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MBIO 345 > Module 11 > Flashcards

Module 11 Flashcards

1
Q

What is the initial identification for streptococcus?

A

Hemolysis

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2
Q

What are the different kinds of hemolysis observed in streptococcus and what organisms exhibit them?

A

Beta- group A, B, C, F, G Strep

Alpha- Streptococcus pneumoniae and viridans

Gamma- D Enterococcus and Nonenterococcus

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3
Q

What are the characteristics of blood agar base for growing Streptococcus?

A

Should support all Strep growth.

Free from fermentable carbs- acid produced can alter acid labile streptolysin S, hemolysis loss

Trypticase soy agar (TSA) recommended

Sheep’s blood- inhibits Haemophilius haemolyticus which resembles Strep

Optimum concentration- 5% with 4-6mm depth

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4
Q

What should be avoided in blood agar for Streptococcus growth?

A

Citrate phosphate dextrose- contains fermentable carbs

Human and horse blood- give different hemolytic reactions for group D Strep

Human blood may contain antistreptolysin O antibodies, could inhibit hemolytic activity

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5
Q

Why must Streptococcus pyogenes be incubated with reduced oxygen tension?

A

Streptolysin O is destroyed by oxygen.

It will appear gamma hemolytic if it’s not.

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6
Q

How can oxygen tension be reduced?

A

Pour plates- obtains subsurface colonies

Cutting the inoculum into the medium and covering with a coverslip.

Anaerobic incubation- gas packs with indicator

Group A Strep DNA (GASD) probe- lysis reagent, DNA extraction and amplification, high volume

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7
Q

What is bacitracin susceptibility testing used for?

A

Presumptive ID of group A Streptococcus (susceptible).

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8
Q

How is bacitracin susceptibility performed?

A

Pure suture incubated overnight at 35°C with 0.04 unit disc.

Any zone of inhibition is susceptible.

Susceptible- presumptive group A Strep

Resistant- B hemolytic Strep not group A

If placed on BAP directly, should be retested with pure culture.

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9
Q

What is the QC for bacitracin susceptibility?

A

Susceptible- S. pyogenes (group A)

Resistant- S. agalactiae (group B)

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10
Q

What does PYR test for?

A

Colourimetric test for L-pyrrolidone aminopeptidase.

Differentiates group D Enterococci from Nonenterococci.

Presumptive S. pyogenes ID.

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11
Q

How is a PYR test performed?

A

Colonies are rubbed on a strip.

Reagent (p-dimethylamino cinnameldehyde) is added.

If the enzyme hydrolyzes L-pyrrolidone-B-naphthylamide) a pink colour is seen.

Must use pure culture.

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12
Q

What is the QC for PYR tests?

A

Positive- group D Enterococcus or group A Streptococcus

Negative- group D Nonenterococcus or group B, C, F, G Streptococcus

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13
Q

How can groups of Streptococcus be definitively identified?

A

Coagglutination

Latex agglutination

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14
Q

How is coagglutination performed?

A

Group specific IgG has killed S. aureus cells linked to the Fc portion.

Fab antibody ends react with Strep antigen causing visible agglutination.

Should occur within 15s-2min.

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15
Q

How is latex agglutination performed?

A

Group specific IgG antibodies have latex particles attached to the Fc portion.

Latex-antibody particles are bound together by cell wall antigen resulting in macroscopic agglutination.

May require extraction of Strep antigen prior to latex reagent addition, this affects group D identification.

Extraction by 10min incubation or one min with acid.

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16
Q

What does the cAMP test determine?

A

The organisms ability to produce cAMP factor.

Presumptive ID of group B Streptococcus.

17
Q

How is the cAMP test performed?

A

Uses sheep or bovine blood- cell membrane is specific for cAMP factor

Plates with a depth of 1.5mm

ATCC 25923 S. aureus

S. aureus and pure Strep cultures are streaked perpendicularly.

Incubated in CO2 (rapid) or air.

cAMP factor and Staph beta hemolysis produce lytic reaction, giving complete hemolysis arrowhead if cAMP factor is present.

18
Q

What is the QC for the cAMP test?

A

Positive- S. agalactiae (group B)

Negative- S. pyogenes (group A)

19
Q

What can affect the results of the cAMP test?

A

Up to 10% of group A are cAMP positive- perform bacitracin to confirm

Anaerobic incubation increases the incidence of positive group A results

20
Q

What is the purpose of the rapid hippurate hydrolysis test?

A

Determines whether an organism can hydrolyze sodium hippurate.

Presumptive ID for group B Strep (positive).

Final product of hydrolysis is benzoic acid but test for glycine (earlier product)

21
Q

How is the rapid hippurate hydrolysis test performed?

A

Sodium hippurate solution is inoculated and incubated for 2-4hr.

0.2mL of ninhydrin reagent is added.

Incubate for 10 more min.

Ninhydrin dean instead glycine, produces ammonia which reacts to give purple colour.

22
Q

What is the QC for the rapid hippurate hydrolysis test?

A

Positive- S. agalactiae (group B) or Campylobacter jejuni

Negative- S. pyogenes (group A) or Campylobacter coli

23
Q

What can cause false positives or negatives in the rapid hippurate hydrolysis test?

A

Positive- contamination with amino acid, bacteria contamination

Negative- technical error, expired reagent

23
Q

What is the purpose of the bile esculin (BEA) test?

A

Determines the ability of an organism to grow in the presence of bike salts and hydrolyze esculin.

ID of group D Strep Enterococci and Nonenterococci (positive)

24
Q

How does the BEA test work?

A

Bacteria break the bond between glucose and esculetin.

Esculetin reacts with ferric salts resulting in a dark brown/black colour.

Bile salts inhibit the growth of other organisms.

25
Q

How is the BEA test performed?

A

40% bike salt medium is inoculated (tube or plate).

Incubated at 35°C for 48hrs, can stop when a positive result is seen.

26
Q

What is the QC for BEA?

A

Positive- Enterococcus faecalis (group D)

Negative- Streptococcus agalactiae (group B), group A Strep, S. pneumoniae, S. viridans

27
Q

What causes false positives and negatives in BEA?

A

Positive- contaminating bacteria

Negative- not inoculated

28
Q

How is BEA done as a rapid test?

A

Buffered solution of esculin in nutrient broth with ferric salts is used.

No bile salts so the culture must be pure.

29
Q

What does bile solubility testing determine?

A

Whether bacteria will be used in the presence of bile salts.

Differentiates between bile soluble Streptococcus pneumoniae and other alpha hemolytic Strep.

30
Q

How is bile solubility testing performed?

A

10% solution of sodium deoxycholate and sodium taurocholate is added to an isolated colony on BAP.

If soluble colony will dissolve partially or completely.

31
Q

What is the QC for bile solubility?

A

Soluble- Streptococcus pneumoniae

Insoluble- Streptococcus viridans

32
Q

What can cause false positives and negatives in bile solubility testing?

A

Positive- contaminating bacteria

Negative- older colonies, some S. pneumoniae strains, expired reagent

33
Q

What are the results of a bile solubility tube test?

A

Soluble- clear

Insoluble- cloudy

34
Q

What is the purpose of the optochin susceptibility test?

A

Differentiation between Streptococcus pneumoniae (susceptible) and other alpha hemolytic Strep.

35
Q

How is optochin susceptibility performed?

A

Single colony is streaked on BAP.

Incubated for 18-24hr in CO2

Zone >14mm is susceptible

36
Q

What is the QC for optochin susceptibility?

A

Susceptible- Streptococcus pneumoniae

Resistant- Streptococcus viridans

37
Q

What can cause false resistance and sensitivity with optochin?

A

Resistance- expired discs, some Streptococcus viridans strains give small zones

Sensitivity- incubated in ambient air, not incubated with 15mjn of inoculation

MBIO 345 (11 decks)

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