Endospore Stain Showing Green Endospores and Pink Vegetative Cells

Endospore Bacterial Stain 
Procedure for Staining Endospores & Vegetative Bacteria Cells

LAB NOTES from Science Prof Online

CELL BIOLOGYMICRO BIO 8-WKMICRO BIO 16-WKINSTRUCTORS



What Is an Endospore?
Endospores are produced by very few types of bacteria, most notably the genera  Clostridium  and Bacillus.  These protective structures are made through a process known as sporulation in response to extreme environmental conditions, such as high temperatures, desiccation, chemicals, changes in pH and lack of food.

Article Summary: Endospore staining involves application of a series of dyes. Malachite green stains endospores and safrinin dyes vegetative cells pink. Here's endospore stain procedure.
Endospore Stain Procedure Lab Notes
Page last updated: 4/2014

Portions of this article originally appeared on Suite101 online magazine.​

Virtual Microbiology
Classroom






You have free access to a large collection of materials used in a college-level introductory microbiology course. The Virtual Microbiology Classroom provides a wide range of free educational resources including PowerPoint Lectures, Study Guides, Review Questions and Practice Test Questions.
Prokaryotic Cell, Mariana Ruiz
More Images of Endospore Stained Bacteria Button
SCIENCE PHOTOS
Endospore Stain Illustration
Challenges Interpreting the Endospore Stain
If the malachite green stain  is allowed to dry out when the slide is on the water bath, a green crust of dye will obscure your specimen. If this has happened, gently tap the crusted dye with your gloved finger while rinsing. This helps dislodge the dried dye.

It should be noted that any debris on the slide can also take up and hold the green stain. Everything that ends up green on the slide is not necessarily an endospore. Endospores are small, typically oval and you should see numerous uniform examples on your slide. Large or irregular globs of green on the slide may be artifacts.

Acid-fast cells, such as members of Mycobacterium and Nocardia have waxy molecules in their cell wall that will take up and retain the malachite green stain when subjected to the endospore staining process. The uniformly green appearance of endospore stained Acid-fast cells doesn’t mean that they produce endospores. These are vegetative cells that have taken up color from the heat driving malachite green into their waxy cell wall.

Helpful Links on Endospores
facebook link
SPO twitter
Tamislinked in page
Photographic Guide to Completing an Endospore Stain
Double click on photo strip for a slideshow of larger images.
The procedure for differentially staining endospores and vegetative cells is as follows:

  • Place the heat-fixed bacterial slide over screened water bath and then apply the primary stain malachite green.
  • Allow the slide to sit over the steaming water bath for 5 minutes, reapplying stain if it begins to dry out.
  • Remove the slide from the water bath and rinse the slide with water until water runs clear.
  • Flood slide with the counterstain safranin for 1 minute, then rinse.
Application of Primary Stain: 1. Endospore stain set-up; 2 & 3. Malachite green being applied to slides on water bath, note in photo 3, clothes pins are used to make handling the slide easier; 4. Rinse. Make sure to rinse thoroughly enough so that there are no "chunks" of green on slide.
Application of Counterstain: 1. Secondary stain safranin; 2 & 3. Flood slide with safranin and leave stain on for 1 minute; 4. Rinse. Click here for more endospore stain photos.
Endospore stained bacterial smear of Bacillus subtilis viewed under oil immersion @1000xTM. Pink rods are vegetative bacillus-shaped cells and smaller blue-green oval objects are endospores.
Endospore stained bacterial smear of Bacillus subtilis under oil immersion @1000xTM. Pink rods are vegetative cells with smaller blue-green oval endospores.
How to Prepare a Bacterial Smear for Endospore Staining
SPO VIRTUAL CLASSROOMS
SPO is a FREE science education website. Donations are key in helping us provide this resource with fewer ads. 
Please help!

(This donation link uses PayPal on a secure connection.)

After this staining procedure, the endospores will appear green, having retained the primary stain, malachite green. Vegetative cells (bacteria are in the active, metabolizing state) will appear pink, having retained the counterstain, safranin.

BACTERIAL SMEAR AND ENDOSPORE STAIN VIDEOS

How to Do an Endospore Stain
Instructor's Corner
In the dormant, inert endospore state, bacteria do not metabolize or reproduce, but exist in a type of suspended animation, much like the seeds of plants do. When environmental conditions again become favorable, the endospore germinates, resulting in a new vegetative cell.

Preparing a Bacterial Sample 
Prior to staining bacteria, a bacterial smear must be heat fixed onto a microscope slide. A smear is a sample of bacteria suspended in a small amount of water on a slide. That sample is then dried using heat. The heat kills the bacteria and attaches the sample to the slide so that it does not easily wash away.

Staining Bacterial Endospores
Normal water-based techniques, such as the Gram stain, will not stain these tough, resistant structures. In order to stain endspores, the dye malachite green must be forced into the spore with heat, in much the same way that carbol fuschsin is forced through the waxy mycolic acid layer of Mycobacterium in the Acid-fast stain.

Microbiology Lab Videos
SCIENCE VIDEOS