Microbiology Lab Techniques: Aseptic, Staining, and Bacterial Morphology

What is the purpose of aseptic technique?

To prevent contamination of cultures, yourself, and the environment.

Define "culture."

Microorganisms growing in a medium.

Define "pure culture."

Contains only one species of microorganism (all cells genetically identical).

Define "mixed culture."

Contains two or more species of microorganisms.

What tool is used to transfer small amounts of bacteria in lab?

Inoculating loop or needle.

Why must the inoculating loop be flamed before and after use?

To sterilize and prevent contamination.

Why should the tube cap never be placed on the bench during inoculation?

To prevent contamination; hold with pinky instead.

Why do we flame the mouth of culture tubes?

To sterilize and reduce contamination.

What method is commonly used to isolate pure colonies?

The streak plate method.

How does the streak plate method work?

Dilutes bacterial cells across 3-4 sectors, producing isolated colonies from single cells.

Define colony morphology.

Visible characteristics of a colony: size, shape, margin, elevation, texture, pigment.

Examples of colony shapes?

Circular, irregular.

Examples of colony margins?

Entire (smooth), undulate (wavy), lobate.

Examples of colony elevation?

Flat, raised, convex.

Colony textures?

Smooth, moist, shiny OR rough, dry.

What are the two types of colony pigments?

Water-soluble (diffuses into agar) and insoluble (stays in colony).

Which bacterium produces red pigment at 25°C?

Serratia marcescens.

How does incubation time affect colonies?

Colonies can change (e.g., Bacillus subtilis smooth at 24h, rough at 48h).

How do nutrients affect colony pigment?

Some bacteria produce more pigment in richer media (e.g., Chromobacterium violaceum).

Why is working near a flame recommended?

Heat creates an updraft that reduces airborne contamination.

Why do we stain bacteria?

To increase contrast between cells and background so they can be seen.

What does a simple stain show?

Bacterial shape, size, and arrangement.

What type of dye is used in simple staining?

Basic (cationic, + charged) dye.

Why do basic dyes stick to bacteria?

Bacterial cells are negatively charged, so they attract positively charged dyes.

Examples of basic dyes for simple staining?

Crystal violet, methylene blue, safranin.

Steps of simple staining?

Smear → air dry → heat-fix → flood with dye → rinse → blot dry → view under microscope.

Why do we heat-fix slides?

Kills bacteria, sticks them to slide, increases dye uptake.

Disadvantage of heat-fixing?

Can distort cell shape.

What type of dye is used in negative staining?

Acidic (anionic, - charged) dye.

Why do acidic dyes not stain the cell?

Both the dye and cell surface are negatively charged → repel each other → background stains instead.

Examples of acidic dyes?

Nigrosin, India ink, Congo red.

Does negative staining use heat-fixing?

No; avoids distortion, shows natural size/shape.

Simple vs Negative staining comparison?

Simple stains the cell with heat-fix (may distort); Negative stains the background, no heat-fix (natural size/shape).

What are the three major bacterial shapes?

Cocci (spherical), Bacilli (rods), Spirilla (spiral).

Variations of bacterial shapes?

Vibrio (comma-shaped), coccobacillus (oval), spirochete (thin flexible spiral), pleomorphic (variable).

Cocci arrangements?

Diplococcus (pairs), Streptococcus (chains), Tetrad (4), Sarcina (8, cube), Staphylococcus (clusters).

Bacilli arrangements?

Single, Diplobacillus (pairs), Streptobacillus (chains), Palisades (side-by-side).

Spiral arrangements?

Usually single (don't form clusters).

Why do we use oil immersion (100x objective)?

Reduces light refraction → increases resolution.

What does "differential staining" mean?

Uses multiple dyes to distinguish between different bacterial groups/structures.

What is the purpose of Gram staining?

Classifies bacteria into Gram-positive and Gram-negative based on cell wall structure.

Gram-positive cell wall features?

Thick peptidoglycan (90%), teichoic acids, no outer membrane.

Gram-negative cell wall features?

Thin peptidoglycan (5-10%), outer membrane with LPS, no teichoic acids.

Steps of Gram stain?

1. Crystal violet 2. Iodine 3. Alcohol 4. Safranin.

Final Gram stain results?

Gram-positive = purple, Gram-negative = pink.

Critical step in Gram staining?

Decolorization (alcohol).

Common Gram-positive and Gram-negative controls?

GP = Staphylococcus aureus; GN = Escherichia coli.

Errors that cause Gram stain problems?

Old culture (>24h), thick smear, over-decolorization, overheated cells.

What is the purpose of acid-fast staining?

Detects bacteria with waxy cell walls (mycolic acid), e.g., Mycobacterium.

Diseases detected with acid-fast stain?

Tuberculosis, leprosy, Nocardia infections.

Steps of acid-fast stain?

1. Carbolfuchsin (with heat) 2. Acid-alcohol 3. Methylene blue.

Results of acid-fast stain?

Acid-fast = red/fuchsia, Non-acid-fast = blue.

What is the purpose of endospore staining?

Detects dormant, resistant endospores in Bacillus and Clostridium.

Steps of endospore stain (Schaeffer-Fulton)?

1. Malachite green (steam) 2. Water rinse 3. Safranin.

Results of endospore stain?

Endospores = green, Vegetative cells = red.

Locations of spores?

Central = Bacillus; Terminal = Clostridium.

Why are endospores resistant?

Keratin coat, dehydrated core, DNA-protecting proteins.

What does capsule staining show?

Capsule appears as a halo around the cell; demonstrates virulence.

What does flagella staining show?

Makes thin flagella visible using mordant and dye.

Types of flagella arrangements?

Monotrichous (1), Amphitrichous (1 at each end), Lophotrichous (tuft), Peritrichous (all around).