staining methods.pptx for bacteria best slides for presentation
- 2. “Bacterial staining is a technique that uses stains to increase the contrast between bacteria and their background, making them easier to see under a microscope”
- 3. OBJECTIVES OF STAINING Improves visibiltiy by greater contrast between the organism and the background, differentiate various morphological types (by shape, size, arrangement, etc.), determine the staining characteristic of organism and, at times, direct diagnosis of disease, and demonstrate the purity of bacterial culture. observe certain structures (flagella, capsules, endospores, etc.),
- 4. STAINS AND DYES A dye is a general-purpose coloring agent, whereas a stain is used for coloring biological material. benzene ring plus a chromophore and A stain is an organic compound containing a an auxochrome group. Chromophore: A chemical group that gives color to benzene. Auxochrome: A group of atoms that is attached to a chromophore in an organic compound, modifying the chromophore’s ability to absorb light. Auxochromomes are colorless by themselves, but they intensify the color of the chromogen when present with the chromophore.
- 5. Chromogen vs chromophore: A chromogen is a colorless chemical compound that can be converted into a colored compound through a chemical reaction, while a chromophore is the part of a molecule that gives it color.
- 8. REQUIREMENTS FOR STAINING Stain – Majority of the stains used for staining bacteria are of the basic type (+ve charged) as nucleic acid of bacterial cells attract the positive ions (-ve charged), e.g. methylene blue, crystal violet. Acidic stains are used for background staining. Mordant – It is a chemical that forms an insoluble complex with the stain and fixes it or causes the stain to penetrate more deeply into the cell. These are used in indirect staining. For example, Gram’s iodine in Gram staining and phenol in Ziehl Neelson’s staining. Accentuater – It is a chemical which when added to a stain to make the reaction more selective and intense. For example, potassium hydroxide added in Loeffler’s methylene blue. Decolorizer – It is a chemical used to remove the excess stain in indirect regressive staining. For example, ethanol in Gram’s staining.
- 9. Types of staining techniques Simple staining (Use of of single stain) Differential staining (Use of two contrasting stains) Direct (Positive) Indirect (Negative) Separation into groups 1. Gram stain 2. Acid Visualization of structures 1. Flagella stain
- 10. SIMPLE STAINING
- 11. SIMPLE STAINING A staining method that uses only a single dye that which does not differentiate between different types of organisms There is only a single staining step and everything is stained with the same color. Simple stains are used to stain whole cells or to stain specific cellular components. Types of simple staining: 1.Direct / Positive staining : stain object 2.Indirect / Negative staining: stain background
- 13. Direct staining (Positive staining) A simple staining technique that stains the bacterial cells in a single color. Many of the bacterial stains are basic chemicals; these basic dyes react with negatively charged cytoplasm (opposite charges attract) and the blue, crystal violet, bacterial organism and basic becomes directly stained Examples are methylene fuchsin.
- 14. Indirect staining (Negative staining) In this staining process, instead of cells background is stained. Here, an acidic dye like nigrosin or Indian ink is used. Acidic stain carries a negative charge and repelled by the bacteria, which also carry a negative charge on their surface. Hence, an acidic dye do not stain bacteria, Instead, it forms a deposit around the organism, leaving the organism itself colorless or transparent upon examination.
- 15. STAINING: GENERAL TECHNIQUE SMEAR AIR DRY HEAT FIX STAI N LOOK
- 16. Importance of fixing the smears “Fixation accomplishes three things: (1) it kills the organisms; (2) it causes the organisms to adhere to the slide; and (3) it alters the organisms so that they more readily accept stains (dyes).
- 18. GRAM’S STAINING
- 19. The Gram Stain In the late 1800’s, Christian Gram observed that some genera of bacteria retained a dye-Iodine complex when rinsed with alcohol, while other genera were easily decolorized with alcohol and could be then visualized by a contrasting counter stain. This staining procedure defines two bacterial groups: those which retain the primary dyes (“Positive by Gram’s Method” or “Gram-Positive”) and those which are easily decolorized (“Negative by Gram’s Method” or “Gram-Negative”). This was the starting point for bacterial identification procedures.
- 20. Counterstain: “A stain that adds color contrast to a microscopic specimen to make parts that were not affected by another stain more visible”. Counterstains differ in color from primary stain
- 21. The Gram Stain The difference in dye retention is dependent on such physical properties as thickness, density, porosity, and integrity of the bacterial cell wall, as well as its chemical composition. Gram-Positive bacteria have thick, dense, relatively non-porous walls, while Gram-Negative bacteria have thin walls surrounded by lipid-rich membranes. Some non-bacterial organisms with thick cell walls (e.g., some yeasts) also stain Gram-Positive. Gram-Positive bacteria which have lost wall integrity through aging or physical or chemical damage may stain Gram-Negative.
- 22. GRAM NEGATIVE Cytoplasm Cytoplasm GRAM POSITIVE Lipoteichoic acid Peptidoglycan-teichoic acid Cytoplasmic membrane Inner (cytoplasmic) membrane Outer Membrane Lipopolysaccharide Porin Braun lipoprotein
- 23. Gram staining – Requirements •Gram-staining is a four part procedure. •The specimen is mounted and heat fixed on a slide before proceeding to stain it. •The reagents required are: Crystal Violet (the Primary Stain) Iodine Solution (the Mordant) Decolorizer (ethanol) Safranin (the Counter stain) Water (preferably in a squirt bottle)
- 24. Gram staining – Procedure 1 The bacteria are first stained with the basic dye crystal violet (primary stain). Both gram-positive and gram-negative bacteria become directly stained and appear purple after this step. 2 The bacteria are then treated with gram's iodine solution (mordant). This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex, called as ‘iodine lake’. Both gram-positive and gram-negative bacteria remain purple after this step. 3 Gram's decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex while gram-negative are decolorized. 4 Finally, the counter stain (secondary stain) safranin (also a basic dye) is applied. Since the gram-positive bacteria are already stained purple, they are not affected by the counter stain. Gram-negative bacteria, that are now colorless, become directly stained by the safranin. Thus, gram- positive appear purple, and gram-negative appear pink.
- 29. Structure of an Acid-Fast Cell Wall
- 30. “Acid fastness is a physiological property of certain bacteria and cells that allows them to resist decolorization by acids during laboratory staining procedures”. Acid-fast bacteria include: Mycobacterium tuberculosis, Macobacterium avium, Mycobacterium leprae and Nocardia species.
- 31. Oncestained the acid fast bacterial cells resist decolorization with acidified organic solvents, e.g acid alcohol and are therefore called ACID FAST. Acid fast staining property of the genus, Mycobacteria, depends upon their lipid-rich (mycolic acid) cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol. The exact method by which the stain is retained is unclear but it is thought that some of the stain becomes trapped within the cell and some forms a complex with the mycolic acids. Acid fast staining - theory
- 32. The procedure for acid-fast staining is as follows: Prepare the sample: Collect a sample from the body, such as urine, stool, sputum, bone marrow, or tissue. Smear the sample: Place a small amount of the sample on a glass slide and smear it in water. Air dry and heat fix: Allow the slide to air dry, then heat fix it by passing it through a flame 3-4 times. Stain: Cover the slide with a paper towel and steam it over a beaker of water for 5-7 minutes. The primary stain is carbol fuchsin (also known as Castellani’s paint, gives RED/PINK color to acid-fast bacteria), which gets embedded into the cell wall. Decolorize: Flood the slide with acid alcohol for 30 seconds or 6 drops. Counterstain: Flood the slide with methylene blue for 30 seconds to 2 minutes. Dry and view: Dry the slide between the pages of a book of bibulous paper. View the organisms under a microscope using the oil immersion objective.
- 33. “In acid-fast staining, acid-fast bacteria stain pink and non-acid-fast bacteria stain blue”.
- 34. Acid-Fast Stain of Mycobacterium tuberculosis in Sputum Note the reddish acid-fast bacilli (AFB) among the blue normal flora and white blood cells in the sputum that are not acid-fast.
- 35. SPECIAL STAINING
- 37. “Flagella staining is a technique that uses chemicals to thicken the coat around bacterial flagella, making them visible under a light microscope. This process is used to identify and mark the morphological features of bacteria”. •Purpose •Flagella staining is used to identify flagella on bacterial cells and differentiate between different types of flagellation. •How it works •The stain coats the flagella with heavy metals or other compounds. The primary stain is often crystal violet in an alcoholic solution. The alcoholic solution evaporates, leaving a precipitate around the flagella that increases their apparent size. •Mordants •A mordant, such as tannic acid, is often used to coat the flagella with stain.
- 38. •Methods •There are many methods for flagella staining, including the Leifson staining method, Ryu method, Fontana method, and Cesares-Gill method. Leifson staining method: •The Leifson staining method is a technique used to stain bacterial flagella for viewing under a microscope. It's a common technique in clinical laboratories and can be used to identify and differentiate prokaryotes.
- 39. Here are the steps for the Leifson staining method: 1.Prepare a bacterial smear on a microscope slide 2.Fix the smear by passing it over a Bunsen burner flame 3.Apply Basic Fuchsin to the smear and let it sit for 2–3 minutes 4.Rinse the slide with distilled water 5.Apply Tannic Acid solution to the smear for about 1 minute 6.Rinse the slide again with distilled water 7.Counterstain with Methylene Blue for 1 minute 8.Rinse and blot the slide 9.Let the slide air dry 10.Examine the slide under a microscope using an oil immersion objective The Leifson method works by using tannic acid and a dye to form a colloidal precipitate that coats the flagellum. This increases the diameter of the flagellum, making it visible under a microscope.
- 41. CAPSULE STAINING
- 42. Capsule staining is diagnostically useful since it is a virulent factor(e.g. pneumococci). Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Capsules are demonstrated either by negative staining (Nigrosin or India ink) or by special staining, e.g. Hiss’ method, Anthony’s method
- 43. Hiss Method • The capsule is non-ionic in nature so it doesn't get stain by a acidic stain but a basic stain, such as crystal violet, stains the cell as well as the capsule. • This is followed by treatment with hypertonic solution - 20% Copper sulphate (blue color) solution, which serves dual role of both the decolorizing agent and counter stain. • Copper sulphate solution, being hypertonic, causes diffusion of stain towards outer surface of cell. • After drying of slide, the stain which is not passed from the capsular layer during diffusion retains in the capsular layer, Copper sulphate then decolorizes the capsule. • Capsule appears as faint blue sorrounding a purple cell.
- 47. Endospore staining Spores are normally impervious (impermeable) to stains. Under the light microscope endospores have a high light refractivity that indicates high protein content. Endospores can be stained by: Modified Zeihl-Nelson's method using 0.25-0.5% sulphuric acid as decolorizing agent, Barthelomew-Mittwar’s method Schaeffer-Fulton stain technique.
- 48. Schaeffer-Fulton method • Malachite green is used to stain the endospores (primary stain) • The malachite green is forced to permeate the spore wall by heating (mordant). • Washing with water remove stain from vegetative cells, but not from spore wall. • The endospores thus retain the primary dye while the vegetative cells lose the primary stain and take the red color of secondary stain (safranin).
- 49. Note green endospores within pink bacilli Endospore stain of Bacillus megaterium