(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
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
3
Q
Citrate Utilization
- Bacteria that can grow on this medium produce an enzyme called ____ capable of converting citrate to pyruvate.
A
Citrate-permease
4
Q
- ____ can then enter the organism’s metabolic cycle for the
production of energy
A
Pyruvate
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
6
Q
Citrate Utilization
- The pH change turns the bromothymol blue indicator from ____ to ____.
A
Green to blue
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
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)
9
Q
Citrate Utilization
At what temperature should we incubate the media?
A
35 to 37 deg C
10
Q
Citrate Utilization
We should observe ____ and the development of ____, denoting alkalinization.
A
Growth and the development of blue color
11
Q
Citrate Utilization
Growth and development of blue color denotes what?
A
Alkalinization
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
13
Q
Citrate Utilization
Negative Result?
A
Absence of growth
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
15
Q
Citrate Utilization
Positive Quality Control?
A
Enterobacter aerogenes: growth with blue color
16
Q
Citrate Utilization
Negative Quality Control?
A
Escherichia coli: little to no growth with no color change
17
Q
- Purpose: This test is used to differentiate decarboxylase-producing Enterobacteriaceae from other gram-negative rods.
A
Decarboxylase Tests (Moeller’s Method)
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
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
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
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
22
Q
Decarboxylase Tests
T or F: Non-fermenting organisms and fermenting organisms utilize the same methods in decarboxylase tests.
A
F (Different methods)
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
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.
25
# 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
Glucose-fermenting Organisms
26
# Decarboxylase Tests
Positive Result?
**Alkaline (purple) color change** compared with the control tube
27
# Decarboxylase Test
Negative Result?
* **No color change or acid (yellow)** color in test and control tube
* Growth in the control tube
28
# 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.
F (it does not mask the alkaline color change)
29
# Decarboxylase Tests
Positive Quality Control?
* **Lysine:** Klebsiella pneumoniae
* **Ornithine:** Enterobacter aerogenes
* **Arginine:** Pseudomonas aeruginosa
| All turns **yellow to purple.**
30
# Decarboxylase Tests
Base Control?
**Positive Glucose Fermenters**
* Klebsiella pneumoniae
* Enterobacter aerogenes
| Both are **yellow.**
31
# Decarboxylase Tests
Negative Quality Control?
* **Lysine:** Citrobacter freundii
* **Ornithine:** Proteus vulgaris
* **Arginine:** Escherichia coli
| All are **yellow.**
32
* **Purpose:** This test is used for the **presumptive identification and differentiation** of Enterobacteriaceae.
Esculin Hydrolysis
33
# Esculin Hydrolysis
* This test is used to determine whether an organism is able to hydrolyze the ____ .
Glycoside esculin
34
* Esculin is hydrolyzed to ____, which reacts with Fe3+ and forms a **dark brown to black precipitate**.
Esculetin
35
# Esculin Hydrolysis
Familiarize yourself with the media inclusions.
* 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**
36
# Esculin Hydrolysis
* The slants of this test are examined for ____ and under the UV rays of a Wood's lamp for ____.
**Blackening** and **esculin hydrolysis** *(under the Wood's lamp)*
37
# Esculin Hydrolysis
Positive Result?
**Blackened medium** which would also show **a loss of fluorescence** under the Wood’s lamp
38
# Esculin Hydrolysis
Negative Result?
* **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
39
# Esculin Hydrolysis
T or F: This medium is a selective agar.
F (non-selective)
40
# Esculin Hydrolysis
The bile esculin hydrolysis test presented in the book is a ____ differential method.
Selective
41
# Esculin Hydrolysis
Positive Quality Control?
Enterococcus faecalis
42
# Esculin Hydrolysis
Negative Quality Control?
Escherichia coli
43
* **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.*
Gelatin Hydrolysis
44
* 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.
Gelatin Hydrolysis
45
# Gelatin Hydrolysis
T or F: Nutrient gelatin medium differs from traditional microbiology media in that the solidifying agent (agar) is replaced with gelatin.
T
46
# Gelatin Hydrolysis
When an organism produces gelatinase, the enzyme ____ the growth medium.
Liquefies
47
# Gelatin Hydrolysis
Familiarize yourself with the media inclusions.
* Enzymatic digest of gelatin (5 g)
* Beef extract (3 g)
* Gelatin (120 g) per 1000 mL
* **pH 6.8**
48
# Gelatin Hydrolysis
The culture is incubated at what temperature for up to how many days?
**35 to 37°C** for **up to 14 days**
49
T or F: We can incubate the medium at 25°C if it grows better at 25°C than 35°C.
T
50
# Gelatin Hydrolysis
The gelatin tube is removed daily from the incubator and placed at what temperature to check for liquefaction?
4°C
51
# Gelatin Hydrolysis
T or F: We are allowed to invert or tip the tube in gelatin hydrolysis.
F
52
# Gelatin Hydrolysis
Why are we not allowed to invert or tip the tube?
Because sometimes the only discernible liquefaction occurs at the **top of the deep** where inoculation occurred
53
# Gelatin Hydrolysis
Liquefaction is determiend only when?
After the control has hardened or gelled.
54
# Gelatin Hydrolysis
Positive Result?
**Partial or total liquefaction** of the inoculated tube (the control tube must be completely solidified) at **4°C within 14 days**
55
# Gelatin Hydrolysis
Negative Result?
**Complete solidification** of the tube at 4°C
56
# Gelatin Hydrolysis
T or F: Some organisms may grow poorly or not at all in this medium.
T
57
# Gelatin Hydrolysis
Gelatin is liquid ____ therefore determination of results
must be **completed after refrigeration.**
above 20°C
58
# Gelatin Hydrolysis
Determination of results in gelatin hydrolysis must be completed ____ refrigeration.
After
59
# Gelatin Hydrolysis
Positive Quality Control?
Bacillus subtilis
60
# Gelatin Hydrolysis
Negative Quality Control?
Escherichia coli
61
# Gelatin Hydrolysis
Uninoculated Control Tube?
Medium becomes solid after refrigeration
62
* **Purpose:** This test is used to **differentiate a pyocyanogenic pseudomonad** from other Pseudomonas spp.
Growth at 42°C
63
* 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.
Growth at 42°C
64
# Growth at 42°C
Tubes are incubated at what two temperatures?
42°C and 35°C
65
# Growth at 42°C
Positive Result?
Good growth at **both 35°C and 42°C**
66
# Growth at 42°C
Negative Result?
**No growth at 42°C** but **good growth at 35°C**
67
# Growth at 42°C
Positive Quality Control?
Pseudomonas aeruginosa
68
# Growth at 42°C
Negative Quality Control?
Pseudomonas fluorescens
69
* **Purpose:** Production of the enzyme **hippuricase** is used for the presumptive identification of a variety of microorganisms.
Hippurate Hydrolysis
70
# Hippurate Hydrolysis
The end products of hydrolysis of hippuric acid by hippuricase
include ____ and ____.
Glycine and benzoic acid
71
# Hippurate Hydrolysis
* Glycine is deaminated by the oxidizing agent ____, which is reduced during the process.
Ninhydrin
72
# Hippurate Hydrolysis
The end products of the ninhydrin oxidation react to form a ____ product.
.
| Clue: Ma'am TJ
Purple-colored product
73
# 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.
T
74
# Hippurate Hydrolysis
The tube is incubated for how long at what temperature?
2 hours at 35°C
75
# Hippurate Hydrolysis
Positive Result?
Deep purple color
76
# Hippurate Hydrolysis
Negative Results?
Colorless or slightly yellow pink color
77
# Hippurate Hydrolysis
A false ____ result may occur if incubation with ninhydrin exceeds 30 minutes.
False-positive
78
# Hippurate Hydrolysis
Positive Quality Control?
Streptococcus agalactiae
79
# Hippurate Hydrolysis
Negative Quality Control?
Streptococcus pyogenes
80
* **Purpose:** This test is used to identify organisms that produce the enzyme **tryptophanase**.
Indole Production
81
* The test is used to determine an organism’s ability to hydrolyze tryptophan to form the compound **indole.**
Indole Production
82
# Indole Production
Tryptophan is present in ____ and ____.
**Casein** and **animal protein**
83
# Indole Production
T or F: Bacteria without tryptophanase are capable of hydrolyzing tryptophan to pyruvate, ammonia, and
indole.
F (with tryptophanase)
84
# Indole Production
Kovach's reagent + broth culture = ?
Red color
85
# Indole Production
Ehrlich's reagent contains ____ which makes it flammable.
Ethyl alcohol
86
# Indole Production
T or F: Ehrlich's reagent is more sensitive for detecting small amount of indole.
T
87
# Indole Production
Familiarize yourself with the media inclusions.
* Casein peptone (10 g)
* NaCl (5 g)
* Tryptophan (10 g) per 1000 mL
88
# 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.
Enterobacteriaceae
89
# 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
Other Gram-Negative Bacilli
90
# Indole Production
Positive Result?
**Pink- to wine-colored ring** after addition of appropriate reagent
91
# Indole Production
Negative Result?
**No color change** after addition of the appropriate reagent
92
# Indole Production
T or F: Ehrlich’s method may also be used to differentiate organisms under aerobic conditions.
F (anaerobic)
93
# Indole Production
Quality Control for Kovac's Method
**Positive:** Escherichia coli
**Negative:** Klebsiella pneumoniae
94
# Indole Production
Quality Control for Ehrlich's Method?
**Positive:** Haemophilus influenzae
**Negative:** Haemophilus parainfluenza
95
# Indole Production
Quality Control for Ehrlich's Method (Anaerobic)?
**Positive:** Porphyromonas asaccharolytica
**Negative:** Bacteroides fragilis
