NEGATIVE STAIN?
NOTE: See below for the Negative vs Capsular Stain discussion...
A photo of a "drying" properly made Negative Prep. - NOTE: the Nigrosin is NOT solid black!
VIEW a NEGATIVE STAIN IN PREPARATION:
View the Preparation of a Negative Stain (or click here)
NEGATIVE (Stain?) can be used as a Capsule Stain see Atlas page 31
The simplest way to prepare bacteria for staining involves NEGATIVE or BACKGROUND STAINING. As bacteria are colorless, this procedure involved mixing the microbes with NIGROSINE (India Ink - is less useful). This is NOT really a stain as the dyes do not penetrate the microbe, instead the obliterate the background leaving the microbes translucent and visible in a darkened field. No SMEAR is made before staining and thus the microbe is alive and experiences no shrinkage from preparation.
Certain microbes in the mouth use sticky sugar capsules to attach themselves to teeth. These capsules hide the microbe from the immune system and are also used as a "glue" to keep the microbe from being swallowed with the saliva. The capsules are impermeable to dyes and thus must be view with a Negative stain.
NOTE: Some CAPSULAR stains use the Negative stain procedure but after preparation, the slide is heat fixed and Crystal Violet is added to the slide for 1 minute and then removed with a gentle stream of water. The slide is blotted with bibulous paper and then viewed under oil immersion
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PHOTO of a typical CAPSULE or Nigrosin background colored slide. The microbes appear as greyish shadows against a gray, purple, or black background. NOTE: LARGE CRACKS are cracks!
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NEGATIVE STAIN PROCEDURE:
1) Obtain 1 clean well-washed slide and one dirty #2 slide.
2) Draw a dime-sized circle 3/4 down the side on the bottom.
3) Add 1 drop of Nigrosine to the circled area (OR PUT TEETH MATERIAL 1ST NP)
4) Take a flat wooden stick and remove the plaque between the gum and tooth (DO NOT GET ROUGH!) and rub the material on the stick onto the slide in the circled area MIXING it with the Nigrosine.
5) Take the other slide #2 and tip it on its narrow edge. Place this edge at the beginning of the drop of mixed ink and microbes and lower slide #2 to almost touching slide #1 then DRAG the #2 slide along the #1 slide smearing the black dye down the whole length of the #1 slide.
6) Wash the #2 slide well.
7) AIR DRY the #1 slide and view it under first 10X and then oil immersion.
When you are observing the slide DO NOT BE FOOLED BY DRYING DYE CRACKS! You are looking for faint greyish-white rods and cocci...
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Photos of OUR Negative Procedure (NOT a stain) without the Capsular Stain (Cry.Violet):
Click for Negative Prep Photos!
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THE CAPSULAR STAIN (see photo above):
One popular CAPSULAR stain procedure uses the Negative background coloration as done above, but after preparation of the Negative background slide, the slide is heat fixed. Subsequently, Crystal Violet is added to the slide for 1 minute and then removed with a gentle stream of water. The slide is blotted with bibulous paper and then viewed under oil immersion. THIS IS AN ACTUAL STAIN while the Nigrosin background coloration is NOT a STAIN!
NOTE the stained microbes in the bottom photo on this page...
| The Capsule Stain is a little similar to the Negative Stain - but shows the capsule more definitivelyPHOTO of a CAPSULE STAIN using the Negative Nigrosin colored background with a Crystal Violet Stain... as discussed below... |  |
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Capsule Stain
1. Place a single drop of India ink on the left-hand end of a clean microscope slide.
2. Using a flamed loop and sterile technique, remove some K. pneumoniae (or the organism you
want to stain) from your slant and mix it into the drop of India ink. Be sure there are no large
clumps of organism, but try to avoid spreading the drop.
3. Place the end of another clean microscope slide at an angle to the end of the slide containing the
organism. Spread out the drop out into a film. This is done by contacting the drop of India ink
with the clean microscope slide and using the capillary action of the dye/ slide to spread the
India ink across the smear. Refer to the Negative Stain portion of this handout for a diagram.
4. Allow the film to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule!
5. Saturate the slide with crystal violet for 1 minute.
6. Rinse the slide gently with water.
7. Allow the slide to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule!
8. Observe the slide under the microscope, using proper microscope technique.
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The background will be dark.
The bacterial cells will be stained purple.
The capsule (if present) will appear clear against the dark background.
background = black or purple
capsule = clear
bacterium = violet or as a dark center
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| Capsule Stain | Capsule Stain |