lab 6: positive stains pt 2, endospore and acid-fast stains

Endospore

Spore produced inside a bacterium. Endospores are associated with gram-positive rods.

Forespore

An endospore that is not fully developed; it is an early stage of endospore formation within the cell.

Free endospore

A fully formed endospore that has been released from the parent cell.

Germination

The process in which an endospore breaks dormancy and returns to the vegetative state.

Sporulation (sporogenesis)

The process of producing endospores.

Central endospore

An endospore located in the middle of the bacterial cell.

Subterminal endospore

An endospore located between the middle and the terminal end of the bacterial cell.

Terminal endospore

An endospore located at one pole or terminal end of the bacterial cell.

Vegetative cell

A bacterial cell that is metabolically active and is not dormant.

Conditions that trigger sporulation

Unfavorable or adverse conditions such as extreme or adverse temperatures, absence of water, presence of toxins, absence of nutrients, unfavorable pH, radiation, and other stressful environmental conditions.

Conditions that trigger germination

Favorable conditions such as availability of water, nutrients, appropriate temperature, and absence of harmful or toxic conditions allow an endospore to break dormancy and return to the vegetative state.

Structure of an endospore

Contains a copy of bacterial DNA surrounded by a spore coat, layers of peptidoglycan, keratin, and multiple membranes. The endospore is dehydrated and dormant.

Bacillus anthracis

Endospore-forming Bacillus species that causes anthrax.

Bacillus cereus

Endospore-forming Bacillus species associated with food poisoning; the notes specifically associate it with rice.

Clostridium tetani

Endospore-forming Clostridium species that causes tetanus.

Clostridium botulinum

Endospore-forming Clostridium species that causes botulism.

Clostridium species

Endospore-forming bacteria; the notes identify Clostridium tetani and Clostridium botulinum as examples and note that they are anaerobic.

Primary stain used in the endospore stain

Malachite green.

Time for malachite green in the endospore stain

15 minutes.

Heat used during the endospore stain

Steam is used during the primary staining step.

Color of vegetative cells after malachite green

Green or teal before the decolorizing step.

Color of endospores after malachite green

Green or teal.

Decolorizer used in the endospore stain

Water.

Time for the water decolorizer in the endospore stain

Rinse until the water runs clear.

Effect of water on vegetative cells during the endospore stain

Water removes the malachite green from vegetative cells, making them colorless.

Effect of water on endospores during the endospore stain

Endospores retain the malachite green and remain green or teal.

Counterstain used in the endospore stain

Safranin.

Time for safranin in the endospore stain

1 minute.

Final color of vegetative cells in the endospore stain

Pink.

Final color of endospores in the endospore stain

Green or teal.

Is the endospore stain a differential stain?

Yes. It uses multiple dyes and a decolorizer to distinguish endospores from vegetative bacterial cells.

Why is heat needed during the endospore stain?

Heat makes the spore coat permeable so that the malachite green dye can enter and stain the endospore.

Appearance of Bacillus megaterium cells in a Gram stain

The vegetative cells would appear purple.

Appearance of endospores in a Gram stain

The endospores would appear colorless.

Disadvantage of the endospore stain

It takes additional time and requires the use of a steam bath.

Mycolic acid

Wax associated with the cell wall of acid-fast bacteria.

BCG

Bacillus of Calmette-Guérin; associated with the vaccine noted on the worksheet.

Tubercle

Nodules that occur in tissues infected with tuberculosis.

Acid-fast bacterial cell wall property

The cell walls of acid-fast bacteria are hydrophobic.

Are acid-fast bacteria easy or difficult to stain?

Acid-fast bacteria are difficult to stain because of their hydrophobic, waxy cell wall.

Ziehl-Neelsen primary stain

Carbol fuchsin.

Ziehl-Neelsen use of heat

Uses steam during staining.

Ziehl-Neelsen decolorizer

Hydrochloric acid plus alcohol.

Ziehl-Neelsen counterstain

Brilliant green or methylene blue.

Final color of acid-fast cells with Ziehl-Neelsen stain

Magenta.

Final color of non-acid-fast cells with Ziehl-Neelsen stain

Blue or green, depending on the counterstain used.

Kinyoun primary stain

Carbol fuchsin containing excess phenol.

Kinyoun use of heat

Does not use heat or steam.

Kinyoun decolorizer

Uses the same acid-alcohol decolorizer as the Ziehl-Neelsen procedure.

Kinyoun counterstain

Uses the same counterstain options as Ziehl-Neelsen: brilliant green or methylene blue.

Final color of acid-fast cells with Kinyoun stain

Magenta.

Final color of non-acid-fast cells with Kinyoun stain

Blue or green, depending on the counterstain.

Is the acid-fast stain a differential stain?

Yes. It uses multiple dyes and a decolorizer to distinguish acid-fast cells from non-acid-fast cells.

Which acid-fast procedure is most similar to the endospore stain?

Ziehl-Neelsen, because both procedures use steam.

How does Ziehl-Neelsen overcome the difficulty of staining acid-fast bacteria?

It uses phenol and steam to help the primary stain penetrate the difficult-to-stain acid-fast cell wall.

How does Kinyoun overcome the difficulty of staining acid-fast bacteria?

It uses an excess amount of phenol rather than steam.

Ziehl-Neelsen advantage

Helps preserve the arrangement of the cells.

Ziehl-Neelsen disadvantages

The procedure takes longer and requires the use of steam.

Kinyoun advantages

It is faster than Ziehl-Neelsen and does not require steam.

Kinyoun disadvantage

The stronger chemical treatment can cause breakdown or damage to cells.

Mycobacterium tuberculosis

Acid-fast pathogen that causes tuberculosis (TB).

Mycobacterium leprae

Acid-fast pathogen that causes leprosy.

autoclave

a device that uses moist heat and pressure to sterilize materials

purpose of an autoclave

sterilization using moist heat and pressure

water boiling point at sea level

100°C

endospores and boiling water

endospores are heat-resistant structures produced by some gram-positive rods and are not killed by water boiling at 100°C

relationship between pressure and boiling point

increasing pressure raises the boiling point of a liquid

standard autoclave temperature

121°C

standard autoclave pressure

15 psi

standard autoclave conditions

121°C and 15 psi

liquid autoclave cycle

pressure should not be released immediately because liquid can steam up or boil over if pressure drops too quickly

solid autoclave cycle

pressure can be released more quickly after sterilization because solid materials do not boil over like liquids

difference between liquid and solid autoclave cycles

liquids require slower pressure release because rapid pressure loss can cause boiling or steaming; solids can tolerate faster pressure release