The Standard Endospore Stain, Atlas pg. 41-42

The standard endospore stain is a vigorous staining procedure using Malachite (măl' -- kīt') Green as the main stain and Safranin as a counter stain (other counter stains may be used but we will ignore them for simplicity).  Endospores are a bacterial structure for survival that do not readily allow stains to get in or attach to the spore.  Heat or steam is used to get Malachite Green into the endospores (and the vegetative cells as well.  All but one species that produce endospores have vegetative bacilli.)  The slide is them washed with water.  As Malachite Green is a water soluble stain, it washes out of vegetative cells, but not out of the endospores.  Safranin is then applied, which stains the vegetative cells redish (it is unable to get into the endospores).  If the species produces endospores, one will likely see roundish green endospores and redish rods.  If the species does not produce endospores, one will only see redish vegetative cells.

Procedure
  1. Obtain a culture that is about a week old.  Such a culture will contain both vegetative cells and endospores.  A culture older than two weeks may have few if any vegetative cells.  A culture younger than a few days may have few if any endospores.  It is suggested that you also stain a positive control, a species that is known to form endospores (such a Bacillus subtilis or B. cereus) to check your staining procedure.
  2. Make a bacterial smear or smears of your bacterial species, let it air dry (do something else while waiting).  It is suggested that you make two smears of differing amounts of bacteria, but remember to keep your smear thin (a single layer of cells) so that it will stain well.
  3. Heat fix the slides by passing them through the flame about three times.
  4. Use Malachite Green and either steam or heat (ask your instructor for which) to stain for endospores.
  5. Apply stain while keeping the slide and perhaps some paper on top of the slide wet for 10 minutes (add more stain to keep it wet).
  6. If using Steam:
  7. If using heat:
  8. Destaining and Counterstaining
Results:
  1. Make sure you view and record your observations using the 10x and 40x objective (color viewing is best under lower powers).
  2. If needed, go to 100x objective with oil (one cannot easily go back to 40x now that oil is present).
  3. Look for round green endospores and red/pinkish rods (vegetative bacteria cells).
  4. If the unknown species has endospores, tell the class and record it in your journal.  Only a few genera produce endospores!
  5. If endospores are inside a parent cell, record their location (centrally located or towards the ends of the parent cell).
  6. If endospores are absent, view some endospores from this link or from your slides (if available).
  7. You will be expected to be able to distinguish if endospores are present or not from an appropriate slide.

Alternate Endospore Stain

There has been some concern about using heat in a teaching environment for safety reasons.  Because of this, an alternative Endospore stain is sometimes used.  It is a simpler but less robust procedure.  For best results, follow this procedure exactly and pay close attention to the timing.

Procedure
  1. Obtain a culture that is about a week old.  Such a culture will contain both vegetative cells and endospores.  A culture older than two weeks may have few if any vegetative cells.  A culture younger than a few days may have few if any endospores.  It is suggested that you also stain a positive control, a species that is known to form endospores (such a Bacillus subtilis or B. cereus) to check your staining procedure.  
  2. Make a bacterial smear or smears of your Unknown species, let it air dry (do something else while waiting).  It is probably a good idea to make two smears of differing amounts of bacteria.
  3. Heat fix the slides about 20 times through a flame or near the heat of an incinerator.  This step is crucial because it puts holes in the endospores so that dye can get in.
  4. Stain with saturated aqueous Malachite Green for 10 minutes.   Pour off the stain into a collection bucket or Malachite Green waste container.
  5. Rinse the slide with water, do not over rinse.
  6. Counterstaining with 0.25% aqueous Safranin for 15 seconds.
  7. Rinse briefly with water, blot dry and observe you cells.

Results:

  1. Look for rounded green endospores and red/pinkish rodish (vegetative bacteria cells).  The cells will be deformed by the excessive heat fixing.
  2. If endospores are inside the parent cells, record their location if you can (centrally located or towards the ends of the parent cell). This may not be possible if the cells are too deformed.

Note to the Instructor: 

This alternate procedure is not very robust.  It relies on the heat fixing to put small holes in the endospores so that Malachite Green can get in the cells within 10 minutes.  Rinsing with water removes the stain from the vegetative cells, but not all of the stain from the endospores.  Counterstaining with Safranin stains the vegetative cells but very little stain enters the endospores within 15 seconds.  Problems may occur from not heat fixing enough, heat fixing too much, over or under staining with one or both of the stains or by over rinsing with water.