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Section III T Thiotone E Peptone, cont. User Quality Control Identity Specifications BBL™ Thiotone™ E Peptone Dehydrated Appearance: Tan, fine, homogeneous, free of extraneous material. Solution: 2.0% solution, soluble in purified water. Solution is clear to moderately hazy. Reaction of 2.0% Solution at 25°C: pH 6.5-7.5 Cultural Response Biochemical Reactions BBL™ Thiotone™ E Peptone Prepare a sterile solution of Thiotone E Peptone as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours. TEST TEST SOLUTION ORGANISM ATCC™ INOCULUM CFU RESULT Fermentable Carbohydrates Indole Production Acetylmethylcarbinol Production Hydrogen Sulfide Production 2% 0.1% 0.1% with 0.5% dextrose 1% Escherichia coli Escherichia coli Enterobacter aerogenes Citrobacter freundii 29552 29552 13048 ~107 0.1 mL, undiluted 0.1 mL, undiluted Positive Positive Positive 8454 0.1 mL, undiluted Positive Growth Response BBL™ Thiotone™ E Peptone Prepare a sterile solution of peptone agar without (plain) and with 5% sheep blood (SB) using 10 g of Thiotone E Peptone, 2.5 g of sodium chloride and 6.5 g of agar in 500 mL of purified water. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at 35 ± 2°C for 2-3 days (incubate streptococci with 3-5% CO2). ORGANISM ATCC™ INOCULUM CFU RECOVERY PLAIN RECOVERY WITH SB HEMOLYSIS Enterococcus faecalis Streptococcus pneumoniae Streptococcus pyogenes 29212 6305 49117 103-104 103-104 104-105 Good N/A Good N/A Good Good – Alpha Beta Directions for Preparation from Dehydrated Product Refer to the final concentration of Thiotone E Peptone in the formula of the medium being prepared. Add product as required. Procedure See appropriate references for specific procedures using Thiotone E Peptone. Expected Results References 1. Tortora. 1984. Appl. Environ. Microbiol. 47:1172. 2. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, DC. 3. Kwinn. 2001. Bug Journal, Biology Department, Massachusetts Institute of Technology. 4:193. 4. Horowitz (ed.). 2000. Official methods of analysis of AOAC International, 17th ed. AOAC International, Gaithersburg, Md. 5. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC International, Gaithersburg, Md. Availability BBL™ Thiotone™ E Peptone AOAC BAM SMWW Cat. No. 212302 Dehydrated – 500 g Refer to appropriate references and procedures for results. Tinsdale Agar Base Tinsdale Enrichment Desiccated Intended Use Tinsdale Agar Base is used with Tinsdale Enrichment Desiccated in isolating and differentiating Corynebacterium diphtheriae. Summary and Explanation Tinsdale Agar Base, supplemented with Tinsdale Enrichment, is employed in the cultural diagnosis of diphtheria. Diphtheria, an acute infectious disease primarily of the upper respira562 tory tract but occasionally of the skin,1 is caused by toxigenic strains of Corynebacterium diphtheriae. The three biotypes are mitis, intermedius and gravis.1 The signs and symptoms of the disease are a pharyngeal membrane, sore throat, malaise, headache and nausea.2 Death can result from respiratory obstruction by the membrane or myocarditis caused by the toxin.2 Tinsdale3 developed a serum-cystine-thiosulfate-tellurite agar medium for the primary isolation and differentiation of Tinsdale Agar Base, cont. Uninoculated Plate User Quality Control Corynebacterium diphtheriae ATCC™ 8028 Identity Specifications Difco™ Tinsdale Agar Base Dehydrated Appearance: Light beige, free flowing, homogeneous. Solution: 4.5% solution, soluble in purified water upon boiling. Solution is light to medium amber, slightly opalescent to opalescent. Prepared Appearance: Light to medium amber, slightly opalescent to opalescent. Reaction of 4.5% Solution at 25°C: pH 7.4 ± 0.2 Difco™ Tinsdale Enrichment Desiccated Desiccated Appearance: Light to dark tan cake; variations may occur. Solution: Soluble in purified water. Solution is light to dark amber, clear to opalescent, may have a slight precipitate. Cultural Response Difco™ Tinsdale Agar Base with Tinsdale Enrichment Desiccated Prepare the medium per label directions. Inoculate to obtain discrete colonies and stab several times using an inoculating needle; incubate at 35 ± 2°C for 18-48 hours. ORGANISM Corynebacterium diphtheriae biotype gravis Corynebacterium diphtheriae biotype mitis Klebsiella pneumoniae Streptococcus pyogenes ATCC™ INOCULUM CFU RECOVERY APPEARANCE 8028 102-103 Good Brown with halos 8024 13883 102-103 102-103 Brown with halos – 19615 102-103 Good Marked to complete inhibition Poor to fair C. diphtheriae. This formulation distinguished between C. diphtheriae and diphtheroids which exhibited similar characteristics. The differential principle is based on the capacity of C. diphtheriae to produce a brown or black halo around the colonies. Billings4 simplified Tinsdale Basal Medium by using Proteose Peptone No. 3 as a nutrient source. This modification improved the differential qualities and recovery of C. diphtheriae. Tinsdale Agar Base and Tinsdale Enrichment are prepared according to the Billings4 modification. Moore and Parsons5 confirmed the halo formation of C. diphtheriae with one exception; C. ulcerans occasionally produced colonies similar to C. diphtheriae and required biochemical identification. Tinsdale Enrichment Desiccated contains bovine serum, sodium hydroxide, L-cystine, sodium thiosulfate and potassium tellurite in the quantity and proportion described by Billings.4 Principles of the Procedure Peptone provides the nitrogen, vitamins, carbon and amino acids in Tinsdale Agar Base. Sodium chloride maintains the osmotic balance of the medium. Agar is the solidifying agent. Tinsdale Enrichment contains bovine serum, which provides essential growth factors. Sodium hydroxide maintains the pH. T Brown to black without halos L-cystine and sodium thiosulfate are H2S indicators. Potassium tellurite is a selective agent. The formation of black to brown halos surrounding the colony results from the reduction of potassium tellurite to metallic tellurite. Stabbing the medium with an inoculating needle accentuates darkening of the medium by C. diphtheriae. Formulae Difco™ Tinsdale Agar Base Approximate Formula* Per Liter Proteose Peptone No. 3 ........................................... 20.0 Sodium Chloride ........................................................ 5.0 Agar ......................................................................... 20.0 g g g Difco™ Tinsdale Enrichment Desiccated Contains Bovine Serum, L-Cystine, Sodium Hydroxide, Sodium Thiosulfate and Potassium Tellurite at pH 8.0-10.0. *Adjusted and/or supplemented as required to meet performance criteria. Directions for Preparation from Dehydrated Product Difco™ Tinsdale Agar Base 1. Suspend 45 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. 563 Section III T Tinsdale Agar Base, cont. 3. Dispense 100 mL amounts into flasks. 4. Autoclave at 121°C for 15 minutes. 5. Aseptically add 15 mL rehydrated Tinsdale Enrichment to each 100 mL at 50-55°C. Mix well. 6. Test samples of the finished product for performance using stable, typical control cultures. Difco™ Tinsdale Enrichment Desiccated 1. Rehydrate with 15 mL sterile purified water. 2. Rotate in an end-over-end motion to dissolve completely. Procedure 1. For a complete discussion on the collection, isolation and identification of C. diphtheriae and other Corynebacterium species, refer to the appropriate procedures outlined in the references.1,2,6 2. Inoculate plates with the test organisms in a manner to obtain discrete colonies and stab the medium several times with an inoculating needle. 3. Definitive identification of a strain of C. diphtheriae as a true pathogen requires demonstration of toxin production.6 Characteristic colonies of C. diphtheriae may be inoculated directly onto KL Virulence Agar enriched with KL Virulence Enrichment and containing Taxo™ KL Antitoxin Strips for toxigenicity tests. Expected Results The appearance of brown-black colored colonies surrounded by brown-black halos is presumptive evidence for C. diphtheriae.1 Limitations of the Procedure 1. Tinsdale Agar is not suitable as a primary plating medium, since it may not support the growth of some strains of C. diphtheriae.1 2. C. ulcerans, C. pseudotuberculosis and (rarely) Staphylococcus species may produce a characteristic halo on Tinsdale Agar.1 3. Do not read Tinsdale Agar early because several organisms may exhibit slight browning on this medium in 18 hours.1 4. Incubation in 5-10% CO2 retards the development of halos on Tinsdale Agar.1 5. On media containing tellurite, diphtheria bacilli are shorter and stain more uniformly; however, granules are less readily observed than when grown on Loeffler’s medium.7 6. Further biochemical tests may be necessary to distinguish between C. diphtheriae and C. ulcerans due to similar reactions on this medium. References 1. Isenberg (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology, Washington, D.C. 2. Funke and Bernard. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 3. Tinsdale. 1947. J. Pathol. Bacteriol. 59:461. 4. Billings. 1956. An investigation of Tinsdale Tellurite medium: its usefulness and mechanisms of halo-formation. M.S. thesis. University of Michigan, Ann Arbor, Mich. 5. Moore and Parsons. 1958. J. Infect. Dis. 102:88. 6. Forbes, Sahm and Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, Inc., St. Louis, Mo. 7. Bailey and Scott. 1966. Diagnostic microbiology, 2nd ed. The C. V. Mosby Company, St. Louis, Mo. Availability Difco™ Tinsdale Agar Base Cat. No. 278610 Dehydrated – 500 g Difco Tinsdale Enrichment Desiccated ™ Cat. No. 234210 Tube – 6 × 15 mL* *Store at 2-8°C. Bacto™ Todd Hewitt Broth • Todd Hewitt Broth Todd Hewitt Broth with Gentamicin and Nalidixic Acid Intended Use Todd Hewitt Broth is a general-purpose medium, which primarily is used for the cultivation of beta-hemolytic streptococci, especially for serological studies. Todd Hewitt Broth with Gentamicin and Nalidixic Acid is used for the selective enrichment of group B streptococci (Streptococcus agalactiae), especially from genital specimens. Summary and Explanation Todd Hewitt broth originally was developed for use in the production of streptococcal hemolysin.1 The modification of Updyke and Nickle2 is used for the growth of beta-hemolytic streptococci for use in fluorescent antibody test procedures3 and for serological typing based on the production of type-specific M protein.4 564 Since its emergence in the 1970s, neonatal group B streptococcal disease has become the major infectious cause of illness and death among newborns. Prior to 1994, an estimated 7,600 episodes of invasive group B streptococcal disease, primarily sepsis and meningitis, occurred in newborns each year in the United States, with approximately 80% of those episodes representing early-onset disease occurring within the first week of life.5 The disease is spread to newborns through vertical transmission from a mother who carries group B streptococci in her anorectum or genital tract. The Centers for Disease Control and Prevention (CDC) has published guidelines for screening and use of intrapartum chemoprophylaxis for prevention of neonatal group B streptococcal disease.6 The use of Todd Hewitt Broth with Gentamicin and Nalidixic Acid (or Lim Broth) is recommended to maximize the likelihood of recovering group B streptococci upon plating on sheep blood agar.