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Clinical Bacteriology > (F) Lesson 11.3: Diagnostic Tests for Gram-Negative Bacilli > Flashcards

(F) Lesson 11.3: Diagnostic Tests for Gram-Negative Bacilli Flashcards

1
Q
  • Purpose/s: This test is used to determine the ability of an organism to produce b-galactosidase, an enzyme that hydrolyzes the substrate o-nitrophenyl-b-D-galactopyranoside (ONPG) to form a visible (yellow) product, ortho-nitrophenol.
  • The test distinguishes late lactose fermenters from non–lactose fermenters of Enterobacteriaceae.
A

o-Nitrophenyl-b-D-Galactopyranoside (ONPG) Test

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2
Q

ONPG Test

  • ____ must be able to transport the carbohydrate (b-galactoside permease) and hydrolyze (b-galactosidase) the lactose to glucose and galactose.
A

Lactose fermenters

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3
Q

ONPG Test

  • Organisms unable to produce b-galactosidase may become genetically altered through a variety of mechanisms and be identified as?
A

Late-lactose fermenters

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4
Q

ONPG Test

Familiarize yourself with the media inclusions for tube method.

A
  • Na2HPO4 (9.46 g)
  • Phenylalanine (4 g)
  • ONPG (2 g)
  • KH2PO4 (0.907 g) per 1000 mL
  • pH 8.0
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5
Q

ONPG Test

Positive Result?

A

Yellow (presence of b-galactosidase)

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6
Q

ONPG Test

Negative Result?

A

Colorless (absence of enzyme)

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7
Q

ONPG Test

Positive Quality Control?

A

Shigella sonnei

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8
Q

ONPG Test

Negative Quality Control?

A

Salmonella typhimurium

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9
Q
  • Purpose: This test determines the presence of cytochrome oxidase activity in microorganisms for the identification of oxidase-negative Enterobacteriaceae, differentiating them from other gram-negative bacilli.
A

Oxidase Test (Kovac’s Method)

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10
Q
  • To determine the presence of bacterial cytochrome oxidase using the oxidation of the substrate tetramethyl-p-phenylenediamine dihydrochloride to indophenol, a dark purple–colored end product.
A

Oxidase Test (Kovac’s Method)

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11
Q

Oxidase Test (Kovac’s Method)

Positive Result?

A

Development of a dark purple color within 10 seconds

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12
Q

Oxidase Test (Kovac’s Method)

Negative Result?

A

Absence of color

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13
Q

Oxidase Test (Kovac’s Method)

Using nickel-base alloy wires containing chromium and iron
(nichrome) to rub the colony paste onto the filter paper may
cause what type of results?

A

False-positive

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14
Q

Oxidase Test (Kovac’s Method)

Positive Quality Control?

A

Pseudomonas aeruginosa

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15
Q

Oxidase Test (Kovac’s Method)

Negative Quality Control?

A

Escherichia coli

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16
Q
  • Purpose: This test is used to determine the presence of the enzyme tryptophanase.
  • It is a rapid method that can be used in lieu of Indole Production.
A

Spot Indole Test

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17
Q

Spot Indole Test

This breaks down tryptophan to release indole, which
is detected by its ability to combine with certain aldehydes to
form a colored compound.

A

Tryptophanase

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18
Q

Spot Indole Test

Positive Result?

A

Development of a blue color within 20 seconds

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19
Q

Spot Indole Test

Negative Result?

A

No color development or slightly pink color

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20
Q

Spot Indole Test

The bacterial inoculum should not be selected from ____ because the color of lactose-fermenting colonies on this medium can interfere with test interpretation.

A

MacConkey agar

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21
Q

Spot Indole Test

Positive Quality Control?

A

Escherichia coli

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22
Q

Spot Indole Test

Negative Quality Control

A

Klebsiella pneumoniae

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23
Q
  • Purpose/s: This used to determine whether a gram-negative rod ferments glucose and lactose or sucrose and forms hydrogen sulfide (H2S).
  • The test is used primarily to differentiate members of the Enterobacteriaceae family from other gram-negative rods.
A

Triple Sugar Iron (TSI) Agar

24
Q

TSI Agar

Composition of TSI

A
  • 10 parts lactose
  • 10 parts sucrose
  • 1 part glucose and peptone
25
Q

TSI Agar

Indicator for acidification?

A

Phenol red

26
Q

TSI Agar

Serves as indicator for H2S formation?

A

Ferrous sulfate

27
Q

TSI Agar

The butt remains acidic after the recommended 18- to 24-hour incubation period because of?

A

The presence of organic acids

From the fermentation of glucose under anaerobic conditions in the butt of the tube

28
Q

TSI Agar

The slant reverts to the alkaline (red) state because of?

A

Oxidation of the fermentation products under aerobic conditions
on the slant

29
Q

TSI Agar

T or F: Reactions in TSI should not be read beyond 24 hours of incubation.

A

T

Aerobic oxidation of the fermentation products from lactose and/or sucrose proceeds, and the slant eventually reverts to the alkaline state.

30
Q

TSI Agar

The formation of CO2 and hydrogen gas (H2) is indicated by?

A

The presence of bubbles or cracks in the agar or by separation of the agar from the sides or bottom of the tube

31
Q

TSI Agar

This requires an acidic environment, and reaction with
the ferric ammonium citrate produces a blackening of the agar
butt in the tube.

A

Production of H2S

32
Q

TSI Agar

Familiarize yourself with the media inclusions.

A
  • : Enzymatic digest of casein (5 g)
  • Enzymatic digest of animal tissue (5 g)
  • Yeast-enriched peptone (10 g)
  • Dextrose (1 g)
  • Lactose (10 g)
  • Sucrose (10 g)
  • Ferric ammonium citrate (0.2 g)
  • NaCl (5 g)
  • Sodium thiosulfate (0.3 g)
  • Phenol red (0.025 g)
  • Agar (13.5 g) per 1000 mL
  • pH 7.3
33
Q

TSI Agar

  • Expected results for glucose, lactose, and sucrose nonutilizer
  • This may also be recorded as K/K (alkaline slant/alkaline butt)
A

Alkaline slant/no change in the butt (K/NC)

34
Q

TSI Agar

Expected results for glucose fermentation only?

A

Alkaline slant/acid butt (K/A)

35
Q

TSI Agar

Expected results for glucose, sucrose, and/or lactose fermenter?

A

Acid slant/acid butt (A/A)

36
Q

TSI Agar

Ⓐ, gas production Quality Control

A

Escherichia coli

37
Q

TSI Agar

K/A, 1/2 gas production, H2S+ Quality Control?

A

Salmonella typhimurium

38
Q

TSI Agar

K/K Quality Control?

A

Pseudomonas aeruginosa

39
Q

TSI Agar

K/A, H2S+ Quality Control?

A

Proteus mirabilis

40
Q

TSI Agar

K/A Quality Control?

A

Shigella flexneri

41
Q
  • Purpose/s: This test is used to determine an organism’s ability to produce the enzyme urease, which hydrolyzes urea.
  • Proteus spp. may be presumptively identified by the ability to rapidly hydrolyze urea.
A

Urease Test (Christensen’s Method)

42
Q

Urease Test

The product of decarboxylation of amino acids

A

Urea

43
Q

Urease Test

Hydrolysis of urea produces ____ and ____

A

Ammonia and CO2

44
Q

Urease Test

Rapid urease-positive organisms turn the entire
medium?

A

Pink within 24 hours

45
Q

Urease Test

T or F: Weakly positive organisms may take several days, and negative organisms produce no color change or purple as a result of acid production.

A

F (yellow not purple)

46
Q

Urease Test

Familiarize yourself with the media inclusions.

A
  • Enzymatic digest of gelatin (1 g)
  • Dextrose (1 g)
  • NaCl (5 g)
  • KH2PO4 (2 g)
  • Urea (20 g)
  • Phenol red (0.012 g) per 1000 mL
  • pH 6.8
47
Q

Urease Test

Positive Result?

A

Change in color of slant from light orange to magenta

48
Q

Urease Test

Negative Result?

A

No color change (agar slant and butt remain light orange)

49
Q

Urease Test

T or F: Acidic reactions may appear after prolonged incubation and may be the result of peptone or other protein utilization raising the pH.

A

F (Alkaline reactions)

50
Q

Urease Test

Positive Quality Control?

A

Proteus vulgaris

51
Q

Urease Test

Weak Positive Quality Control?

A

Klebsiella pneumoniae

52
Q

Urease Test

Negative Quality Control?

A

Escherichia coli

53
Q
  • Purpose: Used to differentiate Haemophilus spp.
    Members of the genus Haemophilus require accessory growth
    factors in vitro.
A

X and V Factor Test

54
Q

X and V Factor Test

Positive Result?

A
  • Growth around the XV disk only shows a requirement for
    both factors
  • Growth around the V disk, no growth around the X disk, and light growth around the XV disk shows a V factor requirement
55
Q

X and V Factor Test

Negative Result?

A

Growth over the entire surface of the agar indicates no
requirement for either X or V factor

56
Q

X and V Factor Test

Positive Quality Control?

A
  • Haemophilus influenza: halo of growth around the XV disk, no growth on the rest of the agar surface
  • Haemophilus parainfluenza: halo of growth around the XV and V disk
  • Haemophilus ducreyi: halo of growth around the XV and X disks

Clinical Bacteriology (33 decks)

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