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Microbiology 1 > Module 8: Laboratory Procedures - Catalase, Nitrate, DNase, and Beta-lactamase > Flashcards

Module 8: Laboratory Procedures - Catalase, Nitrate, DNase, and Beta-lactamase Flashcards

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Biology 101 flashcards
1
Q

What is the genus test for Staphylococcus and Streptococcus?

A

Catalase test

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2
Q

What organisms require the catalase test for genus testing?

A

Staphylococcus and Streptococcus

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3
Q

What does the catalase test demonstrate?

A

It detects the catalase enzyme, which cause the breakdown of the reagent H2O2 into water and O2

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4
Q

What other uses are there for the catalase test other than Staph and Strep differentiation?

A

Identifying Mycobacterium and anaerobes

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5
Q

What reagent is used for the catalase test?

A

3% hydrogen peroxide (H2O2)

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6
Q

How is the catalase reagent made?

How would you prepare 100mL of 3% H2O2 from a 30% stock solution?

A 
Diluting 30% stock solution in water
V1 x C1 = V2 x C2
V1 x 30% = 100mL x 3%
V1 = (100mL x 3%) / 30%
V1 = 300 / 30
V1 = 10mL
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7
Q

What precautions have to be taken with catalase reagent?

A

It is unstable and breaks down easily especially in light and heat
Must be stored in a brown bottle in the fridge and QC’d daily or once per shift

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8
Q

What are the two common methods used for the catalase test?

A

Tube method - H2O2 in a tube, stick tested for negative reaction, then colony picked up and put into tube
Slide method - colony or H2O2 placed on slide, H2O2 dropped on colony or colony added to H2O2

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9
Q

What is one uncommon way a catalase test could be performed?

A

Overnight culture

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10
Q

How is the overnight culture catalase test performed?

A

Pour H2O2 over colonies on nutrient agar slant or plate

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11
Q

What might cause false positives for the catalase test?

A

Platinum wires, blood from agar (contains catalase)

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12
Q

What might cause false negative results for the catalase test?

A

Old colonies (may lose their catalase activity), broken down reagent

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13
Q

What is the nitrate reduction test used for?

A

Not common anymore

Helpful to identify Enterobacteriaceae, non-fermentive GNB, anaerobes, and separating staph from strep

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14
Q

How does the nitrate reduction test work?

A

Nitrate reduction by the bacteria is tested for by detecting end points like nitrogen gas, nitrite, ammonia, nitric oxide, and hydoxylamine
N2 gas and nitrite can be easily detected, the others cannot be detected, and are assumed to be there if no unreduced nitrate is found with other reagents

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15
Q

What will give a positive result in the nitrate reduction test? A negative result?

A

Positive, nitrogen gas (in durham tube), nitrite (red color after reagent a and b added), other reduction products (no gas, no color with a and b, no color with zinc dust)
Negative, unreduced nitrate (red color after zinc dust)

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16
Q

What are reagents a and b for the nitrate reduction test made of?

A

A - sulfanilic acid in acetic acid

B - alpha naphthlamine in acetic acid

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17
Q

How do you set up the nitrate reduction test? How are the rest of the steps performed?

A

Nitrate broth inoculated and Durham tube inserted upside down, incubated
If no gas is present, add reagents a and b
If no red color is formed add zinc salt

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18
Q

What would the expected result be in the nitrate test if the bacteria is staph?

A

No gas, red color after reagents a and b = Nitrite

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19
Q

If the nitrate reduction test is positive for nitrite, what organism is it?

A

Staphylococcus

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20
Q

What is in the nitrate reduction broth?

A

Beef extract, peptone, and potassium nitrate dissolved in water

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21
Q

What is the coagulase test? What does it detect?

A

Detects coagulase enzymes

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22
Q

What is the difference between bound and free coagulase?

A

Bound/clumping factor - attached to cell wall, acts on fibrinogen in plasma to form fibrin
Free - extracellular enzyme, produces thrombin-like substance from plasma which converts into a fibrin clot

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23
Q

Where is bound coagulase detected? Free?

A

Bound - slide coag

Free - tube coag

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24
Q

What reagent is used for the coagulase test?

A

Saline - negative slide control

Rabbit or human plasma with heparin or EDTA

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25
Q

Why would citrate not be used as the anticoagulant for the plasma used in the coagulase test?

A

Bacteria may use the citrate as a nutrient which would cause the plasma to clot on its own = false positive

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26
Q

Why would serum not be used for the coagulase test?

A

There is no fibrinogen (clotting already happened) and thus no clotting will be able to happen even if the organism has coagulase enzymes = false negative

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27
Q

How is rabbit plasma usually received in and stored in lab?

A

Lyophilized and refrigerated

When made up with distilled water it is kept in the fridge for a week or frozen

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28
Q

How is the slide coagulase test done? Tube?

A

Slide - 1 drop of saline, 1 drop of plasma. Add bacteria to both, observe for clumping
Tube - add bacteria to tube, incubate for 4 hours, observe for clumping, if none, incubate at RT for 24 hours

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29
Q

What might cause a false positive coagulase test? Negative?

A

Positive - citrated plasma, not emulsifying slide coag enough
Negative - light suspension, too high incubation temperature, frequent/excess agitation, over incubation, using serum

30
Q

Why must a negative tube coag be incubated at RT for 24 hours?

A

Staph produces and enzyme called staphylokinase which will break down the clot, and may make it appear negative when incubated at 35, but will not at RT

31
Q

What is the DNase test?

A

Determines if the bacteria produces enzymes that depolymerize (unwind) DNA to oligonucleotides, which is a virulence factor

32
Q

What organisms can the DNase test differentiate?

A

S. aureus from other staph
M. catarrhalis from Neisseria
Serratia from other Enterobacteriaceae
Xanthomonas maltophilia from other non-fermenters

33
Q

What are 3 reagents that can detect the oligonucleotide endpoint of the DNase test?

A

Hydrochloric acid, methyl green, and toluidine blue

34
Q

What is the principle behind the hydrochloric acid DNase test?

A

Intact DNA is precipitated by hydrochloric acid but oligonucleotides are soluble in acid

35
Q

What is the hydrochloric acid DNase test procedure? Results?

A

Inoculate DNA containing plate, incubate, flood with HCl

Whole DNA = precipitate, cloudy, DNase negative
Oligonucleotides = clearing around bacteria, DNase positive

36
Q

What is the principle behind the methyl green DNase test?

A

Methyl green is green when bound to whole DNA, and colorless when oligonucleotides are present

37
Q

What is the procedure for the methyl green DNase test? Results?

A

Inoculate Methyl Green DNase agar, incubate, observe for color
Whole DNA = green, negative
Oligonucleotides = colorless, positive

38
Q

What is the procedure for the toluidine blue DNase test? Results?

A

Inoculate DNase agar, incubate, flood with 0.1% toluidine blue

Whole DNA = blue, negative
Oligonucleotides = pink, positive

39
Q

What are positive and negative controls for the DNase test?

A

Positive - S. aureus, Serratia marcescens

Negative - E. coli, S. epidermidis

40
Q

What is the beta-lactamase test?

A

Detects the presence of beta-lactamase enzymes

41
Q

What is induced beta-lactamase?

A

Bacteria can be induced to produce beta-lactamase enzymes by growing them in the presence of beta-lactam antibiotics

42
Q

How is the beta-lactamase test set up?

A

Inoculate plate, place Oxacillin disc in the center, incubate, scrape growth from edge of zone of inhibition and test

43
Q

What are the 3 methods that can be used for the beta-lactamase test?

A

Acidimetric, iodometric, chromogenic Cephalosporin test

44
Q

How does the acidimetric beta-lactamase test work?

A

Beta-lactam hydrolysis yeilds an acid pH, which can be detected by pH indicators
Rub bacteria on a moistened pH strip, color change = acid/positive

45
Q

How does the iodometric beta-lactamase test work?

A

Combines penicillin, starch, and iodine

When penicillin is hydrolyzed to penicilloic acid, it converts iodine to iodide

46
Q

How is the iodometric beta-lactamase test set up?

A

Soak filter paper in benzylpenicillin and starch, add iodine just before use (dark purple), rub colonies on
No purple = positive

47
Q

How does the chomogenic Cephalosporin test for beta-lactamase work?

A

Nitrocefin has a beta-lactam ring, yellow to start and red when hydrolyzed

Bacteria rubbed on moistened disc/strip
Yellow to red color change = positive

48
Q

What are some precautions about the chromogenic Cephalosporin test?

A

Nitrocefin disks must be stored in a dark bottle as they deteriorate in light
During the test it must be kept in a dark drawer

49
Q

The reagent used for the catalase test is

a. sodium hydroxide
b. sodium citrate
c. human plasma
d. hydrogen peroxide

A

d. hydrogen peroxide

50
Q

A positive catalase result is indicated by

a. clotting of the reagent
b. bubbles
c. color change
d. pH change

A

b. bubbles

51
Q

Which of the following would not be suitable for picking colonies for catalase testing?

a. platinum wire
b. nichrome wire
c. applicator stick
d. plastic wire

A

a. platinum wire

52
Q

Which of the following may cause a false positive catalase test?

a. scraping colonies from a chocolate agar
b. scraping colonies from a blood agar
c. scraping colonies from nutrient agar
d. using a nichrome wire

A

b. scraping colonies from a blood agar

53
Q

A suitable organism for negative control in the catalase test is

a. S. aureus
b. S. epidermidis
c. Streptococcus specis

A

c. Streptococcus species

54
Q

Catalase testing may be used to

a. differentiate species of staphylococci
b. differentiate species of streptococci
c. differentiate streptococci from staphylococci
d. all of the above

A

c. differentiate streptococci from staphylococci

55
Q

The end products detected in the nitrate reduction test are

a. hydroxylamine
b. nitrite
c. nitrogen gas
d. b and c

A

d. b and c

nitrite and nitrogen gas

56
Q

Which of the following would be an essential ingredient in media for detection of nitrate reduction?

a. phosphate buffer
b. potassium nitrate
c. sulfanilic acid
d. a pH indicator

A

b. potassium nitrate

57
Q

Reagent B used for nitrate reduction test contains

a. sulfanilic acid
b. acetic acid
c. alpha naphthylamine
d. b and c

A

d. b and c

acetic acid and alpha naphthylamine

58
Q

After incubation, a nitrate broth showed growth and gas in the Durham tube. This would be interpreted as

a. positive for nitrate reduction
b. nitrate reduced to nitrite
c. nitrate reduced to nitrogen gas
d. a and b
e. a and c

A

e. a and c

positive for nitrate reduction and nitrate reduced to nitrogen gas

59
Q

After incubation, nitrate broth showed growth and no gas in the Durham tube. A red color developed after addition of reagent and and b. This would be interpreted as

a. nitrate reduced to nitrogen gas
b. nitrate reduced to nitrite
c. nitrate reduced to unidentified end products
d. nitrate not reduced

A

b. nitrate reduced to nitrite

60
Q

After incubation, a nitrate broth showed growth and no gas in the Durham tube. No color developed after reagent a and b but red color developed after zinc was added. This would be interpreted as

a. nitrate reduced to nitrogen gas
b. nitrate reduced to nitrite
c. nitrate reduced to unidentified end products
d. nitrate not reduced

A

d. nitrate not reduced

61
Q

An isolate of S. aureus tested negative for beta-lactamase when tested from the primary plate. A second test was done on colones of the same orgaism that had been exposed to low concentrations of oxacillin. This test was positive. This indicates

a. penicillin G could be used for treating this patient
b. presence of constitutive beta-lactamase
c. presence of induced beta-lactamase
d. error in the testing procedure

A

c. presence of induced beta-lactamase

62
Q

An isolate of S. aureus tested positive for beta-lactamase. Which of the following antibiotics would not be suitable for inhibiting growth of this organism?

a. penicillin G
b. oxacillin
c. ampicillin
d. and and c

A

d. a and c

penicillin G and ampicillin

63
Q

The results of beta-lactamase testing are useful in predicting the resistance of an organism to

a. benzylpenicillin
b. oxacillin
c. methicillin
d. third generation cephalosporins

A

a. benzylpenicillin

64
Q

Which of the following is not true of the nitrocefin method of detecting beta-lactamase?

a. the reagent is yellow
b. red color indicates a positive test
c. a positive test is indicated by a pH change
d. reagent may be dispensed on paper strips

A

c. a positive test is indicated by a pH change

65
Q

Which of the following may cause a false positive tube coagulase test?

a. over incubation
b. excess agitation during incubation
c. contaminated plasma
d. autoagglutination

A

c. contaminated plasma

66
Q

Which of the following is not an indicator system for the DNase test?

a. methylene blue
b. methyl green
c. toluidine blue
d. dilute hydrochloric acid

A

a. methylene blue

67
Q

The usual reason for a false negative slide coagulase result is

a. autoagglutination
b. contaminated plasma
c. weak bacterial suspension
d. waiting up to five minutes for agglutination

A

c. weak bacterial suspension

68
Q

A gram positive cocci that grew on blood agar had a nitrate done as follows:

  • nitrate broth was cloudy after 18 hours incubation
  • no gas in Durham tube
  • no red color after reagents a and b
  • no red color after zinc dust

Does this organism reduce nitrate?

A

Yes it does, but to end products other than nitrite or nitrogen gas

69
Q

A gram positive cocci that grew on blood agar had a nitrate done as follows:

  • nitrate broth was cloudy after 18 hours incubation
  • no gas in Durham tube
  • no red color after reagents a and b
  • no red color after zinc dust

Is this organism Staphylococcus? Explain.

A

No, because Staphylococcus reduces nitrate to nitrite

70
Q

If a Staphylococcus agglutinates in saline before plasma is added in the slide coagulase test

a. the organism has clumping factor
b. autoagglutination has occurred
c. the organism is S. aureus
d. the organism is a coagulase negative Staphylococcus

A

b. autoagglutination has occurred

Microbiology 1 (11 decks)

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