Bacteriology&immunology lab2


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culture identification through culture characteristics and grams staining reactions

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Bacteriology&immunology lab2

  1. 1. Title: culture identification through culture characteristics, appearance, grams staining reactions and catalase tests of cultures K & J. Aim: To identify the bacteria in culture K & J through culture appearance, staining characteristics and microscopic examination by the gram staining method. Introduction A fundamental method of studying bacteria is by culturing them in liquid media or on the surface of media that have been solidified by agar. Media contain nutrients, varying from simple sugars to complex substances such as meat broth. To purify or isolate a single bacterial species from a mixture of different bacteria, solidified media generally are used. Individual cells dividing on the surface of solidified media do not move away from each other as they do in liquid, and after many rounds of replication they form visible colonies composed of tens of millions of cells all derived by binary fission from a single cell. If a portion of a colony is then transferred to a liquid medium, it will grow as a pure culture free of all other bacteria except the single species that was found in the colony. Many different species of bacteria so closely resemble one another in appearance that they cannot be differentiated from one another under the microscope. Various culture techniques have been developed to aid in species identification. Some media contain substances to inhibit the growth of many bacteria, but not the species of interest. Others contain sugars that some but not all bacteria can utilize for growth. Some media contain pH indicators that change color to indicate that a constituent of the media has been fermented, yielding acid end products. Gas production as an end product of fermentation can be detected by inoculating bacteria in solidified media in tubes rather than on plates. Sufficient gas production will result in the formation in the agar of bubbles that can easily be seen. Still other media are formulated to identify bacteria that produce certain enzymes that can break down constituents in the media; for example, blood agar plates, which can detect whether bacteria produce an enzyme to lyse, that is, dissolve, red blood cells. The various culture media and culture techniques are essential to the hospital laboratory, whose job it is to identify the cause of various infectious diseases.Sterilization is maintained during this experiment: Drying or freezing kills many species of bacteria and causes others to become inactive. Heat or moist heat above a certain temperature kills all bacteria. Sterilization of many different objects, such as spacecraft and surgical instruments, are important facets of bacteriological work.
  2. 2. There are several number of bacteria that is gram positive and these include; STAPHYLOCOCCUS, STREPTOCOCCUS, CORYNRBACTERIUM, LISTERIA, ERYSIPELOTHRIX, CLOSTRIDIUM, BACILLUS, ACTINOMYCES, NOCARDIA, MYCOBACTERIUM and DERMATOPHILUS Culture appearances refer to the physical or grossly viewed features such as colonies on the plate: size, shape, elevation, texture and colonies. Hemolysis in blood agar and lactose fermentation in MacConkey agar were also taken note of by simple gross observation. Besides being differential, MacConkey is used to detect bacteria that produces hemolysin, a substance that is capable of lysing red blood cells. The lysis appears around the colony on the agar plate and it is used to identify the characteristics. Streptococcus exhibits a kind of hemolysis called alpha hemolysis, characterized by a greenish zone around the colonies (Nester, E, L et al, 2004). Some bacteria are covered with a layer of firm gelatinous material in direct contact with the cell wall and visible by light microscopy. This material is the capsule. The capsule is normally composed of complex polysaccharides. Streptococcus pneumoniae (Pneumococcus) is an organism with prominent capsule composed of polysaccharide. Capsules may also take the form of a very thin microcapsule or a loose layer of slime. The form of capsule is influenced by the culture conditions. The bacterial genera streptococcus and staphylococcus belong to the family Enterobacteriaciae together with several other bacteria such as Escherichia, Salmonella, Brucellaceae, Brucella, Bacteroidaceae, Sphaerophorus, Micrococcaceae, Lactobacillaceae, Corynebacteriaceae and Corynebacterium. The structure of the cell wall in bacterial are more complex than those of eukaryotic cells and they contain substances unique to bacteria. Peptidoglycan, formally called mucopeptide is the most important component of the bacterial cell wall; being common to both Gram positive and Gram-negative cells. It surrounds the cell external to the cytoplasmic membrane as a single baglike molecule. It is composed of linear glycan chains of alternating residues of N-acetyl glucosamine and N-acetyl muramic acid. These are linked together by short peptide bridges to form a cross-linked insoluble polymer. Thus, peptidoglycan forms the basic structure of the bacterial cell wall. In addition to peptidoglycan, other accessory polymers are also found in most bacteria. Gram positive organisms such as staphylococci, contain teichoic acids composed of either polyglycerol phosphate or polyribitol phosphate. These occur both within and on the surface of the cell wall and may account for 20-50% of the cell wall mass. In Gram - negative cells, the peptidoglycan is much thinner than in Gram - positive organisms. External to the peptidoglycan lies a membrane (the outer membrane) which contains different proteins and a unique component, lipopolysaccharide (endotoxin). Endotoxin is important in pathogenicity of some disease and is also known as somatic O antigen. The LPS is responsible for many of the biologic activities associated with gram negative bacteria. A major protein of outer membrane is called porin which forms transmembrane pores or diffusion channels.
  3. 3. components Gram positive Peptidoglycan + (thick) Teichoic acid / Teichuronic acid + Lipopolysaccharide - Polysaccharide + Protein Present/absent Lipid - Lipoprotein - Fig1. Principal components of cell wall of gram positive and gram negative components. Gram’s stain is the most widely used differential stain. Gram-positive bacteria stain purple and gram-negative bacteria appear pink. The primary stains (crystal violet and iodine) enter the bacterial cell and, brief treatment with a decolourising agent such as acetone or alcohol removes the stain from Gram-negative bacteria but not from intact Gram-positive bacteria. The decolourised bacteria are then counter stained with a simple stain (carbol fuchsin) which allows the Gram-negative organism to be seen. The time allowed for decolourisation is critical as too little will leave Gram-negative organisms apparently positive and too much will over decolourise Gram-positive organisms so that they appear to be negative. The uses of the Gram’s stain reflects basic differences in cell-wall structure the reaction of bacteria to it is widely used in classification and indicates how an individual species may react to antibiotics such as penicillin. Gram positive bacteria are more susceptible than Gram-negative bacteria to the antibacterial actions of penicillin, acids, iodine, basic dyes, detergents and Lysozyme and less susceptible to alkalines, azide, tellurite proteolytic enzymes and lysis by antibody and complement. The mechanism of the Gram stain is not fully understood. One factor of importance is that the protoplasm of the Gram-positive bacteria is more acidic. Another is that the cell wall of Gram positive bacterium is less permeable Microscopic examination; the microscope is one of the most important tools used in studying bacteria. Dyeing or staining bacterial specimens or cultures was introduced in 1871 by the German pathologist Karl Weigert and has greatly helped the bacteriologist in identifying and observing bacteria under the microscope. A bacterial specimen is first placed on a glass slide. After the specimen has dried, it is stained to render the organism easier to observe. Stains also stimulate reactions in certain bacteria. For example, the tuberculosis bacillus can be recognized only on the basis of its reaction to certain stains. Bacteriologists have been greatly aided by the electron microscope, which has far greater magnification powers than ordinary microscopes. The gram stain is a differential stain method that distinguishes between gram positive and gram negative bacteria. Differences in the cell wall composition of gram +/- bacteria accounts for the characteristic stain. (Encarta, Microsoft student, 2009)
  4. 4. Material             Prepared cultures of K & J on blood agar and MacConkey Microscopic glass slide Light Microscope Loop Normal saline Bunsen burner Staining rack Oil crystal violet dye 70% alcohol Safranin dye Lugols iodine solution Procedure Slide preparation & fixing 1. 2. 3. 4. 5. 6. The slide was cleaned in order to remove all traces of grease A loop full of normal saline or distilled water sterile was transferred onto the glass slide Using a sterile loop, the surface of an independent colony was touched gently The loop full was emulsified/mixed with a drop of normal saline and it was spread The slide was then left to dry on the bench at room temperature The slide was then fixed over a Bunsen burner flame by passing it over the flame 3 times –noting that the surface with bacteria should be on top so as to kill the bacterial thereby making it less viable and also to make bacteria to adhere to the slide. 7. The slide was left to cool and the staining procedure followed. Staining: Gram Stain procedure 1. The slide was flooded with crystal violet solution for 1 minute 2. The stain was washed off under running tap water 3. The slide was Counter stained with lugols iodine; a mordant that does not take part in the actual staining but only enhances the performance of crystal violet 4. The slide was Washed off again under running tap water 5. The slide was decolorized using 70% alcohol /acetone in brief 6. The slide was washed off under running tap water 7. The slide was counter stained with dilute safranin/ carbofushin 8. The slide was washed off under running tap water 9. The slide was then left to dry 10. An oil drop was added on the slide to enhance visibility under the light microscope 11. The slide was examined under a light microscope at x100 oil power
  5. 5. Results Fig2: showing results of different physical/microscopic bacterial parameters Parameter Shape Size Hemolysis color Picture Blood agar K Entire 0.5-1.0mm positive Greyish J Entire 2mm Positive Greyish MacConkey K/J No growth in both media No growth Discussion In this laboratory practical several methods of culture identification were employed and it was observed that some evidence streptococcus and staphylococcus bacteria were noted. On blood agar plate the observations that were made were the same for culture K & J: entire shaped, positive hemolysis and greyish in color. The exceptions were in terms ofsize: where k was 0.51.0mm and J was 2mm. Another difference is in the arrangement of the cocci: k was long chained, while J was clustered like grapes. Most bacteria come in one of three shapes: rod, sphere, or spiral. Rod-shaped bacteria are called bacilli. Spherical bacteria are called cocci, and spiral or corkscrew-shaped bacteria are called spirilla. Some bacteria come in more complex shapes. A hairlike form of spiral bacteria is called spirochete. Streptococci and staphylococci are well-known disease-causing bacteria among the cocci. Streptococcus, genus of spherical, gram-positive, aerobic bacteria. The streptococci occur in pairs or chains, and some species are pathogenic in humans. Streptococcal infections include strep throat, scarlet fever, erysipelas, puerperal fever, and some pneumonias. The drugs of choice for treating such infections are penicillin and erythromycin. Cultures of nonpathogenic lactic streptococci are used in the fermentation of dairy products such as cheese and buttermilk (Microsoft ® Encarta ® 2009). Staphylococcus, genus of round, parasitic bacteria, commonly found in air and water and on the skin and upper part of the human pharynx. These bacteria are known to cause pneumonia and septicemia as well as boils and kidney and wound infections.
  6. 6. The antibiotic drug penicillin was once effective for the treatment and control of staphylococci, but the increase of resistant strains has required use of other antibiotic agents such as semisynthetic penicillins, cephalosporins, or vancomycin. Studies announced in 2006 showed that the antibiotic daptomycin is effective against staphylococcal infections of the bloodstream, heart, and skin. No vaccine against Staphylococcus is currently available. Two common species of Staphylococcus include Staphylococcus aureus, which is commonly responsible for skin infections, and Staphylococcus epidermis, which does not normally cause infection. However, either of these bacteria can cause serious infections under the right conditions. Staphylococcus aureus is found on the skin and in the nostrils of many healthy individuals. These bacteria often give rise to minor superficial diseases, including the formation of pustules or boils in hair follicles. Staphylococcus aureus infections are characterized by the presence of pus and formation of abscesses. In addition to skin pustules, boils, and carbuncles, Staphylococcus aureus is responsible for impetigo, infections of wounds and burns (particularly in a hospital environment), breast abscesses, whitlow (inflammation of a finger or toe near the nail), osteomyelitis, bronchopneumonia, septicemia, bacteremia, acute endocarditis, food poisoning, and scalded skin syndrome. Scalded skin syndrome occurs in newborns and is due to infection by toxigenic strains of Staphylococcus aureus. The toxins cause the skin to exfoliate, which leaves an appearance of having been scalded. In some cases Staphylococcus aureus can also cause necrotizing fasciitis, commonly called flesh-eating bacteria. This severe infection can be life threatening and is more. Conclusion Based on the cultures K and J appearance, staining characteristics of gram stain and the catalase biochemical test. Culture K and J from blood agar exhibited (alpha) hemolysis-incomplete hemolysis. Gram positive bacteria were identifiedin J & K asStaphylococcus & streptococcus respectively. And in MacConkey agar both cultures K and J showed no growth. References Hawkey M.P and Lewis A.D, 1989, medical bacteriology and practical approach, IRC Press at oxford university press, England, page8. Nester W.E et al, 2004, microbiology: A human perspective, 4th edition, McGraw Hill, New York, pages 83, 84, and 94. Microsoft ® Encarta ® 2009. © 1993-2008 Microsoft Corporation. All rights reserved.