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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
# TSI Agar Indicator for acidification?
Phenol red
26
# TSI Agar Serves as indicator for H2S formation?
Ferrous sulfate
27
# TSI Agar The butt remains acidic after the recommended 18- to 24-hour incubation period because of?
The presence of organic acids ## Footnote From the fermentation of glucose under anaerobic conditions in the butt of the tube
28
# TSI Agar The slant reverts to the alkaline (red) state because of?
Oxidation of the fermentation products under aerobic conditions on the slant
29
# TSI Agar T or F: Reactions in TSI should not be read beyond 24 hours of incubation.
T ## Footnote Aerobic oxidation of the fermentation products from lactose and/or sucrose proceeds, and the slant eventually reverts to the alkaline state.
30
# TSI Agar The formation of CO2 and hydrogen gas (H2) is indicated by?
The presence of bubbles or cracks in the agar or by separation of the agar from the sides or bottom of the tube
31
# TSI Agar This requires an acidic environment, and reaction with the ferric ammonium citrate produces a blackening of the agar butt in the tube.
Production of H2S
32
# TSI Agar Familiarize yourself with the media inclusions.
* : 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
# TSI Agar * Expected results for glucose, lactose, and sucrose nonutilizer * This may also be recorded as K/K (alkaline slant/alkaline butt)
Alkaline slant/no change in the butt (K/NC)
34
# TSI Agar Expected results for glucose fermentation only?
Alkaline slant/acid butt (K/A)
35
# TSI Agar Expected results for glucose, sucrose, and/or lactose fermenter?
Acid slant/acid butt (A/A)
36
# TSI Agar **Ⓐ, gas production** Quality Control
Escherichia coli
37
# TSI Agar **K/A, 1/2 gas production, H2S+** Quality Control?
Salmonella typhimurium
38
# TSI Agar **K/K** Quality Control?
Pseudomonas aeruginosa
39
# TSI Agar **K/A, H2S+** Quality Control?
Proteus mirabilis
40
# TSI Agar **K/A** Quality Control?
Shigella flexneri
41
* **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.
Urease Test (Christensen's Method)
42
# Urease Test The product of decarboxylation of amino acids
Urea
43
# Urease Test Hydrolysis of urea produces ____ and ____
Ammonia and CO2
44
# Urease Test Rapid urease-positive organisms turn the entire medium?
Pink within 24 hours
45
# 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.
F (yellow not purple)
46
# Urease Test Familiarize yourself with the media inclusions.
* 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
# Urease Test Positive Result?
Change in color of slant from light orange to magenta
48
# Urease Test Negative Result?
No color change (agar slant and butt remain light orange)
49
# 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.
F (Alkaline reactions)
50
# Urease Test Positive Quality Control?
Proteus vulgaris
51
# Urease Test Weak Positive Quality Control?
Klebsiella pneumoniae
52
# Urease Test Negative Quality Control?
Escherichia coli
53
* **Purpose:** Used to differentiate Haemophilus spp. Members of the genus Haemophilus require accessory growth factors in vitro.
X and V Factor Test
54
# X and V Factor Test Positive Result?
* 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
# X and V Factor Test Negative Result?
Growth over the entire surface of the agar indicates no requirement for either X or V factor
56
# X and V Factor Test Positive Quality Control?
* **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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