Chap 9- Biochemical ID of GN Bacteria

Is this bacteria ID based on phenotypes or genotypes?

• colony morphology

• microscopic morphology

• Gram stain

• Biochemical testing - done in 4 tubes or automated instruments

• Gas liquid chromatography

Phenotypes

Is this bacteria ID based on phenotypes or genotypes?

• nucleic acid assays

• highly, sensitive, specific and rapid

genotypes

What differentiated lactose fermenters (LF) from non lactose fermenters (NLF)?

carbohydrate utlization

Lactose consists of glucose and galactose, making it a…

disaccharide

In lactose degradation, what enzyme transports lactose through the cell wall?

Lactose permease

In lactose degradation, what enzyme breaks lactose into subunits?

beta-galactosidase

If a bacterial species lacks permease, but has beta-galactosidase, they are known as … lactose fermenters

delayed

Fermentation utilizes carbohydrates …

anaerobically

Oxidation utilizes carbohdyrates…

aerobically

Does fermentation or oxidation produce acid products and gases?

fermentations

Do fermentation or oxidation produce weak acids?

oxidation

In the oxidation glycolysis pathway, pyruvate is oxidized to … unlike in the fermentation pathway, where pyruvate is oxidized to …

CO2, acids

The test that determines whether a bacteria is an oxidizer or fermenter is the

O/F Basal medium

In the O/F basal medium test, if both the open and closed tubes are yellow this indicates that the bacteria

Ferments - because it can do so with and without oxygen

In the O/F basal medium test, if both the open tube is yellow this indicates that the bacteria

Oxidizes - needs oxygen

Triple Sugar Iron Agar (TSI) and Kligler Iron Agar (KIA) slants detect…

hydrogen sulfide gas

Glucose, lactose, and sucrose are in the … slant

TSI

Glucose and lactose are in the … slant

KIA

What does a yellow reaction indicate in TSI/KIA?

The organism fermented the sugars, producing acids that turns indicator yellow

What does a red reaction indicate in TSI/KIA?

The organism did not ferment the sugar; it used peptones instead, raising the pH

Why is the butt of TSI/KIA slants important?

It is anaerobic, detects fermentation

Why is the slant of TSI/KIAs important?

It is aerobic- used to detect oxidation of sugars or peptone utlization

What does black precipitate in the butt mean in TSI/KIA slants?

H2S production

What does cracks or bubbles of the media in TSI/KIA slants mean?

gas production

The main purpose of TSI and KIA media is to detect

carbohydrate fermentation

What does a K/A (red slant/yellow butt) mean in TSI/KIA media ?

Only glucose was fermented only. (It was in the butt)

What does a A/A (yellow slant/yellow butt) mean in TSI/KIA media ?

glucose, lactose, and/or sucrose was fermented

What does a K/K (red slant/red butt) mean in TSI/KIA media ?

No sugar fermentation- it only uses peptones for energy

What does K/No (red slant/unchanged butt) mean in TSI/KIA media?

It is a non-glucose fermenter (aerobic only)

What is the end product of the Embden-Meyerhof (glycolysis) pathway?

pyruvic acid (pyruvate)

What does the EMP break down?

Glucose (into 2 molecules of pyruvic acid)

Are the mixed acid and butylene glycol fermentation pathways parts of the EMP?

No, they begin with pyruvic acid- the end product of the EMP

What are the main end products of mixed acid fermentation?

Lactic acid, acetic acid, formic acid, ethanol

What is a major end product of the butylene glycol fermentation pathways?

Acetoin

What test detects mixed acid fermentation?

Methyl Red (MR) test

What test detects Acetoin produced in the butylene glycol fermentation pathway?

the Voges-Proskauer (VP) test

Does the mixed acid pathway or the butylene glycol pathway produce more neutral products?

Butylene Glycol pathway

What color change indicates a positive MR result?

Red (means strong acid production <4.4 pH)

What color change indicates a negative MR result?

Yellow or orange (means acids were neutralized >6.0 pH)

What reagents are used in the VP test?

alpha naphthol and KOH

What color change indicates a positive VP result?

Red color after adding reagents (due to oxidation of acetoin)

Why are MR and VP tests often run together?

Organisms are usually MR+/VP- or MR-/VP+ so they complement them for ID

What is the purpose of the Moeller decarboxylase test?

To detect the production of specific AA decarboxylase enzymes (lysin, ornithine, or arginine) that convert AA into alkaline amines

What pH indicator does Moeller decarboxylase medium contain?

Bromocresol purple - yellow in acidic and PURPLE in alkaline conditions

Why is mineral oil added on top after inoculation for the Moeller decarboxylase test?

To make the medium anaerobic, encouraging glucose fermentation so that acid forms and activates the decarboxylase enzyme

What color indicates a positive decarboxylase reaction in Moeller medium?

Purple (AA was decarboxylated to an amine and medium became alkaline)

What is the purpose of the deaminase test (PAD)?

To determine whether the bacteria contains the deaminase enzyme that metabolizes AAs by the removal of an amine group

What is the reagent for the Deaminase test?

10% ferric chloride

What is the end product in the Phenylalanine deaminase (PAD) test?

Phenylpyruvic acid

What color indicates a positive test in the Deaminase (PAD) test and what does it indicate is present?

Green and Phenylpyruvic acid

What is the purpose of the Lysine Iron Agar (LIA) test?

To detect a bacteria’s ability to decarboxylate or deaminate lysine and to produce hydrogen sulfide (H2S)

What are the two main substrates in the LIA medium?

Glucose (for initial fermentation) and lysine ( to test for decarboxylation or deamination)

What happens first when bacteria grow on LIA?

Organism ferments glucose, producing acid and turning medium yellow (A/A)

What happens after glucose if organism has lysine decarboxylase on LIA?

The acid activates the enzyme and lysine is decarboxylate, the alkaline amine forms, and the butt turns purple

What does K/K (purple slant/purple butt) mean in LIA test?

Glucose was fermented then lysine was decarboxylated

What does K/A (purple slant/yellow butt) mean n LIA?

Organism fermented glucose only, lysine decarboxylation occurred

What does R/A (red slant/yellow butt) indicate on LIA?

The organism deaminated lysine on the slant (aerobic) and fermented glucose in the butt (anaerobic)

what is the purpose of the Citrate Utilization test?

To determine whether the bacteria can utilize sodium citrate as a sole carbon source

A positive result on a citrate utilization test is the color

blue

What is the purpose of the DNase test?

To detect whether an organism produces DNase

How do you interpret a positive DNase test?

A clear zone around the bacterial growth indicates that DNA has been hydrolyzed

What is the purpose of the Indole test?

To determine whether an organism can break down tryptophan into indole using tryptophanase

What reagent is added to detect indole?

Kovac’s reagent (p-dimethylaminobenzaldehyde, HCl)

What does a pink layer at the top of the tube mean after adding Kovac’s reagent on the Indol test?

Positive

Enterobacterales are mostly oxidase…

negative

What is the purpose of the Nitrate Reduction test?

To determine whether an organism can reduce nitrate to nitrite or further to nitrogen gas using nitrate reductase

The spot indole test is a … test by smearing a colony on a filter paper or adding to a swab with a colony

rapid

What reagents are added to detect nitrate reduction to nitrite?

Reagent A (sulfanilic acid) and reagent B (alpha naphthylamine)/ A red color color after reagents indicates nitrite present 

A bright pink color for the urease test indicates a … result

positive