Bacterial Staining

Certain stains can also be used to identify internal structures of the cell, which would otherwise be unseen. Further, in order to use the 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 bacteria do not appear 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 media: 0 Make a smear from the broth. You don't have to add water as the bacteria are 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 smear 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 method 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 Gram's 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 whi ch 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 – Methyl Blue – 2 to 5 minutes – Crystal violet – 30 to 45 seconds 3. Carefully wash 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 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 positively bacteria. Charged. Thes e positive dyes 0 Shape: interact with the slightly 0 Spherical – coca negatively charged bacterial 0 Rod – bacilli cell wall thus lending the color 0 Arrangement : of the dye to the cell wall. 0 Coca in clusters – staphylococci 0 Coca in chains – thyrotrophic 2. A.Gram's Stains most common technique the gram stain is valid only when performed on young (less than 24 hours old) cultures of bacteria Procedure:l b. Gram staining: Steps Purpose 1. Use a clothespin or slide rack to hold the slide. 3 2. Cover 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 directly Primary stain – all bacteria are stained purple. Onto the smear 4. Without drying, cover the smear with Gram's iodine for 30 seconds. 5.Without washing, decolonize with 95% ethyl alcohol. 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. 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 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): 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 Gram's 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 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 stops streaming from the smear. 8. Rinse with distilled water