Tests for Enterobacterales Flashcards
What is the purpose of the ONPG test (o-Nitrophenyl-B-D-Galactopyranoside)?
To determine if organism has B-galactosidase (ferments lactose) regardless of the presence of the enzyme permease. Distinguishes LLF from NLF (permease does not count).
Permease transports lactose into the cell and LLF do not have that enzyme. The ONPG substrate is structurally similar to lactose, and permeates the cell without the enzyme.
Note: B = beta symbol
How do you interpret the ONPG test and what is its limitations?
Pos: Yellow
Neg: No colour change.
If the org has B-galactosidase enzyme, it will cleave ONPG into galactose (structurally similar to lactose) and orthonitrophenol (yellow color).
Test cannot performed on an organism with an obvious yellow pigment.
What are the control of ONPG?
Pos Control: E.coli
Neg Control: Proteus mirabilis
What is the purpose of the ONPG-PAM test?
Used to test an organisms ability to ferment lactose, produce phenylalanine deaminase and motility.
Useful in ID of Morganella, Providencia, and Proteus spp.
PMP group is Phenylalanine +.
How does ONPG-PAM detect LF, Phenylalanine deaminase, and motility?
LF - from ONPG substrate - yellow color means pos.
Phenylalanine is in media, if the org has phenylalanine deaminase it will deaminate it to
phenylpyruvic acid detected by adding 10% FECl3 which turns green if pos.
Motility - detected by lower agar conc. - diffuse growth from line of inoculum.
What is the reagent in the oxidase test?
di(tetra)methyl-p-phenylenediamine dihydrochloride
What is the main purpose of the oxidase test?
Use in ID schemes of most gram neg orgs. RRC: Presumptively IDs Neisseria spp. and separates Enterobacteriacae from Ox pos Pseudomonadaceae and other non-glucose fermenters.
What is the principle of the Oxidase teset?
Cytochrome oxidase (iron containing hemoproteins) participates in the electron transport and in the nitrate metabolic pathways of certain bacteria. The enzyme acts as the last link in the chain of aerobic respiration by transferring electrons to oxygen with the formation of water.
System found in aerobes, facultative anaerobes, microaerophilic orgs but not strict anaerobes.
What are the limitations with performing the Oxidase test?
Cannot be used from colonies from MAC or media containing dyes.
False positives can occur if SS or nichrome loops are used.
False negs may occur in cultures mixed with Pseudomonas with Neisseria (although both are Ox +).
What is the purpose of the Oxidation/Fermentation (O/F) Test?
Differentiate organisms based on their ability to ferment or oxidize carbohydrates (glucose*).
*Many different carbs can be used (maltose, sucrose, lactose, mannitol or xylose).
How are the two different oxidative and fermentative environments created in the O/F test?
- Use of a carb by an oxidative process occurs in an aerobic environment.
- Use of a carb fermentatively occurs in an anaerobic environment.
- Two tubes are used, after inoculation 2/ a straight stab one tube is incubated with the cap loose for oxidative, and the other is overlayed with mineral oil and cap tightened to help create anaerobic environment.
What is the indicator for the O/F test and how does it work?
Indicator is bromothymol blue, which turns yellow in an acid environment. High sugar to peptone ratio is used to reduce alkaline end-products that could neutralize acids produced.
What would be the O/F test results for an only oxidative, fermentative, asaccharolytic (non-saccharolytic) organisms?
Oxidative: +/- (open tube yellow, closed tube green)
Fermentative: +/+ (open tube yellow, closed tube yellow)
Asaccharolytic: -/- (open green or blue, closed tube green or blue)
Yellow means acid products, green alkaline.
What special consideration should be given Pseudomonas, Stenotrophomonas, or Burkholderia for the O/F test?
Should be incubated at 30C. These organisms are more active at lower temperature.
What are the controls for the O/F test?
(-/-) = Alcaligenes faecalis
(+/-) = P. aeruginosa
(+/-) = Enterobacter aerogenes
Controls depends on what carbs are used.
