STAINING Staining- It is defined to ewhanie microscopic level. the specimen as and contrast a technique nohich is werd a biological specimen at Stains and dyes are used to visualize at microscopic level to study at higher magnification for histopathological studies El diagnostic purposes * on basis of chemical Behavior, dyes are classified as Acidic, Basic and neutral. Different types of staining Methods. Simple Staining:- simple stains all alro called direct stains El are singly used to study the shape, size etc of microorganism. Simple stain may be acidic (or) basic. * Basic Strainas stains (Eg-methylene blue, aystal videt, carbol fuchsin, cosin etc) stain microbial cell components that are acidic in nature and are called direct positive strains. *Acidic stains (eg-sodium eosinate, nigrosine, India prode ink etc.) fail to stain acidic components of miciasial cell but deposit around colourless. the boundary of cell leaving the cell + This indicates the shape El size of cell as only surroundy is stained. * Thise simple staine are called Indirect negative stains. In simple staining, the microbial cells are stained by application of a single staining stain which may be either positive Stain or negative stain. 1. Positive (direct) staining:- In positive staining, the stain (ieg-methylene bue, Crystal violet, Carbol fuchsin, eosin etc) is basic having positive charge and attaches to that is negatively charged. surface of object Dack Microbial cell (Microbial ull- the stain char background. Negative (indirect) staining!- In negative (indirect) staining the stain leg-sodium cosinate, nigrosine, India ink, etel is acidic having negative charge and is repelled, thus filling the spaces în direct between (le stains background resulting in Staining of object. * Negative Staining 2 reasons- is said to be advantageous due to (i) Microbial alle appear less shrinelled or distorted as no heat fixing is done (ii) Capsulated bacteria which haldly stain can be obsened by this method. Miaobial cell Clear microbial cell clear background. Procedure for simple Staining 1. Take a chan glass oft slide undevide dey and mark of slide with mackel pen. an area on & Flame the inoculating 100p at portion of flame holding in 60° angle on the apple right hand. 3. Pick up bacterial culture with left hand. A Remove the pligllid war the flame, and flame the mouth of tube I plate and replace the lid I plug- 5. Insat the sterilized loop into culture medium and remove coopful of culture. 6- Dispense on clean slide. 4. Flame the inoculating 100p. Prepare bacterial smear by spreading culture uniformly with Inoculating loop. 9. Add que deops of stain lany one of stain) for 2-3mins. 10. wash the smear gently with running tap watu to remone the excess stain. 11- Blot dry the slide wring blotting paper. 12. Observe under the microscope. Differential staining wwhen more than one staining reagents are used and specific objects exhibit different staining reactions readily distinguishable, Differential staining. the procedure is called Gram Staining The was gram stain, a differential staining technique developed by Dr. Hans Christian Gram. * It is most important and commonly Microbiology Laboratory. *It is used used technique in to distinguish between gram positive f gram negative bacteria, which have distinct differences in their cell walls. *In this, bacterial cells are heat fixed and stained with basic dye, crystal violet which is taken up by bacteria. * The Slides are then treated with Iodine (Mordant) to fix the stain, washed alcohol. and destained with 95%. Finally counter * Gram positive bacteria Stained with Safranin. retain the initial crystal violet Stain, nohile gram-negative bacteria by organic solvent and are decolorized hence shows the pink counterstain. * The staining property is related to physical and chemical differences in their cell walls. PROCEDURE - ← Thin film of smear heat-fixed on clean slide 1. Crystal violet staining for minute 2. watu linse for 1-2 minutes. 1. Daline solution treatment for 30sec-Iminute 2. watu rinse ← Bacteria appear deep purple in color. 1. 95%. ethyl alcohol treatment for 30sec, wash off until no color flows from smear Water rinse. ← Gram negative bacteria decdourize. Gram positine bacteria retain deep purple color. 1. Counter Stain with Safranin for 1-2 minutes 2. watu vinee. ← Gram negativne appear pink (red) ← gram positive bacteria retais deep purple colour Endospore staining:- Certain bacteria under unfavourable conditions produce resistant and dehydrated Structures called Endospores. Generally species of Bacillus (aerobic) and (anaerobic) (lostridiun form spores. They cell- They are position may be They may to and a cell Ther are radiation, be are formed Spherical or taminal insi de parental bacterial oval in shape and in subterminal bulging giving or they may are known be extremely a central. Swollen appearance non bulging as well. resistant forms of bacteria for their resistance to desiccation, nutrition depletion 40 presence of a exosporium. Endospores dipicotinic acid. temperature, chemical disinfecting agente, and others. This resistance is due tough spore cont termed as are rich in Calcium and Endospores However they are make the Spore are resistant to chemical stains. subjected to heat treatment to coat permeable to stains. It is a differential staining technique and employs 2 Stains Malachite green and Saffranin. lupon heat treatment endospore takes up Malachite green and vegetative cells take up counter stain Saffranin. However the spore once stained, they resist decolourizing treatments under normal temperature conditions. PROCEDURE:- Take a grease fue slide and prepare uniform smear 2. Air dry and heat fix the smear. 3 Place the slide on staining rack above boiling water bat 4' cover the smear with tesne paper and flood the slide with malachite green stain. 5. Continue heating for 5 minutes felter paper to dry. keep through out heating. the and donot allow the paper saturated 6. Renrove the Water. paper and wash the slide gently with 7. Counter stain it with Saffranin for Iminute. 8. wash, blot and air dey the slide. 9- Obsesve unda oil immersion lens for size and position of endospore. CAPSULE STAINING Some bacteria produces capsule. It is a mucilaginous covering gelatinous extermal to cell wall. It is or made up of polysaccharides, glycoproteins or a polypeptide. They play an pathogenicity of a important role in detamining the species. They are antigenic in nature, escape phagocytosis and confers protection to cell. All bacteria donot produce capsule. In some cases capsules are only means of pathogenicity. For Eg:- Non-capsulated species & Diplococcus pneumonia Capsulated strains are avmilent while of same yecies are virulent. Capsular material is of water is Also heat of cells and as an when washed avoided in fixing may water soluble, hence usage capsule staining technique. is avoided as it leads to shrinking create clear yone around the ull artefact and will be mistaken as capsule. cells are stained with 11. crystal violet and with 20% copper sulphate, capsule gets decolarized while the colour of vegetative The cells appear deep blue in appears al light blue in colour. cell remains coloured. coleur while capsule PROCEDURE: Take a grease fere slide. 2 Prepare a Smear of bacterial culture and alloro. it today. 3. Donot heat fix the slide. 4. Add 1% crystal videt and allow it to stain for Iminute 5. keep the Slide in angular position and add 10 drops of 20% of copper sulphate gently over the culture. 6. Donot wash with water. 7 Blot dey and examine magnification. the slide under high