Biochemical Identification of Gram-Negative Bacteria

Comprehensive practice flashcards reviewing the biochemical tests, carbohydrate fermentation reactions, amino acid utilization pathways, manual multi-test panels, and automated systems used to identify gram-negative bacteria.

Why is determining lactose utilization considered the most critical test for differentiating gram-negative rods?

It separates organisms that can ferment lactose (lactose fermenters, LFs) from those that cannot (non-lactose fermenters, NLFs).

What do the pink and clear colony morphologies represent on this MacConkey agar plate?

Pink colonies indicate lactose fermenters (LFs), whereas clear/colorless colonies indicate non-lactose fermenters (NLFs).

How do bacteria differ in their utilization of carbohydrates via oxidation versus fermentation?

Oxidation requires oxygen (aerobic respiration) and produces relatively small amounts of acid, whereas fermentation occurs anaerobically and produces large amounts of acid or acid with gas.

Which three media can be used to determine the oxidation-fermentation (O/F) patterns of Enterobacteriaceae?

Triple sugar iron ($\text{TSI}$) agar, Kligler iron agar ($\text{KIA}$), and Hugh Leifson O/F basal medium.

What carbohydrates and their respective concentrations are found in Triple Sugar Iron ($\text{TSI}$) agar?

$1\%$ lactose, $0.1\%$ glucose, and $1\%$ sucrose.

Which ingredients in Triple Sugar Iron ($\text{TSI}$) agar are responsible for detecting hydrogen sulfide gas ($\text{H}_2\text{S}$)?

Ferrous sulfate and sodium thiosulfate.

What pH indicator is included in Triple Sugar Iron ($\text{TSI}$) agar, and what do its color changes indicate?

Phenol red; yellow indicates an acidic environment ($\text{pH} < 6.8$) and red indicates an alkaline environment.

What inoculation procedure for a Triple Sugar Iron ($\text{TSI}$) agar slant is illustrated here?

Inoculating the butt (deep portion) by stabbing with an inoculating needle, followed by fishtailing (a back-and-forth zigzag streak) up the slant.

What incubation parameters are required for a Triple Sugar Iron ($\text{TSI}$) slant?

Incubate with loose caps to allow airflow, in a non-$\text{CO}_2$ incubator, for $18\text{--}24\text{ hours}$.

What metabolic reaction is indicated by an alkaline slant/alkaline butt ($\text{K/K}$, red slant/red butt) on a TSI slant?

No carbohydrate fermentation (the organism cannot ferment glucose, lactose, or sucrose); such organisms are typically not members of the Enterobacteriaceae family.

What metabolic reaction does an alkaline slant/acid butt ($\text{K/A}$, red slant/yellow butt) represent in a TSI slant?

Fermentation of glucose only.

What metabolic reaction does an acid slant/acid butt ($\text{A/A}$, yellow slant/yellow butt) represent in a TSI slant?

Fermentation of glucose, as well as lactose, sucrose, or both sugars.

Under what condition does hydrogen sulfide ($\text{H}_2\text{S}$) precipitate form in TSI agar, and how does it appear visually?

It requires an acidic environment; it appears as a completely black precipitate in the butt of the tube.

How is gas production (aerogenic reaction) detected in a TSI tube?

By the formation of bubbles, splitting of the agar in the butt, or complete displacement of the agar medium from the bottom of the tube.

What enzyme deficiency characterizes delayed lactose fermenters (dLFs) compared to true non-lactose fermenters (NLFs)?

dLFs lack $\beta$-galactoside permease (transport enzyme) but possess $\beta$-galactosidase (cleaving enzyme), whereas NLFs lack both enzymes.

In an ONPG test, what do yellow and colorless results mean?

Yellow means positive (delayed lactose fermenter), and colorless means negative (non-lactose fermenter).

What metabolic pathway does the Methyl Red ($\text{MR}$) test evaluate, and what indicates a positive reaction?

The mixed acid fermentation pathway of glucose; a positive reaction produces stable acid end products that lower the pH, turning the methyl red indicator red.

What does the Voges-Proskauer ($\text{VP}$) test detect?

Acetoin, produced when bacteria ferment glucose via the $2,3\text{-butanediol}$ pathway.

Can an organism yield positive results for both the Methyl Red ($\text{MR}$) and Voges-Proskauer ($\text{VP}$) tests?

No; a culture will only be positive for one pathway (either $\text{MR}^+$ or $\text{VP}^+$).

In the Moeller decarboxylase test, what do the purple and yellow results mean?

Purple means positive (the bacteria broke down the amino acid). Yellow means negative (the bacteria fermented glucose into acid, but did not break down the amino acid).

Why is sterile mineral oil layered over inoculated Moeller decarboxylase tubes?

To create an anaerobic environment, which is required along with an acidic pH for amino acid decarboxylation to occur.

What reagent is added to a phenylalanine deaminase (PAD) slant after incubation, and what confirms a positive result as shown in the middle tube?

$10\%$ ferric chloride; the development of a green color confirms the presence of phenylpyruvic acid (positive result).

What biochemical mechanism causes the Simmons citrate agar slant on the right to turn blue?

The organism utilizes citrate as its sole carbon source and ammonium salts as its nitrogen source, releasing ammonia and creating an alkaline pH that shifts the indicator from green to blue.

What enzyme mediates the deamination of tryptophan to produce indole?

Tryptophanase.

How does the Ehrlich indole test procedure differ from the Kovac's reagent indole test?

The Ehrlich indole test requires prior extraction with $1\,\text{mL}$ of xylene before adding Ehrlich's reagent (PDAB), whereas Kovac's reagent is added directly to the broth culture without xylene extraction.

In the rapid spot indole test shown, what reaction denotes a positive result?

Development of a blue/green color (spot B) on filter paper moistened with $p\text{-dimethylaminobenzaldehyde (PDAB)}$.

What indicates a positive reaction in the sodium malonate utilization test shown?

A color shift from green (negative, right tube) to blue (positive, left tube) using bromothymol blue as the pH indicator.

How is a positive motility test distinguished from a negative motility test in semisolid medium?

A negative result shows growth restricted to the single stab line (middle tube), while a positive result shows diffuse movement/haziness spreading away from the stab line throughout the medium (right tube).

In the nitrate reduction test, what reagents are added after initial incubation, and what does an immediate red color indicate?

Sulfanilic acid (reagent A) (reagent B); an immediate red color indicates that nitrate was reduced to nitrite (positive test).

In a nitrate reduction test that remains colorless after adding reagents A and B, what does a red color after adding zinc dust mean?

It indicates a negative nitrate reduction test; zinc chemically reduces unreduced nitrate to nitrite, proving that the bacteria never reduced nitrate.

What constitutes a positive Kovac's oxidase test as seen on the left square?

Development of a lavender/purple color within $10\text{--}15\text{ seconds}$ after rubbing a colony onto reagent-moistened filter paper.

How does the oxidase test differentiate Enterobacteriaceae from pseudomonads?

Enterobacteriaceae are oxidase-negative, whereas pseudomonads are oxidase-positive.

In a Christensen's urea agar slant, what does a bright pink color indicate?

A positive urease test; the bacteria break down urea to make ammonia, creating a basic (alkaline) environment that turns the dye pink.

What manual multi-test system was released in 1970 to identify gram-negative fermentative bacteria (Enterobacteriaceae)?

The Analytical Profile Index 20E ($\text{API 20E}$) system.

Which automated identification system operates based on fatty acid analysis of microbial cells?

Sherlock Microbial Identification System (manufactured by MIDI).