Microbiology Lab Techniques & Procedures
Pasteur’s Gooseneck Flask Experiment
Nutrient-rich broth in an S-shaped flask was boiled and left open to the air.
Observation: Nothing grew in the broth.
Conclusion: This experiment disproved spontaneous generation, demonstrating that bacteria trapped in the flask’s vent prevented contamination.
Gram Stain
Purpose: To separate bacteria into two categories (Gram-positive and Gram-negative) based on cell wall structure.
Mechanism:
Gram-positive bacteria have a thick peptidoglycan layer in their cell wall that retains the primary stain (crystal violet) even after decolorization.
Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane that is disrupted by decolorization, causing them to lose the primary stain and take up the counterstain (safranin).
Procedure:
- Apply primary stain: Crystal violet (1 minute).
- Wash with water.
- Apply Gram’s iodine (1 minute).
- Rinse with water.
- Decolorize with ethanol or acetone alcohol (8-10 drops).
- Rinse with water immediately.
- Apply counterstain: Safranin (1 minute).
- Rinse with water.
- Blot dry.
- Observe under oil immersion (100x).
Results:
Gram-positive: Violet/Purple
Gram-negative: Red/Pink
Acid-Fast Stain
Purpose: To differentiate bacteria with a waxy lipoidal layer in their cell wall (acid-fast bacteria) from those without.
Mechanism:
Acid-fast bacteria, like those in the genus Mycobacterium, resist decolorization by acid-alcohol due to the waxy layer in their cell walls, retaining the primary stain (carbol fuchsin).
Non-acid-fast bacteria are decolorized and take up the counterstain (methylene blue).
Procedure:
- Smear bacteria across the slide.
- Apply carbol fuchsin.
- Steam for 5 minutes (do not let dry).
- Rinse with water.
- Decolorize with acid-alcohol.
- Rinse with water.
- Apply methylene blue (1 minute).
- Rinse with water, blot dry.
- Observe under oil immersion.
Results:
Acid-fast bacteria: Violet/Red
Non-acid-fast bacteria: Blue
Spore Stain
Purpose: To differentiate bacterial endospores from vegetative cells.
Mechanism:
Malachite green stains both endospores and vegetative cells. However, the heat used during the staining process helps the malachite green penetrate the tough spore coat.
Decolorization with water removes the stain from vegetative cells, but not from the spores. Counterstaining with safranin then stains the vegetative cells pink.
Results:
Endospores: Green
Vegetative cells: Red/Pink
Capsule Stain
Purpose: To visualize the capsule, a gelatinous outer layer, surrounding some bacteria.
Mechanism:
Crystal violet stains both the capsule and the cell wall. Copper sulfate is used as a decolorizing agent, which removes the crystal violet from the capsule but not the cell wall.
Results:
Capsule: White/Light Blue
Cell Wall: Purple
Negative Stain
Purpose: To observe bacteria in their natural size, shape, and arrangement without heat-fixing, which can distort cell morphology.
Mechanism:
Nigrosin or India ink, both negatively charged stains, are repelled by the negatively charged bacterial cell wall, resulting in a dark background and unstained cells.
Results:
Cells: Colorless
Background: Black/Gray
Koch’s Postulates
These postulates are a set of criteria used to establish a causal relationship between a microbe and a disease.
- The organism must be present in all cases of the disease and absent in healthy individuals. (Note: This postulate has limitations, as some pathogens can be present in asymptomatic carriers).
- The organism must be isolated from the diseased host and grown in pure culture in vitro. (Note: Not all organisms can be cultured in vitro).
- The pure culture of the organism must cause the disease when inoculated into a healthy, susceptible animal in vivo.
- The organism must be re-isolated from the experimentally infected animal and shown to be identical to the original isolate.
Prokaryotes vs. Eukaryotes
| Characteristic | Prokaryotes | Eukaryotes |
|---|---|---|
| Nucleus | Absent | Present |
| Cell Membrane | No sterols | Sterols present |
| Ribosomes | 70S (50S + 30S subunits) | 80S (60S + 40S subunits) |
| Organelles | Absent | Present (e.g., mitochondria, chloroplasts) |
| Respiration | Cell membrane | Mitochondria |
| Photosynthesis | Cell membrane (if present) | Chloroplasts |
| Size | <2µm (generally smaller) | 2-100µm+ (generally larger) |
| DNA | Single, circular molecule; no histones | Multiple chromosomes with histones |
| Division | Binary fission (no mitosis) | Mitosis |
| Motility | Flagella (if present; simple structure) or gliding | Flagella (if present; complex 9+2 structure), cilia, or cytoplasmic streaming |
| Microtubules | Absent or some present | Present (especially in animals) |