Introduction bacteria are characterized by the presence or absence of a number of different structures. It is made of polysaccharide, glycoprotein or polypeptide. Capsule stain antonys method principle the capsule or glycocalyx is a gelatinous outer layer that is secreted by the microbe and remains stuck to it. It is nonionic, so the dyes that we commonly use will not bind to it. A rapid method for distinction between gramnegative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 grampositive and 55 gramnegative bacterial strains.
Capsule stain principle, procedure and result interpretation. The second is a directstaining drymount method that precipitates copper sulfate and leaves the capsule as a pale blue zone. The most common clinical use of trypan blue is staining of the anterior tenon capsule to assist in creating a capsulorrhexis during removal of a white or mature cataract. Bacterial capsules are composed of highmolecularweight polysaccharides andor polypeptides, and are associated with virulence and biofilm formation. In your procedure however, the preparation of the smear needs not be sterile but aseptically done to prevent unwanted materialsorganisms on your slide and assuring that only the specimen you intend to examine is on your slide. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Older cultures are more likely to exhibit capsule production. Sample, glass slide, wire loop, burner, malachite green, safranine, distilled water and cedar wood oil, and a microscope. Jan 11, 2020 capsule staining is more difficult than other types of differential staining procedures because the capsular materials are watersoluble and may be dislodged and removed with vigorous washing. Unfortunately, capsules do not stain well with crystal violet, methylene blue, or other simple stains. Most staining techniques are based on fomation of ionic bond between the stain. Introduction as mentioned in lab exercise 6, not all dyes stain the bacterial cell. If you are staining an unknown organism, be sure your culture is several days old. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis tb along with some others of this.
As a result, acidic and basic stains will often fail to adhere on to them. Negative staining technique principle, procedure, results. In capsule staining, a smear of bacteria is made at the center of a slide. Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Wiley online library unit describes two methods of capsule staining. A bacteria, which does not possess capsule is a noncapsulated bacteria. The procedure of negative staining technique take a clean, dry, scratch and grease free microscopic glass slide and place a drop of india ink or nigrosin on it at one end near the edge. Flagella stain principle, procedure and result interpretation. The purpose of capsule staining is to observe bacterial capsule by distinguishing capsular material from the bacteria cell. Capsule staining principle, reagents, procedure and result. It is called a differential stain since it differentiates between grampositive and gramnegative bacteria.
Acidfast stain principle, procedure, interpretation and. Hemosiderin staining occurs when red blood cells are broken down, causing hemoglobin to be stored as hemosiderin. Staining of the tenon capsule with trypan blue during. The terms positive and negative have nothing to do with. History of gram stain the gram stain procedure was developed in 1883 by hans christian gram, a danish physician, who was working as a pathologist on a method he hoped would differentiate all bacteria from mammalian cells. Most capsules are composed of polysaccharides, but some are composed of polypeptides. Capsule stain is a type of differential stain which involves the use of two stains.
The gram stain is a differential method of staining used to assign bacteria to one of two groups grampositive and gramnegative based on the properties of their cell walls. Protocols for endospore stain, capsule stain, motility stab and wet mount. Acknowledgments uk standards for microbiology investigations uk smis are developed under the auspices of public health england phe working in partnership with the national. Take a small portion of the bacterial colony with the help of sterilized inoculating loop. Your white blood cells, or immune system cells, can clear up some of the excess.
When the capsule is too thin to be observed under light microscope, it is called microcapsule and when. Staining techniques read about staining techniques in tortora et. The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. Full text full text is available as a scanned copy of the original print version. Capsule staining is more difficult than other types of differential staining procedures because the capsular materials are watersoluble and may. This unit describes two methods of capsule staining. Here, it is worth noting that for most part, capsules are nonionic. This staining technique divides bacteria into two groups namely acidfast and non acidfast. In some bacteria, the cell wall is surrounded by a viscous cell envelope called capsule. Therefore any time the cell wall is damaged the gram stain characteristics of gram positive cells will change. The area between the 2 dyes is the halo of the capsule surrounding the cell.
Two dyes, one acidic and one basic, are used to stain the background and the cell wall, respectively. Capsule is the rigid, slimy and gummy covering of the bacterial cell which lies external to the cell wall. The pas staining procedure may also be used for the demonstration of fungal organisms in tissue sections. Uk standards for microbiology investigations issued by the standards unit, public health england acknowledgments auspices of public health england phe working in partnership with the national. Flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the gram stain, or a simple stain. For clinical samples follow appropriate techniques for handling specimens as. Prepare smears of organisms to be tested for the presence of endospores on a clean microscope slide and air dry it. Bacteria that stain purple with the gram staining procedure are termed grampositive. Apply 1% crystal violet and allow it to remain on the slide for 2 minutes. The cell that posses the capsule are called as capsulated cell and those who lack the capsule are called as the noncapsulated bacteria. Note that no capsule is visible because no milk or other proteinaceous material was present or added to take up the crystal violet stain and provide a background.
In this type of capsule staining procedure, the primary stain is crystal violet, and all parts of the cell take up the purple crystal violet stain. Capsule stainkit is recommended for staining bacterial capsule against dark. This video was prepared by the teaching support team for the university of western australias school of pathology and laboratory medicine. Also look up all of these stains in your leboffe and pierce book. Bacterial capsules are composed of highmolecularweight polysaccharides andor polypeptides, and. Capsule staining is usually a two or threestage procedure in which trypan blue is injected under air, a viscoelastic device,4 viscoelastic mixed with dye,6 or compartmentalized viscoelastic and. The capsule if present will appear clear against the dark background.
For this reason, it becomes necessary to stain the background using an acidic. Aim to perform capsule staining of bacteria, to observe bacterial capsule. Bacterial capsule is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to the cell wall the capsule stain employs an acidic stain and a basic stain to detect capsule production. Procedure of endospore stain spore staining procedure. It is believed that the gram staining characteristics of an organism is a function of its cell wall. 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. Get a printable copy pdf file of the complete article 127k, or click on a page image below to browse page by page. Young, fresh cultures wont have developed capsules yet. Capsule staining capsule staining purpose of capsule.
Mar 04, 2017 capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Jan 15, 2020 flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the gram stain, or a simple stain. Overheating of smears when heat fixation is used d. Each of these structures is visible with light microscopy if the correct staining procedure is employed. Smears should not be heated because the resultant cell shrinkage may create a clear zone around the organism that is an artifact that can be mistaken for. Using a flamed loop and sterile technique, remove some klebsiella pneumoniae from culture tube or plate and mix it into the drop of india ink. Some bacilli show bipolar staining, with the ends staining deeply and the middle portion very little.
Place a drop of india ink on a slide and mix colonies in it and emulsify put a cover slip over it and press it down so that the film becomes very thin and pale in colour observe wet film under high power objective the capsule is seen as clear refractile,halo around the organism against. Periodic acid schiff pas reagents are for in vitro diagnostic use. There is no mordant in the capsule staining procedure. Stain protocols biol 2420 smith 2010 page 4 of 4 acidfast stain 1.
The capsule stain is a differential stain used to detect cells capable of. Endospores, capsules and flagella are three such examples. Mar 16, 2015 this video was prepared by the teaching support team for the university of western australias school of pathology and laboratory medicine palm. It was developed by danish microbiologist hans christian gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. A 20% copper sulfate solution serves a dual role as both the decolorizing agent and counterstain. Place a small piece of blotting paper absorbent paper over the smear and place the slide smear side up on a wire gauze on a ring stand. The capsule stain employs an acidic stain and a basic stain to detect capsule production. Take a clean, dry, scratch and grease free microscopic glass slide and place a.
Use millipore filtered water in the waterbath and staining procedure. The second is a direct staining drymount method that precipitates copper sulfate and leaves the capsule as a pale blue zone. The capsule stain the capsule is a thick polysaccharide layer around the outside of the cell. Take a clean, dry, scratch and grease free microscopic glass slide and place a drop of crystal violet on it at one end near the edge. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless. This procedure is extensively used in the diagnosis of tuberculosis and leprosy. When performing a capsule stain on your unknown, be sure the culture you take your sample from is at least five days old.
To identify the organism in a given sample requirements. Perform a successful capsule stain to distinguish capsular material from the bacterial cell. Place a single drop of india ink on a clean microscope slide, adjacent to the frosted edge. Get 50% off quizlet plus through monday learn more. Sample, glass slide, wire loop, burner, malachite green, safranine, distilled water. Jan 18, 2014 capsule staining technique by hiss method. Tiana ramer score capsule staining 100% introduction to negativecapsule staining 100% explain the purpose of.
Capsule and capsular staining authorstream presentation. 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. The gram stain procedure is a differential staining procedure that involves multiple steps. 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. Encapsulated klebsiella pneumoniae grown in nutrient broth and stained using the anthonys capsule stain. Prepare thin smears of bacterial culture on a microscope slide. How to perform capsule staining of a bacteria experiment. Learn vocabulary, terms, and more with flashcards, games, and other study tools. The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. Both methods are easily performed within approximately 5 min. The gram stain is the most widely used staining procedure in bacteriology. The basic dye will stain the negative bacterial cell, and an acidic stain will stain the positive background. A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria. Thus, to stain a capsule, we need a special capsule stain that will focus on the capsule a capsule can be easily destroyed by a heat treatment thats why a step of heat fixing is skipped.
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