lab study guide

Microbiology Lab Practical Study Guide

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## Lab Safety

- Always wear PPE (lab coat, gloves, goggles).

- Proper disposal of biohazards, sharps, and chemicals.

- No food, drinks, or open-toed shoes in the lab.

- Report spills and accidents immediately.

- Know emergency procedures (fire extinguisher, eyewash station, safety shower).

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## Journal Article Components

1. Title – Brief and informative.

2. Abstract – Summary of the study.

3. Introduction – Background, hypothesis.

4. Materials & Methods – Experimental procedures.

5. Results – Data presented in figures/tables.

6. Discussion – Interpretation of results.

7. Conclusion – Summary and future directions.

8. References – Cited sources.

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## Microscopy

### Parts of the Microscope

- Ocular lens – Magnification (10x).

- Objective lenses – Varying magnifications (4x, 10x, 40x, 100x).

- Stage – Holds the slide.

- Condenser – Focuses light on the specimen.

- Diaphragm – Controls light intensity.

- Coarse/Fine focus – Adjust clarity.

### Using the Microscope

- Start with 4x objective and coarse focus, then move to higher magnifications.

- Use oil immersion for the 100x lens.

- Adjust light and contrast appropriately.

### Cleaning & Storing the Microscope

- Use lens paper only.

- Rotate scanning objective in place before storage.

- Wrap cord neatly and cover with dust cover.

- oil immersion lens: use lens paper and ethanol

### Resolution & Magnification

- Resolution: Ability to distinguish two close points as separate. - know how to calculate

• wavelength/ 2( numerical aperture)

- Total Magnification = Ocular magnification × Objective magnification.- know how to calculate

### Types of Microscopy

- Brightfield – Common, requires staining.

- Darkfield – Good for live, unstained specimens.

- Phase Contrast – Highlights density differences.

- Electron Microscopy:

- Scanning (SEM) – Surface details.

- Transmission (TEM) – Internal details.

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## Staining Techniques

### Smear Preparation

- Spread culture thinly on slide.

-dry culture: add water

- Air dry and heat fix to adhere bacteria.

### Simple Staining

- Uses one basic dye (e.g., Methylene blue) to visualize bacterial shape/arrangement.

### Negative Staining

- Uses acidic dye (e.g., Nigrosin) to stain the background, leaving bacteria unstained.

- No heat fixing to preserve size and shape.

### Gram Staining

1. Crystal violet – Primary stain (stains all cells purple). (Primary stain)

2. Gram’s iodine – Mordant (fixes stain in Gram-positive bacteria).

3. Alcohol – Decolorizer (removes stain from Gram-negative bacteria). (Stain remover)

4. Safranin – Counterstain (stains Gram-negative cells pink). (Counterstain)

- Gram-positive: Thick peptidoglycan, retains purple stain.

- Gram-negative: Thin peptidoglycan, decolorized, stained pink.

^ if you leave out a step what color would it be

Ex: leaving out the mordant stain

#### Troubleshooting Gram Stain

- Over-decolorization: Gram-positive may appear Gram-negative.

- Under-decolorization: Gram-negative may remain purple.

- Thick smears can trap stain and prevent proper results.

- Old cultures may give incorrect results.

### Spore Staining (Schaeffer-Fulton Method)

- Smear stain

- Malachite green stains endospores.

- Safranin stains vegetative cells.

- Identifies Bacillus and Clostridium species.

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## Bacterial Shapes & Arrangements

- Cocci (spherical) – Staphylococcus (clusters), Streptococcus (chains).

- Bacilli (rod-shaped) – Single, chains.

- Spirilla (spiral-shaped).

### Common Bacteria in Lab

| Bacteria | Shape | Gram Stain |

|----------|--------|-------------|

| Escherichia coli | Rod | Gram-negative |

| Staphylococcus epidermidis | Cocci | Gram-positive |

| Enterobacter aerogenes | Rod | Gram-negative |

| Bacillus megaterium | Rod | Gram-positive |

^ know how to spell

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## Pure Culture Techniques

### Streak Plate Method

- Used to isolate individual colonies from a mixed culture.

- Quadrant streaking: Flame loop between streaks.

- A good streak plate shows well-isolated colonies.

-two different kinds of organisms growing

### Troubleshooting Streak Plates

- Not sterilizing the loop properly can lead to contamination.

- Dragging too much bacteria can prevent isolation.

- Not cooling the loop can kill bacteria before streaking.

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## Flashcard-Style Questions

### Lab Safety

1. What PPE is required for microbiology lab work?

2. How should biohazard waste be disposed of?

### Microscopy

3. What part of the microscope controls light?

4. How do you calculate total magnification?

5. When would you use phase contrast microscopy?

### Staining

6. What is the primary stain in Gram staining?

7. What does a negative stain highlight?

8. Why do Gram-positive bacteria retain the crystal violet stain?

### Bacteria Identification

9. What shape is Staphylococcus epidermidis?

10. Which bacteria are Gram-negative rods?

### Culture Techniques

11. What is the purpose of the streak plate method?

12. How can you tell if a streak plate was done correctly?

Tips for Making Effective Flashcards

1. Keep it Simple: Use brief questions and answers. Avoid cluttering the card with too much information.

2. Use Clear Language: Use simple and straightforward language for better understanding.

3. Incorporate Visuals: Include diagrams or images when possible to enhance memory retention.

4. Organize by Topics: Group flashcards by themes or topics for more focused study sessions.

5. Use Colors for Cues: Color code cards to differentiate between types of information (e.g., definitions, examples).

6. Practice Retrieval: Regularly test yourself with the flashcards to reinforce learning and improve recall.

7. Rotate and Update: Regularly review and update cards to reflect your current knowledge and to include new information.