1. Methylene blue (CI 52015) is a heterocyclic aromatic chemical compound with the molecular formula C16H18N3SCl.

It has many uses in a range of different fields, such as biology and chemistry. At room temperature it appears as a solid, odorless, dark green powder, that yields a blue solution when dissolved in water. The hydrated form has 3 molecules of water per molecule of methylene blue.[1] Methylene blue should not be confused with methyl blue, another histology stain, new methylene blue, nor with the methyl violets often used as pH indicators. The International Nonproprietary Name (INN) of methylene blue is methylthioninium chloride.[2][3] Redox indicator Methylene blue is widely used as a redox indicator in analytical chemistry. Solutions of this substance are blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent. The redox properties can be seen in a classical demonstration of chemical kinetics in general chemistry, the "blue bottle" experiment. Typically, a solution is made of glucose (dextrose), methylene blue, and sodium hydroxide. Upon shaking the bottle, oxygen oxidizes methylene blue, and the solution turns blue. The dextrose will gradually reduce the methylene blue to its colorless, reduced form. Hence, when the dissolved dextrose is entirely consumed, the solution will turn blue again. Peroxide generator Methylene blue is also a photosensitizer used to create singlet oxygen when exposed to both oxygen and light. It is used in this regard to make organic peroxides by a Diels-Alder reaction which is spin forbidden with normal atmospheric triplet oxygen. Sulfide analysis The formation of methylene blue after the reaction of hydrogen sulfide with dimethyl-pphenylenediamine and iron(III) at pH 0.4 0.7 is used to determine by photometric measurements sulfide concentration in the range 0.020 to 1.50 mg/L (20 ppb to 1.5 ppm). The test is very sensitive and the blue coloration developing upon contact of the reagents with dissolved H2S is stable for 60 min. Ready-to-use kits such as the Spectroquant sulfide test[4] facilitate routine analyses. The methylene blue sulfide test is a convenient method often used in soil microbiology to quickly detect in water the metabolic activity of sulfate reducing bacteria (SRB). It should be observed that in this test, methylene blue is a product of reaction and not a reagent. The addition of a strong reducing agent, such as ascorbic acid, to a sulfide-containing solution is sometimes used to prevent sulfide oxidation from atmospheric oxygen. Although it is certainly a sound precaution for the determination of sulfide with an ion selective electrode, it might however hamper the development of the blue color if the freshly formed methylene blue is also reduced, as described here above in the paragraph on redox indicator.

2.counterstaing - a stain of contrasting color that is used when the principal stain does not show the structure clearly stain - (microscopy) a dye or other coloring material that is used in microscopy to make structures visible 3. The presence of acid-fast bacteria indicates the presence of mycobacterium. There are several strains, but at a practical level they are divided into two groups: 1. mycobacterium tuberculosis, and 2. all the rest. Acid-fast bacteria in the sputum is most likely to be mycobacterium tuberculosis, indicating active TB of the lungs. There is a possibility that it might be some other form of mycobacterium causing lung infection, especially if the patient has a defective immune system because of medication or chronic disease. Culture of the bacterium is necessary to distinguish between mycobacterium tuberculosis and non-tuberculous mycobacteria.

4. In biology, a spore is a reproductive structure that is adapted for dispersal and

surviving for extended periods of time in unfavorable conditions. Spores form part of the life cycles of many bacteria, plants, algae, fungi and someprotozoa.[1] According to scientist Dr. Steinn Sigurdsson, "There are viable bacterial spores that have been found that are 40 million years old on Earth - and we know they're very hardened to radiation."[2] A chief difference between spores and seeds as dispersal units is that spores have very little stored food resources compared with seeds. Spores are usually haploid and unicellular and are produced by meiosis in the sporangium by the sporophyte. Once conditions are favorable, the spore can develop into a new organism using mitotic division, producing a multicellulargametophyte, which eventually goes on to produce gametes. Two gametes fuse to create a new sporophyte. This cycle is known as alternation of generations, but a better term is "biological life cycle", as there may be more than one phase and so it cannot be a direct alternation. Haploid spores produced by mitosis (known as mitospores) are used by many fungi for asexual reproduction. Many species of fern, especially those adapted to dry conditions, produce diploid spores. This form of asexual reproduction is called apogamy. It is a form of apomixis. Spores are the units of asexual reproduction, because a single spore develops into a new organism. By contrast, gametes are the units of sexual reproduction, as two gametes need to fuse to create a new organism.

Definition The term spore derives from the ancient Greek word sowing," related to sporos, "sowing," and spora, meaning "seed, speirein, "to sow."

In common parlance, the difference between a "spore" and a "gamete" (both together called gonites) is that a spore will germinate and develop into a sporeling, while a gamete needs to combine with another gamete before developing further. However, the terms are somewhat interchangeable when referring to gametes. A chief difference between spores and seeds as dispersal units is that spores have little food storage compared with seeds, and thus require more favorable conditions in order to successfully germinate. (This is not without its exceptions, however: many orchid seeds, although multicellular, are microscopic and lack endosperm, and spores of some fungi in the Glomeromycota commonly exceed 300 m in diameter.)[3] Seeds, therefore, are more resistant to harsh conditions and require less energy to start mitosis. Spores are produced in large numbers to increase the chance of a spore surviving in a number of notable examples.

5. Spores are one of Nature's miracles -- so tiny and insignificant, but designed to survive incredibly harsh conditions. Part of the design includes a very hard exterior, that is often impervious to acids (sincespore bearing plants grow in acidic soils). Since many stains are acidic, they are ineffective of spores. 6. The antigenic properties of the capsule is to provide the pathogen an antigenic disguises. In this way, they can survive by hiding their unique antigens from being detected by antibodies of the host cell. Some bacteria may be able to coat themselves with host mucuous, Ig molecule protein to hide their own antigenic surface components from the immunological system. Example, E.coli that causes meningitiscomposed a capsule predominantly of sialic acid which provides antigenic disguises. 7. Capsule is a protective layer around bacteria. It has the ability to inhibit phagocytosis by host immune cells lyk macrophages which is the major factor responsible fordisease development. Lose of capsule makes a bacteria avirulent that is,it loses its ability 2 cause disease. Capsule formation is not influenced by environmental conditions. On the other hand, lose of capsule is influenced by stress conditions like high temperature,pressure etc. Source(s): myself a graduate in microbiology

8. The cell capsule is a very large structure of some prokaryotic cells, such as bacterial
cells. It is a layer that lies outside the cell wall of bacteria. It is a well organized layer, not easily washed off, and it can be the cause of various diseases. Composition It usually consists of polysaccharides,[1] but can be composed of other materials (e.g., polypeptide in B. anthracis). Because most capsules are water soluble[citation needed], they are difficult to stain using standard stains because most stains do not adhere to the capsule. For examination under the microscope, the bacteria and their background are stained darker than the capsule, which doesn't stain. When viewed, bacterial cells as well as the surface they are on, are stained dark, while the capsule remains pale or colorless and appears as a ring around the cell. Function The capsule is considered a virulence factor because it enhances the ability of bacteria to cause disease (i.e. prevents phagocytosis). The capsule is slippery and fragile, so when a phagocyte tries to phagocytose the bacteria, it can slip away. A capsulespecific antibody may be required for phagocytosis to occur. Capsules also contain waterwhich protects bacteria against desiccation. They also exclude bacterial viruses and most hydrophobic toxic materials such as detergents. There are 14 different capsule types, which each impart their own specific antigenicity. Immunity to one capsule type does not result in immunity to the other types. Capsules also help cells adhere to surfaces. 9. You can see how they move and interact with other microorganisms. Also, you might be able to detect metabolic rate. Looking at killed microorganisms pretty much only lets you look at their anatomy, not physiology or communication with other microbes (via chemical signlas, etc). For example, there are certain microbes that move in a certain direction based on a magnetic force, chemical gradients (eg oxygen), or even amounts of sunlight. You couldn't see this if they were dead. 10. Staining Techniques Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope. In some cases, staining is unnecessary, for example when microorganisms are very large or when motility is to be studied, and a drop of the microorganisms can be placed directly on the slide and observed. A preparation such as this is called a wet mount. A wet mount can also be prepared by placing a drop of culture on a cover-slip (a glass cover for a

slide) and then inverting it over a hollowed-out slide. This procedure is called the hanging drop. In preparation for staining, a small sample of microorganisms is placed on a slide and permitted to air dry. The smear is heat fixed by quickly passing it over a flame.Heat fixing kills the organisms, makes them adhere to the slide, and permits them to accept the stain. Simple stain techniques. Staining can be performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm. Such a procedure is thesimple stain procedure. An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged dyes. They are repelled by the negatively charged cytoplasm and gather around the cells, leaving the cells clear and unstained. This technique is called the negative stain technique. Differential stain techniques. The differential stain technique distinguishes two kinds of organisms. An example is the Gram stain technique. This differential technique separates bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria. Crystal violet is first applied, followed by the mordant iodine, which fixes the stain (Figure 1 ). Then the slide is washed with alcohol, and the Gram-positive bacteria retain the crystal-violet iodine stain; however, the Gram-negative bacteria lose the stain. The Gram-negative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oil-immersion lens, while Gram-positive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step.

Figure 1 The Gram stain procedure used for differentiating bacteria into two groups. Another differential stain technique is the acid-fast technique. This technique differentiates species of Mycobacterium from other bacteria. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid-alcohol solution. Mycobacterium species resist the effect of the acidalcohol and retain the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene blue stain (blue). Thus, the acid-fast bacteria appear bright red, while the nonacid-fast bacteria appear blue when observed under oil-immersion microscopy.

Other stain techniques seek to identify various bacterial structures of importance. For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Flagella so stained can then be observed. A special stain technique is used to examine bacterial spores. Malachite green is used with heat to force the stain into the cells and give them color. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria. At the end of the procedure, spores stain green and other cells stain red.