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Clinical Bacteriology > (F) Lesson 11.1: Diagnostic Tests for Gram-Negative Bacilli > Flashcards

(F) Lesson 11.1: Diagnostic Tests for Gram-Negative Bacilli Flashcards

1
Q
  • Purpose: To identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source
A

Citrate Utilization

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2
Q
  • The test is part of a series referred to as IMViC which is used to differentiate Enterobacteriaceae from other gram-negative rods
A

Citrate Utilization

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3
Q

Citrate Utilization

  • Bacteria that can grow on this medium produce an enzyme called ____ capable of converting citrate to pyruvate.
A

Citrate-permease

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4
Q
  • ____ can then enter the organism’s metabolic cycle for the
    production of energy
A

Pyruvate

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5
Q

Citrate Utilization

  • Bacteria capable of growth in this medium
    use the citrate and convert ____ to ____ and ____, creating an alkaline pH.
A

Convert ammonium phosphate to ammonia and ammonium hydroxide

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6
Q

Citrate Utilization

  • The pH change turns the bromothymol blue indicator from ____ to ____.
A

Green to blue

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7
Q

Citrate Utilization

Familiarize yourselves with the media inclusions.

A
  • NH4H2PO4 (1 g)
  • K2HPO4 (1 g)
  • NaCl (5 g)
  • Sodium citrate (2 g)
  • MgSO4 (0.2 g)
  • Agar (15 g)
  • Bromothymol blue (0.08g) per 1000 mL
  • pH 6.9
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8
Q

Citrate Utilization

T or F: We should inoculate from a broth culture to ensure that the inoculum is heavy.

A

F (We should not because it will be too heavy)

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9
Q

Citrate Utilization

At what temperature should we incubate the media?

A

35 to 37 deg C

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10
Q

Citrate Utilization

We should observe ____ and the development of ____, denoting alkalinization.

A

Growth and the development of blue color

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11
Q

Citrate Utilization

Growth and development of blue color denotes what?

A

Alkalinization

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12
Q

Citrate Utilization

Positive Result?

A
  • Growth on the medium, with or without a change in the color of the indicator
  • Growth typically results in the bromothymol blue indicator turning from green to blue
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13
Q

Citrate Utilization

Negative Result?

A

Absence of growth

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14
Q

Citrate Utilization

Limitations of Citrate Utilization?

A
  • Some organisms are capable of growth on citrate and do not produce a color change.

Growth is considered a positive citrate utilization test, even in the absence of a color change

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15
Q

Citrate Utilization

Positive Quality Control?

A

Enterobacter aerogenes: growth with blue color

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16
Q

Citrate Utilization

Negative Quality Control?

A

Escherichia coli: little to no growth with no color change

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17
Q
  • Purpose: This test is used to differentiate decarboxylase-producing Enterobacteriaceae from other gram-negative rods.
A

Decarboxylase Tests (Moeller’s Method)

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18
Q
  • This test measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine.
A

Decarboxylase Test

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19
Q

Decarboxylase Tests

  • Decarboxylation, or hydrolysis, of the amino acid results in an ____ pH and a color change from orange to purple.
A

Alkaline pH

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20
Q

Decarboxylase Tests

  • Decarboxylation, or hydrolysis, of the amino acid results in an alkaline pH and a color change from ____ to ____.
A

Orange to purple

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21
Q

Decarboxylase Tests

Familiarize yourself with the media inclusions.

A
  • Peptic digest of animal tissue (5 g)
  • Beef extract (5 g)
  • Bromocresol purple (0.1 g)
  • Cresol red (0.005 g)
  • Dextrose (0.5 g)
  • Pyridoxal (0.005 g)
  • Amino acid (10 g)
  • pH 6.0
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22
Q

Decarboxylase Tests

T or F: Non-fermenting organisms and fermenting organisms utilize the same methods in decarboxylase tests.

A

F (Different methods)

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23
Q

Decarboxylase Tests

This method is used for:
* Prepare a suspension (McFarland No. 5 turbidity standard) in brain-heart infusion broth from an overnight culture (18 to 24 hours old) growing on 5% sheep blood agar.
* Inoculate each of the three decarboxylase broths (arginine, lysine, and ornithine) and the control broth (no amino acid) with four drops of broth.
* Add a 4-mm layer of sterile mineral oil to each tube.
* Incubate the cultures at 35°C to 37°C in ambient air. Examine the tubes at 24, 48, 72, and 96 hours.

A

Glucose Non-Fermenting Organisms

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24
Q

Decaarboxylase Tests

Glucose non-fermenting organisms should be cultured at what temperature and examined every how many hours?

A

35 to 37 deg C and examined at 24, 48, 72, and 96 hours

Same with glucose fermenting but they are incubated for 4 days.

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25
Q

Decarboxylase Tests

This method is used for:
* Inoculate tubes with one drop of an 18- to 24-hour brain-heart infusion broth culture.
* Add a 4-mm layer of sterile mineral oil to each tube.
* Incubate the cultures for 4 days at 35°C to 37°C in ambient air. Examine the tubes at 24, 48, 72, and 96 hours

A

Glucose-fermenting Organisms

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26
Q

Decarboxylase Tests

Positive Result?

A

Alkaline (purple) color change compared with the control tube

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27
Q

Decarboxylase Test

Negative Result?

A
  • No color change or acid (yellow) color in test and control tube
  • Growth in the control tube
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28
Q

Decarboxylase Tests

T or F: The fermentation of dextrose in the medium causes the acid color change which masks the alkaline color change brought about by a positive decarboxylation reaction.

A

F (it does not mask the alkaline color change)

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29
Q

Decarboxylase Tests

Positive Quality Control?

A
  • Lysine: Klebsiella pneumoniae
  • Ornithine: Enterobacter aerogenes
  • Arginine: Pseudomonas aeruginosa

All turns yellow to purple.

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30
Q

Decarboxylase Tests

Base Control?

A

Positive Glucose Fermenters
* Klebsiella pneumoniae
* Enterobacter aerogenes

Both are yellow.

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31
Q

Decarboxylase Tests

Negative Quality Control?

A
  • Lysine: Citrobacter freundii
  • Ornithine: Proteus vulgaris
  • Arginine: Escherichia coli

All are yellow.

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32
Q
  • Purpose: This test is used for the presumptive identification and differentiation of Enterobacteriaceae.
A

Esculin Hydrolysis

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33
Q

Esculin Hydrolysis

  • This test is used to determine whether an organism is able to hydrolyze the ____ .
A

Glycoside esculin

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34
Q
  • Esculin is hydrolyzed to ____, which reacts with Fe3+ and forms a dark brown to black precipitate.
A

Esculetin

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35
Q

Esculin Hydrolysis

Familiarize yourself with the media inclusions.

A
  • NaCl (8 g)
  • K2HPO4 (0.4 g)
  • KH2PO4 (0.1 g)
  • Esculin (5 g)
  • Ferric ammonium citrate (0.5 g)
  • Agar (15 g) per 1000 mL
  • pH 7.0
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36
Q

Esculin Hydrolysis

  • The slants of this test are examined for ____ and under the UV rays of a Wood’s lamp for ____.
A

Blackening and esculin hydrolysis (under the Wood’s lamp)

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37
Q

Esculin Hydrolysis

Positive Result?

A

Blackened medium which would also show a loss of fluorescence under the Wood’s lamp

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38
Q

Esculin Hydrolysis

Negative Result?

A
  • No blackening and no loss of fluorescence under the Wood’s lamp

OR

  • Slight blackening with no loss of fluorescence under the Wood’s lamp
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39
Q

Esculin Hydrolysis

T or F: This medium is a selective agar.

A

F (non-selective)

40
Q

Esculin Hydrolysis

The bile esculin hydrolysis test presented in the book is a ____ differential method.

41
Q

Esculin Hydrolysis

Positive Quality Control?

A

Enterococcus faecalis

42
Q

Esculin Hydrolysis

Negative Quality Control?

A

Escherichia coli

43
Q
  • Purpose: The production of gelatinases capable of hydrolyzing gelatin is used as a presumptive test for the identification of various organisms, including Staphylococcus spp., Enterobacteriaceae, and some gram-positive bacilli.
A

Gelatin Hydrolysis

44
Q
  • This test is used to determine the ability of an organism to
    produce extracellular proteolytic enzymes (gelatinases) that liquefy gelatin, a component of vertebrate connective tissue.
A

Gelatin Hydrolysis

45
Q

Gelatin Hydrolysis

T or F: Nutrient gelatin medium differs from traditional microbiology media in that the solidifying agent (agar) is replaced with gelatin.

46
Q

Gelatin Hydrolysis

When an organism produces gelatinase, the enzyme ____ the growth medium.

47
Q

Gelatin Hydrolysis

Familiarize yourself with the media inclusions.

A
  • Enzymatic digest of gelatin (5 g)
  • Beef extract (3 g)
  • Gelatin (120 g) per 1000 mL
  • pH 6.8
48
Q

Gelatin Hydrolysis

The culture is incubated at what temperature for up to how many days?

A

35 to 37°C for up to 14 days

49
Q

T or F: We can incubate the medium at 25°C if it grows better at 25°C than 35°C.

50
Q

Gelatin Hydrolysis

The gelatin tube is removed daily from the incubator and placed at what temperature to check for liquefaction?

51
Q

Gelatin Hydrolysis

T or F: We are allowed to invert or tip the tube in gelatin hydrolysis.

52
Q

Gelatin Hydrolysis

Why are we not allowed to invert or tip the tube?

A

Because sometimes the only discernible liquefaction occurs at the top of the deep where inoculation occurred

53
Q

Gelatin Hydrolysis

Liquefaction is determiend only when?

A

After the control has hardened or gelled.

54
Q

Gelatin Hydrolysis

Positive Result?

A

Partial or total liquefaction of the inoculated tube (the control tube must be completely solidified) at 4°C within 14 days

55
Q

Gelatin Hydrolysis

Negative Result?

A

Complete solidification of the tube at 4°C

56
Q

Gelatin Hydrolysis

T or F: Some organisms may grow poorly or not at all in this medium.

57
Q

Gelatin Hydrolysis

Gelatin is liquid ____ therefore determination of results
must be completed after refrigeration.

A

above 20°C

58
Q

Gelatin Hydrolysis

Determination of results in gelatin hydrolysis must be completed ____ refrigeration.

59
Q

Gelatin Hydrolysis

Positive Quality Control?

A

Bacillus subtilis

60
Q

Gelatin Hydrolysis

Negative Quality Control?

A

Escherichia coli

61
Q

Gelatin Hydrolysis

Uninoculated Control Tube?

A

Medium becomes solid after refrigeration

62
Q
  • Purpose: This test is used to differentiate a pyocyanogenic pseudomonad from other Pseudomonas spp.
A

Growth at 42°C

63
Q
  • The test is used to determine the ability of an organism to grow at 42°C.
  • Several Pseudomonas spp. have been isolated in the clinical laboratory that are capable of growth at elevated temperatures.
A

Growth at 42°C

64
Q

Growth at 42°C

Tubes are incubated at what two temperatures?

A

42°C and 35°C

65
Q

Growth at 42°C

Positive Result?

A

Good growth at both 35°C and 42°C

66
Q

Growth at 42°C

Negative Result?

A

No growth at 42°C but good growth at 35°C

67
Q

Growth at 42°C

Positive Quality Control?

A

Pseudomonas aeruginosa

68
Q

Growth at 42°C

Negative Quality Control?

A

Pseudomonas fluorescens

69
Q
  • Purpose: Production of the enzyme hippuricase is used for the presumptive identification of a variety of microorganisms.
A

Hippurate Hydrolysis

70
Q

Hippurate Hydrolysis

The end products of hydrolysis of hippuric acid by hippuricase
include ____ and ____.

A

Glycine and benzoic acid

71
Q

Hippurate Hydrolysis

  • Glycine is deaminated by the oxidizing agent ____, which is reduced during the process.
72
Q

Hippurate Hydrolysis

The end products of the ninhydrin oxidation react to form a ____ product.
.

Clue: Ma’am TJ

A

Purple-colored product

73
Q

Hippurate Hydrolysis

T or F: The test medium must contain only hippurate, because ninhydrin might react with any free amino acids present in growth media or other broths.

74
Q

Hippurate Hydrolysis

The tube is incubated for how long at what temperature?

A

2 hours at 35°C

75
Q

Hippurate Hydrolysis

Positive Result?

A

Deep purple color

76
Q

Hippurate Hydrolysis

Negative Results?

A

Colorless or slightly yellow pink color

77
Q

Hippurate Hydrolysis

A false ____ result may occur if incubation with ninhydrin exceeds 30 minutes.

A

False-positive

78
Q

Hippurate Hydrolysis

Positive Quality Control?

A

Streptococcus agalactiae

79
Q

Hippurate Hydrolysis

Negative Quality Control?

A

Streptococcus pyogenes

80
Q
  • Purpose: This test is used to identify organisms that produce the enzyme tryptophanase.
A

Indole Production

81
Q
  • The test is used to determine an organism’s ability to hydrolyze tryptophan to form the compound indole.
A

Indole Production

82
Q

Indole Production

Tryptophan is present in ____ and ____.

A

Casein and animal protein

83
Q

Indole Production

T or F: Bacteria without tryptophanase are capable of hydrolyzing tryptophan to pyruvate, ammonia, and
indole.

A

F (with tryptophanase)

84
Q

Indole Production

Kovach’s reagent + broth culture = ?

85
Q

Indole Production

Ehrlich’s reagent contains ____ which makes it flammable.

A

Ethyl alcohol

86
Q

Indole Production

T or F: Ehrlich’s reagent is more sensitive for detecting small amount of indole.

87
Q

Indole Production

Familiarize yourself with the media inclusions.

A
  • Casein peptone (10 g)
  • NaCl (5 g)
  • Tryptophan (10 g) per 1000 mL
88
Q

Indole Production

This method is used for:
* Inoculate tryptophane broth with one drop from a 24-hour brain-heart infusion broth culture.
* Incubate at 35°C to 37°C in ambient air for 48 hours.
* Add 0.5 mL of Kovac’s reagent to the broth culture.

A

Enterobacteriaceae

89
Q

Indole Production

This method is used for:
* Inoculate tryptophane broth with one drop of a 24-hour broth
culture.
* Incubate at 35°C to 37°C in ambient air for 48 hours.
* Add 1 mL of xylene to the culture.
* Shake the mixture vigorously to extract the indole and allow it to stand until the xylene forms a layer on top of the aqueous phase.
* Add 0.5 mL of Ehrlich’s reagent down the side of the tube

A

Other Gram-Negative Bacilli

90
Q

Indole Production

Positive Result?

A

Pink- to wine-colored ring after addition of appropriate reagent

91
Q

Indole Production

Negative Result?

A

No color change after addition of the appropriate reagent

92
Q

Indole Production

T or F: Ehrlich’s method may also be used to differentiate organisms under aerobic conditions.

A

F (anaerobic)

93
Q

Indole Production

Quality Control for Kovac’s Method

A

Positive: Escherichia coli
Negative: Klebsiella pneumoniae

94
Q

Indole Production

Quality Control for Ehrlich’s Method?

A

Positive: Haemophilus influenzae
Negative: Haemophilus parainfluenza

95
Q

Indole Production

Quality Control for Ehrlich’s Method (Anaerobic)?

A

Positive: Porphyromonas asaccharolytica
Negative: Bacteroides fragilis

Clinical Bacteriology (33 decks)

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