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IDENTIFICATION OF PROKARYOTES

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 .

or protozoan .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.

. Trichomonas vs.g.PHENOTYPIC CHARACTERISTICS Microscopic morphology • Size and shape – Often sufficient for clinical diagnosis e. and other features often sufficient for identification . Candida in vaginal secretions • e. shape.g. Roundworm eggs in stool – Size..

.g.g...PHENOTYPIC CHARACTERISTICS Microscopic morphology • Cell groupings – Cells adhering to one another following binary fission often form characteristic arrangements • e. Most Streptococcus species form long chains • e.g. Neisseria gonorrhoeae typically displays a diplococcus arrangement • e. Most Staphylococcus species form grapelike clusters .

coli and Salmonella gram stains look alike ..g.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. E.

. Gram-positive encapsulated diplococci and numerous white blood cells in sputum  Streptococcus pneumoniae – Sometimes enough for complete diagnosis • e.g. Gram-negative rods in ♀ urine  E.g.g. coli UTI • e...PHENOTYPIC CHARACTERISTICS Microscopic morphology • Gram stain – Sometimes highly suggestive of a particular microorganism • e. Gram-negative diplococci clustered in white blood cells of male urethral secretions  Neisseria gonorrhoeae .

PHENOTYPIC CHARACTERISTICS Microscopic morphology • Special stains – Some microbes have unique characteristics that can be detected with special staining procedures – e. Filobasidiella (Cryptococcus) neoformans is one of a few types of capsule-forming yeast • Capsule stain on cerebrospinal fluid is diagnostic for cryptococcal meningitis ..g.

Mycobacterium species possess cell walls with a high lipid content • Acid-fast stain on sputum is diagnostic for tuberculosis .PHENOTYPIC CHARACTERISTICS Microscopic morphology • Special stains – Some microbes have unique characteristics that can be detected with special staining procedures – e..g.

.g.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 . Colonies of streptococci generally form fairly small colonies • e...PHENOTYPIC CHARACTERISTICS Metabolic differences • Colony morphology – Colonies can exhibit macroscopic differences • e.

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 .

PHENOTYPIC CHARACTERISTICS Metabolic differences • Culture characteristics – MacConkey agar is both selective and differential • Bile salts and dyes inhibit all but certain gramnegative rods – “Selective” • Acid produced by bacteria able to ferment lactose will turn a pH indicator red and form red colonies – “Differential” .

“Strep throat”-causing Streptococcus pyogenes causes beta-hemolysis – Clear zone around colonies .g... Harmless Streptococcus species residing in the throat often cause alpha-hemolysis – Greenish clearing around colonies • e.g.PHENOTYPIC CHARACTERISTICS Metabolic differences • Culture characteristics – Blood agar can be used to detect bacteria producing hemolysins (exotoxins) • e.

. Azotobacter can be identified from soil samples incubated aerobically on such media .g.PHENOTYPIC CHARACTERISTICS Metabolic differences • Culture characteristics – Media lacking nitrogen can be used to detect nitrogen-fixing bacteria • e.

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 .

sucrose.PHENOTYPIC CHARACTERISTICS Metabolic differences • Biochemical tests – Sugar fermentation • e. Lactose.. etc. • Fermentation results in acid production – pH indicator changes color – Pink  yellow • Inverted tube (Durham tube) collects any gas produced . glucose.g.

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 .

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 .

g.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.. Streptococcus » Beta-hemolytic catalase-negative bacteria from a throat culture may be Streptococcus pyogenes .

PHENOTYPIC CHARACTERISTICS Metabolic differences • Biochemical tests – Catalase • Simple assay – H2O2  H2O & O2 – O2 bubbles are visible .

PHENOTYPIC CHARACTERISTICS .

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 .

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

PHENOTYPIC CHARACTERISTICS Serology • Proteins and polysaccharides of some bacteria can function as identifying markers – Generally molecules on surface structures • e. Cell wall..g.. glycocalyx.g. Rapid detection of Streptococcus pyogenes . flagella. pili – Detection is based upon the specific interaction between antibodies and these antigens • e.

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 .

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 .

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 .

• Useful in detecting microbes present in extremely small numbers • Useful in detecting microbes that are difficult to culture – Amplified DNA can be analyzed . etc..g. soil. food. water. Body fluids.PHENOTYPIC CHARACTERISTICS Polymerase chain reaction • Amplifies specific nucleotide sequences – DNA can be obtained from many sources • e.

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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