Why is capsule stain a negative stain

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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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. 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. A few bacteria are gram variable. Trichomonas , Strongyloides , some fungi, and some protozoa cysts also have a Gram reaction. Very small bacteria or bacteria without a cell wall, such as Treponema , Mycoplasma , Chlamydia , or Rickettsia do not have a gram reaction.

The characterization of any new bacteria must include their gram reaction. Typically a differential stain has four components; the primary stain, a mordant that sets the stain, a decolorizing agent to remove the primary stain, and a counter stain. In the Gram stain, the primary stain is crystal violet. This gives the cell an intense purple color. The mordant, iodine, forms a complex with the crystal violet inside the cell wall. Gram positive cells will retain the dye complex and remain purple.

The dye rinses out in gram negative cells. The large iodine-crystal violet complex is retained within the cell walls of gram positive cells because of the molecular structure of the many layers of peptidoglycan in the cell wall. There are lots of cross-linked teichoic acids and the iodine-dye complex cannot physically get out. There is also less lipid in the membrane and the decolorizing agent cannot get to it as well.

Gram negative cells have an outer membrane and only one layer of peptidoglycan, with more lipid. The crystal violet dye is easily washed out. The accuracy of the Gram stain is dependent on the integrity of the bacterial cell wall. There are a variety of things that can influence the cell wall integrity; old cells i. Under these conditions, gram positive cells will come out as gram-negative.

If you de-colorize too long, Gram-positive cells will look like Gram-negative cells. Conversely, if you do not decolorize enough, Gram-negatives will look like Gram-positives. The only way you can trust your results it to always run a known Gram-positive and a known Gram-negative on the same slide. If they stain as predicted you can be pretty sure the result of your unknown sample is reliable. The Gram staining takes practice to get right.

Do not expect to get a good Gram stain on your first try. It is a good idea to hold your slide with a clothespin; your gloves will get pretty psychedelic as will everything you touch! The Congo Red Capsule stain is a modification of the nigrosin negative stain you may have done previously. The bacteria take up the congo red dye and the background is stained then with acid fuchsin dye. The capsule or slime layers, highly hydrated polymers, exclude both dyes.

The background will appear blue, the bacterial cells will appear pink, and the clear halos are the capsules. Clinically, the capsules of some highly pathogenic bacteria i. The bacteria are suspended in the antisera and then mixed with methlyene blue.

In the antisera staining procedure, the bacteria will appear blue surrounded by a clear halo and then surrounded by a thin blue line where the antisera have attached to the capsule. Endospore formation is characteristic of Clostridum and Bacillus spp. The ability to concentrate and coat their protoplasm allows them to survive the adverse environmental conditions they experience in their soil habitat. This also allows the spores to resist staining.

Endospores are typically highly refractile, light striking them is deflected. Many Bacillus species have inclusion bodies that are highly refractile. These inclusion bodies may look like endospores with regular staining. The presence of endospores must be confirmed with endospore specific stains.

The presence, and characteristic shape and position of endospores require special procedures to permeate the endospore coat. By counterstaining with dyes like crystal violet or methylene blue, bacterial cell wall takes up the dye. Furthermore, what are two things that are stained in a capsule stain? In this method two dyes, crystal violet and india ink are used. The capsule is seen as a clear halo around the microorganism against the black background.

This method is used for demonstrating Cryptococcus. The background will be dark color of india ink. Capsules are formed by organisms such as Klebsiella pneumoniae.

Bacterial capsules are non -ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic stain and to stain the cell itself using a basic stain. Because most capsules are so tightly packed, they are difficult to stain because most standard stains cannot adhere to the capsule. For examination under the microscope, the bacteria and their background are stained darker than the capsule , which doesn't stain.

We use nigrosin as our negative stain. Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion and becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. Negative stain. From Wikipedia, the free encyclopedia. Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible.

Why is it that negative stain called a negative stain? Save my name and email in this browser for the next time I comment. Principle of Capsule Staining Capsules stain very poorly with reagents used in simple staining and a capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained.

Congo Red is easier to see, but it does not work well with some strains. India Ink generally works, but it has tiny particles that display Brownian motion that must be differentiated from your bacteria. Nigrosin may need to be kept very thin or diluted.

Using sterile technique, add a loopful of bacterial culture to slide, smearing it in the dye. Use the other slide to drag the ink-cell mixture into a thin film along the first slide and let stand for minutes.