identification of pathogenic bacteria by laboratory methods m. kent froberg, md
TRANSCRIPT
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Identification of Pathogenic Bacteria by Laboratory Methods
M. Kent Froberg, MD
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Laboratory Methods
• ID of pathogenic bacteria using series of differential tests
differential & selective media morphology & staining biochemical properties antibiotic resistance or susceptibility plasmid, ribosomal or DNA analysis
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Gram Positive Cocci
• Staphylococci vs Streptococci – Staph aureus catalase +, coagulase +,
& -hemolytic – Strep , , or non-hemolytic, catalase -
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Catalase
• Enzyme that converts H2O2H2O and O2
• Visible bubbles when drop of 3% H2O2 is added to culture of catalase + bacteria
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Catalse + Catalse -
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Catalase - Catalase +
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Between Staph Species
• S aureus coagulase + (clots plasma)
• S epidermidis & saprophyticus coagulase neg– S saprophyticus novobiocin resistant– S epidermidis novobiocin sensitive
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Lower tube shows fibrin clot following addition of plasma to culture of Stap aureus. Top tube is negative indicating non-pathogenic Staph.
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Left: novobiocin resistant Staph saprophyticus
Right: novobiocin senstitive Staph epidermidis
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Between Strep Species
• Hemolysis:– Strep pyogenes & agalactiae hemolytic – Enterococcus variable hemolysis – Strep viridans & pneumoniae hemolytic
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Beta-hemolysis with clearing of blood agar
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Green discloloration of alpha-hemolysis
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No hemolysis on blood agar
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More Strep Tests
• Strep pyogenes bacitracin sensitive
• Strep agalactiae & Enterococcus bacitracin resistant
• Strep viridans resistant to optochin
• Strep pneumoniae sensitive to optochin
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Zone of inhibition around optochin disk - presumptive identification of Strep pneumoniae
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Motility Tests
• Many bacteria show motility in media • Agar tube inoculated with needle stab • Media contains tetrazolium bacteria
reduces to formazan (red color) • Location of bacteria determined by location
of red color• Useful for Borrelia, Vibrio, Salmonella, E
coli, etc.
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2,3,5-Triphenyltetrazolium chloride (TTC) Formazan (red color)
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Left tube: motile Center: nonmotile Right: control
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Oxidase Tests
• Presence of cytochrome oxidase
• Neisseria & Pseudomonas +
• Enterobacteriaceae neg
• Test converts colorless tetramethyl-p-phenylenediamine to oxidized form (deep purple/blue)
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Oxidase + colony at 12 o’clock, 3 & 9 o’clock cultures are negative
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Oxidase test done on paper saturated with oxidase reagent. Pseudomonas on left (+), Staph aureus on right (-).
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Enterobacteriaceae
• API 20 E series of substrates and indicators that are inoculated with unknown and media
• Tests for enzymes and carbohydrate utilization
• Pattern of + and - results compared to a table of known results to identify unknown
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cultureno.
ONPG
ADH
LDC
ODC
CIT
H2S
URE
TDA
IND
VP
GEL
GLU
MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA
identification
8101 + – + + – – – – + – – + + – + + + + – +Escherichia
coli
5B + – – – + – – – – + – + + + + + – – + +Enterobacteragglomerans
8P44 – – + + – + – – + – – + + – – – + – – +Edwardsiellahoshinae
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Oxidation-Fermentation Tests
• Mostly used to distinguish fermentative Enterobacteriaceae from oxidative Pseudomonas & Bordetella
• Grow isolate in low agar content media in presence of different sugar substrates and pH indicator
• Stab media: one tube sealed with mineral oil to promote anaerobic growth/ other tube unsealed
• Indicator yellow at pH 6.0/ green at pH 7.1, blue at pH 7.6
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Mineral oil covers media in tubes 1 & 3. Pseudomonas in tubes 1 & 2, Shigella in tubes 3 & 4.
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Enterobacteriaceae
• Enterotube II 12 compartments of various media allows determination of 15 different characteristics of organism
• Positive results given a number, added in groups and sums combined to create unique 5-digit identifier in computer databank
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Question #1
1. The laboratory obtains a surgical wound swab from a 56 year old male four days after undergoing a colectomy for colon cancer. The isolate is gram +, catalase +, coagulase + and hemolytic. It is most likely
a. Streptococcus pyogenes b. Enterococcus faecalis c. Staphylococcus aureus d. Staphylococcus saphrophyticuse. Streptococcus viridans
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Question #2
2. The initial data necessary to adequately identify a bacterial unknown are its
a. Ribosomal sequence
b. Antibiotic resistance pattern
c. Motility
d. Ability to ferment glucose
e. Morphology and gram staining
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Question #3
3. Streptococcus viridans and pneumoniae are both hemolytic, catalase -, gram + cocci. Which test may readily distinguish them?
a. Enterotube II
b. Novobiocin resistance
c. Motility test
d. Oxidase test
e. Resistance to optochin