Can you relate endospore staining

Gram-positive bacteria stain purple due to the thick peptidoglycan layer which is not easily penetrated by the decolorizer; Gram-negative bacteria, with their thinner peptidoglycan layer and higher lipid content, destain with the decolorizer and are counterstained red when Safranin is added Figure 3.

Gram staining is used to differentiate cells into two types Gram-positive and Gram-negative and is also useful to distinguish cell shape spheres or cocci, rods, curved rods, and spirals and arrangement single cells, pairs, chains, groups, and clusters 1, 3.

Figure 2: Schematic of the Gram Staining Protocol. The left column shows how Gram-negative bacteria react at each step of the protocol.

The right column shows how Gram-positive bacteria react. Also, shown are two typical bacterial cell shapes: the bacilli or rods and the cocci or spheres. Figure 3: Gram Staining Results.

A Gram stain of a mixture of Staphylococcus aureus Gram-positive purple cocci and Escherichia coli Gram-negative red rods. Some bacteria produce an extracellular viscous outer layer called a capsule 3, 5. Capsules are protective structures with various functions, including but not limited to adherence to surfaces and other bacteria, protection from desiccation, and protection from phagocytosis. Due to their mostly non-ionic composition and tendency to repel stains, simple staining methods do not work with capsule; instead, capsule staining uses a negative staining technique which stains the cells and the background, leaving the capsule as a clear halo around the cells 1, 3 Figure 4.

Capsule staining involves smearing a bacterial sample into an acidic stain on a microscope slide. Unlike Gram staining, the bacterial smear is not heat-fixed during a Capsule Stain. Heat-fixing can disrupt or dehydrate the capsule, leading to false negatives 5. Furthermore, heat-fixing can shrink cells resulting in a clearing around the cell which can be mistaken as a capsule, leading to false positives 3.

The acidic stain colors the slide background; while follow up with a basic stain, Crystal Violet, colors the bacterial cells themselves, leaving the capsule unstained and appearing as a clear halo between the cells and the slide background Figure 5.

Traditionally, India ink has been used as the acidic stain because these particles cannot penetrate the capsule. Therefore, neither the capsule nor the cell is stained by India ink; instead, the background is stained. Congo Red, Nigrosin, or Eosin can be used in place of India ink. Capsule staining can help doctors diagnose bacterial infections when looking at cultures from patient samples and guide appropriate patient treatment.

Common diseases caused by encapsulated bacteria include pneumonia, meningitis, and salmonellosis. Figure 4: Schematic of the Capsule Staining Protocol. The top panel shows the slide smear prior to any stain application. The middle panel shows how the slide and bacteria look after the primary stain, Congo Red. The final panel shows how the slide and bacteria look after the counterstain, Crystal Violet. Figure 5: Capsule Staining Results. Capsule staining of encapsulated Acinetobacter baumannii denoted with black arrows and non-encapsulated Escherichia coli denoted with white arrows.

Notice the background is dark and A. The capsule around A. In adverse conditions for example nutrient limitation, extreme temperatures, or dehydration , some bacteria produce endospores, metabolically inactive structures that are resistant to physical and chemical damage 1, 2, 8, 9.

Endospores allow the bacterium to survive harsh conditions by protecting the genetic material of the cells; once conditions are favorable for growth, spores germinate, and bacterial growth continues. 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. 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. Finally, the mother cell is destroyed in a programmed cell death, and the endospore is released into the environment.

The endospore will remain dormant until it senses the return of more favorable conditions. Some Epulopiscium -like surgeonfish symbionts form mature endospores at night. These spores possess all of the characteristic protective layers seen in B.

These are the largest endospores described thus far, with the largest being over times larger than a Bacillus subtilis endospore. The formation of endospores may help maintain the symbiotic association between these Epulopiscium -like symbionts and their surgeonfish hosts. Since endospore formation coincides with periods in which the host surgeonfish is not actively feeding, the cells do not need to compete for the limited nutrients present in the gut at night.

The protective properties of the endospores also allow them to survive passage to new surgeonfish hosts. Following a decolorization step which removes the dye from the vegetative cells in the smear, the counterstain safranin is applied to provide color and contrast.

When stained by this method, the endospores are green, and the vegetative cells stain pink, as shown in Figure 7. Although endospores themselves are resistant to the Gram stain technique, bacterial cells captured in the process of creating these structures can be stained.

In this case, the endospores are seen as clear oval or spherical areas within the stained cell. Endospores can also be directly observed in cells by using phase contrast microscopy, as shown in Figure 8. Because many differential staining methods require several steps and take a long time to complete, we will not be performing all of the differential staining methods discussed above. Pre-stained slides will be used to visualize bacterial capsules, metachromatic granules, and acid-fast bacilli.

Obtain one slide of each of the three bacteria listed in the table below. Your environmental isolate may have one or more of these cellular features, and learning to recognize them will aid in identification. These should all be viewed using the oil immersion objective lens.

All staining procedures should be done over a sink. The Gram stain procedure will be demonstrated, and an overview is provided in Table 1. A volunteer from your lab bench should obtain cultures of the bacteria you will be using in this lab, as directed by your instructor.

One of the cultures will be a Gram positive bacterium, and the other will be Gram negative. Below, write the names of the bacteria you will be using, along with the BSL for each culture:. Obtain two glass slides, and prepare a smear of each of the two bacterial cultures, one per slide, as demonstrated. Stain both smears using the Gram stain method. Observe the slides with a light microscope at 1,X and record your observations in the table below.

Heat fix the smear and Gram stain it. You should be able to determine the Gram stain reaction, cellular morphology and arrangement of BOTH bacteria in this mixed smear.

Your instructor may ask to see this slide and offer constructive commentary. Only a few genera of bacteria produce endospores and nearly all of them are Gram-positive bacilli. Most notable are Bacillus and Clostridium species, which naturally live in soil and are common contaminants on surfaces. The growth of Clostridium spp. Endospore-forming bacteria are distinct from other groups of Gram positive bacilli and distinguishable by their endospores. After staining, endospores typically appear as light green oval or spherical structures, which may be seen either within or outside of the vegetative cells, which appear pink.

The shape and location of the endospores inside the bacterial cells, along with whether the sporangium is either distending D or not distending ND the sides of the cell, are important characteristics that aid in differentiating among species see Figure 9. A volunteer from your lab bench should obtain bacterial cultures for endospore staining, as directed by your instructor.

Note that these will all be species of Bacillus. Prepare smears and stain each using the endospore staining technique. Observe the slides and note the shape and location of the endospore and the appearance of the sporangium swollen or not swollen in the table below:. In addition, choose ONE of the cultures from above and Gram stain it.

Record your results below in the spaces provided:. Are endospores visible in the Gram stained smear? Skip to content Viewing Bacterial Cells The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Figure 1. Negative stain of Cyptococcus neoformans , an encapsulated yeast Figure 2.

Positive stain of Staphylococcus aureus. Figure 3. Possible bacterial cell arrangements for cocci. Figure 4. Possible bacteria cell arrangements for bacilli.