MCB 2010L – IRSC
Microbiology – Gram Staining and Acid-fast staining
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Gram Stain
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Differential stain
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Tells the difference between gram-positive and gram-negative cells
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Gram positive – appear purple
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Have thick layer of peptidoglycan
Gram negative – alcohol or acetone removes the crystal violet ; must
be counterstained with red dye (safari) so they appear
reddish/pinkish
o
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Have outer membrane that covers thin layer of peptidoglycan
Make very thin smears on your slide – this is very important (don’t want too much of
the sample) – Why?
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Decolorization step is most important step – if done for too long, Gram + will appear
Gram –
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Modify following steps in your lab manual:
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Step 3 – rinse with water after iodine
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Step 4 – 1 to 2 seconds
Acid-Fast Staining
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Bacteria such as Mycobacterium phlei have cell walls that have high lipid content,
one of those lipids is a waxy material called mycolic acid
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Composed of fatty acids and fatty alcohols, affects staining properties and
is important diagnostic tool in identifying Mycobacterium tuberculosis
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Procedure
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Prepare smear containing both acid-fast (Mycobacterium) and non-acid-fast
(S. aureus) on same slide
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First place S. aureus on slide, flame loop, and then small amount of
Mycobacterium (clumps so be sure to break up and spread out on
slide)
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Modify the following steps in your lab manual:
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Step 1 – rinse slide with water before decolorizing with acidalcohol
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DON’T MIX UP ACID-ALCOHOL WITH 95%
ALCOHOL
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Step 2 – decolorize for 15-20 seconds
Be sure you are using proper aseptic techniques!!!!! Be slow and cautious!