Microbiology Lab Techniques and Bacterial Cell Structures: Study Notes

Metachromatic Granules

Definition and Importance

• Metachromatic granules (also called volutin) are large inclusions found inside prokaryotic cells.

• They stain red with certain blue dyes, such as methylene blue, due to their chemical composition.

• These granules act as an inorganic phosphate reserve (polyphosphate), which is important for ATP synthesis.

• Formed by cells in phosphate-rich environments and used when phosphate is scarce.

• The presence of metachromatic granules can help identify certain bacterial species, such as Corynebacterium diphtheriae (the causative agent of diphtheria).

Example: Corynebacterium diphtheriae accumulates metachromatic granules, which aids in its identification in clinical microbiology.

Bacterial Motility

Mechanisms and Observation

• Motility is the ability of bacteria to move toward or away from stimuli (e.g., nutrients or toxins).

• The main mechanism is the flagellum, which enables movement through runs and tumbles.

• Chemotaxis refers to movement in response to chemical gradients:

  • Positive chemotaxis: movement toward an attractant.

  • Negative chemotaxis: movement away from a repellent.

Brownian Movement vs. True Motility

• Brownian movement is the random motion of particles caused by collisions with water molecules; it is not true motility.

• Streaming refers to the appearance of cells moving in one direction due to currents under the coverslip, also not true motility.

Example: In a wet mount, Brownian movement may be observed, but only flagellar movement is considered true motility.

Microscopy Techniques for Observing Bacteria

Hanging Drop Method

• A technique where a drop of liquid culture hangs from a coverslip over a depression slide.

• Advantages:

  • Bacteria are not compressed, allowing for more natural movement.

  • Less heat from the microscope lamp affects the sample.

  • Superior to standard wet mounts for observing motility.

Heat Fixation

• Process of attaching microorganisms to a slide by briefly passing it through a flame.

• Kills the cells and preserves their structure with minimal distortion.

• Essential for staining procedures to ensure cells remain on the slide.

• Steps:

  1. Smear preparation

  2. Fixation (heating)

  3. Staining

Isolation and Enumeration of Bacteria

Streak Plate Method

• Used to isolate pure bacterial cultures from mixed samples.

• A sterile loop is used to streak bacteria over the surface of an agar plate, diluting the sample to obtain isolated colonies.

• Allows for the study of individual bacterial species without interference from others.

Spread Plate Method

• Used to isolate and count bacteria by spreading a small volume (usually 0.1 mL) of diluted sample over the surface of an agar plate.

• Colonies grow only on the surface, making them easy to count and analyze.

• Disadvantages: Only surface growth is observed; not suitable for heat-sensitive organisms due to brief exposure to heat.

Pour Plate Method

• Used to count viable bacteria by mixing a diluted sample with molten agar (at ~50°C) and pouring into a Petri dish.

• Colonies grow both within and on the surface of the medium.

• Useful for detecting anaerobic bacteria and for samples with high bacterial concentrations.

• Disadvantages: Heat can kill sensitive bacteria; colonies within agar are not ideal for diagnostic purposes.

Staining Techniques and Cell Wall Differentiation

Staining Mechanisms

• Dyes are salts composed of positive and negative ions; the ion carrying the color is the chromophore.

• Basic dyes (e.g., crystal violet, safranin): chromophore is positive; stains the negatively charged cell interior.

• Acidic dyes (e.g., nigrosine): chromophore is negative; stains the background, not the cell (used in negative staining).

• Negative staining is best for observing cell shape as it does not require heat fixation, minimizing distortion.

Gram Stain

• Differential stain that classifies bacteria based on cell wall structure.

• Procedure:

  1. Add crystal violet (primary stain).

  2. Add iodine (mordant) to form a crystal violet-iodine (CV-I) complex, which is larger and retained differently by Gram-positive and Gram-negative bacteria.

  3. Decolorize with alcohol.

  4. Counterstain with safranin.

• Gram-positive bacteria retain the CV-I complex and appear purple; Gram-negative bacteria lose the complex and appear pink/red.

Acid-Fast Stain

• Used to identify bacteria with waxy cell walls containing mycolic acid (e.g., Mycobacterium).

Endospore Stain

• Endospores require two elements for germination: calcium and dipicolinic acid.

Media Types and Their Uses

Selective and Differential Media

• Selective media contain ingredients that inhibit the growth of some organisms while allowing others to grow (e.g., mannitol salt agar selects for salt-tolerant bacteria).

• Differential media allow for the distinction between different types of bacteria based on their biological characteristics (e.g., mannitol fermentation changes pH indicator color).

• Mannitol salt agar is both selective (for salt-tolerant organisms) and differential (phenol red indicates mannitol fermentation).

Growth Phases of Bacteria

Phases of Bacterial Growth

• Lag phase: Cells are metabolically active but not dividing; preparing for cell division.

• Log (exponential) phase: Cells divide at a constant rate; population doubles every 10–20 minutes under optimal conditions.

Oxygen Requirements and Enzymes

Oxygen Toxicity and Bacterial Defense Mechanisms

• Some bacteria require oxygen, while others are killed by it or tolerate it using enzymes.

• Catalase converts toxic hydrogen peroxide into water and oxygen:

  • $2H_2O_2 \rightarrow 2H_2O + O_2$

• Bacteria lacking catalase (e.g., Streptococcus) may use peroxidase or other enzymes to detoxify oxygen radicals.

Microscopy Terms and Calculations

Key Terms

• Parfocal: Microscope remains in focus when switching objectives.

• Resolving power: The ability to distinguish two points as separate; higher resolving power means greater detail.

Ocular Micrometer Calibration

• Used to measure microorganisms under the microscope.

• Requires calibration with a stage micrometer and application of a conversion formula.

Colony Counting and Calculations

Colony-Forming Units (CFU)

• CFU/mL is calculated to estimate the number of viable bacteria in a sample.

• Plates with fewer than 30 colonies are considered too few to count (TFTC); more than 300 are too many to count (TMTC).

Absorbance Calculations

• Absorbance (A) can be calculated from percent transmittance (%T) using the formula:

$A = 2 - \log_{10}(%T)$

Media Preparation Calculations

Example Calculations

• To prepare media, use the ratio provided (e.g., 30 g/L for soy broth).

• For 500 mL of broth: $\frac{30\ g}{1000\ mL} \times 500\ mL = 15\ g$

Summary Table: Staining Dyes and Their Properties

Dye	Type	Chromophore Charge	Stains
Crystal Violet	Basic	Positive	Cell interior
Safranin	Basic	Positive	Cell interior
Nigrosine	Acidic	Negative	Background (negative stain)

Additional Info

• Ubiquity of bacteria: Bacteria are found everywhere in the environment.

• Colony color changes: Some bacteria (e.g., Serratia marcescens) produce pigments at certain temperatures (red at room temperature, colorless at 37°C).