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 varying magnifications (e.g., 4x, 10x, 40x, 100x).

• Stage: Holds the specimen slide.

• 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 the product of the ocular lens and the objective lens magnifications.

• 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, making details visible.

• Resolution: The ability to distinguish two close points as separate.

Microscope Care and Storage

• Clean lenses with lens paper only.

• Store with the lowest objective in place.

• Cover the microscope to prevent dust accumulation.

Achieving Good Contrast

• Adjust the diaphragm and condenser.

• Use stains to enhance contrast.

Simple vs. Compound Microscopes

• Simple microscope: Uses a single lens.

• Compound microscope: Uses multiple lenses for higher magnification and resolution.

Aseptic Technique

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

• Flame sterilization of tools.

• Minimizing exposure of sterile items to air.

Streaking Techniques and Isolated Colonies

• Streak plate method: Used to isolate individual colonies from a mixed culture.

• Proper streaking involves spreading bacteria over the surface in a pattern to dilute them.

• Isolated colonies indicate successful separation.

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 biohazardous materials in designated containers.

• Glassware in sharps containers.

• Contaminated media in autoclave bags.

Staining and Cell Morphology

Simple Stains

Simple stains use a single dye to highlight cell shape and arrangement.

• Provides information about morphology and arrangement.

• Common dyes: methylene blue, crystal violet.

Cell Shapes and Arrangements

• Coccus: Spherical.

• Bacillus: Rod-shaped.

• Spirillum: Spiral-shaped.

• Arrangements: Chains (strepto-), clusters (staphylo-), pairs (diplo-).

Gram Stain Procedure

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 Gram 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).

Bacterial Smear Preparation and Heat Fixing

• Spread bacteria on slide, air dry.

• Heat fix to kill and adhere cells.

Capsule and Endospore Stains

• Capsule stain: Detects polysaccharide capsules; uses negative staining.

• Endospore stain: Detects spores; uses malachite green and safranin.

KOH Test

• Potassium hydroxide test distinguishes Gram-negative (stringy DNA released) from Gram-positive (no string).

Microbial Metabolism and Biochemical Tests

Citrate Test

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

• Negative result: Green color; cannot use citrate.

Urea Hydrolysis Test

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

• Negative result: Yellow/orange; no urea breakdown.

Decarboxylation Test

• Purpose: Detects decarboxylation of amino acids.

• Positive: Purple color; alkaline products.

• Negative: Yellow; acidic products.

• Mineral oil: Creates anaerobic conditions.

Gelatinase Test

• Positive: Liquefied gelatin; bacteria produce gelatinase.

• Negative: Solid gelatin.

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

• Sulfur production: Black precipitate.

• Indole production: Red after adding Kovac's reagent.

• Motility: Growth away from stab line.

Phenol Red Broth Test

• Components: Base broth, carbohydrate, phenol red indicator, Durham tube.

• Positive: Yellow; acid produced from carbohydrate fermentation.

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

Advanced Biochemical Tests and Antimicrobial Susceptibility

MR and VP Tests

• Methyl Red (MR): Red color indicates mixed acid fermentation.

• Voges-Proskauer (VP): Red color indicates acetoin production.

Kirby-Bauer Test

• Preparation: Create a lawn of bacteria on agar.

• Purpose: Test antibiotic susceptibility.

• Zone of inhibition: Measure diameter to assess effectiveness.

Oxidase Test

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

• Negative: No color change.

Catalase Test

• Reagent: Hydrogen peroxide.

• Positive: Bubbling (oxygen released).

• Negative: No bubbles.

Oxygen Requirements and Anaerobic Jar

• Obligate aerobe: Requires oxygen.

• Facultative anaerobe: Can grow with or without oxygen.

• Anaerobic jar: Used to determine oxygen requirements.

Nitrate Reduction Test

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

• Important for environmental nitrogen cycling.

Selective and Differential Media

PEA Plates

• Selective agent: Phenylethyl alcohol.

• Growth: Gram-positive bacteria.

MSA Plates

• Selective: High salt; selects for Staphylococcus species.

• Differential: Mannitol fermentation turns medium yellow.

MacConkey Agar

• Selective: Bile salts and crystal violet inhibit Gram-positive.

• Differential: Lactose fermentation turns colonies pink.

EMB Plates

• Selective: For Gram-negative bacteria.

• Differential: Escherichia coli produces metallic green sheen.

Blood Agar and Hemolysis

• Alpha hemolysis: Partial, greenish discoloration.

• Beta hemolysis: Complete, clear zone.

• Gamma hemolysis: No hemolysis.

Leukocytes and Blood Smears

Leukocyte Characteristics

• Granulocytes: Have granules; include neutrophils, eosinophils, basophils.

• Agranulocytes: No granules; include lymphocytes, monocytes.

• Nuclei: Shape and staining help identify cell type.

• Most common: Neutrophils.

• Rarest: Basophils.

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 leukocytes
Hairy cell leukemia	Presence of hairy cells

Quantitative Microbiology: Cell Counting and Dilutions

Original Cell Density (OCD) Calculation

• CFU: Colony Forming Unit; represents a viable cell or group.

• Formula:

$\text{OCD} = \frac{\text{CFU}}{\text{Volume plated (mL)} \times \text{Dilution factor}}$

Dilution Factor Calculation

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

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

• Example:

$\text{Plate dilution} = 10^{-3} \times \frac{0.1}{1} = 10^{-4}$

Counting Colonies

• Count plates with 30-300 colonies for accuracy.

• Too few: <30; too many: >300.

Eukaryotic Microbes: 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.

• Metabolism: Fungi are saprotrophic, absorbing nutrients from decaying matter.

Thermal Dimorphism in Fungi

• Some fungi change form based on temperature.

• Yeast form at 37°C; mold form at 25°C.

Protozoan Forms

• Trophozoite: Active, feeding stage.

• Cyst: Dormant, resistant stage.

Helminth Anatomy

• Scolex: Head of tapeworm, attaches to host.

• Proglottids: Segments containing reproductive organs.

Host Types

• Definitive host: Where parasite reaches maturity.

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

Fungal and Protozoan Reproductive Structures

• Sporangia: Produce spores.

• Sporangiophores: Stalks bearing sporangia.

• Gametangia: Produce gametes.

• Conidia: Asexual spores.

• Conidiophores: Stalks bearing conidia.

Clinically Important Microbes and Diseases

Microbe	Type	Disease
Candida	Fungus	Candidiasis
Aspergillus	Fungus	Aspergillosis
Entamoeba	Protozoan	Amebiasis
Giardia	Protozoan	Giardiasis
Trichomonas	Protozoan	Trichomoniasis
Trypanosoma	Protozoan	Trypanosomiasis (e.g., sleeping sickness)
Plasmodium	Protozoan	Malaria
Toxoplasma	Protozoan	Toxoplasmosis
Flukes	Flatworm	Schistosomiasis
Tapeworms	Flatworm	Taeniasis
Ascaris	Roundworm	Ascariasis
Pinworms	Roundworm	Enterobiasis
Hookworms	Roundworm	Hookworm infection

Additional info: Academic context and definitions were added to clarify brief points and ensure completeness. Tables were reconstructed for leukocyte changes and clinically important microbes. Equations are provided in LaTeX format as required.