Lab 11

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Lab 11 Gram Staining

Purpose: to examine the difference between gram positive and gram negative bacteria by using counterstains and to know the characteristics of bacteria.

Procedure:

• prepare a slide with 3 dots on it and label Se, Bs, and Ec.
• Put a water and the cultures on the slide with a steriled cooled loop
• let it air dry competely
• heat fix it
• flood the slide with Crystal violet for 1 minute
• wash it off with distilled water
• flood the slide with Iodine for 1 minute
• wash it with distilled water
• Wash it with Alcohol for 15 seconds and immediately wash it with distilled water
• Add safranin and wait 1 minute
• wash it with distilled water and dry the slide with bibulous paper.

Observations:

Questions:

• Why do we use controls with the Gram stain?

to evaluate whether or not the criteria have been met each time; to make sure your staining technique is effective.

• What structural characteristic of the cell wall makes bacteria Gram negative or positive?

gram +  has

• Fill in the table below for the four steps of the Gram Stain.

Reagent	Purpose	Color of Gram + cell	Color of Gram – cell
Crystal violet	Stain both cells wall	Purple	Purple
Iodine (mordant)	Stiffens protein walls	Purple	Purple
Alcohol	Decolorant for gram negative	Purple	Colorless
Safranin (pink/red)	Stains both walls	Purple	Pink/reddish

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