W7 Tues

Carbohydrate Catabolism & Fermentation

Metabolism Overview

Metabolism encompasses all chemical reactions occurring within living organisms, crucial for energy production and biosynthesis. These processes are mediated by enzymes, which are specialized proteins that catalyze biological reactions.

• Enzymes: Proteins that accelerate biochemical reactions.

• Endoenzymes: Function inside the cell.

• Exoenzymes: Released from the cell to catalyze reactions outside.

Catabolism and Anabolism

• Catabolism: Breakdown of large molecules (carbohydrates, lipids, proteins) into smaller units, releasing energy for cellular activities. Example: Glycolysis, digestion of food.

• Anabolism: Synthesis of complex macromolecules from smaller units, requiring energy input. Example: Protein synthesis, muscle growth.

Carbohydrates: Structure and Classification

Carbohydrates are organic molecules with the general formula (CH2O)n, classified by size:

• Monosaccharides: Simple sugars (e.g., glucose).

• Oligosaccharides: 2–20 monosaccharide units.

• Polysaccharides: 20 or more monosaccharide units (e.g., starch).

Starch Hydrolysis

Principle and Biochemical Basis

The starch hydrolysis test detects an organism's ability to produce the enzyme amylase, which breaks down starch into simpler sugars. Starch agar is a differential medium used to test for exoenzymes (hydrolytic enzymes) that degrade starch.

• Hydrolysis: Breakdown of large substrates by addition of water.

• Starch: Complex polysaccharide, too large to enter bacterial cells; exoenzymes degrade it into utilizable subunits.

Procedure

1. Divide starch agar plate into sectors and label.

2. Streak bacteria sample in a single line.

3. Incubate inverted at 35°C for 24 hours.

4. Flood plate with Gram’s iodine (indicator).

5. Examine for clear zone around bacterial growth.

Interpretation of Results

• Positive Test: Clear zone around growth after iodine addition indicates starch hydrolysis. Example: Bacillus species show strong amylase activity.

• Negative Test: Dark blue coloration; starch remains intact. Example: Staphylococcus aureus, Staphylococcus epidermidis, E. coli.

Oxidative Fermentative (OF) Test

Principle and Media

The OF test differentiates bacteria based on their ability to metabolize glucose oxidatively (aerobic) or fermentatively (anaerobic and aerobic). OF-glucose deeps contain glucose, peptones, and bromothymol blue (pH indicator).

• Oxidative bacteria: Produce acid only under aerobic conditions.

• Fermentative bacteria: Produce acid under both aerobic and anaerobic conditions.

• Non-saccharolytic bacteria: Do not metabolize glucose; may increase pH by amine production.

Procedure

1. Inoculate two tubes of OF-glucose medium.

2. Overlay one tube with mineral oil (anaerobic), leave the other open (aerobic).

3. Incubate at 35°C for 24–48 hours.

4. Observe growth, color change, and type of metabolism.

Results and Interpretation

• Oxidative Result: Acid production in open tube (yellow), no change in oil-covered tube. Example: Pseudomonas aeruginosa, Acinetobacter species.

• Fermentative Result: Acid production in both tubes (yellow); facultative anaerobes. Example: Enterobacteriaceae.

• Negative Result: No color change or blue coloration (alkaline); non-saccharolytic. Example: Alcaligenes faecalis.

Carbohydrate Fermentation Tests

Principle and Media

Carbohydrate fermentation tests determine whether bacteria can ferment specific carbohydrates, producing acid and/or gas.

• Basal medium: Contains a single carbohydrate (e.g., glucose, lactose, sucrose).

• Phenol red: pH indicator (red = neutral, yellow = acidic).

• Durham tube: Detects gas production (bubble formation).

Interpretation of Results

• Fermenter with acid only: Yellow color, no gas.

• Fermenter with acid and gas: Yellow color, gas bubble in Durham tube.

• Non-fermenter: Red color, no acid or gas.

Methyl Red (MR) and Voges–Proskauer (VP) Tests

Principle and Procedures

The MR and VP tests differentiate Enterobacteriaceae and other Gram-negative rods based on glucose fermentation pathways.

• MR Test: Detects strong acid production from mixed acid fermentation. Positive: Red color (pH < 4.4). Negative: Yellow/orange (pH > 6).

• VP Test: Detects acetoin production (neutral fermentation). Positive: Red color after reagents added. Negative: No color change or copper discoloration.

• Distinguishes: E. coli (MR+) from Enterobacter/Klebsiella (VP+).

Simmons Citrate Agar Test

Principle and Biochemical Basis

Simmons citrate agar is a defined medium used to determine if an organism can utilize citrate as its sole carbon source.

• Citrate lyase: Hydrolyzes citrate to oxaloacetic acid and acetic acid.

• CO2 production: Reacts with medium to form sodium carbonate, raising pH.

• Bromthymol blue: pH indicator (green = neutral, blue = alkaline).

Interpretation of Results

• Citrate positive: Growth and intense blue color (alkaline pH > 7.6). Example: Klebsiella, Enterobacter, Salmonella, most Proteus species.

• Citrate negative: No growth, medium remains green. Example: E. coli, Shigella.

Digestive System (Upper GI) Medical Terms

Key Terms

• Esophago-: Esophagus

• Gastro-: Stomach

• Pyloro-: Pylorus (stomach outlet)

• Cholecysto-: Gallbladder

• Cholangio-: Bile ducts

• Hepato-: Liver

• Pancreato-: Pancreas

• Emesis: Vomiting

• Dysphagia: Difficulty swallowing

• GERD: Gastroesophageal reflux disease (acid reflux)

Additional info: Academic context was added to clarify the principles, procedures, and interpretation of each biochemical test, as well as to provide definitions and examples for key terms and processes.