Microbiology Lab Study Guide: Microscopy, Staining, Bacterial Classification, and Culturing Techniques

Lab Safety and Biosafety Levels

Primary and Secondary Commitment

Lab safety is essential for protecting personnel and the environment from exposure to infectious agents. Understanding biosafety levels is crucial for safe laboratory practice.

• Primary commitment: Protection of personnel and immediate surroundings from infectious agents.

• Secondary commitment: Protection of the environment external to the lab, achieved through facility design and operational procedures.

Biosafety Levels (BSL)

• BSL-1: Minimal risk; suitable for work with non-pathogenic microbes.

• BSL-2: Moderate risk; associated with agents that can cause human disease but are not easily transmitted.

• BSL-3: High risk; agents may cause serious or lethal disease, often with available treatment.

• BSL-4: Extreme risk; agents cause severe disease, often untreatable.

Microscopy: Parts and Use

Compound Light Microscope

The compound light microscope is a fundamental tool for observing microorganisms. Understanding its parts and proper use is essential for accurate observation.

• Body tube: Connects the eyepiece with the rotating nosepiece.

• Eyepiece: Lens the observer looks through.

• Rotating nosepiece: Holds objective lenses and allows switching between them.

• Coarse adjustment: Moves the stage for focusing with low power objectives.

• Fine adjustment: Allows precise focusing, especially with high power objectives.

• Stage: Platform for holding slides.

• Mechanical stage: Clamping device for slide movement.

• Arm: Used to carry the microscope.

• Light source: Mirror or lamp; most have a built-in neutral density filter.

Microscope Resolution and Magnification

• Resolution: Ability to distinguish two close points as separate.

• Magnification: Total magnification = objective lens power × eyepiece power.

• Use oil immersion lens for highest resolution (100x objective).

Proper Use and Care

• Start with low power objective, focus, then switch to higher power.

• Use fine adjustment for high power focusing.

• Adjust condenser and diaphragm for optimal light and contrast.

Lab Techniques: Aseptic Technique and Streaking

Aseptic Technique

Aseptic technique prevents contamination of cultures and the environment. It is essential for reliable results in microbiology.

• Use sterile tools and surfaces.

• Flame loops and needles before and after use.

• Minimize exposure of cultures to air.

Streak Plate Method

• Used to isolate pure colonies from a mixed sample.

• Involves spreading bacteria over the surface of an agar plate in a specific pattern.

Staining Techniques

Gram Stain

The Gram stain is a differential stain used to distinguish between Gram-positive and Gram-negative bacteria based on cell wall structure.

• Gram-positive: Thick peptidoglycan layer, retains crystal violet stain (purple).

• Gram-negative: Thin peptidoglycan layer, does not retain crystal violet, counterstained with safranin (pink).

Steps of Gram Staining

1. Prepare smear and heat fix.

2. Apply crystal violet (primary stain).

3. Add Gram's iodine (mordant).

4. Decolorize with alcohol or acetone.

5. Counterstain with safranin.

Capsule Stain

• Used to visualize bacterial capsules, which are polysaccharide layers outside the cell wall.

• Negative staining with India ink or nigrosin creates a dark background, highlighting the capsule.

Endospore Stain

• Used to detect spores in genera such as Bacillus and Clostridium.

• Common methods: Schaeffer-Fulton (malachite green and heat), Dorner method.

Acid-Fast Stain

• Used for bacteria with waxy cell walls, such as Mycobacterium.

• Primary stain: carbol fuchsin; decolorizer: acid alcohol; counterstain: methylene blue.

Bacterial Classification and Growth Requirements

Shapes of Bacteria

• Coccus: Spherical

• Bacillus: Rod-shaped

• Spiral: Spiral or curved

Fungal Structures

• Hyphae: Filamentous structures

• Mycelium: Mass of hyphae

• Yeast: Unicellular fungi

Bacterial Oxygen Requirements

• Obligate aerobe: Requires oxygen

• Obligate anaerobe: Cannot tolerate oxygen

• Facultative anaerobe: Can grow with or without oxygen

• Microaerophile: Requires low oxygen

• Aerotolerant anaerobe: Tolerates oxygen but does not use it

Media Types

• Complex media: Contains unknown exact composition; supports a wide range of organisms.

• Defined media: Exact chemical composition is known; used for fastidious organisms.

• Selectivity: Selective media inhibit some organisms and allow others to grow (e.g., EMB for Gram-negative bacteria).

• Differential media: Distinguish between organisms based on biochemical reactions (e.g., color change).

Metabolic Categories and Nutritional Requirements

Energy and Carbon Sources

• Chemotrophs: Obtain energy from chemicals.

• Phototrophs: Obtain energy from light.

• Autotrophs: Use CO2 as carbon source.

• Heterotrophs: Use organic compounds as carbon source.

Nutritional Requirements

• Water

• Carbon source

• Energy source

• Growth factors

• Minerals

Specialized Lab Setups

Anaerobic Jar and FTM Tubes

• Anaerobic jar: Used to create an oxygen-free environment for obligate anaerobes.

• FTM (Fluid Thioglycollate Medium): Contains reducing agents to support growth of anaerobes and aerobes.

Summary Table: Bacterial Oxygen Requirements

Type	Oxygen Requirement	Growth Pattern in Tube
Obligate aerobe	Requires oxygen	Growth at top
Obligate anaerobe	Cannot tolerate oxygen	Growth at bottom
Facultative anaerobe	With or without oxygen	Growth throughout, more at top
Microaerophile	Low oxygen	Growth just below surface
Aerotolerant anaerobe	Tolerates oxygen	Growth throughout

Additional info:

• Some details on fungal structures and metabolic categories were expanded for clarity.

• Summary table inferred from context for oxygen requirements.