Microbiology Lab Study Guide: Microscopy, Staining, Metabolic Tests, Selective Media, and Eukaryotic Microbes

Microscopy and Laboratory Techniques

Microscope Parts and Functions

The microscope is an essential tool in microbiology for observing microorganisms. Understanding its parts and their functions is crucial for accurate observation and analysis.

• Ocular lens (eyepiece): Magnifies the image, typically 10x.

• Objective lenses: Provide various magnifications (e.g., 4x, 10x, 40x, 100x).

• Stage: Holds the slide in place.

• Coarse and fine focus knobs: Adjust the clarity of the image.

• Condenser: Focuses light onto the specimen.

• Diaphragm: Controls the amount of light passing through the specimen.

• Light source: Illuminates the specimen.

Estimating Object Size: Field of View Method

To estimate the size of an object under the microscope, use the field of view method:

• Measure the diameter of the field of view at a given magnification.

• Estimate how many times the object fits across the diameter.

• Calculate object size: Object size = Field diameter / Number of objects across

Total Magnification Calculation

Total magnification is determined by multiplying the magnification of the ocular lens by the objective lens.

• Formula: $\text{Total Magnification} = \text{Ocular Magnification} \times \text{Objective Magnification}$

• Example: 10x ocular and 40x objective = 400x total magnification.

Key Terms: Magnification, Contrast, Resolution

• Magnification: The process of enlarging the appearance of an object.

• Contrast: The difference in light intensity between the specimen and background; improved by adjusting light, using stains, or phase contrast.

• Resolution: The ability to distinguish two close points as separate; higher resolution reveals finer details.

Microscope Care and Storage

• Clean lenses with lens paper only.

• Store with the lowest objective in place.

• Cover the microscope to prevent dust accumulation.

• Handle with both hands; avoid abrupt movements.

Achieving Good Contrast

• Adjust diaphragm and condenser.

• Use stains to color cells or background.

• Employ phase contrast or dark-field microscopy.

Simple vs. Compound Microscopes

• Simple microscope: Uses a single lens (e.g., magnifying glass).

• Compound microscope: Uses multiple lenses (ocular and objectives) for higher magnification and resolution.

Aseptic Technique

Aseptic technique involves procedures to prevent contamination of cultures, media, and the environment.

• Flame sterilization of tools.

• Minimize exposure of sterile items to air.

• Work near a flame or in a laminar flow hood.

Streaking Techniques and Isolated Colonies

Streaking is used to separate individual bacteria from a mixed culture to obtain isolated colonies.

• Use a sterile loop to spread bacteria across agar in a pattern (e.g., quadrant streak).

• Isolated colonies indicate successful separation.

• Purpose: To study pure cultures and identify bacteria.

Bacterial Growth Patterns in Broth

• Pellicle: Growth at the surface.

• Sediment: Growth at the bottom.

• Turbidity: Cloudy appearance throughout.

• Flocculent: Clumps suspended in broth.

Proper Disposal in Lab

• Dispose of cultures in biohazard containers.

• Glass slides in sharps containers.

• Contaminated materials in autoclave bags.

Staining and Cell Morphology

Simple Stains

Simple stains use a single dye to color cells, enhancing visibility and allowing observation of cell shape and arrangement.

• Information: Cell morphology, arrangement.

• Common dyes: Methylene blue, crystal violet.

Cell Shapes and Arrangements

• Cocci: Spherical cells; arrangements include diplococci, streptococci, staphylococci.

• Bacilli: Rod-shaped; arrangements include single, diplobacilli, streptobacilli.

• Spirilla: Spiral-shaped.

• Example: Staphylococcus aureus (clusters of cocci).

Gram Stain Procedure and Interpretation

The Gram stain differentiates bacteria based on cell wall structure.

• Steps:

  1. Crystal violet (primary stain)

  2. Iodine (mordant)

  3. Alcohol (decolorizer)

  4. Safranin (counterstain)

• Gram-positive: Purple (thick peptidoglycan retains crystal violet).

• Gram-negative: Pink/red (thin peptidoglycan loses crystal violet, takes up safranin).

• Cell wall structure determines reaction.

Acidic vs. Basic Stains

• Basic stains: Positively charged; bind to negatively charged cell components (e.g., methylene blue).

• Acidic stains: Negatively charged; stain background (e.g., nigrosin).

• Use: Basic stains for cell morphology; acidic stains for capsule visualization.

Bacterial Smear Preparation and Heat Fixing

• Spread bacteria on slide, air dry.

• Heat fix by passing through flame; kills cells, adheres them to slide.

Capsule and Endospore Stains

• Capsule stain: Stains background and cell, leaving capsule clear.

• Endospore stain: Uses malachite green (stains spores) and safranin (stains cells).

• Purpose: Visualize protective structures.

KOH Test

• Mix bacteria with 3% KOH.

• Gram-negative cells lyse, DNA forms stringy material (positive result).

• Gram-positive cells do not lyse (negative result).

Microbial Metabolism and Biochemical Tests

Citrate Test

• Positive: Blue color; bacteria can use citrate as sole carbon source.

• Negative: Green color; cannot use citrate.

Urea Hydrolysis Test

• Positive: Pink color; urea broken down to ammonia by urease.

• Negative: Yellow/orange; no urea breakdown.

Decarboxylation Test

• Positive: Purple color; decarboxylation of amino acids produces alkaline products.

• Negative: Yellow; no decarboxylation.

• Mineral oil creates anaerobic conditions.

Gelatinase Test

• Positive: Gelatin liquefied; bacteria produce gelatinase, breaking down gelatin.

• Negative: Gelatin remains solid.

S.I.M. Test (Sulfur, Indole, Motility)

• Sulfur production: Black precipitate.

• Indole production: Add Kovac's reagent; red color indicates positive.

• Motility: Growth away from stab line.

Phenol Red Broth Test

• Components: Peptone, carbohydrate, phenol red, Durham tube.

• Positive: Yellow color; acid produced from carbohydrate fermentation.

• Negative: Red/magenta; no fermentation, alkaline products.

• Durham tube: Gas production.

Advanced Biochemical Tests and Antimicrobial Susceptibility

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

• MR positive: Red color; mixed acid fermentation.

• VP positive: Red color; acetoin production.

Kirby-Bauer Test (Antibiotic Susceptibility)

• Prepare lawn of bacteria by spreading culture evenly.

• Place antibiotic disks; measure zones of inhibition.

• Step ensures uniform growth for accurate results.

Oxidase Test

• Positive: Purple color within 30 seconds; presence of cytochrome c oxidase.

• Negative: No color change.

Catalase Test

• Add hydrogen peroxide to bacteria.

• Positive: Bubbling (oxygen released); catalase present.

• Negative: No bubbles.

Oxygen Requirements and Anaerobic Jar

• Obligate aerobes: Require oxygen.

• Facultative anaerobes: Grow with or without oxygen.

• Obligate anaerobes: Cannot tolerate oxygen.

• Anaerobic jar creates oxygen-free environment to test growth.

Nitrate Reduction Test

• Tests ability to reduce nitrate to nitrite or nitrogen gas.

• Critical for microbial nitrogen cycling in environment.

Selective and Differential Media

PEA Plates

• Selective for Gram-positive bacteria.

• Selective agent: Phenylethyl alcohol.

MSA Plates (Mannitol Salt Agar)

• Selective for Staphylococcus species (high salt).

• Differential for mannitol fermentation (yellow color indicates fermentation).

MacConkey Agar

• Selective for Gram-negative bacteria (bile salts, crystal violet inhibit Gram-positive).

• Differential for lactose fermentation (pink colonies indicate fermentation).

EMB Plates (Eosin Methylene Blue)

• Selective for Gram-negative bacteria.

• Differential: Escherichia coli produces metallic green sheen.

Blood Agar and Hemolysis

• Used to detect hemolytic activity.

• Types:

  • Alpha hemolysis: Partial (greenish).

  • Beta hemolysis: Complete (clear zone).

  • Gamma hemolysis: None.

Leukocytes and Blood Smears

Leukocyte Characteristics

• Granulocytes: Neutrophils, eosinophils, basophils; have granules, lobed nuclei.

• Agranulocytes: Lymphocytes, monocytes; lack granules, round or kidney-shaped nuclei.

• Staining: Nuclei stain purple, cytoplasm varies.

• Neutrophils most common; basophils rarest.

Leukocyte Changes in Disease

Disease	Leukocyte Change
Acute myeloblastic leukemia	Increased myeloblasts
Eosinophilia	Elevated eosinophils
Bacterial infections	Elevated neutrophils
Lymphoma	Elevated lymphocytes
Infectious mononucleosis	Elevated atypical lymphocytes
Leucopenia	Decreased total leukocytes
Hairy cell leukemia	Presence of "hairy" lymphocytes

Quantitative Microbiology: Cell Counting and Dilutions

Original Cell Density (OCD) Calculation

• OCD is the concentration of cells in the original sample.

• Formula: $\text{OCD} = \frac{\text{CFU}}{\text{Volume plated} \times \text{Dilution factor}}$

• CFU: Colony Forming Units; each colony arises from a single cell or group.

Dilution Factor Calculation

• If tube dilution is $10^{-3}$ and 100µL (0.1mL) is plated from a 1000µL tube:

• Plate dilution = tube dilution × (volume plated / total volume)

• Formula: $\text{Plate dilution} = 10^{-3} \times \frac{100}{1000} = 10^{-4}$

Counting Colonies: Acceptable Range

• Count plates with 30–300 colonies for accuracy.

• Fewer than 30: Too few; unreliable.

• More than 300: Too many; difficult to count.

Fungi, Protozoa, and Helminths

Fungal Structure and Metabolism

• Hyphae: Filamentous structures.

• Mycelium: Network of hyphae.

• Septate: Hyphae with cross-walls.

• Non-septate: Hyphae without cross-walls.

• Fungi are saprotrophic: absorb nutrients from decaying matter.

Thermal Dimorphism in Fungi

• Some fungi change form based on temperature.

• Room temperature: Mold form.

• Body temperature (37°C): Yeast form.

Protozoan Forms

• Trophozoite: Active, feeding stage.

• Cyst: Dormant, resistant stage.

Helminth Anatomy and Hosts

• Scolex: Head of tapeworm, attaches to host.

• Proglottids: Segments containing reproductive organs.

• Definitive host: Where parasite reaches maturity.

• Intermediate host: Where parasite develops but does not mature.

Fungal and Protozoan Reproductive Structures

Structure	Function
Sporangia	Produce spores
Sporangiophores	Stalks supporting sporangia
Gametangia	Produce gametes
Conidia	Asexual spores
Conidiophores	Stalks supporting conidia

Clinically Important Eukaryotic Microbes

• Fungi: Candida (causes candidiasis), Aspergillus (causes aspergillosis).

• Protozoa: Entamoeba (amoebic dysentery), Giardia (giardiasis), Trichomonas (trichomoniasis), Trypanosoma (African sleeping sickness), Plasmodium (malaria), Toxoplasma (toxoplasmosis).

• Helminths: Flatworms (flukes, tapeworms), roundworms (Ascaris, pinworms, hookworms).

Example: Plasmodium causes malaria, transmitted by mosquitoes.