IDENTIFICATION OF PROKARYOTES

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IDENTIFICATION OF PROKARYOTES

• CHAPTER 10

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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

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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

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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

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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

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PHENOTYPIC CHARACTERISTICS

• Microscopic morphology
• 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

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PHENOTYPIC CHARACTERISTICS

• Microscopic morphology
• 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

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PHENOTYPIC CHARACTERISTICS

• Microscopic morphology
• 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

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PHENOTYPIC CHARACTERISTICS

• Microscopic morphology
• 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

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PHENOTYPIC CHARACTERISTICS

• Microscopic morphology
• 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

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PHENOTYPIC CHARACTERISTICS

• Microscopic morphology
• 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

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PHENOTYPIC CHARACTERISTICS

• Microscopic morphology
• 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

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PHENOTYPIC CHARACTERISTICS

• 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

Pseudomonas aeruginosa
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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Culture characteristics
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Culture characteristics
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Culture characteristics
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Biochemical tests
• Generally necessary for more conclusive
  identification
• Most rely on pH indicator or color change when a
  compound is degraded

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Biochemical tests
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Biochemical tests
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Biochemical tests
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Biochemical tests
• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Biochemical tests
• Catalase
• Simple assay
• H2O2 ? H2O O2
• O2 bubbles are visible

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PHENOTYPIC CHARACTERISTICS
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PHENOTYPIC CHARACTERISTICS

• 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

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PHENOTYPIC CHARACTERISTICS

• Metabolic differences
• Biochemical tests
• Commercial modifications of traditional
  biochemical tests
• e.g., APITM system, EnterotubeTM

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PHENOTYPIC CHARACTERISTICS

• 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

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PHENOTYPIC CHARACTERISTICS

• 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

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PHENOTYPIC CHARACTERISTICS

• 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

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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

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PHENOTYPIC CHARACTERISTICS

• 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

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PHENOTYPIC CHARACTERISTICS

• Polymerase chain reaction
• Procedure
• DNA is isolated, then denatured
• Complementary primers are lengthened
• DNA is doubled
• Repeat 30 times

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GENOTYPIC CHARACTERISTICS

• Polymerase chain reaction
• Procedure
• DNA is isolated, then denatured
• Complementary primers are lengthened
• DNA is doubled
• Repeat 30 times

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GENOTYPIC CHARACTERISTICS

• 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

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GENOTYPIC CHARACTERISTICS