Why endospores are so difficult to stain

Small acid-soluble proteins SASPs are also only found in endospores. Other species-specific structures and chemicals associated with endospores include stalks, toxin crystals, or an additional outer glycoprotein layer called the exosporium.

The process of forming an endospore is complex. The model organism used to study endospore formation is Bacillus subtilis. Endospore development requires several hours to complete. Key morphological changes in the process have been used as markers to define stages of development. As a cell begins the process of forming an endospore, it divides asymmetrically Stage II. This results in the creation of two compartments, the larger mother cell and the smaller forespore. These two cells have different developmental fates.

Intercellular communication systems coordinate cell-specific gene expression through the sequential activation of specialized sigma factors in each of the cells. Next Stage III , the peptidoglycan in the septum is degraded and the forespore is engulfed by the mother cell, forming a cell within a cell.

Endospores are difficult to stain with standard staining techniques because they are impermeable to dyes typically used for staining 1, 9. The technique routinely used to stain endospores is the Schaeffer-Fulton Method Figure 6 , which uses the primary stain Malachite Green, a water soluble stain that binds relatively weakly to cellular material, and heat, to allow the stain to break through the cortex of the spore Figure 7.

These steps color the growing cells termed vegetative cells in the context of endospore biology , as well as endospores and any free spores those no longer within the former cell envelope.

Vegetative cells are washed with water to remove Malachite Green; endospores retain the stain due to heating the Malachite Green within the spore. Finally, the vegetative cells are counterstained with Safranin to visualize Figure 8. Staining for endospores helps differentiate bacteria into spore formers and non-spore formers, as well as determines whether spores are present in a sample which, if present, could lead to bacterial contamination upon germination.

Figure 6: Schematic of the Endospore Staining Protocol. The left column shows how spore forming bacteria react at each step of the protocol. The right column shows how non-spore forming bacteria react. Figure 7: Diagram of Endospore Structure. Bacterial cell containing an endospore with the various spore structures labeled. Figure 8: Endospore Staining Results. A typical staining of endospores of Bacillus subtilis. The vegetative cells denoted with the white arrows are stained red, while the endospores denoted with the black arrows are stained green.

These features can all be visualized by staining and aid in the identification and classification of different bacterial species. To examine the first two characteristics of cell shape and arrangement, we can use a simple technique called Gram staining.

Here, crystal violet is applied to bacteria, which have been heat-fixed onto a slide. Next, Gram's iodine solution is added to the slide, resulting in the formation of an insoluble complex between the crystal violet and the Gram's iodine solution. A decolorizer is then applied and any bacteria with a thick peptidoglycan layer will stain purple, as this layer is not easily penetrated by the decolorizer.

These bacteria are referred to as Gram-positive. Gram-negative bacteria have a thinner peptidoglycan layer and will de-stain the decolorizer, losing the purple color. However, they will stain reddish-pink when a safranin counterstain is added, which binds to a lipopolysaccharide layer on their outside. Once stained, the cells can be observed for morphology, size, and arrangement, such as in chains or clusters, which further aids in classification and identification.

Another useful technique in the microbiologist's toolkit is the capsule stain, used to visualize external capsules that surround some types of bacterial cells. Due to the capsule's non-ionic composition and tendency to repel stains, simple staining methods won't work. Instead, a negative staining technique is used, which first stains the background with an acidic colorant, such as Congo red, before the bacterial cells are stained with crystal violet. This leaves any capsule present as a clear halo around the cells.

The final major staining technique covered here can help determine if the bacteria being studied forms spores. In adverse conditions, some bacteria produce endospores, dormant, tough, non-reproductive structures whose primary function is to ensure the survival of bacteria through periods of environmental stress, like extreme temperatures or dehydration. However, not all bacterial species make endospores, and they are difficult to stain with standard techniques because they are impermeable to many dyes.

The Schaeffer-Fulton method uses malachite green stain, which is applied to the bacteria fixed to a slide. The slide is then washed with water before being counterstained with Safranin.

Vegetative cells will appear pinkish-red, while any endospores present will appear green. In this video, you will learn how to perform these common bacterial staining techniques and then examine the staining samples using light microscopy.

To begin the procedure, tie back long hair and put on the appropriate personal protective equipment, including a lab coat and gloves. Then, clean a fresh microscope slide with a laboratory wipe. Next, pipette 10 microliters of 1X phosphate-buffered saline onto the first slide. Then, use a sterile pipette tip to select a single bacterial colony from the LB agar plate.

Smear the bacterial colony in the liquid to produce a thin, even layer. Set the slide on the benchtop, and allow it to fully air dry. Once dried, light a Bunsen burner to heat-fix the bacteria. Using tongs, pass the slide through the burner flame several times, with the bacteria side up, taking care not to hold the slide in the flame too long, which may distort the cells. Now, working over the sink, hold the slide level and apply several drops of Gram's crystal violet to completely cover the bacterial smear and then place the slide onto the bench to stand for 45 seconds.

Next, hold the slide at an angle and gently squirt a stream of water onto the top of the slide, taking care not to squirt the bacterial smear directly. Now, holding the slide level again, apply Gram's iodine solution to completely cover the stained bacteria and then allow it to stand for another 45 seconds.

Next, carefully rinse the iodine from the slide, as shown previously. While holding the slide at an angle, add a few drops of Gram's decolorizer to the slide, allowing it to run down over the stained bacteria, just until the run-off is clear, for approximately 5 seconds.

Immediately, rinse with water as shown previously. This will limit over-decolorizing the smear. Next, holding the slide level again, apply Gram's safranin counterstain to completely cover the stained bacteria. After 45 seconds, gently rinse the Safranin from the slide with water, as shown previously, and then blot dry with paper towels.

Finally, add a drop of immersion oil directly to the slide, and then examine the slide using a light microscope with a X oil objective lens. To begin this staining protocol, first put on the correct personal protective equipment and then ensure that the glass slides that will be used are clean.

Next, prepare the solutions. Now, pipette 10 microliters of the Congo red solution onto the slide. Using a clean, sterile pipette tip, select a single bacterial colony from the LB agar plate. Then, smear the bacterial colony into the dye to produce a thin, even layer. Completely air dry the bacterial slide for minutes. Now, hold the slide at an angle and gently squirt a stream of water onto the top of the slide, taking care not to squirt the bacteria directly. This prevents dyes from entering to stain the structures of the cell.

For this reason, the barrier has to be destroyed, which is why heat is used. Through heat fixing, the cortex of the endospore is penetrated allowing for the dye to interact with the petodoglycan and thus produce desired effects. In this case, heat acts as a mordant.

After heat fixing, the slide is washed using tap water or distilled water. Here, water is used as a decolorizer. Because malachite green binds relatively weakly, it can be washed off easily.

However, it cannot be washed off easily once it is locked in the spore wall. Once they take in the dye, endospores retain the dye and will be resistant to de-staining. However, vegetative cells will easily lose the stain when washed with water because they lack the spore wall. After the initial washing, a counter stain safranin is used. The purpose of the counter stain is to stain the vegetative cells that lost the primary stain.

Here, it is worth noting that the primary stain and the secondary stain are of different colors. As such, they allow the technician to differentiate different types of cells under the microscope.

Sometimes an outer membrane composed of lipid and protein and called an exosporium is also seen Fig. Finally, the remainder of the bacterium is degraded and the endospore is released.

Sporulation generally takes around 15 hours see Fig. The process is summarized in Fig.