Presentation is loading. Please wait.

Presentation is loading. Please wait.

In the name of God By: Dr. S. S. Khoramrooz Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran Yasuj University.

Similar presentations


Presentation on theme: "In the name of God By: Dr. S. S. Khoramrooz Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran Yasuj University."— Presentation transcript:

1 In the name of God By: Dr. S. S. Khoramrooz Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran Yasuj University of Medical Sciences Department of Microbiology

2 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.  Oxgall is used to inhibit gram-positive bacteria other than enterococci. 2 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 hours, the test is positive; if less than half is blackened or no blackening occurs within hours, the test is negative. 3 Dr. S. S. Khoramrooz

4 4

5 5

6 6

7 7

8  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. 8 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 aureus from coagulase-negative staphylococci, and Moraxella catarrhalis from Neisseria species.  DNase Test Agar with Toluidine Blue contains a metachromatic dye to eliminate the necessity of reagent addition to the agar following incubation.  Toluidine blue may be toxic to some gram-positive cocci and, therefore, should be used primarily with Enterobacteriaceae. 9 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. 10 Dr. S. S. Khoramrooz

11  In the absence of DNase activity, 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) DNA to form the green color of the medium. 11 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. 12 Dr. S. S. Khoramrooz

13  For detection of deoxyribonuclease activity in microorganisms including staphylococci 13 Dr. S. S. Khoramrooz

14 14 Dr. S. S. Khoramrooz

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. 15 Dr. S. S. Khoramrooz

16  In the presence of nucleotides produced from the DNase depolymerization, 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. 16 Dr. S. S. Khoramrooz

17 17 Dr. S. S. Khoramrooz

18 18 Dr. S. S. Khoramrooz

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 hours. 19 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. 20 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. 21 Dr. S. S. Khoramrooz

22 Intended Use  Mannitol Salt Agar is used for the selective isolation and enumeration of staphylococci from clinical and nonclinical materials. 22 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. 23 Dr. S. S. Khoramrooz

24 24 Dr. S. S. Khoramrooz

25 Procedure  Incubate plates at 35 ― 2‹C in an aerobic atmosphere for hours, or as instructed in the standard reference. 25 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.  Micrococcus produce large, white to orange colonies, with no color change to the medium.  Most other bacteria will be inhibited. 26 Dr. S. S. Khoramrooz

27 27

28  Intended Use  Bismuth Sulfite Agar is a highly selective medium used for isolating Salmonella spp., particularly Salmonella Typhi, from food and clinical specimens. 28 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 sulfate is 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. 29 Dr. S. S. Khoramrooz

30 30 Dr. S. S. Khoramrooz

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. 31 Dr. S. S. Khoramrooz

32  Expected results  The typical discrete S. Typhi surface 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. Typhi may not show this reaction except near the margin of the mass inoculation. 32 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. 33 Dr. S. S. Khoramrooz

34 34

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. 35 Dr. S. S. Khoramrooz

36 Intended Use  Brilliant Green Agar is a highly selective medium for the isolation of Salmonella other than S. Typhi from 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 indicator and yields a yellow color as a result of acid production in the fermentation of the lactose and/or sucrose in the medium. 36 Dr. S. S. Khoramrooz

37 37 Dr. S. S. Khoramrooz

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 hours.  If negative after 24 hours, reincubate an additional 24 hours. 38 Dr. S. S. Khoramrooz

39 39 Salmonella on BPLS Agar. The colonies are red because the bacterium does not ferment lactose or sucrose. 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.

40 40 Dr. S. S. Khoramrooz

41 Intended Use  Decarboxylase media are used in the biochemical differentiation of gram-negative enteric bacilli based on the production:  Arginine dihydrolase  Lysine decarboxylase  Ornithine decarboxylase  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. 41 Dr. S. S. Khoramrooz

42 Summary and Explanation  Moeller introduced the decarboxylase media for detecting the production of lysine and ornithine decarboxylase and arginine dihydrolase.  These media are a useful adjunct to other biochemical tests for the speciation and identification of the Enterobacteriaceae and 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 ornithine decarboxylase, while most Enterobacter species are motile and, except for E. agglomerans, usually produce this enzyme. 42 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 acids lysine, 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. 43 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. 44 Dr. S. S. Khoramrooz

45 45

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). 46 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. 47 Dr. S. S. Khoramrooz

48 48 Dr. S. S. Khoramrooz

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. 49 Dr. S. S. Khoramrooz

50 50 Dr. S. S. Khoramrooz

51 Intended Use  Eosin Methylene Blue Agar, Levine is a slightly selective and differential plating medium for the isolation of gram- negative enteric bacteria.  Principles of the Procedure  The eosin Y and methylene blue dyes in Levine EMB Agar render the medium slightly selective in that they inhibit gram- positive bacteria to a limited degree. 51 Dr. S. S. Khoramrooz

52  These dyes also play a role in differentiating between lactose fermenters and lactose nonfermenters due to the presence or absence of dye uptake in the bacterial colonies.  Coliforms, as lactose-fermenting organisms, are visualized as blue-black colonies, whereas colonies of Salmonella and Shigella, as lactose nonfermenters, appear colorless, transparent or amber.  Some gram-positive bacteria, such as fecal streptococci, staphylococci and yeasts, will grow on this medium and usually form pinpoint colonies. 52 Dr. S. S. Khoramrooz

53 53 Dr. S. S. Khoramrooz

54  Expected Results  Typical colonial morphology on Eosin Methylene Blue Agar, Levine is as follows: 54 Dr. S. S. Khoramrooz

55 55

56  Intended Use  Hektoen Enteric (HE) Agar is a moderately selective medium used in qualitative procedures for the isolation and cultivation of gram-negative enteric microorganisms, especially Shigella, from a variety of clinical and nonclinical specimens. 56 Dr. S. S. Khoramrooz

57 Principles of the Procedure  The selective nature of Hektoen Enteric Agar is due to the incorporation of bile salts in the formulation.  These substances inhibit gram-positive organisms but also can be toxic for some gram-negative strains. 57 Dr. S. S. Khoramrooz

58  This medium contains three carbohydrates, lactose, sucrose (saccharose) and salicin, for optimal differentiation of enteric pathogens  The lactose concentration is higher than in many other media used for enterics in order to aid in the visualization of enteric pathogens and minimize the problem of delayed lactose fermentation.  Ferric ammonium citrate and sodium thiosulfate in the medium enable the detection of hydrogen sulfide production.  The indicator system, consisting of acid fuchsin and bromthymol blue, has a lower toxicity than that of many other enteric media, resulting in improved recovery of enteric pathogens. 58 Dr. S. S. Khoramrooz

59 Procedure  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 hours. 59 Dr. S. S. Khoramrooz

60 60 DO NOT AUTOCLAVE.

61 Dr. S. S. Khoramrooz 61

62 Dr. S. S. Khoramrooz 62

63 Dr. S. S. Khoramrooz 63 Salmonella mixed with normal fecal flora.

64 Dr. S. S. Khoramrooz 64

65 Intended Use  CLED Agar is used for the isolation, enumeration and presumptive identification of microorganisms from urine. 65 Dr. S. S. Khoramrooz

66 Principles of the Procedure  Lactose is included to provide an energy source for organisms capable of utilizing it by a fermentative mechanism.  The cystine permits the growth of “dwarf colony” coliforms.  Bromthymol blue is used as a pH indicator to differentiate lactose fermenters from lactose nonfermenters. 66 Dr. S. S. Khoramrooz

67  Organisms that ferment lactose will lower the pH and change the color of the medium from green to yellow.  Electrolyte sources are reduced in order to restrict the swarming of Proteus species. 67 Dr. S. S. Khoramrooz

68  Bacteriuria is determined by inoculating the surface of an agar medium using 0.1 mL of a 102 dilution of the urine sample or using a calibrated loop (0.001 mL) of the undiluted sample.  Current guidelines are that for a single isolate a density of >105 CFU/mL indicates infection, <104 CFU/mL indicates urethral or vaginal contamination, and between 104 and 105 CFU/mL needs to be evaluated based on clinical information. 68 Dr. S. S. Khoramrooz

69 Procedure  Inoculate the medium as soon as possible after the specimen is received in the laboratory.  It is recommended that quantitative methods be used for culturing urine specimens.  Incubate at 35 ― 2‹C for hours. 69 Dr. S. S. Khoramrooz

70 Expected Results  Count the number of colonies on the plate or dipstick.  Multiply by an appropriate number to convert the count to CFU per mL of sample.  Contaminant bacteria usually appear in low numbers which vary in colonial morphology.  Urinary pathogens will usually yield high counts having uniform colonial morphology and should be subcultured directly to routine media for identification and susceptibility testing. 70 Dr. S. S. Khoramrooz

71  Typical colonial morphology on CLED Agar is as follows: 71 Dr. S. S. Khoramrooz

72 E. coli on CLED Dr. S. S. Khoramrooz 72

73 Dr. S. S. Khoramrooz 73

74 Intended Use  Lauryl Tryptose Broth and Lauryl Sulfate Broth, which are also known as Lauryl Sulfate Tryptose (LST) Broth, are used for the detection of coliform organisms in materials of sanitary importance. 74 Dr. S. S. Khoramrooz

75  Lactose provides a source of fermentable carbohydrate for coliform organisms.  The fermentation of lactose with gas formation is a presumptive test for coliforms.  Sodium lauryl sulfate inhibits organisms other than coliforms. 75 Dr. S. S. Khoramrooz

76  This medium is used for the detection of coliforms in foods and dairy products.  It is now the medium of choice for use in the presumptive phase of the Standard Total Coliform Multiple-Tube (MPN) Test for the microbiological examination of water. 76 Dr. S. S. Khoramrooz

77 77 Dr. S. S. Khoramrooz

78 Expected Results  After incubation of the tubes with loosened caps at 35 ± 0.5°C for 24 hours, examine for turbidity and for gas production in the Durham fermentation tubes.  If no gas has formed and been trapped in the inverted tube, reincubate and reexamine after 48 hours.  Turbidity of the medium accompanied by formation of gas in any amount in the Durham tubes within 48 hours is a positive presumptive test for the presence of coliforms in the sample.  The result should be confirmed by additional standard testing. 78 Dr. S. S. Khoramrooz

79 79

80 Intended Use  Lysine Iron Agar is used for the differentiation of enteric organisms based on their ability to decarboxylate or deaminate lysine and to form hydrogen sulfide. Principles of the Procedure  Dextrose serves as a source of fermentable carbohydrate.  The pH indicator, bromcresol purple, is changed to a yellow color at or below pH 5.2 and is purple at or above pH Dr. S. S. Khoramrooz

81  Ferric ammonium citrate and sodium thiosulfate are indicators of hydrogen sulfide formation.  Lysine is the substrate for use in detecting the enzymes, lysine decarboxylase and lysine deaminase.  Cultures of enteric bacilli that produce hydrogen sulfide cause blackening of the medium due to the production of ferrous sulfides. 81 Dr. S. S. Khoramrooz

82  Those that produce lysine decarboxylase produce an alkaline reaction (purple color) or neutral reaction in the butt of the medium.  Organisms that deaminate the lysine cause the development of a red slant over an acid butt.  Gas may be formed but its formation is often irregular or suppressed. 82 Dr. S. S. Khoramrooz

83 83

84 Dr. S. S. Khoramrooz 84

85 85 Dr. S. S. Khoramrooz

86 Procedure  Using an inoculating needle, stab the butt twice then streak the slant with growth from a pure culture.  Incubate tubes with loosened caps for hours at 35 ± 2°C in an aerobic atmosphere.  Triple Sugar Iron Agar slants should be inoculated in parallel unless results from this medium have already been obtained to distinguish coliforms from Shigella, for example. 86 Dr. S. S. Khoramrooz

87 Expected Results  Lysine decarboxylation is detected in the butt by an alkaline (purple) reaction.  Lysine deamination is detected by a red slant.  Hydrogen sulfide production is detected by the formation of a black precipitate.  A negative reaction (purple slant and yellow butt) indicates fermentation of dextrose only. 87 Dr. S. S. Khoramrooz

88  Hydrogen sulfide may not be detected in this medium by organisms that are negative for lysine decarboxylase activity since acid production in the butt may suppress its formation.  For this reason H2S-producing Proteus species do not blacken this medium Dr. S. S. Khoramrooz 88

89 Intended Use  MR-VP Medium and MR-VP Broth (Methyl Red- Voges Proskauer Medium/Broth, also known as Buffered Peptone- Glucose Broth) are used for the differentiation of bacteria by means of the methyl red and Voges-Proskauer reactions. 89 Dr. S. S. Khoramrooz

90 Principles of the Procedure  Methyl red-positive organisms produce high levels of acid during fermentation of dextrose, overcome the phosphate buffer system and produce a red color upon the addition of the methyl red pH indicator.  In the Voges-Proskauer test, the red color produced by the addition of potassium hydroxide to cultures of certain microbial species is due to the ability of the organisms to produce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of dextrose.  The acetoin is oxidized in the presence of oxygen and alkali to produce a red color.  This is a positive Voges-Proskauer reaction. 90 Dr. S. S. Khoramrooz

91 91 Dr. S. S. Khoramrooz

92 Procedure  Using a light inoculum, inoculate tubes of MR-VP media with 18- to 24-hour pure cultures.  Incubate tubes aerobically at 35 ― 2‹C for a minimum of 48 hours but preferably for 5 days.  Prepare the methyl red indicator:  0.1 g of methyl red in 300 mL of 95% ethyl alcohol.  Add sufficient purified water to make 500 mL.  After the appropriate incubation period, aseptically remove aliquots (1 mL for the VP test) of the medium and conduct the following tests: 92 Dr. S. S. Khoramrooz

93 1. Methyl Red Test:  Add 5 drops of methyl red indicator to an aliquot of the broth.  Interpret the color result immediately. 2. Voges-Proskauer Test:  Empty the contents (15 drops) from the reagent A dropper  5 drops from the reagent B dropper into 1 mL of broth culture.  Shake well after the addition of each reagent to aerate the sample. 93 Dr. S. S. Khoramrooz

94 Expected Results  1. Methyl Red Test  a. Positive – red color at surface of the medium.  b. Negative – yellow color at surface of the medium. 2. Voges-Proskauer Test  A positive reaction is indicated by the development of a distinct red color which occurs within 5 minutes.  Certain species within Enterobacteriaceae genera may react differently or give variable results.  Consult appropriate texts for reactions of specific species. 94 Dr. S. S. Khoramrooz

95 Limitations of the Procedure 1. Results of the MR and VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae. 2. A precipitate may form in the potassium hydroxide reagent solution.  This precipitate has not been shown to reduce the effectiveness of the reagent. 95 Dr. S. S. Khoramrooz

96 3. Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test. However, certain organisms such as Hafnia alvei and Proteus mirabilis may give a positive result for both tests. 4. Incubation time for the Methyl Red test cannot be shortened by increasing the dextrose concentration in the medium or by heavily inoculating the broth. 5. Incubate MR-negative tests for more than 48 hours and test again. Dr. S. S. Khoramrooz 96

97 6. Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with reading the results. 7. VP reagents must be added in the order and the amounts specified or a weak-positive or false-negative reaction may occur.  A weak-positive reaction may be masked by a copper-like color which may form due to the reaction of KOH and α-naphthol. 8. Read the VP test within 1 hour of adding the reagents. The KOH and α-naphthol may react to form a copper-like color, causing a potential false-positive interpretation. 9. Due to the possible presence of acetoin, diacetyl or related substances in certain raw materials, the use of media low in these substances (such as MR-VP media) is recommended for this test. 97 Dr. S. S. Khoramrooz

98 98 Dr. S. S. Khoramrooz

99 Intended Use  MacConkey agars are slightly selective and differential plating media mainly used for the detection and isolation of gram-negative organisms from clinical, dairy, food, water, pharmaceutical, cosmetic, and other industrial sources.  MacConkey Agar is used for isolating and differentiating lactose-fermenting from lactose-nonfermenting gram- negative enteric bacilli.  MacConkey Agar Base is used with added carbohydrate in differentiating coliforms based on fermentation reactions. 99 Dr. S. S. Khoramrooz

100  MacConkey Agar without Crystal Violet is used for isolating and differentiating enteric microorganisms while permitting growth of staphylococci and enterococci.  The medium can be used also to separate Mycobacterium fortuitum and M. chelonae from other rapidly growing mycobacteria.  MacConkey Agar without Crystal Violet or Salt and MacConkey Agar without Salt are used for isolating and differentiating gram-negative bacilli while suppressing the swarming of most Proteus species. 100 Dr. S. S. Khoramrooz

101 Principles of the Procedure  Lactose is a fermentable carbohydrate.  When lactose is fermented, a local pH drop around the colony cause a color change in the pH indicator (neutral red) and bile precipitation. 101 Dr. S. S. Khoramrooz

102  Bile salts, bile salts no. 3, oxgall and crystal violet are selective agents that inhibit growth of gram-positive organisms.  Magnesium sulfate is a source of divalent cations. Agar is the solidifying agent. 102 Dr. S. S. Khoramrooz

103 103 Dr. S. S. Khoramrooz

104 Expected Results  Lactose-fermenting organisms grow as pink to brick-red colonies with or without a zone of precipitated bile.  Lactose-nonfermenting organisms grow as colorless or clear colonies.  Swarming by Proteus spp. is reduced on MacConkey agars without salt. 104 Dr. S. S. Khoramrooz

105 105

106 Limitations of the Procedure 1. Although MacConkey media are selective primarily for gram-negative enteric bacilli, biochemical and, if indicated, serological testing using pure cultures are recommended for complete identification. 2. Incubation of MacConkey Agar plates under increased CO2 has been reported to reduce the growth and recovery of a number of strains of gram-negative bacilli. 106 Dr. S. S. Khoramrooz

107 Intended Use  Malonate Broth is used for differentiating Enterobacter from Escherichia based on malonate utilization. Principles of the Procedure  Malonate Broth contains ammonium sulfate, which is the sole source of nitrogen in the medium;  Sodium malonate is the sole source of carbon.  Increased alkalinity resulting from malonate utilization causes the indicator, bromthymol blue, to change color from green to blue. 107 Dr. S. S. Khoramrooz

108 108 Dr. S. S. Khoramrooz

109 Procedure 1. Inoculate tubes with a loopful of test organism. 2. Incubate at 35 ― 2‹C for hours. 3. Examine tubes for a change in the color of the medium from green to blue. Expected Results  Malonate utilization is indicated by a change in the color of the medium from green to blue:  Positive: Blue  Negative: Green 109 Dr. S. S. Khoramrooz

110 110 Dr. S. S. Khoramrooz

111 Intended Use  OF (Oxidation Fermentation) media are used for the determination of oxidative and fermentative metabolism of carbohydrates by gram-negative rods on the basis of acid reaction in either the open or closed system. Summary and Explanation  OF Medium was developed by Hugh and Leifson who described the taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by gram-negative bacteria. 111 Dr. S. S. Khoramrooz

112  They showed that when an organism is inoculated into two tubes of OF Basal Medium containing a carbohydrate and the medium in one of the tubes is covered with melted petrolatum prior to incubation, the patterns of metabolism are of differential significance.  Oxidative organisms only produce an acid reaction in the open tube with little or no growth and no acid formation in the covered tube.  Fermentative organisms will produce an acid reaction in both types of tubes.  Changes in the covered agar are considered to be due to true fermentation, while changes in the open tubes are due to oxidative utilization of the carbohydrate present.  If the carbohydrate is not utilized by either method, there is no acid production in either tube. Dr. S. S. Khoramrooz 112

113 Principles of the Procedure  Dextrose is the most important carbohydrate for use in OF Basal Medium; however, certain organisms may metabolize other carbohydrates even if they are unable to utilize dextrose.  Prepared tubed media containing arabinose, dextrose, dulcitol, fructose, galactose, lactose, maltose, mannose, raffinose, rhamnose, salicin, sorbitol, sucrose and xylose are provided. 113 Dr. S. S. Khoramrooz

114 114 Dr. S. S. Khoramrooz

115 Procedure  Inoculate a pair of OF tubes of each carbohydrate used with each organism being tested.  The tubes should be stabbed to approximately 1/4 inch from the bottom using an inoculating needle and a light inoculum.  Overlay one tube of each pair with sterile mineral oil.  Incubate tubes at 35 ± 2°C in an aerobic atmosphere for 48 hours.  Do not discard as negative until after 4 days of incubation. 115 Dr. S. S. Khoramrooz

116  Expected Results  Record results as acid (A) or alkaline/no change (–).  Also record whether or not the organism is motile as evidenced by the appearance of growth away from the line of inoculation.  Typical reaction patterns are as follows. 116 Dr. S. S. Khoramrooz

117 Limitations of the Procedure 1. The acid reaction produced by oxidative organisms is apparent at the surface and gradually spreads throughout the medium. If the oxidation is weak or slow, however, an initial alkaline reaction at the surface of the open tube may persist for several days and eventually convert to an acid reaction. 2. If an organism is unable to grow on OF Basal Medium, Cowan recommends adding either 2% serum or 0.1% yeast extract to each carbohydrate tube. 117 Dr. S. S. Khoramrooz

118 118

119 Intended Use  SIM Medium is used to differentiate enteric bacilli on the basis of sulfide production,indole formation and motility. 119 Dr. S. S. Khoramrooz

120 Summary and Explanation  Hydrogen sulfide production, indole formation and motility are distinguishing characteristics which aid in the identification of the Enterobacteriaceae, especially Salmonella and Shigella.  SIM Medium, therefore,is useful in the process of identification of enteric pathogens. 120 Dr. S. S. Khoramrooz

121 Principles of the Procedure  Sodium thiosulfate and ferrous ammonium sulfate are indicators of hydrogen sulfide production.  The ferrous ammonium sulfate reacts with H2S gas to produce ferrous sulfide,a black precipitate.  The casein peptone is rich in tryptophan,which is attacked by certain microorganisms resulting in the production of indole.  The indole is detected by the addition of chemical reagents following the incubation period. 121 Dr. S. S. Khoramrooz

122  Motility detection is possible due to the semisolid nature of the medium.  Growth radiating out from the central stab line indicates that the test organism is motile. 122 Dr. S. S. Khoramrooz

123 123 Dr. S. S. Khoramrooz

124 Procedure  Loosen caps, boil and cool before use.  Using growth from a pure culture, stab an inoculating needle two-thirds of the distance to the bottom in the center of the tube.  Incubate tubes with loosened caps for18-24 hours at 35±2°C in anaerobic atmosphere. 124 Dr. S. S. Khoramrooz

125 125

126 Intended Use  SS Agar and Salmonella Shigella Agar are moderately selective and differential media for the isolation of pathogenic enteric bacilli, especially those belonging to the genus Salmonella.  This formulation is not recommended for the primary isolation of Shigella. 126 Dr. S. S. Khoramrooz

127 Principles of the Procedure  SS Agar and Salmonella Shigella Agar are designated as moderately selective media based upon the degree of inhibition of gram-positive microorganisms that they inhibit due to their content of bile salts, brilliant green and citrates.  Differentiation of enteric organisms is achieved by the incorporation of lactose in the medium.  Organisms that ferment lactose produce acid which, in the presence of the neutral red indicator, results in the formation of red colonies.  Lactose nonfermenters form colorless colonies. 127 Dr. S. S. Khoramrooz

128  The latter group contains the majority of the intestinal pathogens, including Salmonella and Shigella.  The sodium thiosulfate and ferric citrate enable the detection of hydrogen sulfide production as evidenced by colonies with black centers. 128 Dr. S. S. Khoramrooz

129 Procedure  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 hours.  If negative after 24 hours, reincubate an additional 24 hours. 129 Dr. S. S. Khoramrooz

130 130

131 Expected Results  Typical colonial morphology on Salmonella Shigella Agar is as follows: 131 Dr. S. S. Khoramrooz

132 132 Dr. S. S. Khoramrooz

133 Limitation of the Procedure  Due to the relatively high level of selectivity, some Shigella strains may not grow on SS Agar and Salmonella Shigella Agar and, therefore, these media are not recommended for the primary isolation of Shigella.  Media recommended for the isolation of Shigella are Hektoen Enteric and XLD agars. 133 Dr. S. S. Khoramrooz

134 Intended Use  Selenite Broth (Selenite-F Broth) is used as an enrichment medium for the isolation of Salmonella from feces, urine, water, foods and other materials of sanitary importance. Principles of the Procedure  The peptone provides essential nitrogenous and carbon compounds.  The lactose in the medium serves to maintain a uniform pH. 134 Dr. S. S. Khoramrooz

135  When selenite is reduced by the growth of bacteria, alkali is produced, and such increase in pH would lessen the toxicity of the selenite and result in overgrowth of extraneous bacteria.  The acid produced by lactose fermentation serves to maintain a neutral or slightly decreased pH.  The function of the phosphate is two-fold; it serves to maintain a stable pH and lessens the toxicity of the selenite, thus increasing the capacity of the medium.  Sodium selenite inhibits many species of grampositive and gram-negative bacteria including enterococci and coliforms. 135 Dr. S. S. Khoramrooz

136 136 Dr. S. S. Khoramrooz

137 137

138 Procedure  For feces and other solid materials, suspend 1-2 g of the specimen in the broth (approximately 10-15% by volume) and emulsify with an inoculating needle, if necessary.  Incubate tubes with loosened caps at 35 ± 2°C for up to 24 hours.  Subcultures should be made after hours of incubation, if possible.  Coliforms will tend to overgrow the pathogens if incubated longer than 24 hours. 138 Dr. S. S. Khoramrooz

139 Expected Results  After incubation, there should be an increase in the number of pathogens that the medium is designed to select for and enrich.  Subculture onto appropriate selective and differential media (e.g., MacConkey Agar, Hektoen Enteric Agar, XLD Agar, XLT4 Agar, CHROMagar™ Salmonella) to isolate pathogens for identification. 139 Dr. S. S. Khoramrooz

140 Limitation of the Procedure  Enrichment broths should not be used as the sole isolation medium.  They are to be used in conjunction with selective and nonselective plating media to increase the probability of isolating pathogens, especially when they may be present in small numbers. 140 Dr. S. S. Khoramrooz

141 Intended Use  Simmons Citrate Agar is used for the differentiation of gram negative bacteria on the basis of citrate utilization. Principles of the Procedure  Organisms able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole sources of nitrogen and carbon, respectively, will grow on this medium and produce an alkaline reaction as evidenced by a change in the color of the bromthymol blue indicator from green (neutral) to blue (alkaline). 141 Dr. S. S. Khoramrooz

142

143 Procedure  Inoculate slants with growth from a pure culture using a light inoculum.  Incubate all tubes for 4 days at 35 ± 2°C in an aerobic atmosphere. Expected Results  A positive reaction is indicated by growth with an intense blue color in the slant.  A negative reaction is evidenced by no growth to trace growth with no change in color (medium remains dark green). 143 Dr. S. S. Khoramrooz

144 144

145 Intended Use  Triple Sugar Iron Agar (TSI Agar) is used for the differentiation of gram-negative enteric bacilli based on carbohydrate fermentation and the production of hydrogen sulfide. Principles of the Procedure  TSI Agar contains three sugars (dextrose, lactose and sucrose),  Phenol red for detecting carbohydrate fermentation 145 Dr. S. S. Khoramrooz

146  Ferrous ammonium sulfate for detection of hydrogen sulfide production (indicated by blackening in the butt of the tube).  Carbohydrate fermentation is indicated by the production of gas and a change in the color of the pH indicator from red to yellow.  To facilitate the detection of organisms that only ferment dextrose, the dextrose concentration is one-tenth the concentration of lactose or sucrose.  The small amount of acid produced in the slant of the tube during dextrose fermentation oxidizes rapidly, causing the medium to remain red or revert to an alkaline pH.  In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. 146 Dr. S. S. Khoramrooz

147  After depletion of the limited dextrose, organisms able to do so will begin to utilize the lactose or sucrose.  To enhance the alkaline condition of the slant, free exchange of air must be permitted by closing the tube cap loosely.  If the tube is tightly closed, an acid reaction (caused solely by dextrose fermentation) will also involve the slant. 147 Dr. S. S. Khoramrooz

148 148 Dr. S. S. Khoramrooz

149 Procedure  To inoculate, carefully touch only the center of an isolated colony on an enteric plated medium with a cool, sterile needle, stab into the medium in the butt of the tube, and then streak back and forth along the surface of the slant.  Several colonies from each primary plate should be studied separately, since mixed infections may occur. 149 Dr. S. S. Khoramrooz

150  Incubate with caps loosened at 35°C and examine after hours for carbohydrate fermentation, gas production and hydrogen sulfide production.  Any combination of these reactions may be observed.  Do not incubate longer than 24 hours because the acid reaction in the slant of lactose and sucrose fermenters may revert to an alkaline reaction. 150 Dr. S. S. Khoramrooz

151 151

152 Expected Results  Carbohydrate fermentation is indicated by a yellow coloration of the medium.  If the medium in the butt of the tube becomes yellow (acidic), but the medium in the slant becomes red (alkaline), the organism being tested only ferments dextrose (glucose).  A yellow (acidic) color in the slant and butt indicates that the organism being tested ferments dextrose, lactose and/or sucrose.  A red (alkaline) color in the slant and butt indicates that the organism being tested is a nonfermenter. 152 Dr. S. S. Khoramrooz

153  Hydrogen sulfide production results in a black precipitate in the butt of the tube.  Gas production is indicated by splitting and cracking of the medium. 153 Dr. S. S. Khoramrooz

154 1. Hydrogen sulfide production may be evident on Kligler Iron Agar but negative on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed that the utilization of sucrose could suppress the enzymatic mechanisms responsible for H2S production. Padron and Dockstader8 found that not all H2S-positive Salmonella are positive on TSI. 2. Sucrose is added to TSI to eliminate some sucrose-fermenting lactose-nonfermenters such as Proteus and Citrobacter spp. 3. Further biochemical tests and serological typing must be performed for definite identification and confirmation of organisms. 154 Dr. S. S. Khoramrooz

155 4. Do not use an inoculating loop to inoculate a tube of Triple Sugar Iron Agar. While stabbing the butt, mechanical splitting of the medium occurs, causing a false positive result for gas production. 5. A pure culture is essential when inoculating Triple Sugar Iron Agar. If inoculated with a mixed culture, irregular observations may occur. 6. Tubes should be incubated with caps loosened. This allows a free exchange of air, which is necessary to enhance the alkaline condition on the slant. 155 Dr. S. S. Khoramrooz

156 Intended Use  Urea Agar and Urease Test Broth are used for the differentiation of organisms, especially the Enterobacteriaceae, on the basis of urease production. 156 Dr. S. S. Khoramrooz

157 Principles of the Procedure  The urea medium of Rustigian and Stuart is particularly suited for the differentiation of Proteus species from other gram negative enteric bacilli capable of utilizing urea.  Unable to do so in Urease Test Broth because of limited nutrients and the high buffering capacity of the medium.  To provide a medium with greater utility, Urea Agar was devised by Christensen with peptone and dextrose included and reduced buffer content to promote more rapid growth of many of the Enterobacteriaceae and permit a reduction in incubation time. 157 Dr. S. S. Khoramrooz

158  The complete Urea Agar contains 15.0 g/L of agar in addition to the ingredients in the base medium.  When organisms utilize urea, ammonia is formed during incubation which makes the reaction of these media alkaline, producing a red-pink color.  Consequently, urease production may be detected by the change in the phenol red indicator. 158 Dr. S. S. Khoramrooz

159 Urease medium

160 160 Dr. S. S. Khoramrooz

161 Dehydrated Product  BBL™ Urea Agar Base 1. Dissolve 29 g of the powder in 100 mL of purified water. Mix thoroughly. Sterilize by filtration. 2. Suspend 15 g of agar in 900 mL of purified water. 3. Autoclave at 121°C for 15 minutes. 4. Cool to 50°C and add 100 mL of the sterile Urea Aga Base. 5. Mix thoroughly and dispense aseptically in sterile tubes. 6. Cool tubed medium in a slanted position so that deep butts are formed. 7. Do not remelt the complete medium. 8. Test samples of the finished product for performance using stable, typical control cultures. 161 Dr. S. S. Khoramrooz

162 Procedure  Using a heavy inoculum (2 loopfuls) of growth from an 18- to 24-hour pure culture (TSI Agar or other suitable medium), inoculate the broth or agar (streaking back and forth over the entire slant surface). 162 Dr. S. S. Khoramrooz

163  Do not stab the butt since it serves as a color control.  For broth, shake tubes gently to suspend the bacteria.  Incubate tubes with loosened caps at 35 ― 2‹C in an incubator or water bath.  Observe reactions after 2, 4, 6, 18, 24 and 48 hours.  For agar, continue to check every day for a total of 6 days; even longer incubation periods may be necessary. 163 Dr. S. S. Khoramrooz

164  The production of urease is indicated by an intense pink-red (red-violet) color on the slant or throughout the broth.  The color may penetrate into the agar (butt); the extent of the color indicates the rate of urea hydrolysis. 164 Dr. S. S. Khoramrooz

165  A negative reaction is no color change.  The agar medium remains pale yellow to buff; the broth remains yellowish orange. 165 Dr. S. S. Khoramrooz

166 Urea Agar Base 1. The alkaline reaction produced in this medium after prolonged incubation may not be caused by urease activity. False positive reactions may occur due to the utilization of peptones (especially in slant agar by Pseudomonas aeruginosa, for example) or other proteins which raise the pH due to protein hydrolysis and the release of excessive amino acid residues. To eliminate possible protein hydrolysis, perform a control test with the same test medium without urea. 2. Do not heat or reheat the medium because urea decomposes very easily. 166 Dr. S. S. Khoramrooz

167 3. Urea Agar detects rapid urease activity of only the urease positive Proteus species. For results to be valid for the detection of Proteus, the results must be read within the first 2-6 hours after incubation. Urease-positive Enterobacter, Citrobacter or Klebsiella, in contrast, hydrolyze urea much more slowly, showing only slight penetration of the alkaline reaction into the butt of the medium in 6 hours and requiring 3-5 days to change the reaction of the entire butt. 167 Dr. S. S. Khoramrooz

168 Urea Broth 1. To rule out false positives due to protein hydrolysis (as opposed to urea hydrolysis) that may occur in the medium after prolonged incubation, perform a control test with the same test medium without urea. 2. Do not heat or reheat the medium because urea decomposes very easily. 168 Dr. S. S. Khoramrooz

169 3. The high buffering system in this medium masks urease activity in organisms that are delayed positive. This medium is therefore recommended for the detection of urease activity in all Proteus spp., Providencia rettgeri and urease- positive Providencia stuartii. M. morganii slowly hydrolyzes urea and may require approximately a 36 hour incubation for a strong urease-positive reaction to occur. If in doubt as to a result, compare with an uninoculated tube or incubate for an additional 24 hours. 4. Variations in the size of the inoculum can affect the time required to reach positive (alkaline, pH 8.1) results. 169 Dr. S. S. Khoramrooz

170 Intended Use  XL (Xylose Lysine) Agar Base is used for the isolation and differentiation of enteric pathogens and, when supplemented with appropriate additives, as a base for selective enteric media.  XLD Agar is the complete Xylose Lysine Desoxycholate Agar, a moderately selective medium recommended for isolation and differentiation of enteric pathogens, especially Shigella species. 170 Dr. S. S. Khoramrooz

171 Principles of the Procedure  Xylose is incorporated into the medium because it is fermented by practically all enterics except for the shigellae.  This property enables the differentiation of Shigella species.  Lysine is included to enable the Salmonella group to be differentiated from the nonpathogens.  Without lysine, salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species. 171 Dr. S. S. Khoramrooz

172  After the salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme lysine decarboxylase, with reversion to an alkaline pH, which mimics the Shigella reaction.  To prevent similar reversion by lysine-positive coliforms, lactose and sucrose (saccharose) are added to produce acid in excess.  Degradation of xylose, lactose and sucrose generates acid products, which in the presence of the pH indicator phenol red, causes a color change in the medium from red to yellow. 172 Dr. S. S. Khoramrooz

173  To add to the differentiating ability of the formulation, an H2S indicator system, consisting of sodium thiosulfate and ferric ammonium citrate, is included for the visualization of the hydrogen sulfide produced, resulting in the formation of colonies with black centers.  The nonpathogenic H2S producers do not decarboxylate lysine; therefore, the acid reaction produced by them prevents the blackening of the colonies. 173 Dr. S. S. Khoramrooz

174  XLD Agar is both a selective and differential medium.  It utilizes sodium desoxycholate as the selective agent and, therefore, it is inhibitory to gram- positive microorganisms. 174 Dr. S. S. Khoramrooz

175 175 Dr. S. S. Khoramrooz

176 176 Dr. S. S. Khoramrooz

177  Expected Results  Degradation of xylose, lactose and sucrose generates acid products, causing a color change in the medium from red to yellow.  Hydrogen sulfide production under alkaline conditions causes colonies to develop black centers.  This reaction is inhibited by the acid conditions that accompany carbohydrate fermentation.  Lysine decarboxylation in the absence of lactose and sucrose fermentation causes reversion to an alkaline condition and the color of the medium changes back to red.  Typical colonial morphology and reactions on XLD Agar are as follows: 177 Dr. S. S. Khoramrooz

178 178 Dr. S. S. Khoramrooz

179 179

180 Dr. S. S. Khoramrooz 180

181 Limitations of the Procedure 1. Red, false-positive colonies may occur with some Proteus and Pseudomonas species. 2. Incubation in excess of 48 hours may lead to false- positive results. 3. S. Paratyphi A, S. Choleraesuis, S. pullorum and S. gallinarum may form red colonies without black centers, thus resembling Shigella species. 4. Some Proteus strains will give black-centered colonies on XLD Agar. 181 Dr. S. S. Khoramrooz

182 Intended Use  Transport Medium Amies and Transport Medium (Stuart, Toshach and Patsula) are used for collecting, transporting and preserving microbiological specimens.  Cary and Blair Transport Medium is used for collecting, transporting and preserving microbiological specimens, particularly those containing Vibrio cholerae. 182 Dr. S. S. Khoramrooz

183 Summary and Explanation  Transport media are chemically defined, semisolid, nonnutritive, phosphate buffered media that provide a reduced environment.  Transport media are formulated to maintain the viability of microorganisms without significant increase in growth.  In 1948, Moffett, Young and Stuart described a medium for transporting gonococcal specimens to the laboratory. 183 Dr. S. S. Khoramrooz

184  The ability of Stuart’s medium to maintain the viability of gonococci during transport led other researchers to explore its use with a variety of specimens.  This medium is currently recommended for throat, vaginal and wound samples. 184 Dr. S. S. Khoramrooz

185 185

186  In 1964, Cary and Blair modified Stuart’s medium by substituting inorganic phosphates for glycerophosphate and raising the pH to 8.4.  The modified medium was effective in maintaining the viability of Salmonella and Shigella in fecal samples.  Due to its high pH, Cary and Blair Transport Medium is also effective in maintaining the viability of Vibrio cultures for up to four weeks.  Cary and Blair Transport Medium is currently recommended for fecal and rectal samples. 186 Dr. S. S. Khoramrooz

187  Transport Medium Amies is recommended for throat, vaginal and wound samples.  Amies media are especially suited for specimens containing Neisseria gonorrhoeae. 187 Dr. S. S. Khoramrooz

188 188 Dr. S. S. Khoramrooz

189 Procedure 1. Obtain specimen with sterile swab. Insert specimen swab(s) into the upper third of the medium in the transport container. 2. Cut with sterile scissors or break-off the protruding portion of the swab stick. Tightly screw the lid on the bottle or vial. 3. Label the bottle or vial and send to the laboratory with minimum delay. Specimens may be refrigerated until ready for shipment. 4. Submit to laboratory within 24 hours for culture and analysis. 189 Dr. S. S. Khoramrooz

190 Expected Results  Survival of bacteria in a transport medium depends on many factors including the type and concentration of bacteria in the specimen, the formulation of the transport medium, the temperature and duration of transport and inoculation to appropriate culture media within 24 hours.  Optimal growth and typical morphology can only be expected following direct inoculation and appropriate cultivation. 190 Dr. S. S. Khoramrooz

191 THE END Dr. S. S. Khoramrooz 191


Download ppt "In the name of God By: Dr. S. S. Khoramrooz Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran Yasuj University."

Similar presentations


Ads by Google