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Aerobic Gram+ Cocci Flashcards

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0
Q

What is the principle of the catalase test?

A

H2O2 converted to H2O and O2 in the presence of catalase.

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1
Q

New aerobic gram positive cocci. First test?

A

Catalase.

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2
Q

How is a catalase test performed?

A

On a slide: 1 drop of 3% H2O2 on a colony.

In a tube: 1 ml 3% H2O2 on an overnight culture slant.

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3
Q

What are the results of a catalase test?

A

Positive: bubbles, lots of bubbles.
Negative: no bubbles.

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4
Q

What does a positive catalase test on a gram positive aerobic cocci colony indicate?

A

The colony is Staphylococcus or Micrococcus.

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5
Q

What does a negative catalase test on a gram positive aerobic cocci colony indicate?

A

The colony is Streptococcus.

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6
Q

Aerobic, gram positive, cocci, catalase positive . What is the next test for determining the type of Staphylococcus/Micrococcus?

A

Coagulase test.

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7
Q

What is the principle of the coagulase test?

A

In the presence of coagulase, fibrinogen forms a clot.

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8
Q

What are the two methods of coagulase test and when is each indicated?

A

Slide and tube. Do the cheaper, quicker slide test first. Confirm negative slide test results in a tube. If the slide test shows auto agglutination, then a tube test must be done.

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9
Q

How is a coagulase test performed on a slide?

A

Put some of a colony into saline on a slide. If a clot forms already, then skip to the tube test.

Add a small drop of rabbit plasma (contains fibrinogen)

Mix and observe any visible clumping.

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10
Q

How is a coagulase test performed in a tube.

A

Start with 0.5 ml rabbit plasma in a tube. Inoculate a loop full of the organism into the plasma.

Incubate at 35*C for 4 hours

Observe for clotting. If no clots, incubate overnight and check for clots again.

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11
Q

Why examine a tube coagulase test after 4 hours and then again after 24?

A

A positive response is observed quicker, and some organisms will lyse the 4 hour clots after 24 hours, giving a false negative. A small number of organisms take 24 hours to produce a positive result.

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12
Q

What results are possible in a coagulase test?

A

Positive: agglutination or clotting

Negative: no agglutination or clots.

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13
Q

The coagulase test is performed on a gram positive, aerobic cocci that is catalase positive. The result of the coagulase test is positive. What organisms have not been ruled out?

A

Staphylococcus aureus.

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14
Q

The coagulase test is performed on a gram positive, aerobic cocci that is catalase positive. The result of the coagulase test is negative. What organisms have not been ruled out?

A

S. aureus is ruled out. Micrococcus and CNS are not ruled out.

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15
Q

What is CNS?

A

Coagulase negative staphylococcus.

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16
Q

Gram positive, aerobic cocci, catalase positive, coagulase negative. What’s the next step?

A

Further identification may not be warranted, as S. aureus has been ruled out.

If further tests are warranted, perform a bacitracin disk test.

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17
Q

What is the principle of a bacitracin disk test?

A

A low concentration of bacitracin inhibits the growth of certain organisms.

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18
Q

How is a bacitracin disk test performed?

A

Place a disk with 0.04 units of bacitracin in the middle of a colony on sheep blood agar.

Incubate overnight at 35*C.

Test 3 or 4 colonies for better results.

Observe the colony for a circle of no organisms around the disk. Measure the size of the circle. The diameter is the zone of inhibition.

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19
Q

What are the possible results of a bacitracin disk test?

A

Any zone of inhibition: the organism is susceptible to bacitracin (called a positive result )

No zone of inhibition: the organism is resistant to bacitracin (called a negative result )

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20
Q

If the organism is gram positive, aerobic cocci, catalase positive, coagulase negative and the bacitracin disk test shows resistance, what organisms remain to be ruled in or out?

A

Micrococcus has now been ruled out. The organism is some form of CNS.

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21
Q

If the organism is gram positive, aerobic cocci, catalase positive, coagulase negative and the bacitracin disk test shows susceptiblity, what organisms remain to be ruled in or out?

A

The organism is Micrococcus.

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22
Q

Staphylococcus aureus and Micrococcus have been ruled out. The organism is some form of CNS. What next?

A

If the original specimen was urine, and further identification is desired, perform a Novobiocin disk test.

Otherwise, report CNS.

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23
Q

What is the principle of a Novobiocin disk test?

A

A low concentration of Novobiocin inhibits the growth of certain organisms. Notably, Staphylococcus saprophyticus is not inhibited.

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24
How is a Novobiocin disk test performed?
Prepare a 0.5 McFarland suspension. Inoculate MH agar or SBA plate. Press one Novobiocin disk ( 5ug) into inoculated area. Incubate overnight at 35*C. Measure zone of inhibition, if any.
25
What is a McFarland suspension?
Solutions of standard turbidity have been prepared according to McFarland's method. A suspension of unknown organism that visually matches the turbidity of a McFarland standard should contain a specified concentration of that organism.
26
What are the possible results of a Novobiocin disk test?
Zone of inhibition > 16 mm: the organism is susceptible to Novobiocin (called a positive result ) Zone of inhibition <= 16 mm: the organism is resistant to Novobiocin. (called a negative result )
27
If the organism is gram positive, aerobic cocci, catalase positive, coagulase negative, bacitracin resistant, and a Novobiocin test shows resistance what organisms remain to be ruled in or out?
The organism is presumptively Staphylococcus saprophyticus. There is a small chance of being another CNS, but those are very rarely isolated in urine.
28
If the organism is gram positive, aerobic cocci, catalase positive, coagulase negative, bacitracin resistant, and a Novobiocin test shows susceptibility what organisms remain to be ruled in or out?
The organism is not Staphylococcus saprophyticus. It is another CNS; report CNSNSS.
29
Aerobic, gram positive, cocci, catalase negative . What is the next test for determining the type of Streptococcus?
Hemolysis test.
30
What is a hemolysis test?
Not exactly a test. The original specimen was likely cultured on Sheep Blood Agar or other medium contain ing blood. if not, incubate an inoculation of the organism on such a plate. Observe the hemolysis pattern.
31
What are the possible hemolysis patterns on an agar plate containing whole blood?
Alpha: RBCs are partially destroyed. The media near the colony takes on a greenish brown color. Beta: RBCs are completely destroyed. The media near the colony becomes clear and colorless. Gamma: RBCs remain intact. The media near the colony shows no apparent color change.
32
Aerobic, gram positive, cocci, catalase negative. Hemolysis is beta type. What organisms are under consideration?
Beta strep, S. viridans, and rarely, Enterococcus or S. mutans.
33
Aerobic, gram positive, cocci, catalase negative. Hemolysis is beta type. What next?
PYR test.
34
What is the principle of the PYR test?
Certain organisms have an enzyme that catalyses The hydrolysis of PYR into free beta-naphthalene, which forms a red complex with cinnamaldehyde.
35
How is a PYR test performed?
Using a commercial substrate disk, moisten it and touch the colony to the disk. Incubate 2 minutes, then add a drop of cinnamaldehyde. Observe for color changes.
36
What are the available results of a PYR test?
Positive: red color. Negative: no red color.
37
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. What organisms are still under consideration?
Enterococcus or group A strep (S. pyogenes)
38
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. What organisms are still under consideration?
Strep groups B, C, F, G, and Streptococcus viridans.
39
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. What next?
Cultivate on bile esculin agar slant.
40
What happens on a bile esculin agar slant?
Bile inhibits most gram positive organisms. Those that live may hydrolyse esculin into esculatin and dextrose. Esculatin reacts with ferric citrate to make a black precipitate.
41
What are the results of growth on a bile esculin stant?
Positive: growth and blackening of the agar around a colony that is more than half the slanted area. Negative: no or too little growth.
42
Is the organism stabbed into or streaked on bile esculin slants?
Streaked.
43
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. The organism grows on bile esculin. What might it be?
Enterococcus.
44
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. The organism doesn't grow on bile esculin. What might it be?
Group A beta hemolytic Streptococcus, aka S. pyogenes.
45
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. What next?
Hippurate hydrolysis test or CAMP test.
46
What is the principle of the hippurate hydrolysis test?
In the presence of an enzyme, sodium hippurate is hydrolysed into benzoic acid and glycine. Glycine combined with ninhydrin makes a purple complex.
47
How is a hippurate hydrolysis test performed?
In a test tube, suspend organism in 0.5 ml sterile distilled water. Add a disk of sodium hippurate Incubate 2 hours at 35*C, then add 0.2 ml ninhydrin. Observe any color change.
48
What are the possible results of the hippurate test?
Positive: deep blue-purple color within 5 minutes Negative: no color change within 5 minutes.
49
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The hippurate hydrolysis test is positive. What organisms are still under consideration?
Group B beta- strep, aka S. agalactiae.
50
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The hippurate hydrolysis test is negative. What organisms are still under consideration?
Beta strep of group C, F, or G, or S. viridans.
51
What is the principle of the CAMP test?
CAMP factor is a diffusable protein that synergises beta lysin from Staphylococcus to lyse RBCs.
52
How is a CAMP test performed?
Make a vertical streak of the organism on a SBA plate. Make a perpendicular, but not crossing streak of S. aureus. Incubate overnight at 35*C
53
What are the results of a CAMP test?
Staph aureus will have a zone of hemolysis. Positive: the area between the end of the shorter streak will show enhanced hemolysis in an arrowhead shape pointing toward the S. aureus. Negative: no enhanced hemolysis.
54
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The CAMP test is positive. What organisms are still under consideration?
Group B beta- strep, aka S. agalactiae.
55
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The CAMP test is negative. What organisms are still under consideration?
Beta strep of group C, F, or G, or S. viridans.
56
Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The CAMP or hippurate test is negative. What next?
If further identification is required, use a commercial serogroup test to determine if the organism is Group C, F, or G beta strep. If none, then report S. viridans.
57
Aerobic, gram positive, cocci, catalase negative. Hemolysis is alpha type. What organisms are under consideration?
Streptococcus pneumoniae, Streptococcus viridans, Enterococcus, other group D.
58
Aerobic, gram positive, cocci, catalase negative. Hemolysis is alpha type. What next?
Optochin test or bile solubility test.
59
What is the principle of the optochin test?
Optochin inhibits the growth of certain organisms, notably S. pneumoniae.
60
How is the optochin test performed?
Inoculate colonies onto SBA plate. Add optochin disk, 5ug/ml, called a "P" disk. Incubate overnight at 35*C Observe zone of inhibition if any.
61
What are the possible results of the optochin test?
Susceptible: p disk is 6mm. Zone of inhibition must be > 14 mm. Inconclusive: zone of inhibition less than 14 mm Resistant: no zone of inhibition (?)
62
If an optochin test is inconclusive what other test can confirm?
Bile solubility test.
63
Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Optochin test is positive. What organisms are still under consideration?
Streptococcus pneumoniae.
64
Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Optochin test is negative. What organisms are still under consideration?
Streptococcus viridans, Enterococcus, other group D cocci.
65
What is the principle of the bile solubility test?
Sodium deoxycholate is a bile salt that lyses pneumococci.
66
How is a bile solubility test performed?
On an agar colony: Add 1 drop of 2-10% sodium deoxycholate and incubate right side up for 30 minutes at 35* C In a test tube: Suspend organism in each of two tubes of 1 ml nutrient broth. Add a few drops if sodium desoxycholate to one tube, the same volume of sterile saline to the other. Incubate for 15 minutes at 35*C.
67
What are the possible results of a bile solubility test?
Positive on agar: colony lyses under drop Negative on agar: no lysis of colony Positive in tube: no change in saline tube, bile tube clear Negative in tube: no change in either tube.
68
Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Bile solubility test is positive. What organisms are still under consideration?
Streptococcus pneumoniae.
69
Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Bile solubility test is negative. What organisms are still under consideration?
Streptococcus viridans, Enterococcus, other group D cocci.
70
Aerobic, gram positive, cocci, catalase negative. Streptococcus viridans, Enterococcus, other group D cocci are all still under consideration. What next?
PYR test
71
Aerobic, gram positive, cocci, catalase negative. All but Streptococcus viridans, Enterococcus,or other group D cocci have been ruled out. PYR test is positive. What organisms are still under consideration?
Enterococcus.
72
Aerobic, gram positive, cocci, catalase negative. All but Streptococcus viridans, Enterococcus,or other group D cocci have been ruled out. PYR test is negative. What organisms are still under consideration?
Streptococcus viridans, or other group D cocci.
73
Aerobic, gram positive, cocci, catalase negative. All but Streptococcus viridans, Enterococcus,or other group D cocci have been ruled out. PYR test is negative. What next?
Cultivate on bile esculin agar.
74
Down to S. viridans or non-Enterococcus group D. Bile esculin agar shows no growth. Which is it?
Streptococcus viridans.
75
Down to S. viridans or non-Enterococcus group D. Bile esculin agar shows growth. Which is it?
Non-Enterococcus Group D.

Microbiology (4 decks)

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