In the name of God

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. Blood Agar Base (Infusion Agar) Intended Use • Blood Agar Base (Infusion Agar), with the addition of sterile blood, is used for the isolation, cultivation and detection of hemolytic activity of streptococci and other fastidious microorganisms. Summary and Explanation • Infusion Agar is an all-purpose medium which has been used for many years as a base for the preparation of blood agars. • In a study of viability of streptococci, Snavely and Brahier performed comparative studies of horse, rabbit and sheep blood Dr. S. S. Khoramrooz

3. with Blood Agar Base, and found that sheep blood gave the clearest and most reliable colony and hemolysischaracteristics at both 24 and 48 hours. • In the course of the investigation, about 1,300 isolations of streptococci were made with Blood Agar Base containing 5% sheep blood. • Blood Agar Base media are specified in standard methods for food testing. • Infusion Agar has been largely replaced as a blood agar base by the Tryptic/Trypticase™ Soy Agar formulations, which contain milk and plant peptones in place of the variable infusion component. Dr. S. S. Khoramrooz

4. Principles of the Procedure • Infusion from heart muscle, casein peptone and yeast extract provide nitrogen, carbon, amino acids and vitamins in Blood Agar Base. • Supplementation with blood (5-10%) provides additional growth factors for fastidious microorganisms, and is the basis for determining hemolytic reactions. • Hemolytic patterns may vary with the source of animal blood or type of base medium used. Dr. S. S. Khoramrooz

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6. Directions for Preparation from Dehydrated Product 1. Suspend 40 g of the powder in 1 L of purified water. Mix thoroughly. 2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. 3. Autoclave at 121°C for 15 minutes. 4. For preparation of blood agar, cool the base to 45-50°C and aseptically add 5% sterile, defibrinatedblood. Mix well. 5. Test samples of the finished product for performance using stable, typical control cultures. Dr. S. S. Khoramrooz

7. Expected Results • Colonial morphology on blood agar containing 5% sheep blood is as follows: 1. Hemolytic streptococci may appear as translucent or opaque, grayish, small (1 mm), or large matte or mucoid (2-4 mm) colonies, encircled by a zone of hemolysis. Gram stains should be made and examined to check the macroscopic findings. (Other organisms which may cause hemolysis include Listeria, various corynebacteria, hemolytic staphylococci, Escherichia coli and Pseudomonas.) Approximate quantitation of the number of colonies of hemolytic streptococci may be helpful to the clinician. Dr. S. S. Khoramrooz

8. 2. Pneumococci usually appear as very flat, smooth, translucent,grayish and sometimes mucoidcolonies surrounded by a narrow zone of “green” (alpha)hemolysis. 3. Staphylococci appear as opaque, white to gold-yellow colonies with or without zones of beta hemolysis. 4. Listeriamay be distinguished by their rod shape in stains, and by motility at room temperature. Small zones of beta hemolysis are produced. 5. Other organisms representing minimal flora and clinically significant isolates can also be expected to grow on this nonselective formulation. Dr. S. S. Khoramrooz

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10. Brain Heart Infusion (Broth Media) Intended Use • Brain Heart Infusion (BHI) is a general-purpose liquid medium used in the cultivation of fastidious and nonfastidious microorganisms, including aerobic and anaerobic bacteria, from a variety of clinical and nonclinical materials. • It serves as a base for supplemented media containing 0.1% agar, Fildes enrichment or 6.5% sodium chloride. • A supplemented pre-reduced formulation in tubes is especially recommended for the cultivation of anaerobes. Dr. S. S. Khoramrooz

11. Principles of the Procedure • In the formulation containing 6.5% sodium chloride, the salt acts as a differential and/or selective agent by interfering with membrane permeability and osmoticand electrokineticequilibria in salt-intolerant organisms. • Fildes enrichment (peptic digest of sheep blood) is incorporated into one tubed formulation for the cultivation of fastidious microorganisms, such as Haemophilusinfluenzae. • The addition of 0.1% agar aids in the cultivation of anaerobic microorganisms because its consistency yields conditions of reduced oxygen tension. Dr. S. S. Khoramrooz

12. The pre-reduced medium in Hungate tubes is based on Hungate methods of culturing anaerobic microorganisms outside of an anaerobic chamber. • The tubes provide a reduced medium in a self-contained, anaerobic tube sealed using a Hungate screw cap. Dr. S. S. Khoramrooz

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14. Expected Results • Growth in the tubes is indicated by the presence of turbidity compared to an uninoculated control. • If growth appears, cultures should be examined by Gram stain and subcultured onto appropriate media; e.g., a Trypticase™ Soy Agar with 5% Sheep Blood and/or Chocolate II Agar plate, EMB Agar or MacConkey II Agar plate. • If anaerobes are suspected, subcultures should be incubated anaerobically, as in a GasPak EZ anaerobic system. • Enterococciwill grow in the 6.5% NaCl broth within 24-48 hours. • Nonenterococcal group D streptococci fail to grow in the medium after 48 hours of incubation. Dr. S. S. Khoramrooz

15. Brain Heart Infusion Agars Intended Use • Brain Heart Infusion (BHI) Agar is a general-purpose medium suitable for the cultivation of a wide variety of organism types, including bacteria, yeasts and molds. • With the addition of 5% or 10% sheep blood, it is used for the isolation and cultivation of a wide variety of fungal species, including systemic fungi, from clinical and nonclinical sources. Dr. S. S. Khoramrooz

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17. LB Agar, Lennox • LB Broth, Lennox Intended Use • LB Agar, Lennox and LB Broth, Lennox are used for maintaining and cultivating recombinant strains of Escherichia coli. Summary and Explanation • LB Agar, Lennox and LB Broth, Lennox are nutritionally rich media developed by Lennox for the growth and maintenance of pure cultures of recombinant strains of E. coli. • These strains are generally derived from E. coli K12, which are deficient in B vitamin production. Dr. S. S. Khoramrooz

18. This strain of E. coli has been further modified through specific mutation to create an auxotrophic strain that is not capable of growth on nutritionally deficient media. • LB Agar, Lennox provides all the nutritional requirements of these organisms. Dr. S. S. Khoramrooz

19. Expected Results • After sufficient incubation, the agar medium should show growth as evidenced by formation of colonies and/or a confluent lawn of growth. • In the broth medium, growth is evident by the appearance of turbidity. Dr. S. S. Khoramrooz

20. LB Agar, Miller • LB Broth, Miller Intended Use • LB Agar, Miller and LB Broth, Miller (Luria-Bertani) are used for maintaining and propagating Escherichia coli in molecular microbiology procedures. Expected Results • Growth should be evident on the agar medium by the appearance of colonies and/or a confluent lawn on the surface of the medium. • In the broth medium, growth is evident by the appearance of turbidity. Dr. S. S. Khoramrooz

21. Luria Agar Base, Miller • Luria Broth Base, Miller Intended Use • Luria Agar Base, Miller and Luria Broth Base, Miller are used for maintaining and propagating Escherichia coliin molecular microbiology procedures with or without added glucose. Principles of the Procedure • Peptone and yeast extract provide nitrogen, carbon, vitamins (including B vitamins) and certain trace elements. • Sodium chloride provides essential ions. Agar is the solidifying agent. Dr. S. S. Khoramrooz

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23. Mueller Hinton Agars Intended Use • Each lot of Mueller Hinton Agar and Mueller Hinton II Agar has been tested according to, and meets the acceptance limits of, the current M6 protocol published by the CLSI. • Mueller Hinton Agar is recommended for antimicrobial disc diffusion susceptibility testing of common, rapidly growing bacteria by the Bauer-Kirby method, as standardized by the Clinical and Laboratory Standards Institute (CLSI). Dr. S. S. Khoramrooz

24. Mueller Hinton Agar with 5% Sheep Blood is recommended for antimicrobial disc diffusion susceptibility testing of Streptococcus pneumoniaewith selected agents; i.e., chloramphenicol, erythromycin, ofloxacin, tetracycline and vancomycin, • In addition to oxacillin screening for susceptibility to penicillin, as standardized by the Clinical and Laboratory Standards Institute (CLSI). Dr. S. S. Khoramrooz

25. NOTE: The recommended medium for disc diffusion susceptibility testing of Streptococcus pneumoniaeis Mueller Hinton agar with 5% sheep blood. • The recommended medium for Haemophilusinfluenzae is Haemophilus Test Medium (HTM) gar. • The recommended medium for Neisseriagonorrhoeaeis GC Agar with 1% defined growth supplement (GC II Agar with BBL™ IsoVitaleX™ Enrichment or equivalent). • Interpretive criteria are provided in the CLSI Document M100 (M2),5 which is included with CLSI Document M2, Dr. S. S. Khoramrooz

26. Principles of the Procedure • The Bauer-Kirby procedureis based on the diffusion through an agar gel of antimicrobial substances which are impregnated on paper discs. • In contrast to earlier methods which used discs of high and low antimicrobial concentrations and which used the presence or absence of inhibition zones for their interpretation, this method employs discs with a single concentration of antimicrobial agent and zone diameters are correlated with minimal inhibitory concentrations (MIC) Dr. S. S. Khoramrooz

27. In the test procedure, a standardized suspension of the organism is swabbed over the entire surface of the medium. • Paper discs impregnated with specified amounts of antibiotic or other antimicrobial agents are then placed on the surface of the medium, the plate is incubated and zones of inhibition around each disc are measured. • The determination as to whether the organism is susceptible, intermediate or resistant to an agent is made by comparing zone sizes obtained to those in the CLSI Document M100(M2) Dr. S. S. Khoramrooz

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29. Various factorshave been identified as influencing disc diffusion susceptibility tests. • These include the medium, excess surface moisture on the medium, agar depth, disc potency, inoculum concentration, pH and β-lactamase production by test organisms. Dr. S. S. Khoramrooz

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31. Nutrient Agar Intended Use • Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms in water, sewage, feces and other materials. Principles of the Procedure • This relatively simple formulation provides the nutrients necessary for the replication of a large number of microorganisms that are not excessively fastidious. Dr. S. S. Khoramrooz

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33. Procedure • Liquefy the agar if prepared tubes are used, cool to 45-50°C and pour into Petri dishes. Allow to solidify for at least 30 minutes. • Use standard procedures to obtain isolated colonies from specimens. Incubate plates at 35 ± 2°C for 18-24 hours and 42-48 hours, if necessary. Dr. S. S. Khoramrooz

34. Tubed slants are used primarily for the cultivation and maintenance of pure cultures. • They should be inoculated with an inoculating loop and incubated under the same conditions as the plated medium. Expected Results • Examine plates for growth. • Growth from tubes inoculated with pure cultures may be used for biochemical and/or serological testing. Dr. S. S. Khoramrooz

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37. Nutrient Broth Intended Use • Nutrient Broth is used for the cultivation of many species of nonfastidious microorganisms. • It is one of several nonselective media useful in routine cultivation of microorganisms. Principles of the Procedure • This relatively simple formulation supports the growth of nonfastidious microorganisms due to its content of peptone and beef extract. Dr. S. S. Khoramrooz

38. Procedure • Inoculate tubes of the broth medium with the test samples. • Incubate tubes for 18-24 hours at 35 ± 2°C in an aerobic atmosphere. • Expected Results • After incubation, growth is evidenced by the appearance of turbidity in the broth. • Aliquots of the broth can be used for subculturing to solid media for purification and identification purposes. Dr. S. S. Khoramrooz

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40. Thioglycollate Media Intended Use • Fluid Thioglycollate Medium (FTM) is used for the sterility testing of biologics and for the cultivation of anaerobes, aerobes and microaerophiles. Dr. S. S. Khoramrooz

41. Thioglycollate Medium, Brewer Modified is used for the cultivation of obligate anaerobes, microaerophiles and facultative organisms. • Fluid Thioglycollate Medium with Beef Extract is used in cultivating microorganisms from normally sterile biological products. Dr. S. S. Khoramrooz

42. Principles of the Procedure • Dextrose, peptone, L-cystine and yeast extract provide the growth factors necessary for bacterial replication. • Sodium thioglycollateis a reducing agent that prevents the accumulation of peroxides which are lethal to some microorganisms. • The L-cystine is also a reducing agent, since it contains sulfhydryl groups which inactivate heavy metal compounds and maintain a low redox potential, thereby supporting anaerobiosis. Dr. S. S. Khoramrooz

43. Methylene blue is an indicator of the level of oxidation/reduction in the medium; increased oxidation raises the Eh, causing the methylene blue indicator to become green. • Resazurin is an oxidation-reduction indicator, being pink when oxidized and colorless when reduced. • The small amount of agar assists in the maintenance of a low redox potential by stabilizing the medium against convection currents, thereby maintaining anaerobiosis in the lower depths of the medium. Dr. S. S. Khoramrooz

44. Precautions • Do not reheat the media more than once; continued reheating gives rise to toxicity. Expected Results • After incubation, growth is evidenced by the presence of turbidity compared to an uninoculated control. • Strict aerobes tend to grow in a thin layer at the surface of the broth; obligate anaerobes will grow only in that portion of the broth below the upper oxidized layer. Dr. S. S. Khoramrooz

45. THE END Dr. S. S. Khoramrooz