Bacterial Staining

Certain corrupts can also be used to identify internal structures of the stall, which would otherwise be unseen. Further, in order to use the crude oil immersion objective of the microscope and thereby obtain the iratest degree of magnification, it is convenient to use stained preparations rather than wet mounts. L Although bacterium do not issue greatly different from their surroundings, they differ chemically. It is this chemical difference that enables us to distinguish bacteria by staining, the stain or dye readily reacting with the bacterial cell but not with the background. Preparation of Smear Before Staining (figure 1) 1. Prepare a clean slide. Put a proper label. 2. Heat your loop to sterilize it. 3. For solid media 0 Using a sterile inoculating loop, place a 1 or 2 loophole of distilled water 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 material. For liquid me dia 0 Make a maculation from the broth. You dont have to add water as the bacteria are already suspended in water. use of goods and services 2 or 3 loophole of the culture. 0 shell out the culture on the center of the slide. 4. Reheat the loop to clean it. 5. Let the derogate dry. air dry heat fix- passing the slide through a flame 2 or 3 times Figure 1. Preparation of smear. Classification of Stain Based on Functions 1. Simple Staining Method In this system one aniline dye is used to stain the organism to be studied. 2. Differential Staining Method Under this type of classification, the staining method employed divides the microorganism into groups.The grammes Staining Method and Acid-Fast (Zilch-Nielsen Method) fall under this category. 3. Selective or Special Staining Method Under this category, parts or portion 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, deliver it to remain for the following intervals Carbon-fuchsia 15 to 30 seconds Methyl Blue 2 to 5 minutes Crystal violet 30 to 45 seconds 3. Carefully break the excess stain off with distilled water from a wash bottle. Let the water run down the tilted slide. 4. Gently blot the smear with a root word 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 tout ensemble bacteria in smear takes stain Simple stains use basic dyes All types of and appears in color of stain which are positively bacteria. Charged. These positive dyes 0 Shape int eract with the slightly 0 Spherical coca negatively charged bacterial 0 Rod bacilli cell debate thus bring 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 most putting surface technique the gram stain is valid only when performed on young (less than 24 hours old) cultures of bacteria Procedurel b. Gram staining Steps theatrical role 1. Use a clothespin or slide rack to hold the slide. 3 2. C everyplace the smear with crystal violet and leave for 30 seconds. 3. washables the slide carefully with distilled water from a wash bottle. O do not squirt the water directly Primary stain all bacteria are stained purple. Onto the smear 4. Without drying, cover the smear with Grams iodine for 30 seconds. 5.Without washing, decolonize with 95% ethyl intoxicant. Let the alcohol run through the smear until no large amount of purple wash out. O do not over decolonize 6. Immediately wash with distilled water. 7. Add seafaring for 30 seconds. Mordant 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. dig into 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 Aggregative (-) 0 pink wall is a thin, complex, multilayered structure containing protein, minicomputers and lipids 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 positive bacilli (figure ad) Colos tomies 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 fatty waxes (mycology acid) 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 filter 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 acid alcohol (leave 15 The waxen cell wall then prevents the second s). The acid alcohol contains stain from being removed by the acid 3% HCI and 95% ethanol or piddle 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 stops streaming from the smear. 8. Rinse with distilled water