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Motility Medium (with TTC)

1. Purpose: Detects bacterial movement.

2. Principle: Motile bacteria use flagella to move through semi-solid agar. TTC, a colorless redox indicator, turns red when reduced by bacterial metabolism, making growth visible.

3. Expected Results:

   • Positive: Diffuse red growth spreads from the stab line.

   • Negative: Red growth stays on the stab line.

Oxygen Requirement (Fluid Thioglycollate Medium)

1. Purpose: Determines how microorganisms respond to oxygen.

2. Principle: FTM creates an oxygen gradient using sodium thioglycollate (reducing agent) and agar. Resazurin indicates oxygen: pink (present), colorless (absent).

3. Expected Results:

   • Obligate Aerobe: Grows only at the top (aerobic).

   • Obligate Anaerobe: Grows only at the bottom (anaerobic).

   • Facultative Anaerobe: Grows throughout, densest at the top.

   • Microaerophile: Grows in a narrow band below the surface.

   • Aerotolerant Anaerobe: Grows uniformly throughout.

Carbohydrate Fermentation (Glucose, Lactose, Maltose, Mannitol, Sucrose)

1. Purpose: Checks if bacteria can ferment specific carbohydrates.

2. Principle: Tubes contain a specific carbohydrate, peptone, pH indicator (e.g., phenol red), and a Durham tube. Fermentation produces acid (lowers pH, changes indicator color, typically red to yellow) and sometimes gas (bubble in Durham tube).

3. Expected Results:

   • Acid Production: Indicator changes from red (neutral) to yellow (acidic).

   • Gas Production: A bubble is present in the Durham tube.

   • No Fermentation: Medium stays red or turns pink/alkaline (peptone use), no gas.

Gelatin Hydrolysis (with Nutrient Gelatin)

1. Purpose: Detects gelatinase enzyme production.

2. Principle: Gelatinase hydrolyzes gelatin, causing the solidified medium to liquefy even when refrigerated.

3. Expected Results:

   • Positive: Gelatin medium liquefies after refrigeration.

   • Negative: Gelatin medium remains solid after refrigeration.

Urea Hydrolysis (Urea Broth)

1. Purpose: Detects urease enzyme production.

2. Principle: Urease breaks down urea into ammonia, raising the pH. Phenol red indicator turns pink/fuchsia at alkaline pH.

3. Expected Results:

   • Positive: Medium changes from orange/yellow to pink or fuchsia.

   • Negative: Medium remains orange/yellow or no color change.

Catalase Test (TSA Slant)

1. Purpose: Detects the catalase enzyme.

2. Principle: Catalase detoxifies hydrogen peroxide ($H<em>2O</em>2$) by breaking it into water ($H<em>2O$) and oxygen gas ($O</em>2$).

3. Expected Results:

   • Positive: Immediate bubbles (effervescence) when $H<em>2O</em>2$ is added.

   • Negative: No bubbles produced.

Citrate Utilization (Simmon’s Citrate Agar)

1. Purpose: Determines if an organism uses citrate as its sole carbon source.

2. Principle: Agar contains citrate as the only carbon source and ammonium dihydrogen phosphate as the nitrogen source. Bromothymol blue is a pH indicator (green neutral, blue alkaline). Citrate metabolism produces alkaline byproducts, raising pH and turning the medium blue.

3. Expected Results:

   • Positive: Medium turns blue and/or shows growth.

   • Negative: Medium remains green with no growth.

Nitrate Reduction (Nitrate Broth)

1. Purpose: Detects the ability to reduce nitrate ($NO<em>3^-$) to nitrite ($NO</em>2^-$) or further to $N_2$ gas.

2. Principle: Nitrate reductase forms nitrite from nitrate. Nitrite reacts with Reagents A and B to form a red color. If colorless, zinc dust is added: red indicates unreduced nitrate, colorless indicates complete reduction beyond nitrite.

3. Expected Results:

   • Positive ($NO<em>3^- \rightarrow NO</em>2^-$): Red color after Reagents A and B.

   • Negative (no reduction): No color after A and B, but turns red after zinc dust.

   • Positive (complete reduction to nitrogen gas or other end products): No color after A and B, and no color after zinc dust.

Methyl Red (MR-VP Broth)

1. Purpose: Detects mixed acid fermentation products from glucose.

2. Principle: Bacteria fermenting glucose via the mixed acid pathway produce strong, stable acids that lower pH. Methyl red indicator turns red at pH $\le 4.4$ and yellow at pH $\ge 6.2$ (orange in between).

3. Expected Results:

   • Positive: Red color after adding methyl red (pH $\le 4.4$).

   • Negative: Yellow color after adding methyl red (pH $\ge 6.2$).

Voges-Proskauer (MR-VP Broth)

1. Purpose: Detects acetoin production from glucose fermentation.

2. Principle: Some glucose fermenters produce acetoin. In the presence of oxygen, strong alkali (KOH), and alpha-naphthol, acetoin oxidizes to diacetyl, forming a red color.

3. Expected Results:

   • Positive: Red color develops on the medium surface after VP reagents.

   • Negative: No color change or a copper-like color.

Indole (Tryptone Broth)

1. Purpose: Detects tryptophanase enzyme production.

2. Principle: Tryptophanase breaks tryptophan into indole. Kovacs' reagent reacts with indole to form a cherry-red ring on the broth surface.

3. Expected Results:

   • Positive: A red ring forms on the broth surface.

   • Negative: A yellow/brown ring, or no color change.

Hydrogen Sulfide Production (Triple Sugar Iron Agar)

1. Purpose: Detects hydrogen sulfide gas ($H_2S$) production.

2. Principle: Bacteria reduce sulfur compounds to $H<em>2S$. $H</em>2S$ reacts with iron salts in the agar to form a black ferrous sulfide ($FeS$) precipitate.

3. Expected Results:

   • Positive: A black precipitate appears in the agar butt.

   • Negative: No blackening of the medium.

Phenylalanine Deamination (Phenylalanine Agar)

1. Purpose: Detects phenylalanine deaminase enzyme.

2. Principle: Phenylalanine deaminase removes the amino group from phenylalanine, producing phenylpyruvic acid. Ferric chloride ($FeCl_3$) reacts with phenylpyruvic acid to form a green color.

3. Expected Results:

   • Positive: A green color develops on the slant after adding $FeCl_3$.

   • Negative: No color change (remains yellow/brown).

Lysine Decarboxylase (Lysine Decarboxylase Broth)

1. Purpose: Determines if an organism can decarboxylate lysine.

2. Principle: Broth contains lysine, glucose (for initial acid), pH indicator (e.g., bromocresol purple), and an oil layer for anaerobiosis. Glucose fermentation initially produces acid (yellow). If lysine decarboxylase is present, it forms cadaverine (alkaline), raising pH and turning the indicator back to purple.

3. Expected Results:

   • Positive: Medium turns purple (alkaline) after initial yellowing.

   • Negative: Medium remains yellow (acidic) or orange/brown.

Ornithine Decarboxylase (Ornithine Decarboxylase Broth)

1. Purpose: Determines if an organism can decarboxylate ornithine.

2. Principle: Similar to lysine decarboxylase, broth contains ornithine, glucose, pH indicator, and oil. Glucose fermentation leads to initial acid (yellow). If ornithine decarboxylase is present, it forms putrescine (alkaline), raising pH and turning the indicator back to purple.

3. Expected Results:

   • Positive: Medium turns purple (alkaline) after initial yellowing.