Microscopes are incredible tools but they’re not useful unless you know how to use them. In order to see anything, samples need to be properly mounted and prepared. And one of the stains that’s used to examine bacteria under a microscope is called the endospore stain.
An endospore stain is a differential stain that is used on bacteria to differentiate between vegetative cells and endospores (non-reproductive structures formed by bacteria for protection in hostile environments). This technique is useful in classifying bacteria and detecting dormant bacteria.
But before we can dive into why endospore staining is useful, we should probably take a few steps back. Namely–what are endospores? And how does the endospore stain work? This will be covered below.
What are Endospores?
Endospores are a way for select Gram-positive bacteria to survive in extreme circumstances. When bacterial cells feel a certain amount of “stress” (they don’t have enough nutrients to survive), they will turn their vegetative (living) cells into endospores.
Endospores are dormant cells that are highly resistant to anything that would normally kill a vegetative cell. Because of this, endospores are extremely difficult to kill, and the same antibacterial and antimicrobial chemicals that would kill a living cell won’t do anything to an endospore.
Endospores are formed when the bacteria cell starts to feel a certain amount of stress. If the cell feels threatened (either by lack of nutrients or other outside circumstances), it will begin the process to form an endospore. There are seven main steps to endospore formation: First the asymmetrical division into two different parts of the cell, engulfment of the smaller cell (forespore), synthesis of the forespore’s cortex and different layers, forespore becoming an endospore, and finally, the lysis (disintegration) of the mother cell.
Endospores, unlike regular Gram-positive bacterial cells, don’t just have a peptidoglycan cell wall and cytoplasmic outer membrane. While they’re being formed, endospores gain many more layers of protection. The layers of an endospore outer shell (from outer to inner) are as follows:
- Coat
- Outer Membrane
- Cortex
- Germ Cell Wall
- Inner Membrane
All five of those layers play different roles in the protection and survival of the endospore cell. The outer coat is directly exposed to the elements, so it protects the cell from most enzyme and chemical attacks. The cortex is there to make sure the endospore is fully dehydrated, which might not sound great to us, but it’s fantastic for the endospore. Dehydration helps protect it from high temperatures.
The germ cell wall isn’t super protective, but it will become the outer cell wall once again when the endospore becomes a vegetative cell once more. And the inner membrane keeps damaging chemicals from getting to the core.
Once you get past all those shields, you’ll find the inner core. The core of an endospore contains the bacteria’s DNA, RNA, acid-soluble proteins (SASPs), and a high concentration of dipicolinic acid, which is the reason the endospore can remain dormant. The SASPs not only protect the DNA and RNA strands, but they are also responsible for protecting the endospore cell from UV light. To learn more about endospores, visit the College of Agriculture and Life Sciences website, and check out the YouTube video below to see how an endospore is formed.
Staining Procedure
Since endospores are so hardcore, it’s been difficult to find dyes that will permeate endospores. The most common method of endospore staining is the Schaeffer-Fulton method because they use a malachite green stain that does show up on endospores. Once the Schaeffer-Fulton method is completed, endospores will be bright green, whereas their vegetative counterparts will appear reddish-pink.
This is a summary of the endospore staining procedure, however, it is not an in-depth procedure, and should NOT be used as instructions for an experiment. This is just to summarize how staining works. If you would like to follow some endospore staining procedures, you can use the one from austincc.edu, microbiologyinfo.com, or samples.jbpub.com
Materials:
- Primary Stain: Malachite Green
- Decolorizing Agent: Distilled Water (dH2O)
- Counter Stain: Safranin
- Petri Dishes
- Agar
- Sterile Swabs or Inoculating Loop
- Microscope
- Blotting Paper
- Glass Slide
- Boiling Water
- Parafilm
- Fume hood
- Bunsen Burner
- Bacteria (specific species being tested for endospores)
Endospore Staining Procedure:
- Prepare Petri dishes with agar gel (to learn how to do that, click here)
- Take the fresh Petri dishes and swab them with the desired bacteria
- Close and seal the Petri dishes with parafilm and label with bacteria name and date
- Put Petri dishes in a warm, dark place to allow the bacteria to grow for a few days until colonies are visible
- Bring Petri dishes back out and unseal them
- Smear some of bacteria organism on a glass slide and cover with blotting paper
- Saturate the slide with malachite green stain
- Steam the stained slide for five minutes; if the slide stops steaming, add more malachite green (MALACHITE GREEN FUMES ARE TOXIC: CONDUCT THIS STEP UNDER A FUME HOOD)
- Wash the slide with distilled water
- Counterstain with Safranin for thirty seconds
- Wash the slide with distilled water
Once these steps are completed, there should be a slide of stained bacteria. Under the microscope, all the endospore cells will look green and the rest of the bacteria will look pink.
If the bacteria species has endospores, you’ll be able to tell by the green bacteria visible in the sample. If some of the bacteria are stained pink but have a greenish hue on the inside, that also means that it contains endospore cells, but the endospore cells are just still within the original mother cell.
Why do Scientists Stain Endospores?
The most obvious answer to this is to discover what kinds of bacteria will form endospores. Almost all bacteria can be tested for endospores using the Schaeffer-Fulton stain, with the exception of the Mycobacterium, which will be stained green at the end of the experiment whether it’s vegetative or not due to its waxy cell wall. Scientists use the Ziehl–Neelsen acid-fast stain on Mycobacterium.
Endospore staining is incredibly important; it only tests for one thing but knowing which bacterium form endospores is extremely useful when it comes to developing antibacterial medications. If a bacterial species forms endospores, then scientists will know that the bacteria will not respond to heat, radiation, chemicals, or desiccation. Other than that, there aren’t many uses for endospore staining.
