Bacterial Staining

Certain stigmas can likewise be used to identify internal structures of the cell, which would early(a)wise be unseen. Further, in order to use the oil immersion objective of the microscope and thereby control the iratest degree of magnification, it is convenient to use posted preparations rather than wet mounts. L Although bacteria do non appear greatly disaccordent from their surroundings, they differ chemically. It is this chemical difference that enables us to distinguish bacteria by patch, the befoul or dye readily reacting with the bacterial cell but not with the background. Preparation of Smear Before detection (figure 1) 1. Prep ar a scant(p) soaring. Put a proper label. 2. Heat your twine to sterilize it. 3. For real media 0 Using a sterile inoculating loop, place a 1 or 2 loophole of distilled piddle on the enter of the slide. 0 Scrape a small amount of the culture off the slant. 0 Smear on the center of the slide, with the distilled water, the scraped materi al. For liquid media 0 Make a discoloration from the broth. You dont have to add water as the bacteria atomic number 18 already suspended in water. Use 2 or 3 loophole of the culture. 0 Spread the culture on the center of the slide. 4. Reheat the loop to clean it. 5. let the pip dry. air dry heat fix- freeing the slide through a flame 2 or 3 times Figure 1. Preparation of smear. Classification of Stain Based on Functions 1. Simple imperfectioning Method In this method one phenylamine dye is used to smut the organism to be studied. 2. variantial speckle Method Under this type of classification, the staining method employed divides the micro-organism into groups.The Grams Staining Method and Acid-Fast (Zilch-Nielsen Method) fall downstairs this category. 3. Selective or Special Staining Method Under this category, parts or component part of the cell are stained differently from the rest of the cell. 4. Indirect Staining Method Indirect stains are also relief stains because it is the background which takes up the taint, not the organism and the organism are only seem by contrast. 2 Examples of Staining Simple Staining Methods Use only 1 stain. Use to determine cell morphology, size and arrangement.Procedure a. Make a smear. B. Staining 1. Place the slide on a staining rack. 2. Flood the smear with several drops of the dye, allow it to remain for the following intervals Carbon-fuchsia 15 to 30 seconds m ethyl radical Blue 2 to 5 minutes Crystal majestic 30 to 45 seconds 3. Carefully wash the excess stain off with distilled water from a wash bottle. Let the water run down the lean slide. 4. Gently defacement the smear with a paper towel or absorbent paper and let it dry. C. View the prepared slide under the microscope. Results and reaction Reaction / Results Principle Samples of Bacteria All bacteria in smear takes stain Simple stains use basic dyes All types of and appears in color of stain which are compulsively bacteria. Charged. The se positive dyes 0 take form interact with the slightly 0 Spherical coca plant prohibitly aerated bacterial 0 Rod bacilli cell surround thus lend the color 0 Arrangement of the dye to the cell wall. 0 coca in clusters staphylococci 0 Coca in chains thyrotrophic 2. A.Grams Stains just about common technique the gram stain is valid only when performed on young (less than 24 hours old) cultures of bacteria Procedurel b. Gram staining Steps Purpose 1. Use a clothespin or slide rack to hold the slide. 3 2. C over the smear with crystal violet and leave for 30 seconds. 3. Wash the slide carefully with distilled water from a wash bottle. O do not squirt the water right off Primary stain all bacteria are stained royal. Onto the smear 4. Without drying, cover the smear with Grams iodine for 30 seconds. 5.Without washing, decolonize with 95% ethyl inebriant. Let the alcohol run through the smear until no humongous amount of purple wash out. O do not over decolonize 6. Immedia tely wash with distilled water. 7. Add seafaring for 30 seconds. inglorious this intensifies the ionic bond between the primary stain and the Primary stain is washed out of some bacteria, while others are unaffected. Secondary stain or countersink stains the decolonize bacteria red. 8. Wash with distilled water and blot the slide with a paper towel or absorbent paper. Let dry. C. Examine under the microscope. 9.Results Reactions / Result Gram cell wall are thick and chemically costive (+) simple, composed mainly of 0 purple protein and cross-linked colored minicomputers alcohol causes dehydration and shrinkage of the gram+ cell wall reducing the loss of substances such as crystal violet aggregated (-) 0 pink wall is a thin, complex, multilayered structure containing protein, minicomputers and lipides when treated with alcohol, the lipid dissolves and the primary stain is wash out Samples of Bacteria Gram positive coca in clusters (figure AAA) Staphylococci species Gram posit ive bacilli (figure ad) Colostomies species Crematoriums Bacillus anthracicGram negative coca in chains Streptococci Gram negative coca (figure 4 Engineers species Gram negative bacilli (figure c) Escherichia coli Kielbasa pneumonia b d Figure 2. Different observations in Grams Staining. (a)gram+ coca in clusters (b)gram + coca in chains (c)gram- bacilli (d)gram+ bacilli (e)gram- coca. (f)gram stain mixed 5 B. Acid Fast Stain (Zilch Nielsen Stain)l ,5 0 to stain Mycobacterium species especially M. Tuberculosis Contain large amount of productive waxes (mycology harsh) within their cell wall resists staining by ordinary methods 0 Procedure 1. Flood smear with Carbon Fuchsia Carbon Fuchsia is a lipid soluble, stain. Heinlein compound, which is able to penetrate the cell wall. 2. Cover flooded smear with reach paper 3. Steam for 10 minutes. Add more Carbon Fuchsia stain as needed. 4. Cool slide. 5. Rinse with distilled water. 6. Flood slide with venereal disease alcohol (leav e 15 The waxy cell wall then prevents the seconds). The acid alcohol contains stain from being removed by the acid 3% HCI and 95% ethanol or H2O alcohol (decolonize) once it has SIS. Penetrated the cell wall. The acid alcohol decolonize will remove the stain from all other cells. . Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop by drop) until the red color clams streaming from the smear. 8. Rinse with distilled water