Transcript 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

III. CELL WALL TYPES
Two different types of cell walls are found in
eubacteria
1. Bacteria cells containing peptidoglycan
2. Bacteria with an outer layer of lipid and
carbohydrate molecules and thin layer of
peptidoglycan
B. Gram staining helps to tell them apart using
two different types of dye
1. Violet - primary stain
2. Red - counterstain
A.
TYPES OF GRAM STAINS
 Gram
positive
Bacteria cells containing a thick layer of
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 and a thin layer of
peptidoglycan
 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
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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