial susceptibility testing (AST)
of isolates.
 Early preliminary indicators of
identity of suspected bacterial
pathogen involved in the
infection :
 Direct microscopy of clinical
specimen’s smear.
 Culture media & incubation
conditions
supporting
its
growth.
 Indicators
significance
of
of
clinical
bacterial
isolate:
 Source (body site) of clinical
specimen.
 Direct smear results.
 Virulence vs Predominance
(Relative quantities of each
colony type).
Bacterial growth on solid media
• Colony = The resulting visible mass of
sufficiently large bacterial numbers (population)
that is considered to be originating from a single
mother bacterial cell.
• All bacterial cells within a single colony are a
single clone (belong to the same genus &
species, having identical genetic & phenotypic
characteristics).
• Bacterial cultures derived from a single
individual colony or clone are considered pure.
Bacterial culture
Pure culture
• Growth containing one
type of bacteria only.
Mixed culture
• Growth containing > one
type of bacteria.
Bacterial colonies
Description
• Size (diameter): usually measured in millimeters or described in
relative terms as tiny pinpoint (Punctiform), small, medium, large.
• Morphology (Shape): includes:
1- Form (basic morphology) of whole colony.
2- Elevation (side view).
3- Margin/border (edge).
• Surface e.g. smooth, rough, wrinkled, glistening, dull, dry, powdery.
• Opacity e.g. transparent (clear), opaque, or translucent (like looking
through frosted glass).
• Colour (pigmentation) e.g. white, buff, yellow, red, purple, etc.
• Odor: e.g. Proteus, Pseudomonas etc.
• Changes in agar media resulting from bacterial growth (e.g.
hemolytic pattern on blood agar, changes in color of pH indicators,
pitting of agar surface).
Colony Shape
GLISTEN or FLAT
Colony color & size
Pigments & agar changes
Bacterial growth in liquid media
Patterns
Principles of Identification
• Identification scheme = set & order-in-use of
laboratory tests that provide the characteristic
profiles of bacterial isolate leading to its definite
identification.
• There are two identification schemes of bacteria:
 Genotypic.
 Phenotypic.
Genotypic Criteria
• involve characterization of some portion of
bacterium’s genome using molecular
techniques for DNA or RNA analysis.
• highly specific & very sensitive.
• presence of specific gene or particular
nucleic acid sequence → definitive
identification.
Phenotypic Criteria
• based on observable physical or metabolic
characteristics of bacteria.
• provides preliminary identification & information
necessary for determining what other identification
procedures should follow to confirm the final
identification.
• most commonly used include:
 Microscopic
cellular
morphology
&
staining
characteristics.
 Macroscopic (colony) morphology.
 Environmental requirements for growth.
 Resistance or susceptibility to antimicrobial agents.
 Nutritional requirements & metabolic capabilities.
1- Microscopic Cellular Morphology
& Staining Characteristics
• Gram stain of bacterial growth from isolated
colonies on various media is usually the first
step in any identification scheme.
• Most pathogenic bacteria can be classified
into four distinct groups:
1- Gram-positive cocci.
2- Gram-positive bacilli.
3- Gram-negative bacilli.
4- Gram-negative cocci.
• Some bacterial species are morphologically
indistinct & are described as “Gram-negative
coccobacilli,”
“Gram-variable
bacilli,”
or
pleomorphic (i.e. various shapes).
• Other morphologies include curved and/or rods
and spirals.
• Even without staining, examination of wet
preparation of bacterial colonies under oil
immersion (1000× magnification) can provide
clues to possible identity.
2- Macroscopic (Colony) Morphology
• Evaluation
includes
considering
colony size, shape, color (pigment),
surface appearance, & any changes
that colony growth produces in the
surrounding agar medium (e.g.
hemolysis of blood in blood agar
plates).
3- Environmental Requirements for Growth
• Organisms growing only in bottom of tube containing
thioglycollate broth are NOT likely to be strictly aerobic
bacteria.
• Organisms growing on blood agar plates incubated in ambient
(room) atmosphere are NOT likely to be anaerobic bacteria.
• Organism’s requirement, or preference, for increased CO2
concentrations e.g. Strep pneumoniae, Haemophilus
influenzae, & Neisseria gonorrhoeae.
• Organism’s ability for survival or growth in temperatures that
exceed or are well below normal body temperature of 37°C
e.g. survival of Yersinia enterocolitica at 0°C & growth of
Campylobacter jejuni at 42°C.
4- Resistance or Susceptibility to
Antimicrobial Agents
• Organism’s ability to grow in presence of certain
antimicrobial agents or specific toxic substances
is widely used to establish preliminary
identification information.
 This is accomplished by using agar media supplemented
with inhibitory substances or antibiotics.
 Or by directly measuring organism’s resistance to
antimicrobial agents that may be used to treat infections.
• Nature of media on which organism is growing
provides the first clue for identification of isolated colony:
 Gram-negative bacteria grow well on MacConkey agar.
 Columbia-CNA agar (with colistin & naladixic acid), will
support growth of Gram-positive organisms by inhibiting
growth of most Gram-negative bacilli.
 Chocolate agar will support growth of all Neisseria spp.,
but antibiotic-supplemented Thayer-Martin formulation
will almost exclusively support growth of pathogenic spp.
N. meningitidis & N. gonorrhoeae.
• Organism’s uncertain Gram stain
“status”:
Susceptibility to vancomycin: truly Gramnegative bacteria are resistant & many
Gram-positive bacteria are susceptible.
Susceptibility to colistin or polymyxin:
most
Gram-negative
bacteria
are
susceptible & Gram-positive bacteria are
frequently resistant.
5- Nutritional Requirements &
Metabolic Capabilities
• the most common approach used for
determining the genus & species of an
organism.
• In general, all methods use combination of tests
to establish:
 Enzymatic capabilities of a given bacterial
isolate.
Isolate’s ability to grow or survive in presence of
certain inhibitors (e.g. salts, surfactants, toxins,
& antibiotics.
A- Establishing Enzymatic Capabilities
• Enzymatic content of an organism is a direct reflection of
the organism’s genetic makeup, which, in turn, is specific
for individual bacterial species.
• In diagnostic bacteriology, enzyme-based tests are
designed to measure:
 Either Presence of one specific enzyme(Single
Enzyme Tests):
 Although most single enzyme tests do not yield sufficient information
to provide species identification, they are used extensively to
determine which subsequent identification steps should be followed
 Complete
metabolic
pathway
contain several different enzymes.
that
may
 Assays for metabolic pathways can be classified into
three general categories:
A. Carbohydrate oxidation & fermentation.
B. Amino acid degradation.
C. Single substrate utilizations.
Bacterial Enzymes
•
•
•
•
•
•
Intracellular
Catalse.
Oxidase.
IMViC.
Urease.
Carbohydrate
fermentation.
Nitrate reduction.
•
•
•
•
•
Extracellular
Blood hydrolysis
(Streptolysin).
Starch hydrolysis
(Amylase).
Casein hydrolysis
(protease).
Lipid hydrolysis (Lipase).
Gelatin hydrolysis
(Gelatinase).
B- Establishing Inhibitor Profiles
• Ability of bacterial isolate to grow in presence of one or
more inhibitory substances can provide valuable
identification information. Examples:
 Growth in presence of various NaCl concentrations
(identification of enterococci & Vibrio spp.).
 Susceptibility to optochin & solubility in bile (identification
of Strep pneumoniae).
 Ability to hydrolyze esculin in presence of bile
(identification of enterococci).