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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. Whatā€™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
Q

How is a Novobiocin disk test performed?

A

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.

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25
Q

What is a McFarland suspension?

A

Solutions of standard turbidity have been prepared according to McFarlandā€™s method. A suspension of unknown organism that visually matches the turbidity of a McFarland standard should contain a specified concentration of that organism.

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26
Q

What are the possible results of a Novobiocin disk test?

A

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 )

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27
Q

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?

A

The organism is presumptively Staphylococcus saprophyticus. There is a small chance of being another CNS, but those are very rarely isolated in urine.

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28
Q

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?

A

The organism is not Staphylococcus saprophyticus. It is another CNS; report CNSNSS.

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29
Q

Aerobic, gram positive, cocci, catalase negative . What is the next test for determining the type of Streptococcus?

A

Hemolysis test.

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30
Q

What is a hemolysis test?

A

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
Q

What are the possible hemolysis patterns on an agar plate containing whole blood?

A

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
Q

Aerobic, gram positive, cocci, catalase negative. Hemolysis is beta type. What organisms are under consideration?

A

Beta strep, S. viridans, and rarely, Enterococcus or S. mutans.

33
Q

Aerobic, gram positive, cocci, catalase negative. Hemolysis is beta type. What next?

A

PYR test.

34
Q

What is the principle of the PYR test?

A

Certain organisms have an enzyme that catalyses The hydrolysis of PYR into free beta-naphthalene, which forms a red complex with cinnamaldehyde.

35
Q

How is a PYR test performed?

A

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
Q

What are the available results of a PYR test?

A

Positive: red color.
Negative: no red color.

37
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. What organisms are still under consideration?

A

Enterococcus or group A strep (S. pyogenes)

38
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. What organisms are still under consideration?

A

Strep groups B, C, F, G, and Streptococcus viridans.

39
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. What next?

A

Cultivate on bile esculin agar slant.

40
Q

What happens on a bile esculin agar slant?

A

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
Q

What are the results of growth on a bile esculin stant?

A

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
Q

Is the organism stabbed into or streaked on bile esculin slants?

A

Streaked.

43
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. The organism grows on bile esculin. What might it be?

A

Enterococcus.

44
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR positive. The organism doesnā€™t grow on bile esculin. What might it be?

A

Group A beta hemolytic Streptococcus, aka S. pyogenes.

45
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. What next?

A

Hippurate hydrolysis test or CAMP test.

46
Q

What is the principle of the hippurate hydrolysis test?

A

In the presence of an enzyme, sodium hippurate is hydrolysed into benzoic acid and glycine. Glycine combined with ninhydrin makes a purple complex.

47
Q

How is a hippurate hydrolysis test performed?

A

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
Q

What are the possible results of the hippurate test?

A

Positive: deep blue-purple color within 5 minutes

Negative: no color change within 5 minutes.

49
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The hippurate hydrolysis test is positive. What organisms are still under consideration?

A

Group B beta- strep, aka S. agalactiae.

50
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The hippurate hydrolysis test is negative. What organisms are still under consideration?

A

Beta strep of group C, F, or G, or S. viridans.

51
Q

What is the principle of the CAMP test?

A

CAMP factor is a diffusable protein that synergises beta lysin from Staphylococcus to lyse RBCs.

52
Q

How is a CAMP test performed?

A

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
Q

What are the results of a CAMP test?

A

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
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The CAMP test is positive. What organisms are still under consideration?

A

Group B beta- strep, aka S. agalactiae.

55
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The CAMP test is negative. What organisms are still under consideration?

A

Beta strep of group C, F, or G, or S. viridans.

56
Q

Aerobic, gram positive, cocci, catalase negative, beta hemolytic. PYR negative. The CAMP or hippurate test is negative. What next?

A

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
Q

Aerobic, gram positive, cocci, catalase negative. Hemolysis is alpha type. What organisms are under consideration?

A

Streptococcus pneumoniae, Streptococcus viridans, Enterococcus, other group D.

58
Q

Aerobic, gram positive, cocci, catalase negative. Hemolysis is alpha type. What next?

A

Optochin test or bile solubility test.

59
Q

What is the principle of the optochin test?

A

Optochin inhibits the growth of certain organisms, notably S. pneumoniae.

60
Q

How is the optochin test performed?

A

Inoculate colonies onto SBA plate.

Add optochin disk, 5ug/ml, called a ā€œPā€ disk.

Incubate overnight at 35*C

Observe zone of inhibition if any.

61
Q

What are the possible results of the optochin test?

A

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
Q

If an optochin test is inconclusive what other test can confirm?

A

Bile solubility test.

63
Q

Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Optochin test is positive. What organisms are still under consideration?

A

Streptococcus pneumoniae.

64
Q

Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Optochin test is negative. What organisms are still under consideration?

A

Streptococcus viridans, Enterococcus, other group D cocci.

65
Q

What is the principle of the bile solubility test?

A

Sodium deoxycholate is a bile salt that lyses pneumococci.

66
Q

How is a bile solubility test performed?

A

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
Q

What are the possible results of a bile solubility test?

A

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
Q

Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Bile solubility test is positive. What organisms are still under consideration?

A

Streptococcus pneumoniae.

69
Q

Aerobic, gram positive, cocci, catalase negative, alpha hemolysis. Bile solubility test is negative. What organisms are still under consideration?

A

Streptococcus viridans, Enterococcus, other group D cocci.

70
Q

Aerobic, gram positive, cocci, catalase negative. Streptococcus viridans, Enterococcus, other group D cocci are all still under consideration. What next?

A

PYR test

71
Q

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?

A

Enterococcus.

72
Q

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?

A

Streptococcus viridans, or other group D cocci.

73
Q

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?

A

Cultivate on bile esculin agar.

74
Q

Down to S. viridans or non-Enterococcus group D. Bile esculin agar shows no growth. Which is it?

A

Streptococcus viridans.

75
Q

Down to S. viridans or non-Enterococcus group D. Bile esculin agar shows growth. Which is it?

A

Non-Enterococcus Group D.

Microbiology (4 decks)

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