Gram Staining

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When preparing for the Gram stains, make sure the slides are

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clean.

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Be sure to label your slides using

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black sharpie or tape

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It is best to mark slides on the bottom. Why?

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the marker will easily erase with alcohol during washing steps.

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What occurs if you heat fix smears which are not dry

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you are essentially boiling the bacteria. The cell walls will be broken, and the cell shapes will be distorted.

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Gram positive cultures which are over 24 hours old often will begin losing

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cell wall integrity

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Gram positive cultures which are over 24 hours old and begin losing cell wall integrity will

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destain easily and may result in a Gram negative appearance

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You can easily determine the Gram reaction of an organism by consulting

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Bergy's manual or the text.

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Too much destaining will also result in the decolorization of Gram positive cells and can lead to

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mistaken identity.

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members of the genus Micrococcus are Gram _____ cocci

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Gram positive cocci

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This genus of Gram positive cocci destain very easily with alcohol

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genus Micrococcus

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This genus destains easily with alcohol which maked it almost impossible to see completely purple Gram stains of this organism.

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Genus Microcossus

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When it appears that both pink and purple cells are in the same cluster, this is a clue that what has happened

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too much decolorization has occurred.

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Remembering to close all of the Gram staining reagent bottles before placing them back on the shelf prevents

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prevents evaporation (especially of the alcohol) and formation of crystals in the stains.

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The Gram stain is called a

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Differential Stain

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Steps in gram stain

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1. Generate smear.
2. Air dry!
3. Heat fix!
4. Crystal violet (basic) stand for 30 seconds.
5. Gram’s iodine stand 1 minute.
6. Decolorize using acetone alcohol for aprox 2 sec.
7. Safranin (basic) stand for 2 mins.

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A mordant intensifies the Gram stain by forming complexes with

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crystal violet

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The intense complexes between a mordant and the crystal violet will be trapped in the thick walls of Gram _________ cells but can easily be washed out of the thinner walls of Gram _____ cells

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Trapped in the Gram Positive cells but washed out of the Gram negative cells

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During decolorization, wash with acetone alcohol only until

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color leaches out of smear

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Order of Gram Stain

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Crystal Violet, Grams Iodine, Decolorizer, Safranin

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E. Coli, Gram +/-

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Gram Negative Bacillus

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S. Aureus, Gram +/-

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Gram Positive staphylococcus

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Gram positive organisms are

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Purple

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Gram Negative organisms are

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pink