How does endospore staining work

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.

Against this dark background, the capsules of the bacteria, if present, will appear as a clear halo around the cells. Lastly, in endospore staining, Vegetative cells will be stained red by the Safranin counterstain.

If endospores are present in the sample, these will retain the malachite green stain and appear bluish-green in color. Subscription Required.

Please recommend JoVE to your librarian. Bacteria have distinguishing characteristics that can aid in their identification. Some of these characteristics can be observed by staining and light microscopy. Three staining techniques useful for observing these characteristics are Gram staining, Capsule staining, and Endospore staining. Each technique identifies different characteristics of bacteria and can be used to help physicians recommend treatments for patients, identify potential contaminants in samples or food products, and verify sample sterility.

Bacteria are microscopic living organisms that have many distinguishing characteristics such as shape, arrangement of cells, whether or not they produce capsules, and if they form spores. To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove.

Your access has now expired. Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team. Login processing This is a sample clip. Sign in or start your free trial. Previous Video Next Video. Overview Source: Rhiannon M. Log in or Start trial to access full content. Gram Staining Set-up Wear gloves and a non-flammable lab coat, as dyes will stain hands and clothing. A Bunsen burner is used to heat-fix the bacteria.

Use care when working with flame; tie back long hair. Commercially available Gram stain reagents will be used. Clean microscope slides with laboratory wipes.

Smear a bacterial colony into the liquid to produce a thin, even layer. Note: Do not use cultures older than 24 hours, as bacteria too old could have changes in their cell wall, which will affect the Gram stain results 1, 4.

Completely air-dry slide. Once dried, heat-fix bacteria by passing slide through the flame bacteria side up times. Note: Do not hold the slide in the flame too long or you might distort the bacterial cells 1.

Working over the sink, hold the slide level and apply Gram's Crystal Violet to completely cover the heat-fixed bacteria, allow to stand 45 seconds.

Rinse excess Crystal Violet by holding the slide at an angle and squirting a gentle, indirect stream of water onto the slide and letting it run down over the stained bacteria.

Do not squirt water directly onto the bacteria. Holding slide level again, apply Gram's Iodine Solution to completely cover the stained bacteria, allow to stand 45 seconds.

Rinse excess Iodine as in step 1. While holding the slide at an angle, add a few drops of Decolorizer onto the slide, letting it trickle down over the stained bacteria just until the runoff is clear; typically, about 5 seconds.

Immediately rinse with water as in step 1. Note: This is a critical step in the protocol. Allowing Decolorizer to trickle too long or not long enough will result in false Gram-staining 4. Holding the slide level again, apply Gram's Safranin to completely cover the bacteria, allow to stand 45 seconds.

Rinse excess Safranin as in step 1. Blot, do not rub, excess water from slide using paper towels. Examine slide on the microscope using oil-immersion with a X objective. Results and Data Analysis Gram-positive bacteria will stain purple.

Gram-negative bacteria will stain red. Endospores are produced by a few genera of Gram-positive bacilli such as Bacillus and Clostridium , in response to adverse environmental conditions. Endospores are highly resistant to environmental conditions such as heat, chemicals also stains and dyes and therefore require special techniques for staining. Endospores can also be demonstrated in unstained wet films under a phase-contrast microscope. They appear as large refractile oval or spherical bodies within the mother cell.

It is the most widely used technique for endospore staining. The technique was first described by Alice B. Schaeffer and MacDonald Fulton in the s.

The method utilizes malachite green as the primary stain and safranin as counterstain. When a heat-fixed smear is flooded with aqueous malachite green solution the primary stain and steamed, the heat assists the stain to penetrate through the spore. In this technique, heating acts as a mordant. Once the endospore has absorbed the stain, it is resistant to decolorization, but the vegetative cells are easily decolorized with water leaving the vegetative cells colorless.

When counter-stained with safranin , the vegetative cells take the color of safranin and appear red or pink, in contrast to the endospores that appear green. Spores may be spherical or elliptical. Primary Stain: Malachite green 0. Counter Stain: Safranin Stock solution 2. Endospores : Endospores are bright green.

Vegetative Cells: Vegetative cells are brownish red to pink. Carbolfuchsin stain 0. Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water. Mix and let stand for several days. Filter before use. Vegetative cells are colorless, endospores are red, and the background is black. Positive Clostridium perfringens, C. I am currently taking a microbiology course at the local community college.

I have an unknown only known to my professor. I performed 3 staining procedures and determined that my unknown is gram-positive cocci, non-acid fast, endospore former. I thought it might be a Mycobacterium. From the information I provided, is that correct?