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Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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Page 1: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Identification of Pathogenic Bacteria by Laboratory Methods

M. Kent Froberg, MD

Page 2: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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

Page 3: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD
Page 4: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD
Page 5: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD
Page 6: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Gram Positive Cocci

• Staphylococci vs Streptococci – Staph aureus catalase +, coagulase +,

& -hemolytic – Strep , , or non-hemolytic, catalase -

Page 7: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Catalase

• Enzyme that converts H2O2H2O and O2

• Visible bubbles when drop of 3% H2O2 is added to culture of catalase + bacteria

Page 8: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Catalse + Catalse -

Page 9: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Catalase - Catalase +

Page 10: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Between Staph Species

• S aureus coagulase + (clots plasma)

• S epidermidis & saprophyticus coagulase neg– S saprophyticus novobiocin resistant– S epidermidis novobiocin sensitive

Page 11: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Lower tube shows fibrin clot following addition of plasma to culture of Stap aureus. Top tube is negative indicating non-pathogenic Staph.

Page 12: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Left: novobiocin resistant Staph saprophyticus

Right: novobiocin senstitive Staph epidermidis

Page 13: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Between Strep Species

• Hemolysis:– Strep pyogenes & agalactiae hemolytic – Enterococcus variable hemolysis – Strep viridans & pneumoniae hemolytic

Page 14: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Beta-hemolysis with clearing of blood agar

Page 15: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Green discloloration of alpha-hemolysis

Page 16: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

No hemolysis on blood agar

Page 17: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

More Strep Tests

• Strep pyogenes bacitracin sensitive

• Strep agalactiae & Enterococcus bacitracin resistant

• Strep viridans resistant to optochin

• Strep pneumoniae sensitive to optochin

Page 18: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Zone of inhibition around optochin disk - presumptive identification of Strep pneumoniae

Page 19: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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.

Page 20: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

2,3,5-Triphenyltetrazolium chloride (TTC) Formazan (red color)

Page 21: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Left tube: motile Center: nonmotile Right: control

Page 22: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Oxidase Tests

• Presence of cytochrome oxidase

• Neisseria & Pseudomonas +

• Enterobacteriaceae neg

• Test converts colorless tetramethyl-p-phenylenediamine to oxidized form (deep purple/blue)

Page 23: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Oxidase + colony at 12 o’clock, 3 & 9 o’clock cultures are negative

Page 24: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Oxidase test done on paper saturated with oxidase reagent. Pseudomonas on left (+), Staph aureus on right (-).

Page 25: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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

Page 26: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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

Page 27: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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

Page 28: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

Mineral oil covers media in tubes 1 & 3. Pseudomonas in tubes 1 & 2, Shigella in tubes 3 & 4.

Page 29: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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

Page 30: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD
Page 31: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD
Page 32: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD
Page 33: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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

Page 34: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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

Page 35: Identification of Pathogenic Bacteria by Laboratory Methods M. Kent Froberg, MD

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


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