Good Morning!
Please pick up the yellow GramStaining Lab worksheets from the
overhead cart.
 Grab some goggles and a lab apron
(in box underneath goggles).
 Start reading the introduction and the
procedure.
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Today’s Objective:
We’re going to be gram-staining today!
I didn’t get to do this until my college
microbiology lab, so you guys are very
lucky.
 This lab experience will be very helpful if
you plan on taking AP Biology or become
a science major in college.
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Introduction to Gram-stains
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In 1884, Hans Christian Gram, a
Danish doctor accidentally stumbled on
a method that differentiates bacteria.
While examining lung tissue from
patients who had died of pneumonia,
he discovered that certain stains were
preferentially taken up and retained
by bacterial cells.
Gram developed a staining procedure
which divided almost all bacteria into
two large groups - the Gram stain.
Why is this important?
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Bacterial cells without a stain are almost
transparent. Adding a stain to these cells helps us
see the bacteria much better than just looking at
them without a stain.
Gram-staining also helps us differentiate types of
bacteria. Knowing whether they are gram-positive
or gram-negative gives us information about their
cell walls.
 Gram-positive:
thicker layer of peptidoglycan
 Gram-negative: thin layer of peptidoglycan
 Peptidoglycan gives the cell structural strength
Gram-Staining in the Real World
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Gram-staining used in hospitals and clinics when
trying to diagnose infections.
Some antibiotics target specific types of bacteria.
For example, penicillin only affects gram positive
bacteria. Knowing whether the infection is caused
by a gram positive or gram negative bacteria can
help the doctor determine the best antibiotic to
prescribe.
Method is also used in scientific research.
Gram-Staining Procedure
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http://www.youtube.com/watch?v=syJTy
8C_BG0
Your product:
Gram Positive: purple
Gram Negative: pink
Station 1: Making a Bacterial Smear
Step 1 on the lab procedure, station is
at the back of the classroom.
Write down your bacteria on your
paper.
1.
2.
3.
Get a microscope slide and place one drop of water on the
slide.
Choose a type of bacteria and open the vial. Take inoculating
loop and scrape some of the bacteria from the vial onto the
loop. Do not pierce into the agar.
Spread the bacteria on your loop into the water droplet. You do
not need to cover the entire slide.
Heat fixing the bacteria kills the bacterial cells.
Station 2: Crystal Violet
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Step 2 on the lab procedure.
Station is next to the pencil sharpener.
Place 1 drop of crystal violet on the slide. Let it sit
for one minute and then wash with water from the
sink.
Tilt slide away from you so you don’t get any of the
stain on your hands.
Crystal violet is the primary stain in this procedure.
If the cell is gram positive it will take in this stain.
Station 3: Iodine
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Step 3 on your lab procedure. Station is located
next to the sink closest to your grades.
Place 1-2 drops of iodine on the smear. Wait one
minute then wash the slide.
Iodine is called a mordant in this procedure. This
means it allows the stain to stick very well to the
bacterial cells.
Station 4: Alcohol
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Step 4 on your lab procedure. Located near the
goggles.
Tilt slide at 45 degree angle away from you. Add
alcohol onto the slide until the color stops flowing.
(You should get runoff that is clear at the end)
The alcohol is a de-colorizer. If the cell is gram
positive, the stain will stick. If it is gram negative the
stain will wash away.
Station 5: Safranin
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Step 5 of lab procedure. Located next to the
notebook cabinet.
Add 1 drop of safranin to the smear, wait one
minute then wash.
Safranin is the counterstain. It will only stain gram
negative bacteria, and dyes the cell pink.
Station 6: Microscope
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Steps 6 of lab
procedure. Located at
the back of the
classroom.
Blot dry the slide. Then
place it on the stage of
the microscope.
Start at the lowest
magnification (4X) then
move to the highest
(100X).
Question to Answer
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Would penicillin be an effective antibiotic
to prevent the spread and growth of your
bacteria?
When you are done with the lab, please answer the
analysis questions on your worksheet.
If you are waiting for your turn or are finished with the
questions please work on the build-a-virus activity and
the viruses and bacteria worksheet.