Biochemical Identification Of Gram Negative Bacteria

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All Enterobacteriaceae

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• Ferment Glucose
• Reduce Nitrate
• Oxidase Negative

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Enterobacteriaceae that is not oxidase negative

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Plesiomonas shigelloides

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Motility of Enterobacteriacae

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Most are

Except: Klebsiella, Shigella, Yersinia

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Colony Morphology on Nonselective Media

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Enterobacteriacae cannot be identified by colony morphology on nonselective media

Large grey colonies, Klebsiella (mucoid wet colonies)

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Reduction of NO3

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to NO2

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What reagent is added to nitrate test?

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N,N-dimethyl-1 naphthylamine

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If no red color after the first reagent in nitrate test what do you add?

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Zinc

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Red color after addition of zinc in nitrate test

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Nitrate was not reduced and is still in the tube

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No color after addition of zinc in nitrate test

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That mean nitrate was reduced all the way to N2 gas

Positive reaction

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Nitrate chemical formula

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NO3-->NO2-->N2

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Oxidase test is looking for

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The presence of cytochrome oxidase

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Enterobacteriaceae is oxidase

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Negative

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Oxidase positive organism

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• Neisseria
• Moraxella
• Eikenella
• Pseudomonas, etc

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Traditional Biochemical tests for Gram negative bacteria

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• TSI slant
• MR/VP- glucose fermentation
• Indole- if indole is formed by tryptophan
• Urease- hydrolysis of urea
• Carbohydrate fermentation
• Lysine iron agar (LIA)
• Sulfide-indole-motility (SIM) or motility-indole ornithine (MIO) meadia
• Citrate-carbon source

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Two enzymes for lactose degradation

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• Beta-galactosidase permease
• Beta-galactosidase

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Enterobacteriacae oxygen use; can be called?

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Facultative anaerobe

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Slow lactose fermenters

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lack permease but have Beta-galactosidase

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Lactose

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glucose and galactose

Useful for identifying enteric pathogens

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Embden-Meyerhoff pathway

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Two molecules of pyruvic acid

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Enter-Doudoroff pathway

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Glyceraldehyde-3-phosphate

Pyruvic acid

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Oxidation

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utilize carbohydrate aerobically

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Fermentation

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utilize carbohydrates anaerobically

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Asaccharolytic

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do not utilize carbohydrates

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Fermentation

Glycolysis pathway:

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Glucose to pyruvate: oxidized to other acid products and gases

pH indicators detect acid production

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Oxidation

Glycolysis pathway

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Glucose to pyruvate--> CO2

weak acid; neutralized by alkaline reactions from peptones

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Lower peptones in O/F basal media

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help to classify as oxidizer or fermenter

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Two tubes for O/F basal media

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• Aerobic
• Anaerobic

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O/F basal media Anaerobic tube outcomes

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• Aerobic- only open tube
• Anaerobic- only closed tube
• Facultative- both tubes

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TSI agar contains the following sugars

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• Lactose
• Sucrose
• Ratio of Lactose and sucrose present in 10:1 to glucose
• KIA is similar but no sucrose

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What detects the production of H2S in TSI

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Ferrous sulfate and sodiu thiosulfate

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Indicator in TSI

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phenol red

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MacConkey Agar selects for Gram negative enter by:

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Bile salts and crystal violet

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MAC agar differentiats

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lactose fermentors from non-fermenters

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MAC red/pink colonies

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fermentation of lactose

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MAC not red or clear

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no fermentation of lactose

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E. coli on Eosin Methylene Blue Agar

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Metallic green sheen: dye precipitate indicating lactose fermenting

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K. pneumoniae on EMB

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Mucoid colonies (due to capsule)

Dark center: Lactose fermentation

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Ortho-Nitrophenyl-beta-D-Galactopyranoside (ONPG) Test what enzyme is it looking for?

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Beta-galactosidase

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ONPG is a test focusing on which type of organism

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Slow lactose fermentors: ONPG is easily transferred inside the cell

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ONPG is hydrolyzed into

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galactose

O-nitrophenol (yellow color)

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ONPG non-lactose fermentor

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clear

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IMViC

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• Indole
• Methyl Red
• Voges Proskauer
• Citrate

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IMViC allows for the differentiation of:

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Enterobacteriaceae

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The indole test looks for

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Tryptophanase

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Enzymatic degradation in the indole test leads to the production of :

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• Pyruvic acid
• Indole
• Ammonia

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Ehrlich's Indole test

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Indole extracted by xylene:

Ehrlich's reagent: PDAB

Positive: Red color

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Kovac's

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Reagent in the indole test

Add five drops

Positive: Red

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Incubation in VP/MR

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2 days

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Methyl Red

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Glucose is metabolized by mixed acid pathway

Red- Positive

pH 4.4

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Glucose usage in MR

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Utilizes dropping the pH to 4.4

Methyl Red is a pH indicator: Turns red in strongly acidic reactions

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Glucose is metabolized by butylene glycol pathway

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Voges Proskauer

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End product in VP

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2,3- butanediol hard to detect

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What is being detected in VP

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acetylmethylcarbinol (acetoin)

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Reagents in VP

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KOH

alpha-naphthol

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End products in VP

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not as acidic would not be positive in MR

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Use of citrate results in:

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alkaline pH

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Positive citrate

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blue

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Citrate inoculum

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must be light (organisms will grow off carbon of dead bacteria)

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The use of citrate as a sole source of carbon will

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Increase the pH (alkaline)

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Indicator in citrate

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bromothymol blue

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Urease agar contains

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urea and phenol red

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Urea is converted to

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CO2 and NH3

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The ammonia in the urea reaction:

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combines with water to increase the pH causing a pink color

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Decarboxylase tests

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the presence of enzymes capable of removing carboxyl groups

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Decarboxylase needs

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anaerobic conditions (oil)

acidic conditions (glucose fermentation)

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Lysine -->

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cadaverine + CO2

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Ornithine-->

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putrescine

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Dihydrolase

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degrades arginine to ornithine

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Arginine -->

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cutrulline-->ornithine--> putrescine

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The final product of a decarboxylase reaction

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alkaline amines and carbon dioxideIn

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Indicator decarboxylase

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bromocresol purple

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Malonate

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Can be used as a sole carbon source

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Indicator in Malonate

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bromothymol blue

+: green to blue

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Deaminase test

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amino acids are metabolized by deaminases that remove an amine group

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PAD

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phenylalanine deaminase test: produces phenylpyruvic acid

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Reagent to add to PAD

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10% ferric chloride

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PAD is useful in differentiating

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Proteus, Providencia, Morganella

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L-arabinose

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CTA agar containing L-arabinose

Id Serratia marcescens from other members of the genus serratia

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DNase

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Endonucleases produced by bacteria that break phosphodiester bonds

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Gelatin liquefaction

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Gelatinase production: loss of gelling