#2 SMEAR IS GOOD (DRY)
A Great Gram Stain video from the U of Wisc. - they decolorize longer than we do but still this is a GREAT studyguide for the Gram stain!
Click on the YELLOW for another video on Gram Staining (U of Wisc Video) AFTER you make a GREAT SMEAR!
So you want a visual of the THEORY of GRAM STAINING (Vid 1 of 2; Click here)
Theory of Gram Staining (vid 2 of 2 - CLICK here for Vid 2)
THE GRAM STAIN: Atlas page 27
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| A GRAM Positive Cell | An OLD Gram Pos Cell |
The Gram staining process involves two dyes and several other solutions. Crystal Violet is known as the PRIMARY STAIN and Safranin is the SECONDARY STAIN. Iodine solution is used as a fixative or MORDANT and the DECOLORIZER is Ethanol. Water is used to wash the excess solutions off the slide.
PROCESS:
A Gram NegativeCell
1) Place the slide with a prepared SMEAR right-side-up on the staining rack in your sink.2) Cover the circled smear with the PRIMARY STAIN for 1 minute.
3) Obtain a spray water bottle and gently aim the water spray ABOVE the circled smear...allowing the water stream to flow down over the smear and gently washing the majority of the excess primary stain off the slide - DO NOT OVERWASH!
4) SHAKE the excess water off the slide.
5) Return the slide to the staining rack and cover the smear with the MORDANT. Immediately shake the Mordant off the slide and reapply the Mordant. Wait for one minute. Shake the excess mordant off the slide.
6) Return the slide to the staining rack and drop 1-3 drops of ETHANOL on the smear. QUICKLY and immediately shake it off. Wash the smear with water as before and shake the excess water off the slide - DO NOT OVER WASH!
7) Return the slide to the staining rack and cover the smear with the COUNTER or SECONDARY STAIN shake it off the slide and re-apply. Leave the Counter stain on the smear for 1 minute and then wash GENTLY.
8) Shake the excess water off the slide and blot the slide dry with BILBOUS PAPER. DO NOT RUB THE SLIDE!
Take the Gram Stained slide to the microscope, center the smear over the "light" and add immersion oil to the top of the smear. Next focus under 10X move the oil immersion objective into place and fine focus.
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THE GRAM STAIN - THEORETICALLY...
Gram positive staining (PURPLE-BLUE) microbes have a VERY thick cell wall made up of either a huge thick layer of peptidoglycan or in a the case of one Genus, a thin peptidoglycan layer with a large thick LIPID layer on top (the Genus MYCOBACTERIUM). As Crystal Violet dye molecules are very large and the mordant makes them LARGER by cross linking several, the primary stain is IRREVERSIBLY trapped behind the THICK cell wall where the decolorizing ethanol cannot penetrate and remove. The colorless microbe is stained BLUISH, the secondary pink stain only making the final microbe color PURPLE-BLUE by adding a small amount of pink tint.
Gram negative staining (PINK) microbes have two membranes but a VERY thin cell wall made up of only a few layers of peptidoglycan. As Crystal Violet dye molecules are very large and the mordant makes them LARGER by cross linking several, the primary stain REVERSIBLY moves behind the THIN cell wall where later the decolorizing ethanol penetrates and removes it. The colorless microbe is not stained and is now colorless again. The secondary pink stain is added and NOT removed with a decolorizer making the final microbe color lightPINKISH RED.
INTERPRETATION CAUTIONS:
CAUTION: The Mycobacteria may take-up the primary stain and loose it at the same time. This microbe is listed as Gram variable on the positive side meaning MOST of the rods will stain purple...
CAUTION: The Bacillus will often stain as purple but have debris underneath the rods that are staining pink. As these cells get older (more than 24 hrs old) they fall apart and the pieces stain pink... the stain should be read as Gram positive
Sin!!!!
