Transcript 5. Gram Staining

GRAM STAINING DISCUSSION
June 2, 2016
LET’S PRACTICE!
Use the sheets given to you and complete a Gram
stain on the bacteria cultures in the petri dishes
 Use what we have already mentioned in class to
determine which bacteria are Gram positive and
which are Gram negative

TYPES OF GRAM STAINS
 Gram
positive
Bacteria cells containing peptidoglycan absorb
only the violet dye
 Appear purple in the microscope

TYPES OF GRAM STAINS (CONT.)
 Gram
negative
Bacteria with a second (outer) layer of lipid &
carbohydrate molecules
 Extra layer allows purple dye to be removed during
decolorizing stage and red dye to be absorbed
 Appear pink in the microscope

WHY IS THERE A DIFFERENCE?

Thick
peptidoglycan of
the Gram +
retains the
purple dye even
after decolorizing
step
WHY DO WE USE GRAM STAINING?
Almost always the first step in the identification of a
bacterial organism
 Allows a doctor to prescribe treatment with an
appropriate antibiotic while waiting for more specific
tests, such as a culture, to be completed
 Not all bacteria can be definitively classified by this
technique

STEPS OF GRAM STAINING
1.
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10.
Place a slide with a BACTERIAL smear
STAIN the slide with crystal violet for 1-2 min.
Pour off the stain
Flood slide with Gram's IODINE for 1-2 min.
Pour off the iodine
DECOLORIZE by washing slide with acetone
WASH slide with water to remove the acetone
Flood slide with safranin COUNTERSTAIN for 2
min.
WASH with water
Blot excess water and DRY in hand
Gram Stain Animation