Microbio Lab Practical

Gram Stain

*Staphylococcus epidermidis and Spirosoma linguale

1a) make thin smear and heat fix on slide warmer

1b) flood with crystal violet for one minute

2a) rinse with water

2b) flood with Gram’s iodine for one minute

3a) rinse with water

3b) add 95% ethanol and let run off smear till purple is gone

4a) rinse with water and add counterstain safranin for one minute

4b) rinse with water and blot dry with bibulous paper

Staphylococcus epidermidis

Gram Positive, dark purple

Spirosoma linguale

Gram negative, pink

Endospore staining

*Bacillus subtilis

1a) Use one loopful of water and make smear of culture and heat fix on slide warmer

1b) leaving on slide warmer, place paper towel and cover with malachite green dye for one minute

2a) Remove from slide warmer and let cool

2b) rinse with water till dye no longer flows

3a) counterstain with safranin dye for one minute

3b) rinse with water, blot dry

Vegetative cells

no endospore, pink color from safranin

Sporulating cells and free spores

spores will be green and the sporulating cells with be pink on outside and green on inside

Acid Fast Cells

Magenta

Non Acid Fast Cells

Blue

Acid Fast Staining

*Mycobacterium smegmatis - acid fast

*Escherichia coli - non acid fast

1a) Add drop of egg albumin to slide, place Mycobacterium smegmatis and mix well. 

1b) Add 1-2 loopfuls of E. coli and stir, heat fix using slide warmer

1c) Leaving slide on warmer, place paper towel and cover with carbofuchisin dye and let sit for one minute

2a) Remove from warmer and allow slide to cool

2b) rinse with water until dye no longer flows

2c) decolorize with acid alcohol until slide is slightly pink (10-30s)

3a) rinse with water and counterstain with alkaline methylene blue for 2 minutes

3b) rinse with water and blot dry

Capsule Stain

*Azotobacter vinelandii

1a) add small drop of Congo red stain

1b) mix small loopful of culture and spread gently over the slide

1c) allow suspension to dry at room temp DO NOT LET IT TOUCH THE BENCH

2a) flood with Maneval’s stain solution for 2 minutes

2b) Pour off Maneval’s Stain Solution, blot dry

Capsule Stain Bacteria

Azotobacter vinelandii

Acid Fast Staining Bacteria

*Mycobacterium smegmatis - acid fast

*Escherichia coli - non acid fast

Endospore Bacteria

Bacillus subtilis

Gram Staining Bacteria

Staphylococcus epidermidis (Gram Positive) and Spirosoma linguale (Gram Negative