Endospore Staining: A Comprehensive Guide (Principle, Reagents, Procedure & Results)

Endospores may be microscopic, but they sure are tough.

Whenever the going gets rough for certain bacteria, these dormant spores wake up (so to speak) and spring to action, helping the organism survive environmental stressors.

Endospore Staining: A Comprehensive Guide (Principle, Reagents, Procedure & Results)

In short, they help to preserve the genetic materials of a bacterium cell in times of peril. As impressive as this resolve is, it can be a real threat, as it can make harmful bacteria difficult to extinguish.

Dorner, in 1922, published his method of staining and studying endospores, a method that would be improved upon just over a decade later by Shaeffer and Fulton.

Their addition to Dorner’s foundational work streamlined the staining process. But why was anyone trying to stain these microscopic spores?

Well, the primary objective in staining endospores is to differentiate them from other cellular entities, such as vegetative cells.

Moreover, Shaeffer and Fulton also wanted to distinguish entities that were capable of forming spores from those that couldn’t.

Endospore Staining: The Core Principle

As mentioned above, endospores are metabolically dormant structures brave in the face of adverse conditions, the last line of bacterial defense against threatening external factors.

When a bacterium capable of forming endospores is at risk, the spores will wake from dormancy, plunging the bacterium into a suspended state, almost like it’s bracing itself.

Once the threat has passed and the environment is more habitable, the bacterium will return to a vegetative state in which it can germinate.

As you can see, it’s worth our while as researchers to observe these robust spores, but to do so, we need to isolate them — enter the stain! But what stain is suitable for such a job? 

For a primary stain, Shaeffer and Fulton utilized malachite green and a steaming period.

Malachite green was picked for its water soluble qualities, as well as the fact that it doesn’t really bind to cellular matter.

Endospore Staining: The Core Principle

This means that they could essentially wash the stain of the vegetative material, all whilst leaving the endospores tattooed and visible for observation. But that’s not the end of the procedure.

In their study, Shaeffer and Fulton didn’t want to omit the vegetative cells from the equation; they just wanted to differentiate them during the observations, which necessitates the introduction of a secondary stain, or a “counterstain”.

For this, safranin was chosen. Safranin, a substance that stains pink, would then stain the vegetative material without having an effect on the darker stained endospores, and voilà; both objects of the study are differentiated and clearly visible through a microscope.

  • The endospores will be a deep green color.
  • And the vegetative cells will be pink.

In terms of location, you may find spores anywhere between the very end and middle of the cell, and shape-wise, they can be either elliptical or spherical, details that can be instrumental to certain diagnostics.

Endospore Staining: Essential Reagents

For the uninitiated, in chemistry, a reagent is a substance that is used to facilitate a chemical reaction. In this scenario, our reagents are as follows:

Primary stain – Malachite green (aqueous concoction: 0.5% water/volume)

  • Malachite green = 0.5 gm
  • Distilled water = 100 ml

Decolorizing agent – Water (tap or distilled)

Counterstain – Safranin O (alcoholic concoction: 2.5% water/volume)

  • Safranin = 2.5 gm
  • Ethanol (95%) = 100 ml

Staining Endospores: Procedure

There are four primary phases of this study:

  1. Application of the primary stain
  1. Thermal intervention
  1. Application of the decolorizer
  1. Application of the counterstain

Now let’s take a look at some more detailed steps.

Step 1 – Grab yourself a slide, and make sure it’s clean and grease-free. Using a sterile method, make a smear on said slide.

Step 2 – Next, you’re going to want to air dry the slide and heat fix the specimen. This can be done by briefly passing the slide through a flame, so have your Bunsen burner (or more sophisticated heating technique) at the ready. The reason we do this is to set the bacteria sample in place on the glass.

Step 3 – Take some blotting paper, trim it to fit the slide, and cover it.

Step 4 – Soak the blotting paper with malachite green, then steam it for approximately 5 minutes — it’s best to use a stop clock here. Make sure to keep the blotting paper nice and moist, and, if need be, don’t hesitate to add more malachite green as you go. For the steaming process, you can simply hold the slide over a pot of water and bring it to the boil.

Step 5 – Rinse your steamed slide with your tap or distilled water.

Step 6 – Now’s the time to break out your counterstain. Apply your 0.5% safranin over the course of 30 seconds. Again, wash with a bit of tap or distilled water, and blot the slide dry.

Step 7 – Load your slide onto your favorite microscope and focus up. You’ll need a microscope capable of magnification to the power of 1000. Through this objective magnification, you will be able to observe vividly green endospores and pinkish vegetative cells.

Staining Endospores: Procedure

Endospore Staining: Results

As we’ve already established, the results of our experiment will be…

  1. The endospores will be stained green, making them easy to observe.
  1. The vegetative cells will be stained pink, making them just as easy to study.

And just to reiterate, spores can be situated both towards the middle or end of a cell, or indeed anywhere in between these poles. They can also be elliptical (as in oval) or spherical (as in more circular).

What Was Dorner’s Method?

Although the original method dreamt up by Dorner for staining endospores follows the exact same principles as the one we’ve discussed here today, their approach was quite different.

Primary stain – Carbolfuchsin

  • Simple fuchsin = 0.3 gm
  • Ethanol (95%) = 10 ml
  • Heat melted phenol crystals = 5 ml
  • Water (distilled) = 95 ml

Process for mixing solution: Combine the basic fuchsin with the ethanol, dissolve the phenol in the water, add the phenol/water concoction to the ethanol solution, mix, then leave to rest for a few days. Before continuing, filter the solution.

Decolorizer – Acid and alcohol

  • Ethanol (95%) = 97 ml
  • Concentrated hydrochloric acid = 3 ml

Counter stain – Nigrosin (diluted)

  • Nigrosin = 10 ml
  • Water (distilled) = 100 ml

The Process

The process:
  1. Much like in our own procedure, you’d make a smear on a clean slide using a sterile method.
  1. Air dry and heat fix (again, like our own experiment).
  1. Wrap in blotting paper (more of the same).
  1. Use the carbolfuchsin to saturate the paper, then steam for up to 10 minutes, adding more stain where appropriate.
  1. Discard the paper, then add the acid/alcohol decolorizer for 60 seconds.
  1. Wash with tap water and dry using more blotting paper.
  1. Using another slide to help spread, apply a thin film of nigrosin over the smear.
  1. Allow the slide to air dry.
  1. Observe the slide with a microscope (oil immersion).

As the process is different, so are the results: Endospores are stained red, vegetative cells are left colorless, and the backdrop is completely black.

Endospore Staining: Positive And Negative Examples

Positive examples:

C. botulinum, clostridium perfringens, Bacillus anthracis, Bacillus cereus, C. tetani, Sporosarcina spp, Desulfotomaculum spp, Sporolactobacillus spp, and so on.

Negative examples:

Salmonella spp, E. coli, and so on.

Endospore Staining: Quality Control

Positive control:

Clostridium perfringens (ATCC 13124)

Negative control:

Escherichia coli (ATCC 25922)

Final Thoughts

There you have it — the principle, reagents, procedure and results of the modern endospore staining experiment, with the vintage Dorner approach added in for good measure. 

Jennifer Dawkins