Simple Stains  (Atlas, p 27-28)

Because cells are mostly water, they are largely transparent unless they contain a pigment such as chlorophyll.  Viewing transparent cells is rather difficult under a microscope because the cytoplasm has little contrast from the cell's background.  Staining cells, especially small bacteria, greatly aids viewing because it increases the contrast between the cell and its background.  Once you can clearly see the cells, the cell morphology (shape), size, and growth pattern may be determined.  There are two types of simple stains, basic stains and negative stains.  A basic stain stains the bacteria (the stain is a positively charged molecule that attaches to cells which are negatively charged or to negatively charged molecules in cells.)  A negative stain does not stain cells (why its called a negative stain), it stains the background around the cells.  Procedure for Simple Basic Stains:
  1. Obtain a bacterial smear that has been dried and heat fixed.
  2. Obtain a simple Basic Stain.  Recall that nearly all stains can be a simple stain if it only uses one dye.
  3. Flood the smear with the stain for about 1 minute, depending on the stain.  Some require more or less time.  It is best to do this step over a slide holder placed over a sink.
  4. Rinse the slide with water, removing excess stain.
  5. Blot the smear dry using bibulous paper, paper towels, or simply air dry.
  6. Observe specimen under the microscope, usually a little moisture does not interfere with your observations.

Negative Stains and Capsule Stains

A negative stain may be used  similar to a basic simple stain or as a simple capsule stain.  We will only use it as a simple capsule stain.  A capsule is an extracellular structure that some species make that is destroyed by normal staining procedures (especially heat fixing and washing).  Capsule staining is a gentle staining procedure that does not destroy the bacterial capsule.  This procedure stains the background but does not stain the bacterial capsule or the bacterial cell.  (Note, some other capsule stains use a second stain as a counter-stain which does stain the cell or even the capsule with another color.)  

Purpose: to make the cells show up better (by adding higher contrast to the background) and to allow for viewing the capsule.

Procedure for a Simple Capsule Stains (or a Wet Mount using a Negative Stain):
  1. Do not start with a bacterial smear!  Set up your bacteria, two to four slides, two cover slips, a Bunsen burner, and your loop.  It is also a good idea to make a third slide containing a positive control (a bacterial strain that has a capsule) if one is available so that you may view a positive result.  (One may find bacteria with capsules by gently inserting a clean toothpick under your gumline next to a tooth.) 
  2. Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on your slides.  Congo Red is easier to see, but it does not work well with some strains.  India Ink generally works, but it has tiny particles that display Brownian motion that must be differentiated from your bacteria.  Nigrosin may need to be kept very thin or diluted.
  3. Sterilize your loop and add an almost invisible amount of bacteria to one slide, smearing it in the dye.  You may spread your drop with the loop, or with a second slide that is brought into the stain drop and is then spread across the first slide.  It is recommended that you make more than one slide of a specimen, slightly increasing the amount of bacteria added to the second.
  4. Reflame your loop.  
  5. Add cover slips at a 45 degree angle to each slide.  You are essentially making a wet mount using a negative stain instead of water.
  6. First observe the slide with the greater number of bacteria, it is probably too many to work with, but look at it first to more easily observe your bacteria, look for an area of clearing caused by a capsule around the cells.  Otherwise the dye should come right up to the cell.  When you think you know what you are seeing, switch to the slide with the fewer bacteria that hopefully will contain only a single layer of cells to be certain of your conclusions.
  7. If your bacteria are very motile, you may need to wait for them to die under the light before making your observations about a capsule.  You can turn up the light to speed this.
  8. Remember that a slide stained with a capsule stain contains live bacteria that should be handled with care and disposed of properly.

Procedure for a Dried Negative Stain:

We will not perform this procedure, it is detailed here just for your knowledge benefit.  Essentially, it is the similar to a Simple Basic Stain, except it uses a Negative Stain, and there are no washing and blotting steps.  Recall that negative stains are simple stains that do not stain the cell, they stain the background.

  1. Obtain a clean slide that has no fingerprints or dust on it (your instructor will inform you how to clean it if necessary.)
  2. Obtain a simple Negative Stain (India Ink, Congo Red, Nigrosin, or Eosin).  
  3. Place a small drop of a Negative Stain on one end of your slide.  
  4. Add a small amount of your bacterial culture to the drop and mix evenly without spreading.
  5. Now, bring a second slide held at a 45 degree angle into the stain drop and with this slide, spread the drop across the first slide.
  6. Dispose of the second spreader slide in disinfectant.
  7. Allow the slide to air dry.  Do not heat fix or wash.  View the slide under the microscope, a coverslip may be added if desired when using oil.
  8. Note, this procedure should not be used to look for capsules as the stain may shrink away from the cell during drying.