Download Chapter 3

Bell Ringer
 Why is C. diff a difficult microbe to treat in an
infected hospital patient?
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Objectives
 Students will describe and perform the Gram stain
technique on two different species of bacteria.
 Why is this important?
 It is always the first step done in order to identify an
unknown pathogen
 Graded on
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

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Gram stain results
Morphology
Arrangement
Relative Size
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Differential Stains
 Used to distinguish between bacteria
 Gram stain
 Gram positive = purple
 Gram negative = pink
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Differential Stain
 3 steps to a differential stain
 1) Primary stain
 Colorize cells
 2)Decolorizing agent
 Has the ability to remove the primary stain
 3) Counterstain
 Contrasting color to the primary stain
 If the primary stain was removed, the cell absorbs the
counterstain
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Gram Stain
 Based on differences in the bacterial cell walls
 Both gram-positive and gram-negative have
peptidoglycan
 Repeating disaccharides attached by polypeptides
 Surrounds and protects cell
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Gram-Positive vs. Gram-Negative Cells
 Gram-positive
 Many layers of peptidoglycan
 Teichoic acid spanning the peptidoglycan layer
 Gram-negative
 One or a few layers of peptidoglycan
 Outer membrane consists of lipopolysaccharide (LPS)
 Resists enzymes, detergents, and heavy metals,
antibiotics
 Harder to kill
Copyright © 2010 Pearson Education, Inc.
Copyright © 2010 Pearson Education, Inc.
Gram Stain
 Utilizes four reagents
 1) Primary stain – Crystal Violet (purple)
 Stains all of the cells purple
 2) Mordant – Gram’s Iodine
 Increases cells affinity for primary stain
 Binds primary stain and forms (CV-I) complex
 Intensify color of stain
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Gram Stain
 3) Decolorizing agent – Acid alcohol (Ethanol)
 Acts to dehydrate proteins and dissolve lipids
 Gram-negative cells - alcohol dissolves outer lipid layer
 CV-I complex becomes easy to remove from thin
peptidoglycan layer
 Cells become colorless and unstained
 Thicker peptidoglycan layer in gram-positive cells traps
CV-I complex
 Alcohol dehydrates pore causing thick peptidoglycan
layer to trap stain
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Gram Stain
 4) Counterstain – Safranin (pink)
 Decolorized, gram-negative, cells stain pink
 Gram-positive cells retain purple stain
Copyright © 2010 Pearson Education, Inc.
Gram Stain
Color of
Gram-positive cells
Color of
Gram-negative cells
Primary stain:
Crystal violet
Purple
Purple
Mordant:
Iodine
Purple
Purple
Decolorizing agent:
Alcohol-acetone
Purple
Clear
Counterstain:
Safranin
Purple
Pink
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Common Errors
 Old culture
 Do not want to exceed 24 hours
 G+ appear pink or mixed
 Thick smear
 Thick smears trap the primary stain
 Cannot decolorize properly
 Over-decolorize
 Most important step
 Alcohol can remove the primary stain from G+ cells
 G+ appear pink
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While You Are Waiting
 1) Complete the questions in the lab report
 2) P. 68 in textbook
 Describe acid-fast, endospore, flagella, and negative
staining procedures
 Include
 Explain what the stain is used for
 Describe what a positive and negative cell look like
 Explain the various stains used in the procedure
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