Microbiology Lab Techniques and Concepts

General Lab Rules

  • Safety Equipment: Wear a lab coat, goggles, and closed-toed shoes; tie back hair.

  • Personal Items: Remove loose jewelry, and do not eat or drink in the lab.

  • Handling Culture Tubes: Always carry culture tubes in a test tube rack.

  • Sanitation Practices: Wipe down the bench before and after the lab with disinfectant using circular motions.

  • Backpack Policy: Backpacks should NOT go on the floor.

  • Hand Hygiene: Wash hands with soap and water, as sanitizers do NOT kill spores.

  • Pipetting: Never pipette by mouth; this practice is considered old, dangerous, and not permitted.

Petri Dishes, Slides & Labeling

  • Slide Cleaning: Clean slides with soap and water, rinse, apply alcohol, and let air dry.

  • Labeling: Always label Petri dishes on the bottom (not the lid) to ensure the label stays with the sample and to avoid condensation interference. All cultures, plates, and tubes should be labeled prior to incubation.

Subculturing

  • Definition: Transferring microorganisms from one medium to another.

Inoculating Tools

  • Inoculating Loop: Used for broth cultures and streak plating.

  • Inoculating Needle: Used for agar deep stabs or precise transfers. Always sterilize tools before AND after use.

Transfer Techniques

  • Slant to Broth:

    • Use a loop or needle.

    • Lightly touch or slightly shake along the slant surface to pick up organisms.

  • Broth to Slant:

    • Dip loop into broth.

    • Streak slant surface in a zig-zag pattern from bottom to top.

  • Slant to Deep (Agar Deep Tube):

    • Use needle only.

    • Pick up cells from slant.

    • Stab straight down into the deep tube to the bottom and withdraw quickly.

Culture Types

Pure Culture

  • Definition: Contains 1 species only.

  • Characteristics: Colonies look identical.

  • Obtaining: Achieved through streak plate isolation.

Mixed Culture

  • Definition: Contains 2+ microbial species.

  • Characteristics: Colonies vary in shape, color, size.

  • Source: Often from natural environments or contamination.

Streak Plate vs. Spread Plate

Streak Plate

  • Purpose: Isolate individual colonies for pure culture.

  • Technique:

    • Use an inoculating loop.

    • Dilute sample by streaking across 4 quadrants, flaming the loop between each.

    • Rotate plate 90° between quadrants.

Spread Plate

  • Purpose: Count viable bacteria (CFUs) and create a uniform lawn (e.g. for antibiotic testing).

  • Steps:

    1. Perform dilutions.

    2. Add a measured volume onto agar.

    3. Use a sterile glass spreader, disinfected in ethyl alcohol and flamed.

    4. Spread evenly while the plate spins.

  • Results: Even growth distribution.

Microscope Basics

Oil Immersion Lens (100×)

  • Usage: Only for bacteria.

  • Function: Oil reduces light refraction to provide a clearer image.

  • Setup: Keep the condenser close to the stage.

Objective Lenses

  • 4×: Scanning

  • 10×: Low power

  • 40×: High power

  • 100×: Oil immersion only

Important Microscope Parts

  • Head: Holds eyepieces & objectives

  • Arm: Supports structure

  • Base: Foundation

  • Stage: Holds slides

  • Illuminator: Light source

  • Condenser + Iris Diaphragm: Controls light intensity

  • Nosepiece: Rotates objectives

  • Coarse/Fine Adjustment Knobs: Focuses the image

Bacterial Smear Preparation

From Broth

  • Steps:

    • Tap the tube.

    • Use a loop to transfer a small drop onto the slide.

    • Air dry completely then heat fix by waving slide over a burner.

From Solid Media

  • Steps:

    • Place 1–2 drops of water on the slide.

    • Use a loop or needle to add bacteria.

    • Spread thinly, air dry, and heat fix.

  • Correct Smear: A correct smear is thin and evenly spread.

Bacterial Shapes

Cocci

  • Shape: Spherical.

  • Arrangements:

    • Single

    • Diplo-

    • Chains (strepto-)

    • Clusters (staphylo-)

Bacilli

  • Shape: Rod-shaped.

  • Arrangements:

    • Single

    • Diplo-

    • Chains (streptobacilli)

Spirilla / Spirochetes

  • Shape: Spiral or corkscrew.

  • Movement: Able to move with flagella (motile).

Gram Stain

Purpose

  • Differential Stain: Separates bacteria based on cell wall structure.

Gram-Positive Bacteria

  • Characteristics:

    • Thick peptidoglycan layer.

    • Retains crystal violet stain resulting in a purple coloration.

    • More sensitive to antibiotics.

Gram-Negative Bacteria

  • Characteristics:

    • Thin peptidoglycan layer plus an outer membrane.

    • Loses purple color during staining and turns pink/red due to safranin.

    • Outer membrane confers more resistance to antibiotics.

Staining Steps

  1. Primary Stain: Crystal violet.

  2. Mordant: Gram’s iodine, which binds the dye to the microorganism.

  3. Decolorizer: Alcohol

    • Gram+: Thick peptidoglycan retains dye and stays purple.

    • Gram–: Thin peptidoglycan + outer membrane, dye washes out and becomes colorless.

  4. Counterstain: Safranin, which stains gram-negative microorganisms pink.

Results Explanation

  • Positive (Purple): Thick peptidoglycan traps crystal violet tightly.

  • Negative (Pink): The alcohol dissolves the outer membrane and washes out the primary stain.

Acid-Fast Stain

Purpose

  • Identification: Specifically identifies Mycobacterium (e.g., TB, leprosy).

  • Reason for Use: The cell wall contains mycolic acids, which are waxy, hydrophobic, and resist normal stains.

Staining Steps

  1. Apply carbolfuchsin: Heat may assist in penetration.

  2. Wash with acid-alcohol: This removes the stain from non-acid-fast cells.

  3. Add methylene blue counterstain.

Results

  • Acid-fast (+): Red/pink color retains carbolfuchsin due to the presence of mycolic acid that prevents decolorization.

  • Acid-fast (−): Blue color indicates no waxy layer; therefore, it loses the red stain and takes up the blue counterstain.

Spectrophotometer

  • Function: Measures turbidity (bacterial growth density) of a culture.

  • Measurement:

    • Measures turbidity (cloudiness) to estimate growth.

    • High absorbance = more bacteria (less light passes through).

    • High % transmittance = fewer bacteria (more light passes).

    • More cloudy = more bacteria.

Media Classifications

Selective Media

  • Function: Suppress some microbes while allowing others to grow.

  • Example: EMB (Eosin Methylene Blue):

    • Selective for Gram-negatives, inhibits growth of Gram-positive bacteria.

    • E. coli appears as a green metallic sheen.

    • Differential: Lactose fermentation results in acid production that interacts with dyes, precipitating and changing colony color.

Results and Meaning of EMB

  • Green Metallic Sheen: Indication of E. coli as a strong lactose fermenter.

  • Dark Purple Colonies: Indication of lactose fermenter.

  • Colorless/Pale Colonies: Indication of non-fermenter.

Differential Media

  • Function: Exhibits visible differences between organisms.

    • Indicators: Include phenol red, eosin Y, methylene blue.

Selective + Differential Media

  • Example: Mannitol Salt Agar (MSA):

    • Selective for Staphylococcus due to high salt concentration.

    • Differential for mannitol fermentation

    • Phenol red indicator detects pH change.

Results Interpretation of MSA

  • Growth: Indicates it is a salt-tolerant organism (usually Staphylococcus) since high salt inhibits most other bacteria.

  • Yellow Medium: Indicates positive mannitol fermentation (acid production lowers pH).

  • Pink/Red Medium: Indicates negative fermentation (no acid production).

Different Staphylococcus Species

  • S. aureus: Mannitol fermentation positive (yellow).

  • S. epidermidis: Mannitol fermentation negative (pink).

Enriched Media

  • Definition: Contains special nutrients to support the growth of fastidious organisms.

  • Example: Blood Agar: Tests for hemolysis, which indicates the ability to lyse red blood cells.

Types of Hemolysis on Blood Agar

  • Alpha: Green halo, indicating partial hemolysis (partial RBC breakdown).

  • Beta: Clear zone, indicating complete hemolysis (complete RBC lysis).

  • Gamma: No hemolysis or change (no hemolysis).

Streptococcus vs. Staphylococcus on Blood Agar

  • Streptococcus: Catalase negative.

    • Many species showcase:

    • Alpha: Streptococcus pneumoniae

    • Beta: Streptococcus pyogenes (Group A strep)

    • Gamma: Enterococcus species

  • Staphylococcus: Catalase positive.

    • Most species show gamma or beta hemolysis.

    • S. aureus: Beta hemolysis (clear zone).

Temperature Classifications

Type

Range (°C)

Where found

Psychrophiles

0–20°C

Arctic, deep ocean

Mesophiles

20–45°C

Human body, soil

Thermophiles

40–70°C

Hot springs

Oxygen Requirements

  • Anaerobes: Grow at the bottom (no oxygen, toxic to them).

  • Aerobes: Grow at the top (require oxygen).

  • Facultative Anaerobes: Grow throughout the tube (can grow with or without oxygen).

Anaerobic Chamber

  • Purpose: Creates an oxygen-free environment (often has a bad smell).

  • Application: Used specifically for anaerobic growth.

Biochemical Tests

Starch Hydrolysis (Starch Agar)

  • Purpose: Tests for the enzyme amylase, which breaks starch into simple sugars.

  • Reaction: Iodine binds to intact starch, producing a dark blue/black coloration.

Results Interpretation of Starch Hydrolysis

  • Positive: Clear halo around growth indicates organism produces amylase (starch is broken down).

  • Negative: No clearing (plate remains dark) indicates starch is not broken down.

MRVP Test

  • Setup: Create two tubes from the same organism.

Methyl Red (MR)

  • Interpretation:

    • Red = positive (acid fermentation, stable acids maintain pH < 4.4).

    • Yellow = negative (organism neutralizes acids, resulting in a higher pH).

Voges-Proskauer (VP)

  • Steps: Add VPA + VPB reagents.

  • Interpretation:

    • Red at the top = positive (acetoin oxidized reacts with reagents).

    • No color = negative (no acetoin pathway).

SIM Tube Tests

  • S: Sulfur production (hydrogen sulfide).

  • I: Indole production (via tryptophanase enzyme).

  • M: Motility.

Results of SIM Test

  • H₂S (S) Production:

    • Positive: Black precipitate indicates H₂S reacts with iron salts.

    • Negative: No blackening.

Indole Test

  • Reagent: Kovac's reagent.

  • Results:

    • Red ring = positive (tryptophan → indole).

    • Yellow ring = negative.

Motility

  • Results:

    • Growth spreading from stab = motile.

    • Growth only along stab line = non-motile.

Citrate Test

  • Purpose: Tests the organism's ability to use citrate as the sole carbon source.

  • Requirement: Enzyme citrate permease.

  • Citrate metabolism: Produces alkaline byproducts that raise pH; bromothymol blue shifts color.

Results Interpretation of Citrate Test

  • Blue Slant: Positive (alkaline reaction; growth occurred).

  • Green: Negative (no growth; no pH change).

Urease Test

  • Function: Detects urease enzymes breaking down urea into ammonia.

  • Result: Ammonia raises pH, causing phenol red to turn bright fuchsia.

Urease Test Results

  • Fuchsia/Pink: Positive (urease present).

  • Yellow/No Change: Negative (urease absent).

Carbohydrate Fermentation

  • Setup: Durham tube captures gas during fermentation.

Interpretation of Results

  • Yellow: Acid positive (fermentation occurred).

  • Red: Negative (no fermentation).

  • Gas Bubble: Indicates gas production.

  • Substrates Used: Glucose (dextrose), lactose, sucrose.

Litmus Milk Reactions

Reactions, Appearance, and Meaning

  • Acid (Pink): Indicates lactose fermentation (pink appearance).

  • Alkaline (Blue/Purple): Indicates protein breakdown (causes ammonia production; milk turns blue/darker purple).

  • Reduction: Appearance of white at the bottom indicates litmus is reduced by bacteria.

  • Curd/Clot: Solidified milk indicates acid coagulation of casein.

  • Peptonization: Watery or cleared appearance indicates complete digestion of casein.

  • Gas Reaction: Results in cracks or bubbles indicating fermentation producing CO₂.

Catalase Test

  • Function: Add hydrogen peroxide to assess the presence of the catalase enzyme, which breaks it down into water and oxygen.

Results Interpretation for Catalase Test

  • Bubbles: Positive result indicates presence of Staphylococcus.

  • No Bubbles: Negative result indicates presence of Streptococcus.

Kirby-Bauer Antibiotic Susceptibility Test

Purpose

  • Tests for sensitivity or resistance to antibiotics.

  • Medium: Mueller-Hinton agar plate used for testing.

  • Albert: Creates zones of inhibition around antibiotic disks.

Interpretation of Results

  • Sensitive: Large zone of inhibition (good response to antibiotic).

  • Intermediate: Moderate zone of inhibition.

  • Resistant: Small/no zone of inhibition (poor response to antibiotic).

Strep vs Staph Testing

Streptococcus

  • Characteristics:

    • Catalase negative (no bubbles).

    • Exhibits alpha, beta, or gamma hemolysis.

    • No growth on MSA (not salt-tolerant).

    • Alpha Hemolysis: Streptococcus pneumoniae.

    • Beta Hemolysis: Streptococcus pyogenes (Group A strep).

    • Gamma Hemolysis: Enterococcus or non-hemolytic strains.

Staphylococcus

  • Characteristics:

    • Catalase positive (bubbles present).

    • Usually exhibits beta or gamma hemolysis.

    • Grows on MSA (salt tolerant).

    • MSA Fermentation:

    • S. aureus: Positive (yellow).

    • S. epidermidis: Negative (pink).