Summary of Microbial Identification Biochemical Tests

## Introduction Bacterial biochemical tests are laboratory procedures used to identify bacteria by detecting specific enzymes, metabolic pathways, or the ability to use certain substrates. These tests are essential in clinical microbiology, food safety, and environmental monitoring because different bacteria produce characteristic reactions that help distinguish species and guide treatment. > Definition: Biochemical tests are assays that detect metabolic activities (enzyme production, substrate utilization, redox reactions) of bacteria to aid identification. ## How to approach biochemical testing Break testing into three practical steps: 1. Choose tests based on suspected organisms (Gram reaction, morphology, clinical context). 2. Inoculate appropriate media and incubate under correct conditions (temperature, oxygen requirement). 3. Interpret results using color changes, gas production, precipitates, or growth patterns. ### Important test categories - **Hydrolysis tests** (starch, gelatin, urea): detect enzymes that break large molecules into smaller ones. - **Fermentation and oxidation tests** (phenol red broths, MR-VP, citrate): determine carbohydrate metabolism and organic acid production. - **Reduction and respiratory tests** (nitrate reduction, litmus milk reduction): follow electron transfer and terminal acceptor use. - **Enzyme detection tests** (catalase, coagulase, oxidase): rapid identification of key virulence or metabolic enzymes. - **Selective/differential media** (MSA, blood agar): combine growth requirements with visible reactions (mannitol fermentation, hemolysis). ## Individual tests — what they detect and how to read them ### 1. Starch hydrolysis (Starch agar) - Substrate: starch - Enzyme: amylase - Indicator: iodine - Positive: clear zone around growth after adding iodine (starch hydrolyzed to glucose/maltose) - Negative: dark blue/black around growth ### 2. Gelatin hydrolysis (Gelatin stab) - Substrate: gelatin - Enzyme: gelatinase - Read: incubate, then refrigerate. If medium remains liquid → positive (gelatin degraded). If solid → negative. ### 3. Carbohydrate fermentation (Phenol red broth) - Substrate: single carbohydrate (glucose, lactose, etc.) - Indicator: phenol red - Outcomes: - Acid production: yellow (phenol red turns yellow), gas: bubble in Durham tube - No fermentation: red (or magenta for deamination) ### 4. Litmus milk reactions - Multiple possible outcomes from one medium: - Fermentation: acidification → pink - Alkalinization: ammonia production → purple/blue - Peptonization (proteolysis): casein breakdown → brown/clear - Reduction: reduced litmus → white/bleached - Interpretation depends on color and consistency changes ### 5. Nitrate reduction (Nitrate broth) - Pathway: nitrate to nitrite (or further to N2 gas) - Reagents: sulfanilic acid + alpha-naphthylamine (DAN sometimes used), then zinc if needed - Positive: red after reagents (nitrite present) or gas in Durham tube (denitrification) - If no color, add zinc; if turns red after zinc → negative (nitrate not reduced); if still colorless → positive (reduced beyond nitrite) ### 6. Decarboxylase tests - Substrate: specific amino acid (e.g., lysine, ornithine) - Enzyme: decarboxylase - Indicator: usually a pH indicator that turns purple when alkaline products (amines) form - Positive: purple (alkaline) - Negative: yellow or original color ### 7. Urease test (Urea broth) - Enzyme: urease - Reaction: urea -> ammonia + CO2 - Indicator: phenol red - Positive: bright pink (alkaline) - Negative: yellow/orange ### 8. SIM medium (Sulfur, Indole, Motility) - Sulfur: H2S production forms black precipitate (FeS) - Indole: tryptophanase detected with Kovac’s reagent → red layer on top indicates indole positive - Motility: diffuse growth away from stab line indicates motility ### 9. MR-VP broth - Methyl Red (MR): detects stable acid end products from glucose fermentation; add methyl red indicator; red at pH < 4.4 = positive - Voge