Bacterial Identification

IDENTIFICATION OF
PROKARYOTES
CHAPTER 10
TAXONOMY
• The study and grouping of organisms
• Three separate but interrelated areas
– Identification
• Process of characterizing organisms
– Classification
• Arrangement of organisms into groups
– Nomenclature
• Assignment of a specific name
TAXONOMY
• Initial identification of microorganisms results in
their classification
– Based on evolutionary relationships
• Identification of microorganisms in particular
environments remains important
– e.g., Microbial contaminants can spoil food
– e.g., Identification of microbes present in a
clinical patient is important in determining
treatment
PROKARYOTE IDENTIFICATION
• Various techniques are employed to characterize
and identify microorganisms
– Phenotypic characteristics
• Microscopic morphology
• Metabolic differences
• Serology
• Fatty acid analysis
– Genotypic characteristics
• Nucleic acid probes
• DNA amplification
• rRNA sequencing
PHENOTYPIC CHARACTERISTICS
Microscopic morphology
• Size and shape
– Readily determined by microscopic examination
of a wet mount
– Can determine whether the microbe is a
prokaryote, fungus, or protozoan
• Size and shape
– Often sufficient for clinical
diagnosis e.g., Trichomonas
vs. Candida in vaginal
secretions
• e.g., Roundworm eggs in
stool
– Size, shape, and other
features often sufficient
for identification
• Cell groupings
– Cells adhering to one another following binary
fission often form characteristic arrangements
• e.g., Neisseria gonorrhoeae typically displays a
diplococcus arrangement
• e.g., Most Streptococcus species form long chains
• e.g., Most Staphylococcus species form grapelike
clusters
• e.g., Sarcina species for cubical packets
• Cell groupings
– Cells adhering to one another
following binary fission often
form characteristic arrangements
• e.g., Neisseria gonorrhoeae
typically displays a diplococcus
arrangement
• e.g., Most Streptococcus species form long chains
• e.g., Most Staphylococcus species form grapelike clusters
• e.g., Sarcina species for cubical packets
• Gram stain
– Differential stain
distinguishing between
gram-positive and
gram-negative bacteria
– Narrows possible identities of an organism
• Excludes many possibilities
– Generally insufficient alone for diagnosis
• e.g., E. coli and Salmonella gram stains look alike
• Gram stain
– Sometimes highly suggestive of
a particular microorganism
• e.g., Gram-negative rods in ♀ urine
 E. coli UTI
• e.g., Gram-positive encapsulated
diplococci and numerous white
blood cells in sputum
 Streptococcus pneumoniae
– Sometimes enough for complete diagnosis
• e.g., Gram-negative diplococci clustered in white blood
cells of male urethral secretions  Neisseria gonorrhoeae
• Special stains
– Some microbes have
unique characteristics
that can be detected
with special staining
procedures
– e.g., Filobasidiella (Cryptococcus) neoformans is
one of a few types of capsule-forming yeast
• Capsule stain on cerebrospinal fluid is diagnostic
for cryptococcal meningitis
• Special stains
– Some microbes have unique characteristics that
can be detected with special staining procedures
– e.g., Mycobacterium
species possess cell
walls with a high lipid
content
• Acid-fast stain on
sputum is diagnostic
for tuberculosis
Metabolic differences
• Colony morphology
– Colonies can exhibit
macroscopic
differences
• e.g., Colonies of streptococci generally form fairly
small colonies
• e.g., Colonies of Serratia marcescens produce a
pigment and are often red when incubated at 22oC
• e.g., Colonies of Pseudomonas aeruginosa often
produce a soluble greenish pigment
• Culture characteristics
– Selective and differential media can aid in the
identification of microbes
• Selective media favors the growth of certain
types of microbes by inhibiting the growth of
others
• Differential media contains a substance that
certain bacteria change in a recognizable way
– MacConkey agar is both
selective and differential
• Bile salts and dyes inhibit
all but certain gram-
negative rods
– “Selective”
• Acid produced by bacteria able to ferment lactose
will turn a pH indicator red and form red colonies
– “Differential”
– Blood agar can be used to detect
bacteria producing hemolysins
• e.g., Harmless Streptococcus
species residing in the throat often
cause alpha-hemolysis
– Greenish clearing around colonies
• e.g., “Strep throat”-causing
Streptococcus pyogenes causes
beta-hemolysis
– Clear zone around colonies
– Media lacking nitrogen can be used to detect
nitrogen-fixing
bacteria
• e.g., Azotobacter
can be identified
from soil samples
incubated
aerobically on
such media
• Biochemical tests
– Generally necessary for more conclusive
identification
– Most rely on pH indicator or color change when a
compound is degraded
– Sugar fermentation
• e.g., Lactose, sucrose,
glucose, etc.
• Fermentation results in
acid production
– pH indicator changes color
– Pink  yellow
• Inverted tube (Durham tube) collects any gas
produced
– Urease detection
• Enzyme degrading urea
– Urea  CO2 & NH3
• pH indicator turns
bright pink in alkaline
conditions
• Helicobacter pylori can be detected using a breath
test
– Urease detection
• Helicobacter pylori can be detected using a breath
test
– Causative agent of most stomach ulcers
– Culturing not necessary
– Patient drinks solution containing 14C-labeled urea
– 14C in expired are indicates presence of urease
– Catalase
• Commonly occurring enzyme
– Possessed by most bacteria growing in the
presence of oxygen
– Absent in lactic acid bacteria
» e.g., Streptococcus
» Beta-hemolytic catalase-negative bacteria from a
throat culture may be Streptococcus pyogenes
– Catalase
• Simple assay
– H2 O 2  H 2 O & O 2
– O2 bubbles are visible
• Organisms are identified
using a dichotomous key
– Multiple biochemical and
other tests are typically
required
• Multiple tests are
generally run concurrently
– Avoids waiting for
incubation time for each
test
– Commercial modifications of traditional
biochemical tests
• e.g., APITM system, EnterotubeTM
Serology
• Proteins and polysaccharides of some bacteria
can function as identifying markers
– Generally molecules on surface structures
• e.g., Cell wall, glycocalyx, flagella, pili
– Detection is based upon the
specific interaction between
antibodies and these
antigens
• e.g., Rapid detection of
Streptococcus pyogenes
Fatty Acid Analysis
• Bacteria differ in the type and relative quantity
of fatty acids that comprise their membranes
– Can function as an identifying marker
• Gram-negative bacteria possess fatty acids in both
of their membranes
• Gram-positive bacteria possess only a single
membrane
Fatty Acid Analysis
• Cells are treated with NaOH and methanol
– Fatty acids are released and converted into
methyl esters
– Methyl esters
analyzed via gas
chromatography
• Profile compared
to those of known
species
GENOTYPIC CHARACTERISTICS
Nucleic acid probes
• Used to locate unique sequences
– Single-stranded DNA (or RNA)
• Generally labeled (radioactive or fluorescent)
– Complementary to the sequence of interest
– Observe and identify intact microorganisms
• Fluorescence in situ hybridization (FISH)
– Observe and identify samples
• Generally preceded by DNA amplification
Polymerase chain reaction
• Amplifies specific nucleotide sequences
– DNA can be obtained from many sources
• e.g., Body fluids, soil, food, water, etc.
• Useful in detecting microbes present in extremely
small numbers
• Useful in detecting microbes that are difficult to
culture
– Amplified DNA can be analyzed
• Procedure
– DNA is isolated, then
denatured
– Complementary primers
are lengthened
• DNA is doubled
– Repeat ~30 times
• Procedure
– DNA is isolated, then denatured
– Complementary primers are lengthened
– DNA is
doubled
– Repeat
~30 times
Sequencing ribosomal RNA genes
• Three rRNAs present in 70S bacterial ribosomes
– 5S, 16S, and 23S
• Evolutionarily highly
conserved genes
– Variable regions are used
to identify an organism
• Particularly useful in
identifying microbes that
are difficult to culture

Bacterial Identification

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Bacterial Identification

Uploaded by

Copyright:

Available Formats

IDENTIFICATION OF

You might also like