Why are endospores difficult to 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.

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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. Shape cocci, bacilli, curved rods, spirals of bacteria will be visible. Arrangement of bacterial cells single cells, paired cells, chains of cells, clusters, groupings will be visible.

Capsule Staining Set-up Wear gloves and a lab coat as dyes stain hands and clothing. Clean slides with laboratory wipes. Using a pipet tip, smear a bacterial colony into the dye to produce a thin even layer. Note: Do not heat fix as heating can dehydrate or distort the capsule.

Rinse excess stain 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. Hold the slide at a degree angle until completely air-dried. Examine smear on the microscope under oil immersion with a X objective. Results and Data Analysis Bacterial cells will stain purple.

The background of the slide will stain dark. Capsules will be a clear halo around cells against a dark background. Endospore Staining Schaeffer-Fulton method Set-up Wear gloves and a non-flammable lab coat to protect hands and clothing from dyes and flame.

A Bunsen burner is used to heat fix the bacteria. To prepare 0. Use commercially available Gram's Safranin reagent solution. Using aseptic technique, smear a bacterial colony into the liquid to produce a thin, even layer. Note: Endospores generally do not form in young cells; therefore, the culture is recommended to be between 18 and 36 hours old 9.

Heat fix by passing slide bacteria side up through flame times. To help contain the dye, place a piece of lens paper cut to fit the bacterial smear over the heat-fixed smear.

Saturate lens paper with Malachite Green solution. Place slide on top of the beaker of boiling water on a hot plate, and steam slide for 5 minutes, keeping lens paper moist by adding more dye a drop at a time as needed.

Note: Avoid overheating and drying out the dye solution. Remove slide from beaker, remove and discard lens paper, allow slide to cool 2 minutes. Holding slide at an angle, rinse thoroughly by squirting a gentle, indirect stream of water onto slide, allowing it to drain down over smear.

Holding slide level, flood smear with Safranin, allow to stand 1 minute. Rinse excess Safranin as in step 3. Allow to air-dry. Examine slide on microscope under oil-immersion with a X objective. The entire process takes 8 to 10 hours.

Whereas endospores reside inside the original cell, free spores spores exist outside the cell on their own. More on Chromosomes. Essentially, endospore stain is a differential stain. As such, it allows for the differentiation of structures and thus the characterization of a cell based on its physical and chemical nature. In this case, endospore stain as a differential staining technique is largely used for the purposes of distinguishing between vegetative cells and endospores.

For endoscopes, there are two major staining techniques used. These include:. This is the most common technique for endospore staining. Staining Procedure. Steps in the Procedure. Unlike the vegetative cells, endospores possess a permeability barrier. 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.

A second layer of cytoplasmic membrane then forms around one of the DNA molecules the one that will become part of the endospore to form a forespore. Both of these membrane layers then synthesize peptidoglycan in the space between them to form the first protective coat, the cortex.

Calcium dipocolinate is also incorporated into the forming endospore. A spore coat composed of a keratin-like protein then forms around the cortex.

Sometimes an outer membrane composed of lipid and protein and called an exosporium is also seen Fig.