Prepare a sterile solution of Bacto Tryptose as directed below. Adjust final ph to Inoculate and incubate at 35 ± 2 C for hours.

Transcription

1 ryptose Bacto ryptose Bacto ryptose is an enzymatic diest of protein used in preparin microbioloical culture media. ryptose was oriinally developed as a peptone particularly adapted to rowth requirements of Brucella. ryptose is very useful for cultivation of streptococci, pneumococci, meninococci and other fastidious oranisms, and was found to be superior to meat infusion peptone media previously used for these oranisms. 1,2 Mobley et al. 3 reported that ryptose Broth was the preferred medium for strains of Bordetella bronchiseptica in studies of phosphatase activity. ryptose has been reported as beneficial for cell culture applications. Litwin 4 found ryptose to be suitable for supplementin a serum-free medium to row human diploid fibroblasts. Vauhn and Fan 5 established that ryptose provided free amino acids necessary for rowth of Spodoptera fruiperda and Lymantria dispar insect cell lines. ryptose is often used as a biomass enhancer for recombinant E. coli production. ryptose is the major inredient and only peptone in the formulation for ryptose Phosphate Broth (PB), an oftenused medium for various culture applications. Hata and Kojima 6 have shown PB to be a useful supplement in culturin the nematode, Aniostronylus cantonensis. PB was also reported as a supplement to a medium for cultivatin a protozoan parasite, which parasitizes vectors of Chaas disease, on its insect cell host. 7 Spodoptera fruiperda, a cotton pest in Arentina 8 and several tick cell lines have also been rown usin a PB-supplemented medium. 9 ryptose Phosphate Broth has been reported as a suitable supplement for rowth of baby hamster kidney cells 10 and porcine kidney cells. 11 Media formulations containin Bacto ryptose are specified in standard methods for various applications Bacto ryptose is a mixed enzymatic hydrolysate with distinctive nutritional properties. he diestive process of Bacto ryptose results in assorted peptides of hiher molecular weiht suitable for lon chain amino acid requirements. Bacto ryptose Bacto ryptose Dehydrated Appearance: Reaction of 1.0% Solution at 25 C: ph an, free-flowin, ranules. 1.0%, 2.0% and 10.0% solutions, soluble in purified water. 1.0% solution is liht amber, clear. 2.0% solution is medium amber, clear to slihtly opalescent. 10.0% solution is medium to dark amber, very slihtly opalescent to opalescent, may have a precipitate. Biochemical Reactions Bacto ryptose Prepare a sterile solution of Bacto ryptose as directed below. Adjust final ph to Inoculate and incubate at 35 ± 2 C for hours. ES ES SOLUION ORGANISM ACC INOCULUM CFU RESUL Fermentable Carbohydrates 2% Escherichia coli ~10 7 Neative Indole Production 0.1% Escherichia coli ml, undiluted Positive Acetylmethylcarbinol 0.1% with Enterobacter aeroenes ml, undiluted Positive Production 0.5% dextrose Hydroen Sulfide Production 1% Salmonella choleraesuis ml, undiluted Positive subsp. choleraesuis serotype yphimurium Growth Response Bacto ryptose Prepare a sterile solution with 2% Bacto ryptose, 0.5% sodium chloride and 1.5% aar. Adjust final ph to Inoculate and incubate plates at 35 ± 2 C for hours. ORGANISM ACC INOCULUM CFU RECOVERY Brucella suis 4314* Undiluted Good Staphylococcus aureus Good pneumoniae Good pyoenes Good *If this strain is not available, verify performance with a known isolate. 596

2 ryptose Blood Aar Base provides nitroen, amino acids and vitamins in microbioloical culture media. ypical Analysis Refer to Product ables in the Reference Guide section of this manual. Precautions Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical specimens of human or animal oriin containin or potentially containin pathoenic Brucella spp. 2. Biosafety Level 3 practices, containment equipment and facilities are recommended for all manipulations of cultures of the pathoenic Brucella spp. and for experimental animal studies. Refer to the final concentration of Bacto ryptose in the formula of the medium bein prepared. Add product as required. See appropriate references for specific procedures usin Bacto ryptose. Refer to appropriate references and procedures for results. 1. Casman J. Bacteriol. 43: Casman Am. J. Clin. Pathol. 17: Mobley, Chenappa, Kadel and Stuart Can. J. Comp. Med. 48: Litwin Dev. Biol. Stand. 60: Vauhn and Fan In Vitro Cell. Dev. Biol. Anim. 33: Hata and Kojima Exp. Parasitol. 70: Reduth, Schaub, and Pudney Parasitoloy 98: Deutschmann and Jaer Enzyme Microb. echnol. 16: Munderloh and Kurtti Exp. Appl. Acarol. 7: Prodafikas and Plavsic Focus 22: Sakoda and Fukusho In Vitro Cell Dev. Biol. Anim. 34: Horowitz (ed.) Official methods of analysis of AOAC International, 17th ed. AOAC International, Gaithersbur, Md. 13. U.S. Food and Dru Administration Bacterioloical analytical manual 8th ed. AOAC International, Gaithersbur, Md. 14. Downes and Ito (ed.) Compendium of methods for the microbioloical examination of foods, 4th ed. American Public Health Association, Washinton, D.C. 15. U.S. Environmental Protection Aency (USEPA) Improved enumeration methods for the recreational water quality indicators: Enterococci and Escherichia coli. EPA-821/R-97/004. Office of Water, USEPA, Washinton, D.C. 16. Clesceri, Greenber and Eaton (ed.) Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washinton, D.C. 17. U.S. Department of Ariculture Microbioloy laboratory uidebook, 3rd ed. Food and Safety Inspection Service, USDA, Washinton, D.C. 18. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health Biosafety in microbioloical and biomedical laboratories, 4th ed. HHS Publication No. (CDC) U.S. Government Printin Office, Washinton, D.C. Bacto ryptose AOAC BAM COMPF EPA SMWW USDA Cat. No Dehydrated Dehydrated 10 k ryptose Blood Aar Base ryptose Blood Aar Base is used with blood in isolatin, cultivatin and determinin the hemolytic reactions of fastidious microoranisms. Investiations of the nutritive properties of tryptose demonstrated that culture media prepared with this peptone were superior to the meat infusion peptone media previously used for the cultivation of Brucella, streptococci, pneumococci, meninococci and other fastidious bacteria. Casman 1,2 reported that a medium consistin of 2% tryptose, 0.3% beef extract, 0.5% NaCl, 1.5% aar and 0.03% dextrose equaled fresh beef infusion base with respect to rowth of oranisms. he small amount of carbohydrate was noted to interfere with hemolytic reactions, unless the medium was incubated in an atmosphere of carbon dioxide. ryptose Blood Aar Base is a nutritious infusion-free basal medium typically supplemented with 5-10% sheep, rabbit or horse blood for use in isolatin, cultivatin and determinin hemolytic reactions of fastidious pathoenic microoranisms. Without enrichment, this base can be used as a eneralpurpose medium. ryptose Blood Aar Base is included in the FDA Bacterioloical Analytical Manual (ph adjusted to 6.8 ± 0.2). 3 ryptose is the source of nitroen, carbon and amino acids in ryptose Blood Aar Base. Beef extract provides additional nitroen. Sodium chloride maintains osmotic balance. Aar is the solidifyin aent. Supplementation with 5-10% blood provides additional rowth factors for fastidious microoranisms and is used to determine hemolytic patterns of bacteria. Formula Difco ryptose Blood Aar Base Approximate Formula* Per Liter ryptose Beef Extract Sodium Chloride Aar *Adjusted and/or supplemented as required to meet performance criteria. 597

3 ryptose Blood Aar Base, cont. Difco ryptose Blood Aar Base Dehydrated Appearance: Beie, free-flowin, homoeneous. 3.3% solution, soluble in purified water upon boilin. Solution is liht amber, very slihtly to slihtly opalescent. Prepared Appearance: Plain Liht amber, slihtly opalescent. With 5% sheep blood Cherry red, opaque. Reaction of 3.3% Solution at 25 C: ph 7.2 ± 0.2 Difco ryptose Blood Aar Base Prepare the medium per label directions without (plain) and with 5% sterile defibrinated sheep blood (SB). Inoculate and incubate at 35 ± 2 C for hours (blood plates under 5-10% CO 2 ). INOCULUM RECOVERY RECOVERY HEMOLYSIS ORGANISM ACC CFU PLAIN WIH SB HR Escherichia coli Good Good Beta Neisseria meninitidis None to poor Good None Staphylococcus aureus Good Good Beta pneumoniae Fair to ood Good Alpha pyoenes Fair to ood Good Beta Escherichia coli ACC pneumoniae ACC 6305 Staphylococcus aureus ACC pyoenes ACC Suspend 33 of the powder in 1 L of purified water. Mix thorouhly. 2. Heat with frequent aitation and boil for 1 minute to completely dissolve the powder. 3. Autoclave at 121 C for 15 minutes. 4. o prepare blood aar, aseptically add 5% sterile defibrinated blood to the medium cooled to C. Mix well. 5. est samples of the finished product for performance usin stable typical control cultures. 1. Process each specimen as appropriate, and inoculate directly onto the surface of the medium. Streak for isolation with an inoculatin loop, then stab the aar several times to deposit beta-hemolytic streptococci beneath the aar surface. Subsurface rowth will display the most reliable hemolytic reactions of both oxyen-stable and oxyenlabile streptolysins Incubate plates aerobically, anaerobically or under conditions of increased CO 2 (5-10%) in accordance with established laboratory procedures. 3. Examine plates for rowth and hemolytic reactions after and 48-hour incubation. Four different types of hemolysis on blood aar media can be described: 5 a. Alpha (α)-hemolysis is the reduction of hemolobin to methemolobin in the medium surroundin the colony. his causes a reenish discolorization of the medium. b. Beta (β)-hemolysis is the lysis of red blood cells, resultin in a clear zone surroundin the colony. c. Gamma (γ)-hemolysis indicates no hemolysis. No destruction of red blood cells occurs, and there is no chane in the medium. d. Alpha-prime (ὰ)-hemolysis is a small zone of complete hemolysis that is surrounded by an area of partial lysis. Limitations of the 1. Blood Aar Base Media are intended for use with blood supplementation. Althouh certain dianostic tests may be performed directly on this medium, biochemical and, if indicated, immunoloical testin usin pure cultures are recommended for complete identification. Consult appropriate references for further information. 2. Hemolytic reactions of some strains of roup D streptococci have been shown to be affected by differences in animal blood. Such strains are beta-hemolytic on horse, human and rabbit blood aar and alpha-hemolytic on sheep blood aar

4 ryptose Aar 3. Colonies of Haemophilus haemolyticus are beta-hemolytic on horse and rabbit blood aar, and must be distinuished from colonies of beta-hemolytic streptococci usin other criteria. he use of sheep blood has been suested to obviate this problem since sheep blood is deficient in pyridine nucleotides and does not support rowth of H. haemolyticus he atmosphere of incubation has been shown to influence hemolytic reactions of beta-hemolytic streptococci. 3 For optimal performance, incubate blood aar base media under increased CO 2 or anaerobic conditions. 5. Hemolytic patterns may vary with the source of animal blood or type of base medium used Casman J. Bacteriol. 43: Casman Am. J. Clin. Pathol. 17: Harmon, Kautter, Golden and Rhodehamel In FDA bacterioloical analytical manual, 8th ed. AOAC International, Gaithersbur, Md. 4. Ruoff, Whiley and Beihton In Murray, Baron, Pfaller, enover and Yolken (ed.), Manual of clinical microbioloy, 7th ed. American Society for Microbioloy, Washinton, D.C. 5. Isenber. (ed.) Clinical microbioloy procedures handbook, vol. 1. American Society for Microbioloy, Washinton, D.C. 6. Baron, Peterson and Fineold Bailey & Scott s dianostic microbioloy, 9th ed. Mosby-Year Book, Inc., St. Louis, Mo. Difco ryptose Blood Aar Base BAM Cat. No Dehydrated Dehydrated 2 k ryptose Aar ryptose Broth ryptose Aar is used for cultivatin a wide variety of fastidious microoranisms, particularly for isolatin Brucella accordin to Huddleson and Castañeda. ryptose Broth is used for cultivatin Brucella and other fastidious microoranisms. Difco ryptose Aar Dehydrated Appearance: Prepared Appearance: Liht beie, homoeneous, free-flowin. 4.1% solution, soluble in purified water upon boilin. Solution is liht amber, slihtly opalescent. Liht amber, slihtly opalescent. Reaction of 4.1% Solution at 25 C: ph 7.2 ± 0.2 Difco ryptose Broth Dehydrated Appearance: Beie, homoeneous, free-flowin. 2.6% solution, soluble in purified water. Solution is liht amber, clear. Prepared Appearance: Liht amber, clear. Reaction of 2.6% Solution at 25 C: ph 7.2 ± 0.2 Difco ryptose Aar or ryptose Broth Prepare the medium per label directions. Inoculate and incubate at 35 ± 2 C under 5-10% CO 2 for hours. ORGANISM ACC INOCULUM CFU RECOVERY Brucella abortus 11192* Good Brucella melitensis 4309* Good Brucella suis 9843* Good *Minimally one strain of Brucella should be used for performance testin. hese ACC strains should be used if available. ryptose media, prepared without extract or infusion of meat, are recommended for the cultivation and isolation of pathoenic and saprophytic bacteria. Historically, it was considered necessary to include meat extract or infusion as a nutritional supplement in culture media. ryptose was developed while studyin the rowth requirements of Brucella. Huddleson 1 found tryptose media to be equal or superior to meat infusion media, providin uniformity for the cultivation and differentiation of fastidious oranisms. ryptose media are particularly well suited for the isolation of Brucella from blood. Castañeda 2 studied the isolation of Brucella species usin a broth containin 2% tryptose and 2% sodium citrate. Sodium citrate serves as an anticoaulant and assists in inactivatin complement in the blood specimen. ryptose Broth can be used as a complete basal medium or supplemented with enrichments. Huddleson 3 used a broth containin 2% tryptose as an enrichment medium in the isolation of Brucella from clinical specimens. McCullouh et al. reported that addition of thiamine, dextrose and iron salts increased rowth of Brucella suis. 4 Addition of 0.1% aar to ryptose Broth can increase rowth of aerobes and anaerobes in liquid media. Blood aar may be prepared by addin 5% sterile, defibrinated sheep, horse or rabbit blood to the sterile medium. he hih productivity of tryptose media in the isolation and cultivation of Brucella supports use of these formulas as eneral-purpose media, especially when avoidance of animal tissue products is desired. ryptose Aar with 5% bovine serum, with or without antibiotics, remains a standard platin medium for the isolation of brucellae. 5 For isolation of Brucella stains from contaminated milk, crystal violet (entian violet) can be added to ryptose Aar to suppress ram-positive oranisms. 6 ryptose media can be supplemented with thiamine or citrate for the cultivation and maintenance of fastidious aerobic and facultative microoranisms

5 ryptose Aar, cont. ryptose Aar is specified in the Compendium of Methods for the Microbioloical Examination of Foods. 8 ryptose media are recommended in the FDA Bacterioloical Analytical Manual for seroloical testin. 9 ryptose peptone is a source of nitroen and carbon. Dextrose is a source of carbohydrate. Sodium chloride maintains osmotic balance. Aar is the solidifyin aent in ryptose Aar. Formulae Difco ryptose Aar Approximate Formula* Per Liter ryptose Dextrose Sodium Chloride Aar Difco ryptose Broth Consists of the same inredients without the aar. *Adjusted and/or supplemented as required to meet performance criteria. Precautions Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical specimens of human or animal oriin containin or potentially containin pathoenic Brucella spp. 2. Biosafety Level 3 practices, containment equipment and facilities are recommended for all manipulations of cultures of the pathoenic Brucella spp. and for experimental animal studies. Difco ryptose Aar 1. Suspend 41 of the powder in 1 L of purified water. Mix thorouhly. 2. Heat with frequent aitation and boil for 1 minute to completely dissolve the powder. 3. Autoclave at 121 C for 15 minutes. 4. est samples of the finished product for performance usin stable, typical control cultures. NOE: o prepare blood aar, aseptically add 5% sterile defibrinated sheep, horse or rabbit blood. Dispense into sterile Petri dishes. Difco ryptose Broth 1. Dissolve 26 of the powder in 1 L of purified water. 2. Autoclave at 121 C for 15 minutes. 3. est samples of the finished product for performance usin stable, typical control cultures. Refer to appropriate references and procedures for results. Limitations of the 1. ryptose media are eneral-purpose, non-selective media. Althouh certain dianostic tests may be performed directly on the medium, biochemical and, if indicated, immunoloical testin usin pure cultures are recommended for complete identification. 2. When preparin blood aar, hemolytic reactions of some strains of roup D streptococci have been shown to be affected by differences in animal blood. 3. Atmosphere of incubation has been shown to influence hemolytic reactions of beta-hemolytic streptococci. 11 For optimal performance, incubate tryptose media supplemented with blood under increased CO 2 or anaerobic conditions. 4. Dextrose has been shown to inhibit hemolysin production by some oranisms. 1. Huddleson Brucellosis in man and animals, rev. ed. he Commonwealth Fund, New York, N.Y. 2. Castañeda Proc. Soc. Exp. Biol. Med. 64: Huddleson Brucellosis in man and animals. Oxford University Press, Oxford, Enland. 4. McCullouh, Mills, Herbst, Roessler and Brewer J. Bacteriol. 53:5. 5. Moyer and Holcomb In Murray, Baron, Pfaller, enover, and Yolken (ed.), Manual of clinical microbioloy, 6th ed. American Society for Microbioloy, Washinton, D.C. 6. MacFaddin Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams & Wilkins, Baltimore, Md. 7. Atlas Handbook of microbioloy media for the examination of food. CRC Press, Boca Raton, Fla. 8. Downes and Ito (ed.) Compendium of methods for the microbioloical examination of foods. 4th ed. American Public Health Association, Washinton, D.C. 9. U.S. Food and Dru Administration Bacterioloical analytical manual, 8th ed. AOAC International, Gaithersbur, Md. 10. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health Biosafety in microbioloical and biomedical laboratories, 4th ed. HHS Publication No. (CDC) U.S. Government Printin Office, Washinton, D.C. 11. Ruoff, Whiley and Beihton In Murray, Baron, Pfaller, enover and Yolken (ed.), Manual of clinical microbioloy, 7th ed. American Society for Microbioloy, Washinton, D.C. Difco ryptose Aar BAM CCAM COMPF Cat. No Dehydrated Dehydrated 2 k Difco ryptose Broth BAM CCAM COMPF Cat. No Dehydrated Dehydrated 10 k Methodoloies for the multiple applications usin tryptose media are outlined in the references. 600

6 ryptose Phosphate Broth Bacto ryptose Phosphate Broth Bacto ryptose Phosphate Broth is used for cultivatin fastidious microoranisms. ryptose Phosphate Broth is an infusion-free buffered medium recommended for the cultivation of fastidious, pathoenic microoranisms. It can be used in a procedure for the serodianosis of Listeria monocytoenes. 1 It is valuable in tissue culture procedures, 2 where the peptone content is considered to be a stimulatin factor for cells. Peptone provides carbon and nitroen. Dextrose is a carbon source. Sodium chloride maintains osmotic balance. Bufferin capacity is provided by disodium phosphate. he addition of % aar to ryptose Phosphate Broth facilitates anaerobic rowth and aids in dispersion of reducin substances and CO 2 formed in the environment. 3 he low aar concentration provides suitable conditions for both aerobic rowth in the upper zone and for microaerophilic and anaerobic rowth in the lower zone. Bacto ryptose Phosphate Broth Dehydrated Appearance: Beie, free-flowin, homoeneous. 2.95% solution, soluble in purified water. Solution is liht amber, clear to very slihtly opalescent, may have a very sliht precipitate. Prepared Appearance: Liht amber, clear to very slihtly opalescent, may have a very sliht precipitate. Reaction of 2.95% Solution at 25 C: ph 7.3 ± 0.2 Bacto ryptose Phosphate Broth Prepare the medium per label directions. Inoculate and incubate at 35 ± 2 o C for hours. ORGANISM ACC INOCULUM CFU RECOVERY Neisseria meninitidis Good Staphylococcus epidermidis Good pneumoniae Good pyoenes Good Formula Bacto ryptose Phosphate Broth Approximate Formula* Per Liter ryptose Dextrose Sodium Chloride Disodium Phosphate *Adjusted and/or supplemented as required to meet performance criteria. 1. Dissolve 29.5 of the powder in 1 L of purified water. (If a medium containin % aar is desired, add 1-2 of aar; heat with frequent aitation and boil for 1 minute to completely dissolve the powder.) 2. Autoclave at 121 C for 15 minutes. 3. est samples of the finished product for performance usin stable, typical control cultures. See appropriate references for specific procedures. Refer to appropriate references and procedures for results. Uninoculated ube pneumoniae ACC

7 ryptose Phosphate Broth, cont. 1. Bennett and Weaver In FDA bacterioloical analytical manual, 8th ed. AOAC International, Gaithersbur, Md. 2. Ginsber, Gold and Jordan Proc. Soc. Exp. Biol. Med. 89: MacFaddin Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams & Wilkins, Baltimore, Md. Bacto ryptose Phosphate Broth BAM Cat. No Dehydrated Dehydrated 2 k Dehydrated 10 k ween 80 Water ween * 80 Water may be used to restore and/or inoculate microdilution panels. *ween is a trademark of ICI Americas. ween 80 (polysorbate 80) is a surface active aent that is recommended for use at a 0.02% concentration in routine inoculum preparation and dispensin procedures in various microdilution systems. 1 ween 80 Water is a 0.02% concentration of polysorbate 80 in purified water that is convenient for use in dispersin microoranisms durin inoculum preparation and for reconstitutin antimicrobial aents in microdilution plates. Follow those procedures or test methods requirin the use of water with 0.02% polysorbate 80. Reference 1. hrupp In Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. Williams & Wilkins, Baltimore, Md. BBL ween 80 Water Cat. No Prepared ubes (D ubes), 12.5 ml Ctn. of Prepared ubes (A ubes), 25 ml Pk. of Prepared ubes (A ubes), 25 ml Ctn. of 100 yrosine Aar (See Nocardia Differentiation Media) 602