Skills 9

Simple stains

Use a single dye. Ex staining a slide with crystal violet

Differential stain

Uses two dyes. Ex. gram stain, acid fast stain and endospores stains

Special stains

highlight a specific structure. Ex. a capsule or a flagella

Main steps in staining

Smearing

air drying

Heat fixing

staining

Instructions for broth cultures/ Agar cultures

Heat fix your slide by passing through flame 3 times

Don’t heat too long becasue the organism can change shape

Escherichia Coil gram negative rods (stains pink to red)

Staphylococcus aureus- gram postive cocci (stain purple)

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Bacillus subtils- gram positive rods (stain purple)

Neisseria sicca gram negative cocci (stain pink)

What is the name of the primary stain

Crystal violet

What is the name of the mordant stain

Gram iodine

What is used as the decolorizer

Acetone alcohol

What is the name of the counterstain

Safranin

Tips Gram stain

You should use fresh cultures, if cultures are old the shape of the organisms may have changed

Make sure to heat fix the organism so it is not washed off during the staining process

You should use a paper towel to blot dry the slides or air dry the slides if you have time. Do not blow on the slides, it can release the bacteria from the slide

Ziehl-Neelsen acid fast stain

Mycobacterium gordonae and staphylococcus aureus mixed together and heat fixed on slides

This stain used for Mycobacterium. This genus has a waxy layer made of mycolic acid outside the cell wall

Name the primary stain ziehl-Neelsen acid fast stain

Carbolfuchsin

What is the decoloirzer that is used ziehl-Neelsen acid fast stain

acid-alcohol

Name the counterstain ziehl-Neelsen acid fast stain

Loefflers methylene blue

Discussion

The bacteria are stained with carbolfuchsim under heat, decolorized, and then counterstained with methylene blue

Mycobacteria remain pink, acid-fast, bacteria decolorize, counterstain blue and are termed non-acid fast

Mycobacterium gordonae

red

Staphylococcus epidermidis

blue

Schaeffer-Fulton endospore stain

The endospore is very good at protecting the DNA from ultra-violet light, chemicals. and desiccation. When conditions are again favorable for growth, and the bacterium germinates from the spore

Capsule stain

In this staining procedure, a thin bacterial smear is prepared on a slide, stained with crystal violet and washed with copper sulfate

It is not heat fixed as it would destory the capsule

Therefore, the bacteria will look as if they are inside of a bubble or appear to have halo