(F) Lesson 11.1: Diagnostic Tests for Gram-Negative Bacilli Flashcards
- Purpose: To identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source
Citrate Utilization
- The test is part of a series referred to as IMViC which is used to differentiate Enterobacteriaceae from other gram-negative rods
Citrate Utilization
Citrate Utilization
- Bacteria that can grow on this medium produce an enzyme called ____ capable of converting citrate to pyruvate.
Citrate-permease
- ____ can then enter the organism’s metabolic cycle for the
production of energy
Pyruvate
Citrate Utilization
- Bacteria capable of growth in this medium
use the citrate and convert ____ to ____ and ____, creating an alkaline pH.
Convert ammonium phosphate to ammonia and ammonium hydroxide
Citrate Utilization
- The pH change turns the bromothymol blue indicator from ____ to ____.
Green to blue
Citrate Utilization
Familiarize yourselves with the media inclusions.
- 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
Citrate Utilization
T or F: We should inoculate from a broth culture to ensure that the inoculum is heavy.
F (We should not because it will be too heavy)
Citrate Utilization
At what temperature should we incubate the media?
35 to 37 deg C
Citrate Utilization
We should observe ____ and the development of ____, denoting alkalinization.
Growth and the development of blue color
Citrate Utilization
Growth and development of blue color denotes what?
Alkalinization
Citrate Utilization
Positive Result?
- 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
Citrate Utilization
Negative Result?
Absence of growth
Citrate Utilization
Limitations of Citrate Utilization?
- 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
Citrate Utilization
Positive Quality Control?
Enterobacter aerogenes: growth with blue color
Citrate Utilization
Negative Quality Control?
Escherichia coli: little to no growth with no color change
- Purpose: This test is used to differentiate decarboxylase-producing Enterobacteriaceae from other gram-negative rods.
Decarboxylase Tests (Moeller’s Method)
- This test measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine.
Decarboxylase Test
Decarboxylase Tests
- Decarboxylation, or hydrolysis, of the amino acid results in an ____ pH and a color change from orange to purple.
Alkaline pH
Decarboxylase Tests
- Decarboxylation, or hydrolysis, of the amino acid results in an alkaline pH and a color change from ____ to ____.
Orange to purple
Decarboxylase Tests
Familiarize yourself with the media inclusions.
- 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
Decarboxylase Tests
T or F: Non-fermenting organisms and fermenting organisms utilize the same methods in decarboxylase tests.
F (Different methods)
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.
Glucose Non-Fermenting Organisms
Decaarboxylase Tests
Glucose non-fermenting organisms should be cultured at what temperature and examined every how many hours?
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.
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
Decarboxylase Tests
Positive Result?
Alkaline (purple) color change compared with the control tube
Decarboxylase Test
Negative Result?
- No color change or acid (yellow) color in test and control tube
- Growth in the control tube
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)
Decarboxylase Tests
Positive Quality Control?
- Lysine: Klebsiella pneumoniae
- Ornithine: Enterobacter aerogenes
- Arginine: Pseudomonas aeruginosa
All turns yellow to purple.
Decarboxylase Tests
Base Control?
Positive Glucose Fermenters
* Klebsiella pneumoniae
* Enterobacter aerogenes
Both are yellow.
Decarboxylase Tests
Negative Quality Control?
- Lysine: Citrobacter freundii
- Ornithine: Proteus vulgaris
- Arginine: Escherichia coli
All are yellow.
- Purpose: This test is used for the presumptive identification and differentiation of Enterobacteriaceae.
Esculin Hydrolysis
Esculin Hydrolysis
- This test is used to determine whether an organism is able to hydrolyze the ____ .
Glycoside esculin
- Esculin is hydrolyzed to ____, which reacts with Fe3+ and forms a dark brown to black precipitate.
Esculetin
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
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)
Esculin Hydrolysis
Positive Result?
Blackened medium which would also show a loss of fluorescence under the Wood’s lamp
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
Esculin Hydrolysis
T or F: This medium is a selective agar.
F (non-selective)
Esculin Hydrolysis
The bile esculin hydrolysis test presented in the book is a ____ differential method.
Selective
Esculin Hydrolysis
Positive Quality Control?
Enterococcus faecalis
Esculin Hydrolysis
Negative Quality Control?
Escherichia coli
- 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
- 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
Gelatin Hydrolysis
T or F: Nutrient gelatin medium differs from traditional microbiology media in that the solidifying agent (agar) is replaced with gelatin.
T
Gelatin Hydrolysis
When an organism produces gelatinase, the enzyme ____ the growth medium.
Liquefies
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
Gelatin Hydrolysis
The culture is incubated at what temperature for up to how many days?
35 to 37°C for up to 14 days
T or F: We can incubate the medium at 25°C if it grows better at 25°C than 35°C.
T
Gelatin Hydrolysis
The gelatin tube is removed daily from the incubator and placed at what temperature to check for liquefaction?
4°C
Gelatin Hydrolysis
T or F: We are allowed to invert or tip the tube in gelatin hydrolysis.
F
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
Gelatin Hydrolysis
Liquefaction is determiend only when?
After the control has hardened or gelled.
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
Gelatin Hydrolysis
Negative Result?
Complete solidification of the tube at 4°C
Gelatin Hydrolysis
T or F: Some organisms may grow poorly or not at all in this medium.
T
Gelatin Hydrolysis
Gelatin is liquid ____ therefore determination of results
must be completed after refrigeration.
above 20°C
Gelatin Hydrolysis
Determination of results in gelatin hydrolysis must be completed ____ refrigeration.
After
Gelatin Hydrolysis
Positive Quality Control?
Bacillus subtilis
Gelatin Hydrolysis
Negative Quality Control?
Escherichia coli
Gelatin Hydrolysis
Uninoculated Control Tube?
Medium becomes solid after refrigeration
- Purpose: This test is used to differentiate a pyocyanogenic pseudomonad from other Pseudomonas spp.
Growth at 42°C
- 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
Growth at 42°C
Tubes are incubated at what two temperatures?
42°C and 35°C
Growth at 42°C
Positive Result?
Good growth at both 35°C and 42°C
Growth at 42°C
Negative Result?
No growth at 42°C but good growth at 35°C
Growth at 42°C
Positive Quality Control?
Pseudomonas aeruginosa
Growth at 42°C
Negative Quality Control?
Pseudomonas fluorescens
- Purpose: Production of the enzyme hippuricase is used for the presumptive identification of a variety of microorganisms.
Hippurate Hydrolysis
Hippurate Hydrolysis
The end products of hydrolysis of hippuric acid by hippuricase
include ____ and ____.
Glycine and benzoic acid
Hippurate Hydrolysis
- Glycine is deaminated by the oxidizing agent ____, which is reduced during the process.
Ninhydrin
Hippurate Hydrolysis
The end products of the ninhydrin oxidation react to form a ____ product.
.
Clue: Ma’am TJ
Purple-colored product
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
Hippurate Hydrolysis
The tube is incubated for how long at what temperature?
2 hours at 35°C
Hippurate Hydrolysis
Positive Result?
Deep purple color
Hippurate Hydrolysis
Negative Results?
Colorless or slightly yellow pink color
Hippurate Hydrolysis
A false ____ result may occur if incubation with ninhydrin exceeds 30 minutes.
False-positive
Hippurate Hydrolysis
Positive Quality Control?
Streptococcus agalactiae
Hippurate Hydrolysis
Negative Quality Control?
Streptococcus pyogenes
- Purpose: This test is used to identify organisms that produce the enzyme tryptophanase.
Indole Production
- The test is used to determine an organism’s ability to hydrolyze tryptophan to form the compound indole.
Indole Production
Indole Production
Tryptophan is present in ____ and ____.
Casein and animal protein
Indole Production
T or F: Bacteria without tryptophanase are capable of hydrolyzing tryptophan to pyruvate, ammonia, and
indole.
F (with tryptophanase)
Indole Production
Kovach’s reagent + broth culture = ?
Red color
Indole Production
Ehrlich’s reagent contains ____ which makes it flammable.
Ethyl alcohol
Indole Production
T or F: Ehrlich’s reagent is more sensitive for detecting small amount of indole.
T
Indole Production
Familiarize yourself with the media inclusions.
- Casein peptone (10 g)
- NaCl (5 g)
- Tryptophan (10 g) per 1000 mL
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
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
Indole Production
Positive Result?
Pink- to wine-colored ring after addition of appropriate reagent
Indole Production
Negative Result?
No color change after addition of the appropriate reagent
Indole Production
T or F: Ehrlich’s method may also be used to differentiate organisms under aerobic conditions.
F (anaerobic)
Indole Production
Quality Control for Kovac’s Method
Positive: Escherichia coli
Negative: Klebsiella pneumoniae
Indole Production
Quality Control for Ehrlich’s Method?
Positive: Haemophilus influenzae
Negative: Haemophilus parainfluenza
Indole Production
Quality Control for Ehrlich’s Method (Anaerobic)?
Positive: Porphyromonas asaccharolytica
Negative: Bacteroides fragilis
