They were identified in the s John Tyndall developed a process for destroying them with intermittent heat procedure , although the stain procedures to identify them did not develop until the early twentieth century. Bacterial endospores are the most resistant structures of all living organisms, and they can live in this dormant dehydrated state for hundreds and hundreds of years even some documented at thousands of years.
Endospores are not for reproduction: One spore forms inside of the vegetative cell. When the spore germinates, one vegetative cell will be produced. The stimulus for sporulation can vary—nutrient depletion, desiccation, chemicals, heat, etc.
As a spore forms inside of the vegetative cell, the spore wall chemically changes and thicken. The primary dye malachite green is a relatively weakly binding dye to the cell wall and spore wall.
In fact, if washed well with water, the dye comes right out of the cell wall, however not from the spore wall once the dye is locked in. That is why there does not need to be a decolorizer in this stain: it is based on the binding of the malachite green and the permeability of the spore vs.
The steaming helps the malachite green to permeate the low-permeability spore wall. A variety of chemicals comprise the spore wall keratin protein, calcium , but deeper in the wall is peptidoglycan.
The keratin forming the outer portion of the endospore wall resists dye. The heating of the bacteria will make the spore wall more permeable to the malachite green, and it then attaches to the peptidoglycan.
Once in, the malachite green will not come out because the overlying spore wall becomes less permeable when the smear cools.
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. Continue holding the slide at a degree angle until completely air-dried.
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. To perform endospore staining, first, prepare a 0. Next, pipette 10 microliters of 1X PBS onto the center of the slide. Smear the bacteria into the liquid to produce a thin, even layer. Now, set the slide on the benchtop, and allow it to fully air dry. Pass the slide through the blue burner flame several times, with the bacteria side facing up.
Then, once the slide has cooled, place a piece of precut lens paper over the heat-fixed smear. Next, turn on a hotplate to the highest setting, and bring a beaker of water to a boil. Saturate the lens paper with the malachite green solution and, using tongs, place the slide on top of the beaker of boiling water to steam for 5 minutes.
Keep the lens paper moist by adding more dye, one drop at a time, as needed. Next, again using tongs, pick up the slide from the beaker and remove and discard the lens paper. Allow the slide to cool for 2 minutes. Working over the sink, hold the slide at an angle, and gently squirt a stream of water onto the top of the slide. Now, hold the slide level and apply Safranin to completely cover the slide. Then, allow it to stand for 1 minute. Next, hold the slide at an angle and rinse as previously shown.
Allow the slide to air dry on the benchtop. Finally, add a drop of immersion oil directly to the slide, and then examine the slide with a light microscope, with a X oil objective.
In the Gram staining protocol, two different colored stains can result. Dark purple staining indicates that the bacteria are Gram-positive and that they have retained the crystal violet stain. In contrast, reddish-pink staining is a characteristic of Gram-negative bacteria, which instead will be colored by the Safranin counterstain.
Additionally, different shapes and arrangements of bacteria can be visualized after Gram staining. For example, it is possible to differentiate Cocci, or round bacteria, from rod-shaped Bacillus, or identify bacteria, which forms strands, compared to those which typically aggregate as clumps or occur singly. In a capsule stained microscope image, the bacterial cells will typically be stained purple, and the background of the slide should be darkly stained. 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. When viewed under the microscope, endospores will be red in colour. In biology, endospore staining is used for the purposes of differentiating and classifying bacteria. On the other hand, it is also very important in medicine and the food industry. Because they are tough and hard to destroy it is very important to determine whether they are present in canned food and thus avoid food poisoning to protect consumers.
Cell Staining in Microscopy. Gram Staining - Purpose, Procedure and Preparation. Capsule Stain - Definitions, Methods and Procedures. Marise A. Hussey, Anne Zayaitz. Endospore stain protocol. Methanobacteria is a class of the phylum Euryarchaeota within the domain Archaea.
Read more here. The Islets of Langerhans is an endocrine tissue located within the pancreas. It consists of a variety of cells capable of producing different hormones.
Hydrogen-oxidizing Bacteria are species that can use gaseous hydrogen as the electron donor to oxidize hydrogen. The material on this page is not medical advice and is not to be used for diagnosis or treatment. Although care has been taken when preparing this page, its accuracy cannot be guaranteed.
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