Transcript Gram Staining Lab

Gram staining
Techniques
Some history
Bacteria are translucent
 Staining make them
visible under
microscope
 1884 Hans Christian
Gram  stained cells
and found that some
lost their color when
excess stain was
washed off
 Differential stain 
distinction between 2
types of bacteria
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http://www2.bvs.org.ve/img/fbpe/rsvm/v23n2/Image140.jpg
Bacteria cell walls
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Peptidoglycan: network of sugars crosslinked by short peptides
Forms the rigid part of the cell wall
 Protects the bacteria against mechanical
damage
 Part that picks up the stain in the gram
procedure
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www-micro.msb.le.ac.uk/ video/graphics/wall.gif
Gram - bacteria
Small peptidoglycan
 Are stained with
crystal violet but
decolorized with
alcohol after which
they pick up the red
stain
 LPS on outer
membrane  toxic
for host
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http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/toll/u1fig10b.html
Gram + bacteria
Large, highly crosslinked petidoglycan
 No outer membrane
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http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/u1fig9b.html
gsbs.utmb.edu/microbook/ images/fig2_6.jpg
Bacteria shape
 Long
rod: bacillus
 Round shaped: cocci
 Spiral: Spirochete
Bacteria shapes
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http://ibri.org/RRs/RR051/51bacterialshapes-Merc.gif
Some bacteria shapes
spirochete
bacillus
cocci
coccobacillus
http://www.spcollege.edu/hec/vt/VTDE/ATE2639LGS/images/image11.jpg
http://textbookofbacteriology.net/B.anthracis.Gram.CDC.jpeg
http://www.furetti.com/images/spirochete%20leptospirosi%201.jpg
Gram stain
Is the most commonly used technique
to stain bacteria
 Almost always first step into
identification
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Tell what other tests to perform
Gram – bacteria stain red-pink
 Gram + bacteria stain blue-purple
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Gram
Positive
Bacteria
(blue/purple)
Gram
Negative
Bacteria
(Red/pink)
Gram stain recipe
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Smear
Heat fix
Crystal violet (30 sec)  wash
Iodine (1 minute)  wash
Ethanol (varies ~15 sec)  wash
Safranin (30 seconds)  wash
Blot dry
How to prepare a smear
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Label your slide with a pencil
Use a clean slide: manipulate by the edges
Using a sterilized loop spread of drop of
bacterial culture on the slide
If solid culture  put a drop of water on the
slide  add a little bit of bacteria (using loop)
A thin film is better
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Dry faster, distribution of dye and decolorizer
more even
No coverslip on! let dry (until most water
evaporated)
Fixation
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Pass the slide through the flame of the
bunsen burner
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Moving in a circular motion (to avoid localized
overheating)
The slide should not be too hot to touch,
slightly warm but no more!
 If too hot  overfixed  burn bacteria
everything will be black
 If underfixed  bacteria do not stick to slide
and will be washed away during the staining
procedure
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Why bacteria need to be fixed
Denature bacterial enzyme  prevent
them from digesting the cell (autolysis)
 Make the bacteria stick to the slide so
they are not washed away during
staining.
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1st stain
Crystal violet
 Colorize all cells
 Flood the slide with crystal violet
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***make sure that the bacteria are on top
30 seconds
 Rinse gently with running tap (or distilled)
water
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Do not squirt water directly onto the smear
Shake off the excess water
Mordant
Iodine
 Make the dye stick to the cell wall
 Crystallize the dye in the peptidoglycan
 Flood the slide for 1 minute
 Wash
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Decolorization
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Ethanol 15-30 seconds, until dye doesn’t run out
anymore
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Critical step***** Washing after is very important (stop
alcohol action)
Cell that have thin peptidoglycan decolorize
 Long story:
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Dissolve the lipid layer from the gram negative bacteria
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Enhance the leaching from the primary stain
In gram + bacteria: alcohol dehydrate the thicker cell
wall
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Prevent diffusion of the violet iodine complex
Ethanol
Sink
Counter stain
Safranin
  color pink
 Flood the slide for 30 seconds
 Rinse gently with water and shake off
the excess water from surface
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Blotting
Slides can be air dried or blotted
 Blotting  put between 2 sheets of
absorbent paper (we will use bibulous
paper).
 DO NOT RUB THE SMEAR (bacteria will
come off)…just blot between papers.
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Oil immersion
Light is refracted when goes trough
slide (change in media from glass to air)
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Oil has same refractive index than glass  light
ray goes with no refraction
Use only one drop of oil
 Only use the 40X objective (greatest power
objective) with oil immersion.
 Clean the objective with paper lens after
use
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Oil immersion
http://www.bmb.psu.edu/courses/micro107/microscopy/oil-lens.jpg
Gram Staining Video
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http://www.youtube.com/watch?v=YvZ
HHNZ8cdo
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http://www.bio.upenn.edu/computing/
media/Instructional.Stain.Gram.php
Objectives for Tomorrow’s Lab
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You have 2 bacteria
S. Urea
 P. fluorescens
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For each bacteria:
 Stain each bacteria
 Observe and identify the bacteria shape and
gram results.
 You are doing this lab in team but make sure
that each student perform at least 1 gram stain
Resources
www.microvet.arizona.edu/Courses/MIC
205/lab/Lab_3_Gram_stain_spring_07.
ppt
 www.google.com/images/
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