Lab 7 - Endospore Stain and Bacterial Motility, Lab Exam 2

What are endospores?

Endospores are highly resistant survival structures formed by certain bacteria that allow them to survive extreme conditions such as heat, drying, radiation, and chemical disinfectants.

Which two bacterial genera produce endospores?

• Bacillus
• Clostridium

What is the metabolically active, growing form of a bacterium that forms an endospore called?

The vegetative cell.

What happens to the vegetative cell after an endospore forms?

The vegetative cell degrades and releases the endospore.

When do endospores become vegetative cells again?

When environmental conditions become favorable, the endospore germinates into a vegetative cell, usually within about 90 minutes.

What triggers endospore formation (sporulation)?

Harsh environmental conditions such as nutrient depletion.

Why are endospores difficult to stain?

Because of their thick, resistant spore coat.

What two important conditions are required to stain endospores?

Strong dye (malachite green) and heat (steam) to allow dye penetration.

What is the primary stain used in endospore staining?

Malachite green.

What is the secondary (counterstain) used in endospore staining?

Safranin.

What stain do endospores take up?

Malachite green.

What stain do vegetative cells take up?

Safranin.

What is used as the decolorizing agent in endospore staining?

Water.

Why is the slide steamed during staining with malachite green?

To force the stain through the tough spore coat so the endospore can be stained.

What color do endospores appear after staining?

Green.

What color do vegetative cells appear after staining?

Red / Pink (safranin).

How do you identify endospores under a microscope?

• Green oval structures inside or outside cells.

How do vegetative cells appear under the microscope in an endospore stain?

Red/pink rod-shaped cells.

What is the basic procedure for endospore staining?

Prepare and heat-fix smear, place blotting paper over smear, saturate with malachite green, steam for ~5 minutes, rinse with water, counterstain with safranin (1 minute), rinse and blot dry.

What is bacterial motility?

The ability of bacteria to move under their own power.

What structures allow bacterial motility?

Flagella.

What are the three basic parts of a flagellum?

Filament, Hook, Basal body.

What does the filament do?

It is the long helical structure that extends from the cell surface.

What does the hook do?

It connects the filament to the basal body.

What does the basal body do?

It anchors the flagellum in the cell membrane and acts as a rotary motor.

What is the behavior of motile bacteria called?

Taxis.

What is chemotaxis?

Movement of bacteria toward or away from chemicals.

What happens when flagella rotate counterclockwise?

The bacteria swim forward (run).

What happens when flagella rotate clockwise?

The bacterium tumbles and changes direction.

What is biased random walk?

A movement pattern where bacteria alternate between swimming and tumbling but overall move toward an attractant.

What is monotrichous flagella arrangement?

Single flagellum at one pole.

What is amphitrichous arrangement?

One flagellum at both poles.

What is lophotrichous arrangement?

Cluster of flagella at one or both poles.

What is peritrichous arrangement?

Flagella distributed all around the cell.

What are axial filaments?

Bundles of endoflagella found in spirochetes.

Where are axial filaments located?

Between the peptidoglycan cell wall and outer membrane.

How are axial filaments arranged?

They attach to both ends of the cell and wrap around the cell body.

What are ways to detect bacterial motility?

Phase contrast microscopy, Dark-field microscopy, Flagella staining, Motility test medium.

Why are special stains required to see flagella with a light microscope?

Because flagella are thinner than the resolution limit of a normal microscope.

How does flagella staining work?

Dyes precipitate along the flagella, making them thicker and visible.

What is a motility test medium?

A semi-solid medium used to detect bacterial motility.

What is the special feature of motility medium?

It contains low agar concentration (0.3%), creating a soft gel that allows bacteria to move.

What inoculation method is used for motility test medium?

Stab inoculation.

How deep should the inoculation stab be?

½–¾ inch into the agar.

How do you detect motility in motility medium?

Observe whether growth spreads away from the stab line.

What result indicates a motile organism?

Cloudy growth spreading throughout the medium.

What result indicates a non-motile organism?

Growth only along the stab line.

What organisms were used in the motility test in this lab?

• Staphylococcus aureus
• Pseudomonas aeruginosa

Would motility testing work if the medium had 3% agar instead of 0.3%?

No. The agar would be too solid for bacteria to move through, preventing detection of motility.