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  1. Yasuj University of Medical Sciences Department of Microbiology In the name of God • Types of culture media • In medical microbiology By: Dr. S. S. Khoramrooz Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran

  2. Bile Esculin Agar Intended Use • Bile Esculin Agar is used to differentiate enterococci and the Streptococcus bovis group from other streptococci. Principles of the Procedure • Enterococci and certain streptococci hydrolyze the glycoside esculin to esculetin and dextrose. • Esculetin reacts with an iron salt to form a dark brown or black complex. • Ferric citrate is incorporated into the medium as an indicator of esculin hydrolysis and resulting esculetin formation. • Oxgallis used to inhibit gram-positive bacteria other than enterococci. Dr. S. S. Khoramrooz

  3. Expected results • Any blackening of the plated medium indicates a positive result; if no blackening occurs, the test is negative. • For slants, if more than half of the slant is blackened within 24-48 hours, the test is positive; if less than half is blackened or no blackening occurs within 24-48 hours, the test is negative. Dr. S. S. Khoramrooz

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  8. DNase Test Agar • Intended Use • DNase Test Agar, DNase Test Agar with Methyl Green and DNase Test Agar with Toluidine Blue are differential media used for the detection of deoxyribonuclease activity to aid in the identification of bacteria isolated from clinical specimens. Dr. S. S. Khoramrooz

  9. Summary and Explanation • The DNase test is used to detect the degradation of deoxyribonucleic acid (DNA). • The test is useful for differentiating Serratia from Enterobacter,Staphylococcus aureusfrom coagulase-negative staphylococci, and Moraxellacatarrhalis from Neisseria species. • DNase Test Agar with Toluidine Blue contains a metachromatic dyeto eliminate the necessity of reagent addition to the agar following incubation. • Toluidineblue may be toxic to some gram-positive cocciand, therefore, should be used primarily with Enterobacteriaceae. Dr. S. S. Khoramrooz

  10. Principles of the Procedure • DNA is the substrate for DNase activity. • DNase is an extracellular enzyme that breaks the DNA down into subunits composed of nucleotides. • The depolymerization of the DNA may be detected by flooding the surface of the medium with 1 N HCl and observing for clear zones in the medium surrounding growth. Dr. S. S. Khoramrooz

  11. In the absence of DNaseactivity, the reagent reacts with the intact nucleic acid, resulting in the formation of a cloudy precipitate. • The HCl reagent is not needed to detect DNase activity on DNase Agar with Methyl Green. • Methyl green forms a complex with intact (polymerized) DNAto form the green color of the medium. Dr. S. S. Khoramrooz

  12. DNase activity depolymerizes the DNA, breaking down the methyl green-DNA complex, which results in the formation of colorless zones around colonies of the test organism. • A negative test is indicated by the absence of a colorless zone around the colonies. Dr. S. S. Khoramrooz

  13. DNase Test Agar w/Toluidine Blue • For detection of deoxyribonuclease activity in microorganisms including staphylococci Dr. S. S. Khoramrooz

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  15. The HCl reagent is not needed to detect DNase activity on DNase Agar with Toluidine Blue. • Toluidine blue forms a complex with intact (polymerized) DNA. • In the intact DNA complex, the toluidine blue has the normal blue color. • DNase activity depolymerizes the DNA, breaking down the dye-DNA complex. Dr. S. S. Khoramrooz

  16. In the presence of nucleotides produced from the DNasedepolymerization, the dye takes on its metachromatic color, forming pink to red zones around bacterial growth. • A negative test is indicated when the medium remains blue. Dr. S. S. Khoramrooz

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  19. Procedure • Inoculate by making a single streak line using inoculum from an agar slant or plate. • One plate may be inoculated with up to eight isolates by spot inoculation (1/8 to 1/4 inch) or streak inoculation (a single 1- to 2-inch line). • Incubate at 35 ± 2°C for 24-48 hours. Dr. S. S. Khoramrooz

  20. Plates should be incubated in an inverted position. Incubate tubes with loosened caps. • Following incubation, flood DNase Test Agar plates with 1N HCl reagent and observe for reaction. • Reagent addition is not required with DNase Test Agar with Methyl Green or with DNase Test Agar with Toluidine Blue. Dr. S. S. Khoramrooz

  21. Expected Results • A clear area surrounding growth (band/spot inocula) on DNase Test Agar after the addition of 1N HCl indicates a positive reaction, DNase activity. • A negative reaction is indicated by no clearing and a cloudy precipitate around colonies and throughout medium due to precipitated salts in the medium. • A positive reaction on DNase Test Agar with Methyl Green is a distinct clear zone surrounding growth in an otherwise green-colored medium. • The color of the medium remains unchanged if the test is negative. • On DNase Test Agar with Toluidine Blue, DNase activity is indicated by pink to red zones surrounding growth. • The color of the medium remains unchanged if the test is negative. Dr. S. S. Khoramrooz

  22. Mannitol Salt Agar Intended Use • Mannitol Salt Agar is used for the selective isolation and enumeration of staphylococci from clinical and nonclinical materials. Dr. S. S. Khoramrooz

  23. Principles of the Procedure • The 7.5% concentration of sodium chloride results in the partial or complete inhibition of bacterial organisms other than staphylococci. • Mannitol fermentation, as indicated by a change in the phenol red indicator, aids in the differentiation of staphylococcal species. • Agar is a solidifying agent. Dr. S. S. Khoramrooz

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  25. Procedure • Incubate plates at 35 ― 2‹C in an aerobic atmosphere for 24-48 hours, or as instructed in the standard reference. Dr. S. S. Khoramrooz

  26. Expected Results • Coagulase-positive staphylococci produce growth of yellow colonies with yellow zones. • Coagulase negative staphylococci produce small red colonies with no color change to the medium. • Micrococcusproduce large, white to orange colonies, with no color change to the medium. • Most other bacteria will be inhibited. Dr. S. S. Khoramrooz

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  28. Bismuth Sulfite Agar • Intended Use • Bismuth Sulfite Agar is a highly selective medium used for isolating Salmonella spp., particularly Salmonella Typhi, from food and clinical specimens. Dr. S. S. Khoramrooz

  29. Principles of the Procedure • Dextrose is an energy source. • Bismuth sulfite indicator and brilliant green are complementary in inhibiting gram-positive bacteria and members of the coliform group, while allowing Salmonella to grow luxuriantly. • Ferrous sulfateis included for detection of H2S production. • When H2S is present, the iron in the formula is precipitated, giving positive cultures the characteristic brown to black color with metallic sheen. Dr. S. S. Khoramrooz

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  31. For isolation of Salmonella spp. from clinical specimens, inoculate fecal specimens and rectal swabs onto a small area of one quadrant of the Bismuth Sulfite Agar plate and streak for isolation. • This will permit the development of discrete colonies. • Incubate plates at 35°C. • Examine at 24 hours and again at 48 hours for colonies resembling Salmonella spp. Dr. S. S. Khoramrooz

  32. Expected results • The typical discrete S. Typhisurface colony is black and surrounded by a black or brownish-black zone which may be several times the size of the colony. • By reflected light, preferably daylight, this zone exhibits a distinctly characteristic metallic sheen. • Plates heavily seeded with S. Typhimay not show this reaction except near the margin of the mass inoculation. Dr. S. S. Khoramrooz

  33. In these heavy growth areas, this organism frequently appears as small light green colonies. • This fact emphasizes the importance of inoculating plates so that some areas are sparsely populated with discrete S. Typhi colonies. • Other strains of Salmonella produce black to green colonies with little or no darkening of the surrounding medium. Dr. S. S. Khoramrooz

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  35. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. • DO NOT AUTOCLAVE. • Use the medium the same day it is prepared. Dr. S. S. Khoramrooz

  36. Brilliant green agar Intended Use • Brilliant Green Agar is a highly selective medium for the isolation of Salmonella other than S. Typhifrom feces and other materials. Principles of the Procedure • Brilliant green dye inhibits gram-positive bacteria and a majority of gram-negative bacilli. • Phenol red serves as a pH indicatorand yields a yellow color as a result of acid production in the fermentation of the lactose and/or sucrose in the medium. Dr. S. S. Khoramrooz

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  38. Procedure • A less selective medium and a nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen. • Incubate plates, protected from light, at 35 ± 2°C for 18-24 hours. • If negative after 24 hours, reincubate an additional 24 hours. Dr. S. S. Khoramrooz

  39. Escherichia coli on BPLS Agar. The colonies are yellow due to the low pH which is caused by the production of acid during fermentation of lactose and/or sucrose. Salmonella on BPLS Agar. The colonies are red because the bacterium does not ferment lactose or sucrose. Dr. S. S. Khoramrooz

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  41. Decarboxylase Differential Media Intended Use • Decarboxylase media are used in the biochemical differentiation of gram-negative enteric bacilli based on the production: • Argininedihydrolase • Lysine decarboxylase • Ornithinedecarboxylase • Decarboxylase Medium Base, with added arginine, lysine or ornithine is used for the same purpose. • Lysine Decarboxylase Broth is used for differentiating microorganisms based on lysine decarboxylation. Dr. S. S. Khoramrooz

  42. Summary and Explanation • Moeller introduced the decarboxylase media for detecting the production of lysine and ornithinedecarboxylaseand argininedihydrolase. • These media are a useful adjunct to other biochemical tests for the speciation and identification of the Enterobacteriaceaeand other gram-negative bacilli. • The production of OD is particularly useful for differentiating Klebsiella and Enterobacter species. • Klebsiella species are non-motile and, except for K. ornithinolytica,do not produce ornithinedecarboxylase, while most Enterobacter species are motile and,except for E. agglomerans, usually produce this enzyme. Dr. S. S. Khoramrooz

  43. Principles of the Procedure • Pyridoxal is an enzyme co-factor for the amino acid decarboxylase. • Dextrose is a fermentable carbohydrate. • Bromcresol purple and cresol red are pH indicators. • The amino acidslysine, ornithine or arginine are added to the basal medium at a concentration of 10.0 g/L to detect the production of the enzyme specific for these substrates. Dr. S. S. Khoramrooz

  44. When the medium is inoculated with a bacterium that is able to ferment dextrose, acids are produced that lower the pH of the medium and change the color of the indicator from purple to yellow. • The acidic condition also stimulates decarboxylase activity. • If the organism produces the appropriate enzyme, the amino acid in the medium is degraded, yielding a corresponding amine. Dr. S. S. Khoramrooz

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  46. Decarboxylation of lysine yields cadaverine. • while decarboxylation of ornithine yields putrescine. • Arginine is first hydrolyzed to form ornithine, which is then decarboxylated to form putrescine. • The production of these amines elevates the pH of the medium, changing the color of the indicator from yellow to purple or violet. • If the organism does not produce the appropriate enzyme, the medium remains acidic (yellow). Dr. S. S. Khoramrooz

  47. Each isolate to be tested must also be inoculated into a tube of the basal medium that does not contain the amino acid. • If this tube becomes alkaline, the test is invalid. • To obtain the appropriate reactions, the inoculated tubes must be protected from air with a layer of sterile mineral oil. • Exposure to air may cause alkalinization at the surface of the medium, which could cause a decarboxylase-negative organism to appear positive. Dr. S. S. Khoramrooz

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  49. Expected Results • Compare the color of tubes of media containing the specific amino acids with the color of control tubes of basal media (without amino acid) that have been inoculated with the same isolate. • If inoculated control tubes show an alkaline reaction, the test is invalid; i.e., • Improperly performed or the test organisms • Degrade the peptone sufficiently to produce an alkaline reaction in the absence of a specific amino acid. • The medium becomes purple to violet if the reaction is positive (alkaline). • A yellow color indicates a negative test; i.e., the organism does not produce the appropriate enzyme. Dr. S. S. Khoramrooz

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