Exercises 4-5

What is the acid-fast stain also called?

ziehl-neelsen method

What is a type of structural stain?

endospore staining

What are the types of differential stains?

• gram stain

• acid-fast stain

What are the reagents used in acid-fast staining?

• carbol fuchsin

• steam

• acid alcohol

• methylene blue

Carbol fuchsin =

primary dye

Acid alcohol =

decolorizer

Methylene blue =

secondary dye/counterstain

Is steam used in acid-fast staining?

yes

Acid-fast microorganisms have

high mycolic acid content in their cell walls

• waxy and resists staining

Why is steam used in acid-fast staining?

to force the primary dye to penetrate the waxy cell wall

Fastness means

to retain the dye when challenged by a decolorizer

Acid-fast cells retain the dye after

decolorization because of the high wax content

Cerise =

acid-fast positive

Blue =

acid-fast negative

Non-acid-fast cells will

readily decolorize and be counterstained with methylene blue

Acid fast genera =

mycobacterium

• M. tuberculosis

• M. leprae

M. tuberculosis is the caustivie agent of

tuberculosis

M. leprae is the causitive agent of

Hansens disease (leprosy)

Spore staining is also called

Schaeffer-fulton method

Structural stain is used to

detect dormant forms of bacteria

• endospores

What are the reagents of spore staining?

• malachite green

• steam

• water

• safranin

Malachite green =

primary dye

Water = 

decolorizer

Safranin =

secondary dye/counterstain

What are endospores that have been released from the cell called?

free spores

How is the primary dye forced into the resistant endospore?

by steaming the sample

What is the purpose of steaming in the endospore stain?

it drives the primary dye into the tough, resistant spore coat

After staining, how do endospores react to decolorization?

stained spores are resistant to decolorization

Endospores =

spores are green inside pink bacterial cells

Free spores =

small green oval bodies

If no spores are present,

then only pink cells will be observed

Genera of spore forming bacteria

• bacillus antracid (gram pos rod)

• clostridium botulinum, tetani, difficile (gram pos rods)

A pure culture contains

a single microbial species

Pure cultures aim to

dilute bacterial samples in order to separate individual cells that can grow into isolated colonies when plated

Environmental and patient samples almost always

contain a mix of microbes

A variety of different types of media are available in microbiology

• selective purpose

• selective

• differential

General purpose media are often used when

trying to isolate microbes from a mixed culture

Solid media are often preferred when

trying to obtain pure cultures

Agar is a solidifying agent derived from

seaweed

Microbes are unable to

metabolize agar

Pour plate method can be used in

both a quantitative and non-quantitative way depending on the dilution method used

Colonies can be found

on top of agar, embedded in agar, and underneath agar

Serial dilution =

broth culture diluted in a series of tubes

• quantitative or non-quantitative depending on dilution procedure

On spread plate, cultures will only form on

the surface of the agar

Streak plate technique method is to

separate a single species from a mixed species population

Streak plate technique

• sample is plated across the surface of the agar in three zones

• isolation of 3 mixed species

• primary streak

Secondary streak (streak plate technique)

subculture of white colonies from the primary streak

• will now be a pure culture

Zone I is

the smallest, and each zone increases in size up to zone IIIB

Zone IIIB is

where you should obtain isolated colonies

Why are there zones in the streak plate technique?

it helps distribute the bacteria evenly in order to achieve isolation of all possible organisms present