Bacteria Stains and Staining Techniques PDF

BACTERIA STAINS AND STAINING TECHNIQUES  Introduction. Bacteria, also called germs, are microscopic organisms not visible with the naked eye. They are everywhere, inside and outside of your body. They can live in different environments, from hot water to ice. Some bacteria...

BACTERIA STAINS AND STAINING TECHNIQUES  Introduction. Bacteria, also called germs, are microscopic organisms not visible with the naked eye. They are everywhere, inside and outside of your body. They can live in different environments, from hot water to ice. Some bacteria are good for you(normal flora), while others can make you sick(Pathogenic bacteria). Bacteria are single-celled, powerful and complex, have a tough protective coating that boosts their resistance to white blood cells in the body, thus they can survive in extreme conditions.  Continue Some bacteria have a tail, called a flagellum. The flagellum helps a bacterium to move around. Other bacteria have sticky hair-like appendages (pili or fimbriae) that help bacteria stick to one another, hard surfaces, and human body cells. There are many bacteria in the human body, especially in the stomach and mouth. Bacteria are found on surfaces and in substances such as water, soil, and food.  Continue Microscopical examination is the first step in examination of the specimen or identification of an unknown bacterium. The morphological feature of an importance are size, shape, and arrangements of cells with respect to each other. possession or non possession of structure like capsules, spores, flagella and inclusion granules. Microscopical bacteria examination may be either in unstained or stained preparation of bacteria.  Stained preparation Bacteria structural details are clearly seen following staining which produces contrast. Bacteria may be stained in living state, however, many dyes are toxic thus kills the cells on staining. Routine staining procedure involve drying and fixation of smear that kills bacteria. Advantage of staining the bacteria is to produce contrast and the killed bacteria due to staining minimizing contamination. And one disadvantage is that it alters the morphology parts of bacteria, like flagella, capsules which may lost. Many bacteria can be stained easily but some are difficulty to stain like: Spirochaetes, rickettsiae and chlamydiae.  Types of the stain Acidic stain: Negatively charged acid radicals combine with positively charged particles in nucleus and gives color. Example: eosin, acid fuchsine, malachite green, nigrosin, Indian ink. Basic stain: Positive charged basic radicals combine with negatively charged particles in cytoplasm and gives color. Example: Haematoxyllin, methylene blue, crystal violet, gentian violet. Neutral stain: Both positively and negatively charged imparts different colors to different components. Example: Giemsa stain, Leishman’s stain Wright’s stain  Continue Staining techniques in common use in microbiology includes: 1. Simple staining: Kind of stain that impart same color to all bacteria, affected by application of a single basic dye such as methylene blue, methyl violet and basic fuchsine. 2. Negative or background staining: This stains the background or the environment around the bacterial cell, remaining the bacteria unstained. Example of this stain are; Indian ink, nigrosin. Capsule, spirochetes (very slender) is demonstrated better by negative stain not by simple stain.  Continue 3.Impregnation methods: This deals with very thin cells and structures which can not be seen by ordinary microscope, are rendered visible by impregnation with silver on the surface. The method is useful for spirochaetes, and bacteria flagella. 4. Differential staining: This stain imparts different color to different bacteria structures. Example of the stains are: Gram stain and Ziehl-Neelsen stain.  GRAM STAIN Material and reagents: a. Primary stain or basic stain example Crystal violet, methyl violet or gentian violet. b. Lugol’s iodine(Mordant), c. 10% acetone alcohol, or ethanol or aniline(decolourizer), d. Neutral red or safranine or carbol fuschin(counter stain).  Principle of Gram stain Those organisms are called gram positive as having large amount of peptidoglycan and teichoic acids that retain the basic dye (crystal violet CV-1) even after decolourization with acetone alcohol which dehydrates the cell and cause a closure of the cell wall pore thus retaining the crystal violet(CV-1) and makes a cell appear purple(gram positive). And those organisms are termed gram negative as they have thin multilayered complex with high content of lipids, when decolourized with  Continue 10% acetone alcohol(a lipid solvent), makes an outer lipopolysacharides membrane of a cell wall to dissolve and damages the cytoplasmic membrine to which peptidoglycan is attached, and leave the cells with large pores which does not close thus allow the CV-1 to drain out of the cell in the process of decolourisation and washing, hence when a counter stain is added, they pink up the color of the stain and appear pink(gram negative).  Gram stain procedure Requirements: See provided materials on the bench. Note them in your note book. Prepare thin smear of bacteria on the slide. Follow the aseptic procedure. 1. Sterilize the loop to red hot, leave to cool, using the loop transfer small drop of saline on to a clean grease free glass slide. 2. Sterilize the loop, cool, touch the bacteria colone(pure colone) and transfer to the drop of saline on the slide, make thin smear. Leave to air dry. 3. Fix the smear by passing quickly through Bunsen flame five times. 4. Put slide on staining rack, smear upward and cover with crystal violet, leave for 1 minute. 5. Wash with clean tap water and tip off all water then cover the smear with lugol’s iodine, leave for 1 minute, and wash off  Continue 6. Decolorize rapidly (Few seconds..10-15 seconds) with 10% acetone alcohol and wash immediately. (DO NOT USE ACETONE CLOSE TO AN OPEN FLAME). 7. Cover the smear with neutral red or safranine for 2 minutes. 8. Wash with clean water., wipe the back of the slide and place in a drying rack for a smear to air dry.  Continue 9. Examine smear microscopically, first with 40x objective to check staining and cell distribution, then with the 100x objective to check the bacteria and cells. Results: Gram positive bacteria: Dark purple Yeast cell: Dark purple Gram negative bacteria: Pale to dark red. Nuclei of pus cell: Red. Epithelial cells: Pale red.  Reporting of the gram smear. Include the following information into your report: Number of bacteria present: Many, moderate, few or scanty. The gram reaction of the bacteria: whether gram positive or gram negative. The morphology of the bacteria: whether cocci, diplococci, Streptococci, or rods, Coccobacilli, also whether bacteria are intracellular. The presence and number of the pus cells Presence of yeast cells and epithelial cells. Example: Moderate number of gram negative intracellular diplococi and many pus cells.  Exercise. With the provided material and reagent on the bench, perform a gram stain of the bacteria colone in culture plate labeled ‘A’. Report your microscopic finding.  Ziehl-Neelsen stain Also called acid fast stain, developed by Franz Ziehl and Friederich group KA Neelsen and was first described by Ehrlich in 1882. This stain detect groups of bacteria described ‘acid fast’, are organisms which do not stained by ordinary stain but need exposure to the strong stain like strong carbol –fuchsin with application of heat, and resists decolorization with mineral acid like H2SO4. The property of acid fastness is due to the large amounts of lipids and fatty acid particularly mycolic acid wax which these organisms contain. There are three types of acid fast bacteria: 1. Mycobacteria group(M. tuberculosis, M.leprphytic, Saprophytic acid fast bacilli. 2.Bacteri spore 3. Nocardia.  Principle Acid fastness of acid fast bacilli is attributed to the presence of large quantities of unsaponifiable wax fraction called mycolic acid in their cell wall and also the intactness of the cell wall. The degree of acid fastness varies in different bacteria. Application of heat helps the dye to penetrate the tubercle bacillus. Once stained, the stain cannot be easily removed. The tubercle bacilli resist the decolorization action of acid alcohol which confers acid fastness.  Staining technique-AFB Smear is prepared under protection of the safety cabinet(HOOD) as this is an air born disease, Dry and heat fixing. Cover the smear with undiluted strong carbo fuchsin, heat till steam rises but don’t boil. Leave to stain for 5 minute, heat at interval making stain hot, and smear be covered with carbo fuchsin all the time. Decolorize with 3% acid in alcohol till no red stain comes out. Wash well with running water. Counter stain with dilute carbo fuchsin for 10-15 second or neutral red for 2 minute Wash with water, allow to dry or blot but not rub. Result: Acid fast:……………Red Tissue cells and other organisms:……………….Green or Blue. NOTE: If tissue cell be red, preparation was not properly decolorized. CDC guidelines on how to report smear Smear results Smear Infectiousness of (Number of AFB interpretation patient observed at 100x magnification) 4 +(>9/field) Strong positive Probably very infectious 3 +(1-9? Field) Strong positive Probably very infectious 2 +(1-9 /10 field) Moderate positive Probably infectious 1 +(1-9/100 field) Moderate positive Probably infectious +/- (1-2/300 field) Weakly positive Probably infectious No acid fast bacilli Negative Probably not infectious  Spore staining (endospore) Spore are highly resistant and metabolically inactive forms. The morphology of bacteria endospore is best observed in unstained wet films under the phase contrast microscope, where they appear as large, refractile, oval or spherical bodies within the bacterial mother cells or else free from the bacteria. Different staining technique are available for staining of spores. A modified ZN stain in which weak, 0.25% sulphuric acid is used as decolorizer, yield red spore in blue-stained bacteria. Lipid granules also stain red, appearing like small spherical spores.