Lab # 3 Gram and Acid Fast
stain
Medgar Evers College
Biology 261
Prof. Santos
• The Gram stain is a differential stain used
to distinguish between gram positive and
gram negative bacteria. This is base on
the biochemistry of the cell wall.
• Gram – bacteria have a thin layer of
peptidoglycan while gram + bacterial have
a thicker layer of peptidoglycan.
Steps
Step 1 make a smear of the sample and
heat fix
Step 2 the primary stain, crystal violet for
20 seconds. Gram – and gram + cells will
pick up this stain and appear purple.
Step 3 Apply the mordant or Gram’s iodine
for 1 minute. This forms a tight complex with the
crystal violet in gram + cells. Both cells still appear
purple.
Step 4 the decolorizer is used to remove the
crystal violet/Iodine from the gram – cells. The
decolorizer, ethyl alcohol, is applied for 20
seconds. The gram + cells continue to appear
purple while the others have become colorless.
Step 5 the counter stain safranin is
applied for 1 minute. This is used to stain the
gram – cells. At this point they will appear
pink.
Things to consider when doing
Gram stain
• When doing the Gram stain, it is important
to use fresh cultures to minimize false
results such as a gram + staining pink due
to the fact that it’s so old it has problems
picking up the crystal violet. Also keep in
mind that gram – never convert to gram +.
• It is critical to prepare a thin smear to allow
you to see single cells instead of layers of
cells superimposed on top of each other.
Acid Fast Stain
• The reason we do the acid fast stain is
because some members of the genus
Mycobacterium and some members of the
genus Nocardia have a layer of mycolic
acid that prevents them from being
properly stained.
• Mycolic acid is a waxy material in their cell
wall.
• The important thing is that the primary
stain used is Carbolfuchsin is applied over
heat. This allows the stain to penetrate the
layer of mycolic acid.
• The counter stain used is methylene blue.
• The acid fast cells tend to appear red or
pink and the non acid fast cells appear
blue.
Steps
1- prepare smear of Mycobacterium
smegmatis and S. aureus and cover
smear with carbolfuchsin.
2- steam over boiling water for 5 minutes
3- after slide has cooled, decolorize with
acid alcohol for 15-20 seconds
4- rinse briefly with water
5- Counter stain with methylene blue for 30
seconds
6- briefly rinse with water
7- blot dry
8-Look under microscope