Introduction to Lab: Differential Stains

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Introduction to Lab Differential
Stains Gram Staining
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Introduction to Lab Differential Stains Gram
Staining

• Basic classification of bacteria is based on the
  cell wall structure.
• There are 2 main groups Gram positive and Gram
  negative.
• Gram staining is a differential staining
  technique that provides an easy differentiation
  of bacteria into one of two groups.

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Differential Stains Gram Staining

• The staining technique, developed in the late
  1700s by Christian Gram classifies the rigid
  cell walled bacteria into one of two groups.
• based on whether they are able to resist the
  decolorizing action of an alcoholic solution.

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Differential Stains Gram Staining

• Those that resist decolorization by 95 ethanol
  are arbitrarily termed Gram positive and those
  that do not are Gram negative
• (the terms positive and negative have nothing to
  do with charges
• of the cell but based on differences in the cell
  wall structure of
• these two groups of bacteria).

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Gram-positive cell walls Gram-negative cell walls
The characteristic compound found in all true
bacterial cell walls is peptidoglycan. The
amount of PPG is among one of the differences
between the GP and GN cell walls.

• Thin peptidoglycan
• 5-10 peptidoglycan
• No teichoic acids
• 3 layers
• Outer membrane has lipids, polysaccharides
• No acid- fast cells (mycolic acid)

• Thick peptidoglycan
• 90 peptidoglycan
• Teichoic acids
• 1 layer
• Not many polysaccharides
• In acid-fast cells, contains mycolic acid

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Figure 4.13b, c
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The process includes the use of a primary stain
(crystal violet) a mordant (helper) iodine
solution, a decolorizer (95 ethanol), a
counterstain (safranin).
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The Gram stain
Thin smear/heat fix Gram stain a. Flood
slide with crystal violet and let stain for 1
minute. b. Drain off crystal violet and rinse
off with distilled water flood slide with
Gram's iodine for 1 minute.  c. Rinse off Gram's
iodine with distilled water. d. Hold the slide
on an angle (preferably with a clothes pin) and
drop 95 ethyl alcohol onto it until the
alcohol leaving the slide no longer has a
purple tint be sure to drop the alcohol onto
the upper portion of the slide so that the
smears are subjected to uniform decolorization.
e. Rinse with distilled water and flood the
slide with safranin and let stain for 2-3
minutes.  f. Rinse with distilled water and
blot dry with bibulous paper.
Gram positive
Gram negative
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The crucial step in the staining process is the
decolorizing step. The most accepted theory
relies on the fact that the PPG is found in
layers and the stain molecules are trapped within
the many layers of the GP CW when they form the
complex with the mordant Iodine molecules.
Since the GN CWs lack much PPG the amount of
stain captured in those CWs is much lesser.
When the cells are treated with the decolorizer
the ethanol this causes denaturation of the
proteins in the outer membrane of the GN CWs
resulting in gaping holes in these CWs that lead
to the removal of the crystal violet-iodine
complexes easily, leaving these cells unstained.
The counterstain -safranin- thus is used to
make these cells visible.
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There are 4 conditions to be followed for a valid
Gram staining procedure  Young cultures -
must be young within 18-24hrs old (older
cultures lose their Gram staining properties
due to changes in the CWs as the cells get
older) Thin smear thicker or uneven smears
will result in uneven staining and
decolorization Fresh reagents - of proper
strength Control cultures - for a known GP
bacterium and GN culture (S.aureus E.coli)
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Demos Gram stained slides of Neisseria,
Streptococcus, Pseudomonas, Actinomyces species.
Pseudomonas
Neisseria
Streptococcus