Tryptic Soy Agar/Trypticase™ Soy Agar (Soybean-Casein Digest Agar)

Tryptic Soy Agar

Formula Presumptive negative nitrate reduction reaction: Lack Difco™ Tryptic Nitrate Medium of color development denotes an absence of nitrite in the Approximate Formula* Per Liter medium; this should be confirmed by addition of Nitrate C Tryptose ...... 20.0 g Reagent (zinc dust). Dextrose ...... 1.0 g Disodium Phosphate ...... 2.0 g 2. After adding Nitrate C Reagent: Potassium Nitrate ...... 1.0 g Positive nitrate reduction reaction: Lack of color development Agar ...... 1.0 g indicates that nitrate has been reduced to nitrogen gas. *Adjusted and/or supplemented as required to meet performance criteria. Negative nitrate reduction reaction: Development of a red- Directions for Preparation from violet color within 5-10 minutes indicates that unreduced Dehydrated Product nitrate is still present. 1. Suspend 25 g of the powder in 1 L of purified water. Mix thoroughly. Limitation of the Procedure 2. Heat with frequent agitation and boil for 1 minute to com- This medium is not recommended for indole testing of coliforms pletely dissolve the powder. and other enterics.1 3. Autoclave at 121°C for 15 minutes. 4. Test samples of the finished product for performance using References 1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, stable, typical control cultures. vol. 1. Williams & Wilkins, Baltimore, Md. 2. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC Inter- national, Gaithersburg, Md. Procedure 3. Pezzlo. 1992. In Isenberg (ed.), Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology, Washington, D.C. 1. Obtain a pure culture of the organism to be tested from a 4. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C. solid culture medium. Select well-isolated colonies. 5. Flowers, Andrews, Donnelly and Koenig. 1993. In Marshall (ed.), Standard methods for the examination of dairy products, 16th ed. American Public Health Association, Washington, D.C. 2. Inoculate a tube of Tryptic Nitrate Medium and incubate 6. Murray, Baron, Pfaller, Tenover and Yolken. 1999. Manual of clinical microbiology, 7th ed. Ameri- aerobically or anaerobically, as appropriate, at 35 ± 2°C can Society for Microbiology, Washington, D.C. 7. Forbes, Sahm and Weissfeld. 1998. Bailey and Scott’s diagnostic microbiology, 10th ed. Mosby, for 18-24 hours. Inc., St. Louis, Mo. 3. Read tubes for growth. 4. Test for nitrate reduction using Difco™/BBL™ Nitrate A, B Availability ™ and C Reagents or equivalents per reagent instructions. Difco Tryptic Nitrate Medium Cat. No. 236710 Dehydrated – 500 g Expected Results Difco™/BBL™ Nitrate A Reagent T 1. After adding Nitrate A and B Reagents: Cat. No. 261197 Droppers – 50 × 0.5 mL Positive nitrate reduction reaction: Development of a red- Difco™/BBL™ Nitrate B Reagent violet color within 1-2 minutes indicates that nitrate has Cat. No. 261198 Droppers – 50 × 0.5 mL been reduced to nitrite. Difco™/BBL™ Nitrate C Reagent Cat. No. 261207 Vials – 50 × 1 g

Tryptic Soy Agar/Trypticase™ Soy Agar (Soybean-Casein Digest Agar)

Intended Use Hycheck™ hygiene contact slides are used for assessing the Tryptic Soy Agar and Trypticase™ Soy Agar conform with speci- microbiological contamination of surfaces and fluids. fications of The United States Pharmacopeia (USP). Summary and Explanation Tryptic (Trypticase) Soy Agar (TSA) is used for the isolation The nutritional composition of TSA has made it a popular and cultivation of nonfastidious and fastidious microorgan- medium for many years. It is the medium specified as isms. It is not the medium of choice for anaerobes. Soybean-Casein Digest Agar Medium in the USP for the total The 150 × 15 mm-style plates of Trypticase Soy Agar are aerobic microbial count portion of the microbial limit testing convenient for use with Taxo™ factor strips in the isolation procedures.1 The medium is used for a multitude of purposes and differentiation of Haemophilus species. including maintenance of stock cultures, plate counting, isolation of microorganisms from a variety of specimen types Sterile Pack and Isolator Pack plates are useful for monitoring and as a base for media containing blood.2-4 It is included surfaces and air in clean rooms, Isolator Systems and other in the compendia of methods for the examination of water, environmentally-controlled areas when sterility of the medium wastewater and foods,5, 6 in the Bacteriological Analytical is of importance. Manual 7 and is used for testing bacterial contaminants in cosmetics.8 577 Section III T Tryptic Soy Agar, cont.

User Quality Control NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.

Identity Specifications Identity Specifications Difco™ Tryptic Soy Agar BBL™ Trypticase™ Soy Agar Dehydrated Appearance: Light beige, free-flowing, homoge- Dehydrated Appearance: Fine, homogeneous, free of extrane- neous. ous material. Solution: 4.0% solution, soluble in purified Solution: 4.0% solution, soluble in purified water upon boiling. Solution is light water upon boiling. Solution is light amber, slightly opalescent. to medium, yellow to tan, clear to Prepared Appearance: Plain – Light amber, slightly opalescent. slightly hazy. With 5% sheep blood – Bright red, Prepared Appearance: Plain – Light to medium, yellow to tan, opaque. clear to slightly hazy. Reaction of 4.0% With 5% sheep blood – Bright red, Solution at 25°C: pH 7.3 ± 0.2 opaque. Reaction of 4.0% Cultural Response Solution at 25°C: pH 7.3 ± 0.2 Difco™ Tryptic Soy Agar Prepare the medium per label directions, without (plain) and with 5% Cultural Response sheep blood (SB). Inoculate and incubate at 35 ± 2°C for 18-48 hours BBL™ Trypticase™ Soy Agar

under 5-10% CO2. Prepare the medium per label directions, without (plain) and with 5% sheep blood (SB). Inoculate and incubate at 35 ± 2°C for 2 days INOCULUM RECOVERY RECOVERY (incubate streptococci with 3-5% CO ). ORGANISM ATCC™ CFU PLAIN WITH SB HEMOLYSIS 2 Escherichia coli 25922 102-103 Good Good Beta INOCULUM RECOVERY RECOVERY ™ Neisseria ORGANISM ATCC CFU PLAIN WITH SB HEMOLYSIS meningitidis 13090 102-103 Good Good None Candida albicans 10231 103-104 N/A Good None Staphylococcus Escherichia coli 25922 103-104 N/A Good Beta aureus 25923 102-103 Good Good Beta Listeria Streptococcus monocytogenes 19115 103-104 N/A Good Beta (+/-) pneumoniae 6305 102-103 Good Good Alpha Pseudomonas Streptococcus aeruginosa 10145 103-104 Good N/A pyogenes 19615 102-103 Good Good Beta Shigella flexneri 12022 103-104 Good N/A CAMP Test medium with 5% sheep blood – Perform using S. aureus ATCC 33862, Streptococcus Staphylococcus sp. group B ATCC 12386 (positive) and S. pyogenes ATCC 19615 (negative). aureus 25923 103-104 Good Good Beta Streptococcus 3 4 Escherichia coli Staphylococcus aureus pneumoniae 6305 10 -10 Good Good Alpha ATCC™ 25922 ATCC™ 25923 Streptococcus pyogenes 19615 103-104 Good Good Beta CAMP Test medium with 5% sheep blood – Perform using S. aureus ATCC 25923, Streptococcus sp. group B ATCC 12386 (positive) and S. pyogenes ATCC 19615 (negative).

Since TSA does not contain the X and V growth factors, it can conveniently be used in determining the requirements for these growth factors by isolates of Haemophilus by the addition of X, V and XV Factor Strips to inoculated TSA plates.3 The 150 mm plate provides a larger surface area for inoculation, making the “satellite” growth around the strips easier to read. With the Sterile Pack and Isolator Pack plates, the entire double- wrapped (Sterile Pack) or triple-wrapped (Isolator Pack) product is subjected to a sterilizing dose of gamma radiation, so that the contents inside the outer package(s) are sterile.9 This allows the inner package to be aseptically removed without introducing contaminants. Since the agar medium has been sterilized after packaging, the presence of microbial growth after sampling and incubation can be relied upon to represent true recovery and Streptococcus Streptococcus not pre-existing medium contaminants. A third rolled sterile pneumoniae pyogenes ATCC™ 6305 ATCC™ 19615 bag is included as a transport device. Isolator Pack plates have been validated to protect the medium from vaporized hydrogen peroxide when used in an Isolator System.

578 Tryptic Soy Agar, cont.

The Hycheck hygiene contact slide is a double-sided paddle Trypticase™ Soy Agar (150 mm plates) for Haemophilus containing two agar surfaces for immersing into fluids or The initial specimens should be inoculated onto Chocolate II sampling surfaces. There are three slides containing TSA along Agar or another suitable medium and incubated for 18-24 with another medium: D/E Neutralizing Agar; Violet Red hours in an aerobic atmosphere supplemented with carbon Bile Glucose Agar; or Rose Bengal Chloramphenicol Agar. A dioxide. Choose one or two well-isolated colonies that fourth slide contains TSA with 0.01% TTC and Rose Bengal resemble Haemophilus species and perform a Gram stain to Chloramphenicol Agar. confirm that the isolate is a gram-negative rod or cocco- bacillus. Suspend 1-2 colonies in 5 mL sterile, purified water Principles of the Procedure or Trypticase Soy Broth and vortex to mix. Dip a swab in the The combination of casein and soy peptones in TSA renders suspension and inoculate the entire surface of the plate with the medium highly nutritious by supplying organic nitrogen, the swab. With sterile forceps, place a Taxo X factor strip, a particularly amino acids and longer-chained peptides. The V factor strip and a XV strip on the plate, at least 20 mm sodium chloride maintains osmotic equilibrium. Agar is the apart. solidifying agent. Incubate plates at 35 ± 2°C for 24 hours in an aerobic atmo- Haemophilus species may be differentiated by their require- sphere supplemented with carbon dioxide. ments for X and V factors. Paper strips impregnated with these factors are placed on the surface of the medium after Expected Results inoculation with the test organism. Following incubation, a After incubation, it is desirable to have isolated colonies of zone of growth around the strip indicates a requirement for organisms from the original sample. Subculture colonies of the factor(s). interest so that positive identification can be made by means of biochemical and/or serological testing.3,10,11 Formulae Consult appropriate texts for the growth patterns produced Difco™ Tryptic Soy Agar by the various strains of Haemophilus.3,12 Approximate Formula* Per Liter Pancreatic Digest of Casein ...... 15.0 g Enzymatic Digest of Soybean Meal ...... 5.0 g References Sodium Chloride ...... 5.0 g 1. United States Pharmacopeial Convention, Inc. 2001. The United States pharmacopeia 25/The national formulary 20 – 2002. United States Pharmacopeial Convention, Inc., Rockville, Md. Agar ...... 15.0 g 2. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, BBL™ Trypticase™ Soy Agar vol. 1. Williams & Wilkins, Baltimore, Md. 3. Forbes, Sahm and Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby Inc., Approximate Formula* Per Liter St. Louis, Mo. T Pancreatic Digest of Casein ...... 15.0 g 4. Nash and Krenz. 1991. In Balows, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. Papaic Digest of Soybean Meal ...... 5.0 g 5. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and Sodium Chloride ...... 5.0 g wastewater, 20th ed. American Public Health Association, Washington, D.C. Agar ...... 15.0 g 6. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C. *Adjusted and/or supplemented as required to meet performance criteria. 7. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC International, Gaithersburg, Md. 8. Curry, Joyce and McEwen. 1993. CTFA microbiology guidelines. The Cosmetic, Toiletry and Directions for Preparation from Fragrance Association, Inc., Washington, D.C. 9. Association for the Advancement of Medical Instrumentation. 1984. Process control guidelines for Dehydrated Product gamma radiation sterilization of medical devices. AAMI, Arlington, Va. 10. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed. 1. Suspend 40 g of the powder in 1 L of purified water. Mix American Society for Microbiology, Washington, D.C. 11. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology, thoroughly. 9th ed. Williams & Wilkins, Baltimore, Md. 2. Heat with frequent agitation and boil for 1 minute to 12. Campos. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. completely dissolve the powder. 3. Autoclave at 121°C for 15 minutes. DO NOT OVER- Availability HEAT. Difco™ Tryptic Soy Agar (Soybean-Casein Digest Agar) 4. For preparation of blood plates, add 5-10% sterile, AOAC BAM CCAM COMPF EP ISO SMD SMWW USDA USP defibrinated blood to the sterile agar which has been cooled Cat. No. 236940 Dehydrated – 100 g to 45-50°C. 236950 Dehydrated – 500 g 236920 Dehydrated – 2 kg 5. Test samples of the finished product for performance using 236930 Dehydrated – 10 kg stable, typical control cultures. BBL™ Trypticase™ Soy Agar (Soybean-Casein Digest Agar) AOAC BAM CCAM COMPF EP ISO SMD SMWW USDA USP Procedure Cat. No. 211043 Dehydrated – 500 g Use standard procedures to obtain isolated colonies from 211046 Dehydrated – 5 lb (2.3 kg) specimens. Since many pathogens require carbon dioxide on 211047 Dehydrated – 25 lb (11.3 kg) primary isolation, plates may be incubated in an atmosphere containing approximately 3-10% CO2. Incubate plates at 35 ± 2°C for 18-24 hours.

579 Section III T Tryptic Soy Agar, cont.

BBL™ Trypticase™ Soy Agar (Soybean-Casein Digest Agar) BBL™ Trypticase™ Soy Agar, Sterile Pack AOAC BAM CCAM COMPF EP ISO SMD SMWW USDA USP Cat. No. 221236 Prepared Settling Plates – Pkg. of 10* United States and Canada 222205 Prepared Settling Plates – Ctn. of 100* Cat. No. 221185 Prepared Plates – Pkg. of 20* 221237 Prepared Settling Plates 221283 Prepared Plates – Ctn. of 100* (150 × 15 mm-style) – Pkg. of 5* 221803 Prepared Plates (150 × 15 mm-style) – Pkg. of 24* 222206 Prepared Settling Plates 221082 Prepared Pour Tubes, 20 mL – Pkg. of 10 (150 × 15 mm-style) – Ctn. of 45* 221086 Prepared Tubes (K Tubes) – Pkg. of 10 292257 Prepared Plates, heavy fill – Pkg. of 10* 221087 Prepared Tubes (K Tubes) – Ctn. of 100 292396 Prepared Plates, heavy fill – Ctn. of 100* 299099 Prepared Bottles, 500 mL – Pkg of 10 BBL™ Trypticase™ Soy Agar, Isolator Pack Europe Cat. No. 292651 Prepared Plates – Pkg. of 10* Cat. No. 254051 Prepared Plates – Pkg. of 20* 292652 Prepared Plates – Ctn. of 100* 254086 Prepared Plates – Ctn. of 120* 292272 Prepared Plates (150 × 15 mm-style) – Pkg. of 5* 25007 Prepared Contact Plates – Pkg. of 33* Difco™ Hycheck™ Hygiene Contact Slides Japan Cat. No. 251167 Prepared Plates (5 × 4) – Pkg. of 20* Cat. No. 290391 Tryptic Soy Agar//D/E Neutralizing Agar (20 slides)* 251185 Prepared Plates – Pkg. of 20* 290371 Tryptic Soy Agar//Violet Red Bile Glucose Agar 251540 Prepared Plates – Pkg. of 20* (20 slides)* 251260 Prepared Plates (150 × 15 mm-style) – Pkg. of 24* 290381 Tryptic Soy Agar//Rose Bengal Chloramphenicol 251812 Prepared Plates (60 × 15 mm-style) – Ctn. of 240* Agar (20 slides)* 251355 Prepared I Plate™ Dishes – Pkg. of 20* 290461 Tryptic Soy Agar with 0.01% TTC//Rose Bengal Chloramphenicol Agar (20 slides)* *Store at 2-8°C.

Tryptic Soy Agar with Lecithin and Polysorbate 80 (Microbial Content Test Agar) • Trypticase™ Soy Agar with Lecithin and Polysorbate 80 • Trypticase™ Soy Agar with Penicillinase • Trypticase™ Soy Agar with Lecithin, Polysorbate 80 and Penicillinase

Intended Use determined by the appearance of colonies on the surface of These media are recommended for the detection and enumeration the medium following application to the test surface and incu- of microorganisms present on surfaces of sanitary importance. bation.7,8 The RODAC plate has a marked grid to facilitate Prepared plates are provided for environmental monitoring. counting organisms. ™ Sterile Pack and Isolator Pack RODAC prepared plates are The 100 × 15 mm and the 150 × 15 mm style plates can be particularly useful for monitoring surfaces in clean rooms, used for active and passive air sampling. These plates are also Isolator Systems and other environmentally-controlled areas designed for personnel monitoring of finger tips (Finger Dab). and are also recommended for use in air sampling equipment such as the Surface Air System. Finger Dab™ Sterile Pack and Principles of the Procedure Isolator Pack plates are intended for sampling gloved hands. Casein and soy peptones are a source of nutrients required for the replication of microorganisms. Sodium chloride maintains Summary and Explanation osmotic equilibrium. Lecithin and polysorbate 80, two These media may be employed to establish and monitor cleaning commonly used neutralizers, are reported to inactivate residual 1-4 techniques and schedules. Collection of “samples” from disinfectants when the sample is being collected.7 Lecithin is identical areas before and after treatment with disinfectant incorporated to neutralize quaternary ammonium compounds yields data useful in evaluating cleaning procedures in and polysorbate 80 is used to neutralize substituted phenolic environmental sanitation. Tryptic (Trypticase) Soy Agar with disinfectants.9-12 Agar is the solidifying agent. Lecithin and Polysorbate 80 is recommended for the Aerobic Plate Count (Microbial Limit Test) for water-miscible cosmetic Trypticase Soy Agar with Penicillinase and Trypticase Soy Agar products containing preservatives.5 with Lecithin, Polysorbate 80 and Penicillinase contain 50 mL/L of penicillinase, which inactivates antibiotics such as RODAC (Replicate Organism Detection and Counting) and penicillins and cephalosporins. contact plates are used in a wide variety of surface sampling programs and may be employed to establish and monitor With the Sterile Pack and Isolator Pack plates, the entire double- cleaning techniques and schedules.1-4,6 The presence and wrapped (Sterile Pack) or triple-wrapped (Isolator Pack) number of microorganisms on a flat impervious surface is product is subjected to a sterilizing dose of gamma radiation,

580 TSA with Lecithin & Poly 80, cont.

Identity Specifications Identity Specifications Difco™ Tryptic Soy Agar with Lecithin and BBL™ Trypticase™ Soy Agar with Lecithin and Polysorbate 80 (Microbial Content Test Agar) Polysorbate 80 Dehydrated Appearance: Beige, free-flowing, homogeneous, Dehydrated Appearance: Medium fine, softly lumped powder, may appear moist. free of extraneous material. Solution: 4.57% solution, soluble in purified NOTE: The dehydrated medium has water upon boiling with frequent a characteristic “brown sugar” gentle swirling. When hot, solution is appearance and may seem moist. medium amber, slightly opalescent Solution: 4.57% solution, soluble in purified with a resuspendable precipitate. water upon boiling. Solution is light Prepared Appearance: Light to medium amber, slightly opal- to medium, yellow to tan, slightly to escent, may have a precipitate. moderately hazy. Reaction of 4.57% Prepared Appearance: Light to medium, yellow to tan, Solution at 25°C: pH 7.3 ± 0.2 slightly to moderately hazy. Reaction of 4.57% Cultural Response Solution at 25°C: pH 7.3 ± 0.2 Difco™ Tryptic Soy Agar with Lecithin and Polysorbate 80 (Microbial Content Test Agar) Cultural Response Prepare the medium per label directions. Test the medium in parallel BBL™ Trypticase™ Soy Agar with Lecithin and with Plate Count Agar, using the pour plate method. Apply disks im- Polysorbate 80 pregnated with varying dilutions of a quaternary ammonium compound Prepare the medium per label directions. Inoculate and incubate at ± ° to the medium surface. Incubate plates at 35 2 C for 40-48 hours 35 ± 2°C for 2 days (incubate A. niger at 25 ± 2°C for 4 days). and inspect for zones of inhibition. ORGANISM ATCC™ INOCULUM CFU RECOVERY INOCULUM ORGANISM ATCC™ CFU GROWTH* Aspergillus niger 16404 Undiluted Good 3 4 Escherichia coli 11229 102-103 Smaller zone of inhibition Bacillus subtilis 6633 10 -10 Good of growth compared to Candida albicans 001 103-104 Good Plate Count Agar Enterococcus faecalis 29212 103-104 Good Staphylococcus aureus 6538P 102-103 Smaller zone of inhibition Micrococcus luteus 9341 103-104 Good of growth compared to Pseudomonas aeruginosa 10145 103-104 Good T Plate Count Agar Salmonella choleraesuis *Interpretation: The smaller zones of inhibition indicate neutralization of the quaternary subsp.choleraesuis ammonium compound by the medium. serotype Typhimurium 13311 103-104 Good Serratia marcescens 13880 103-104 Good Staphylococcus aureus 25923 103-104 Good Staphylococcus epidermidis 12228 103-104 Good Streptococcus pyogenes 19615 103-104 Good so that the contents inside the outer package(s) are sterile.13 BBL™ Trypticase™ Soy Agar with Lecithin and This allows the inner package to be aseptically removed Polysorbate 80 Approximate Formula* Per Liter without introducing contaminants. Since the agar medium has Pancreatic Digest of Casein ...... 15.0 g been sterilized after packaging, the presence of microbial growth Papaic Digest of Soybean Meal ...... 5.0 g after sampling and incubation can be relied upon to represent Sodium Chloride ...... 5.0 g Lecithin ...... 0.7 g true recovery and not pre-existing medium contaminants. A third Polysorbate 80 ...... 5.0 g rolled sterile bag is included as a transport device. Isolator Pack Agar ...... 15.0 g plates have been validated to protect the medium from vapor- *Adjusted and/or supplemented as required to meet performance criteria. ized hydrogen peroxide when used in an Isolator System. Directions for Preparation from Formulae Dehydrated Product Difco™ Tryptic Soy Agar with Lecithin and Polysorbate 1. Suspend 45.7 g of the powder in 1 L of purified water. 80 (Microbial Content Test Agar) Mix thoroughly. Approximate Formula* Per Liter 2. Heat with frequent agitation and boil for 1 minute to Pancreatic Digest of Casein ...... 15.0 g completely dissolve the powder. Soy Peptone ...... 5.0 g Sodium Chloride ...... 5.0 g 3. Autoclave at 121°C for 15 minutes. Cool to approximately Lecithin ...... 0.7 g 45°C. Polysorbate 80 ...... 5.0 g Agar ...... 15.0 g

581 Section III T TSA with Lecithin & Poly 80, cont.

4. In RODAC plates, use 16.5-17.5 mL per plate. Expected Results 5. Test samples of the finished product for performance using Because interpretations are relative, each laboratory should stable, typical control cultures. establish its own values for what constitutes a clean area. Procedure Count all developing colonies. Spreading colonies should be 100 × 15 mm and 150 × 15 mm-Style Plates counted as one but care should be taken to observe other distinct colonies intermingled in the growth around the plate periphery 1. If specimen is being cultured from a swab, roll the swab or along a hair line. These should also be counted as one colony, directly on the medium surface. as should bi-colored colonies and halo type spreaders. 2. Incubate all plates at 35-37°C for 48 hours, and 25°C for 7 days or as required. It is generally agreed that 200 colonies is the approximate 3. When incubation has been completed, count the colonies. maximum that can be counted on contact plates. RODAC™/Contact Plates Colony counts may be recorded by: Selected surfaces are sampled by firmly pressing the agar 1. Simply keeping individual counts. medium against the test area. Hold the plate with thumb and 2. Number of viable particles per square foot (agar area is second finger and use index finger to press plate bottom firmly 3.97 square inches). against surface. Pressure should be the same for every sample. 3. Means and standard deviations. Do not move plate laterally; this spreads contaminants over the agar surface making resolution of colonies difficult. Slightly Subculture colonies of interest so that positive identification can curved surfaces may be sampled with a rolling motion. be made by means of biochemical and/or serological testing. Areas (walls, floors, etc.) to be assayed may be divided into Limitation of the Procedure sections or grids and samples taken from specific points within The effectiveness of preservative neutralization with this the grid. medium depends on both the type and concentration of the Grid method: preservative(s). 1. Subdivide surface (floor or wall) into 36 equal squares per References 100 square feet of area by striking five equidistant dividing 1. Vesley and Michaelson. 1964. Health Lab. Sci. 1:107. 2. Pryor and McDuff. 1969. Exec. Housekeeper, March. lines from each of the two adjacent sides. 3. Dell, L. A. 1979. Pharm. Technol. 3:47. 2. These dividing lines intersect at twenty-five points. 4. Hickey, Beckelheimer and Parrow. 1993. In Marshall (ed.), Standard methods for the examination of dairy products, 16th ed. American Public Health Association, Washington, D.C. 3. Number these intersections consecutively in a serpentine 5. Orth. 1993. Handbook of cosmetic microbiology. Marcel Dekker, Inc., New York, N.Y. 6. Hall and Hartnett. 1964. Public Health Rep. 79:1021. configuration. 7. McGowan. 1985. In Lennette, Balows, Hausler and Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 4. Use red numerals for odd numbers, black numerals for even 8. Bryan. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C. numbers. 9. Favero, Gabis and Vesley. 1984. In Speck (ed.), Compendium of methods for the microbiological 5. Omit number 13 which falls in the center of the total area. examination of foods, 2nd ed. American Public Health Association, Washington, D.C. 10. Quisno, Gibby and Foter. 1946. Am. J. Pharm. 118:320. 6. Sample odd points at one sampling period, even points at 11. Erlandson and Lawrence. 1953. Science 118:274. 12. Sveum, Moberg, Rude and Frank. 1992. In Vanderzant and Splittstoesser (ed.), Compendium of the next sampling period. methods for the examination of foods, 3rd ed. American Public Health Association, Washington, D.C. 7. For areas greater than 100 square feet, extend grid to in- 13. Association for the Advancement of Medical Instrumentation. 1984. Process control guidelines for clude entire area. gamma radiation sterilization of medical devices. AAMI, Arlington, Va. 8. For areas smaller than 25 square feet, divide the areas into Availability twenty-five equal squares (sixteen intersections). Sample Difco™ Tryptic Soy Agar with Lecithin and eight even-numbered or odd-numbered intersections at each Polysorbate 80 (Microbial Content Test Agar) sampling period. Cat. No. 255320 Dehydrated – 500 g* 9. For areas between 25 and 100 square feet, divide into 36 255310 Dehydrated – 2 kg* equal squares as in #1. BBL™ Trypticase™ Soy Agar with Lecithin and 10.Mark plates with intersection numbers. Polysorbate 80 Cat. No. 211764 Dehydrated – 500 g* Incubate exposed plates at 35-37°C for 48 hours, and 25°C 212263 Dehydrated – 5 lb (2.3 kg)* for 7 days or as required. 296033 Dehydrated – 25 lb (11.3 kg)*

582 TSA Blood Agars

United States and Canada Europe Cat. No. 221943 Prepared Plates (Double Bag) – Ctn. of 100* Cat. No. 254038 Contact Plates – Pkg. of 33*

221945 Contact Plates (Double Bag) – Pkg. of 20* ™ ™ 221288 Prepared RODAC™ Plates – Pkg. of 10* BBL Trypticase Soy Agar with Penicillinase, Sterile 221287 Prepared RODAC™ Plates – Ctn. of 100* Pack 221961 Sterile Pack Contact Plates – Pkg. of 10* Cat. No. 221839 Prepared Plates – Pkg. of 10* 222208 Sterile Pack Contact Plates – Ctn. of 100* 221837 Prepared Plates (150 × 15 mm-style) – Pkg. of 5* 221238 Sterile Pack RODAC™ Plates – Pkg. of 10* 292256 Prepared Plates (Deep Fill) – Pkg. of 10* ™ 222207 Sterile Pack RODAC Plates – Ctn. of 100* BBL™ Trypticase™ Soy Agar with Lecithin, Polysorbate 80 ™ 299896 Isolator Pack RODAC Plates – Pkg. of 10* and Pencillinase, Sterile Pack 292335 Isolator Pack RODAC™ Plates – Ctn. of 100* ™ Cat. No. 221987 Contact Plates – Pkg. of 10* 292273 Sterile Pack Finger Dab Plates – Pkg. of 10* ™ ™ 221234 Prepared RODAC Plates – Pkg. of 10* 292271 Sterile Pack Finger Dab Plates – Ctn. of 100* ™ 292305 Sterile Pack Finger Dab™ Plates 292301 Prepared Finger Dab Plates – Ctn. of 100* (150 × 15 mm-style) – Pkg. of 5* *Store at 2-8°C. 292648 Isolator Pack Finger Dab™ Plates – Pkg. of 10* 292649 Isolator Pack Finger Dab™ Plates – Ctn. of 100* 292650 Isolator Pack Finger Dab™ Plates (150 × 15 mm-style) – Pkg. of 5* TSA Blood Agars Tryptic Soy Blood Agar Base No. 2 • Tryptic Soy Blood Agar Base EH • Trypticase™ Soy Agar, Modified (TSA II) Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) Trypticase™ Soy Agar with 10% Sheep Blood (TSA II) Trypticase™ Soy Agar with 5% Horse Blood (TSA II) ™ Trypticase Soy Agar with 5% Rabbit Blood (TSA II) T Intended Use Blood agar base media are specified in standard methods for Tryptic Soy Blood Agar Base No. 2, Tryptic Soy Blood Agar food testing.1,2 Base EH and Trypticase Soy Agar, Modified (TSA II) supplemented with blood are used for cultivating fastidious microorganisms and Principles of the Procedure for the visualization of hemolytic reactions produced by many The combination of casein and soy peptones in Trypticase Soy bacterial species. Agar, Modified (TSA II) renders the medium highly nutritious by supplying organic nitrogen, particularly amino acids and Summary and Explanation larger-chained peptides. The sodium chloride maintains os- The nutritional composition of TSA (Tryptic Soy Agar/ motic equilibrium. Agar is the solidifying agent. Trypticase Soy Agar) has made it a popular medium, both Tryptic Soy Blood Agar Base No. 2 and Tryptic Soy Blood unsupplemented and as a base for media containing blood. Agar Base EH are similar in composition to TSA II. These Trypticase Soy Agar, Modified (TSA II) is an improved version formulations utilize the peptones Tryptone H and Tryptone H of the original TSA formulation for use with animal blood Plus to enhance hemolysin production while minimizing supplements. With 5 or 10% sheep blood, it is extensively antagonism or loss in activity of streptococcal hemolysins. used for the recovery and cultivation of fastidious microbial species and for the determination of hemolytic reactions that Defibrinated sheep blood is the most widely used blood for 3 are important differentiating characteristics for bacteria, espe- enriching agar base media. Hemolytic reactions of strepto- cially Streptococcus species. Some investigators prefer the use cocci are proper and growth of Haemophilus hemolyticus, a of rabbit or horse blood, but Trypticase Soy Agar with 5% nonpathogen whose hemolytic colonies are indistinguishable Horse Blood is not recommended for use with throat cultures. from those of beta-hemolytic streptococci, is inhibited. Tryptic Soy Blood Agar Base No. 2 and Tryptic Soy Blood Trypticase Soy Agar with 5% Sheep Blood (TSA II) prepared Agar Base EH (enhanced hemolysis) are additional options plates provide excellent growth and beta hemolysis by when hemolytic reactions are important. Tryptic Soy Blood Streptococcus pyogenes (Lancefield group A) and also Agar Base No. 2 provides clear hemolytic reactions with group provide excellent growth and appropriate hemolytic reactions A streptococci, while Tryptic Soy Blood Agar Base EH with other fastidious organisms. This medium is suitable for provides dramatic, improved hemolysis. performing the CAMP test for the presumptive identification 583 Section III T TSA Blood Agars, cont.

Identity Specifications Identity Specifications Difco™ Tryptic Soy Blood Agar Base No. 2 or Tryptic BBL™ Trypticase™ Soy Agar, Modified (TSA II) Soy Blood Agar Base EH Dehydrated Appearance: Fine, homogeneous, free of extrane- Dehydrated Appearance: Light beige, free-flowing, homoge- ous material. neous. Solution: 4.0% solution, soluble in purified Solution: 4.0% solution, soluble in purified water upon boiling. Solution is light water upon boiling. Solution is light to medium, yellow to tan, clear to to medium amber, clear to slightly slightly hazy. opalescent. Prepared Appearance: Plain – Light to medium, yellow to tan, Prepared Appearance: Plain – Light to medium amber, clear clear to slightly hazy. to slightly opalescent. With 5% sheep blood – Bright red, With 5% sheep blood – Bright cherry opaque. red, opaque. Reaction of 4.0% Reaction of 4.0% Solution at 25°C: pH 7.3 ± 0.2 Solution at 25°C: pH 7.3 ± 0.2 Cultural Response Cultural Response BBL™ Trypticase™ Soy Agar, Modified (TSA II) Difco™ Tryptic Soy Blood Agar Base No. 2* or Tryptic Prepare the medium per label directions with 5% sheep blood. Inocu- Soy Blood Agar Base EH late and incubate at 35 ± 2°C for 18-48 hours (incubate streptococci

Prepare the medium per label directions with 5% sheep blood. Inocu- with 3-5% CO2). late and incubate at 35 ± 2°C for 18-48 hours under 5-10% CO . 2 ORGANISM ATCC™ INOCULUM CFU RECOVERY HEMOLYSIS INOCULUM HEMOLYSIS HEMOLYSIS Candida albicans 10231 103-104 Good None ORGANISM ATCC™ CFU RECOVERY TSA NO. 2 TSA EH Escherichia coli 25922 103-104 Good, within Beta 2 3 Escherichia coli 25922 10 -10 Good Beta Beta 24 hours Neisseria Listeria 2 3 meningitidis 13090 10 -10 Good None None monocytogenes 19115 103-104 Good Beta (+/-) Staphylococcus Shigella 2 3 aureus 25923 10 -10 Good Beta Beta dysenteriae 9361 103-104 Good None Streptococcus Staphylococcus 2 3 pneumoniae 6305 10 -10 Good Alpha Alpha aureus 25923 103-104 Good, within Beta Streptococcus 24 hours 2 3 pyogenes 19615 10 -10 Good Beta Beta Staphylococcus *CAMP Test – Perform using S. aureus ATCC 25923, Streptococcus sp. group B ATCC 12386 aureus 6538P 103-104 Good Beta (positive) and S. pyogenes ATCC 19615 (negative). Streptococcus pneumoniae 6305 103-104 Good, within Alpha TSA II with 5% Sheep Blood 24 hours Escherichia coli Staphylococcus aureus ATCC™ 25922 ATCC™ 25923 Streptococcus pyogenes 19615 103-104 Good, within Beta 24 hours Streptococcus pyogenes 49117 103-104 Good Beta

CAMP Test – Perform using S. aureus ATCC 25923, Streptococcus sp. group B ATCC 12386 (positive) and S. pyogenes ATCC 19615 (negative).

of group B streptococci (S. agalactiae), and for use with low concentration (0.04 unit) bacitracin discs (Taxo™ A) for presumptive identification of group A streptococci (S. pyogenes). The CAMP test is based on the formation of a zone of synergistic hemolysis at the junction of perpendicular streak inocula of Staphylococcus aureus and group B streptococci. The reaction is caused by the sphingomyelinase (beta-toxin) of S. aureus reacting with sphingomyelin in the sheep erythrocyte membrane to produce ceramide. A non-enzymatic protein (CAMP protein), produced by S. agalactiae, binds to the ceramide and leads to disorganization of the lipid bilayer of the sheep erythrocyte 4 Enterococcus Streptococcus membrane resulting in complete lysis. faecalis pyogenes ATCC™ 29212 ATCC™ 19615

584 TSA Blood Agars, cont.

Trypticase Soy Agar with 10% Sheep Blood (TSA II) prepared Directions for Preparation from plates are provided for those laboratories preferring the Dehydrated Product increased blood content. This medium is not recommended 1. Suspend 40 g of the powder in 1 L of purified water. Mix for performance of the CAMP test. Additionally, the increased thoroughly. blood content can make hemolytic reactions less distinct and 2. Heat with frequent agitation and boil for 1 minute to more difficult to read. completely dissolve the powder. Trypticase Soy Agar with 5% Horse Blood (TSA II) prepared 3. Autoclave at 121°C for 15 minutes. DO NOT OVER- plates supply both the X and V factors which are growth HEAT. requirements for certain organisms; e.g., Haemophilus 4. For preparation of blood plates, add 5-10% sterile, influenzae. Sheep and human blood are not suitable for defibrinated blood to sterile agar which has been cooled to this purpose because they contain enzymes that inactivate 45-50°C. Mix well. the nicotinamide adenine dinucleotide (NAD), which is the 5. Test samples of the finished product for performance using V factor.5 stable, typical control cultures. Defibrinated horse blood may give hemolytic reactions different Procedure 3 from sheep blood. Some streptococci (e.g., group D) give Use standard procedures to obtain isolated colonies from hemolytic reactions on horse blood, but not on sheep blood specimens. After streaking, stab the agar several times to and may be mistakenly reported as group A. If a hemolytic deposit beta-hemolytic streptococci beneath the agar surface. reaction is obtained, the organisms should be tested with Subsurface growth will display the most reliable hemolytic a Taxo A disc and grouped serologically or tested by the reactions owing to the activity of both oxygen-stable and 6 fluorescent method. Beta-hemolytic streptococci and oxygen-labile streptolysins.9 Haemophilus hemolyticus may be differentiated by perform- ± ° ing a Gram stain on a smear prepared from the colony.7 Incubate plates at 35 2 C for 18-72 hours. Since many pathogens require carbon dioxide on primary isolation, plates Defibrinated rabbit blood is also used for enriching agar-based may be incubated in an atmosphere containing approximately 8 media. Hemolytic reactions on Trypticase Soy Agar with 5% 5% CO . Rabbit Blood (TSA II) prepared plates are similar to those on 2 10 sheep blood. However, rabbit blood does not inhibit CAMP Test Haemophilus haemolyticus, a bacterium inhibited by sheep Non-hemoytic, bile-esculin negative streptococci or bacitracin- blood that produces colonies indistinguishable from those of resistant beta-hemolytic streptococci may be tested by the T beta-hemolytic streptococci. CAMP test for presumptive identification as S. agalactiae (Lancefield group B). The inoculum may be taken from an Formulae overnight broth culture or from colonies picked from a blood Difco™ Tryptic Soy Blood Agar Base No. 2 agar plate. Make a single streak of Staphylococcus aureus Approximate Formula* Per Liter ATCC 33862 across the center of a blood agar plate. If a loop Tryptone H ...... 15.0 g is used, do not use it parallel to the agar surface, since the Soytone ...... 5.0 g Sodium Chloride ...... 5.0 g streak will be too wide and the results will not be satisfactory. Agar ...... 15.0 g The streptococcal isolates to be tested are inoculated by Difco™ Tryptic Soy Blood Agar Base EH making a simple streak perpendicular to the S. aureus line Approximate Formula* Per Liter coming as close as possible (2-3 mm), but not touching it. Tryptone H Plus ...... 15.0 g Several streptococcal isolates may be tested on the same plate. Soytone ...... 5.0 g Sodium Chloride ...... 5.0 g Perpendicular streptococcal streaks should be 5-8 mm apart. Agar ...... 15.0 g Include a known S. agalactiae for a positive control and BBL™ Trypticase™ Soy Agar, Modified (TSA II) S. pyogenes as a negative control. The procedure should be Approximate Formula* Per Liter practiced with known cultures before using it to identify Pancreatic Digest of Casein ...... 14.5 g unknown isolates. Papaic Digest of Soybean Meal ...... 5.0 g Sodium Chloride ...... 5.0 g NOTE: Studies on the CAMP Test have shown that the Agar ...... 14.0 g reaction is most reliable early in the shelf life of some lots of Growth Factors ...... 1.5 g *Adjusted and/or supplemented as required to meet performance criteria. the prepared plated medium. It is recommended that S. agalactiae ATCC 12386 be included along with patient isolates to verify satisfactory performance. Incubate plates in an aerobic atmosphere at 35 ± 2°C for

18-24 hours. Do not incubate anaerobically or in a CO2 incubator. False-positive results may occur with group A strep-

tococci when incubation is in an anaerobic or CO2-enriched atmosphere.10,11 585 Section III T TSA Blood Agars, cont.

Expected Results Availability Hemolytic streptococci may appear translucent or opaque, Difco™ Tryptic Soy Blood Agar Base No. 2 grayish, small (1 mm), or large matt and mucoid (2-4 mm) BAM COMPF colonies, encircled by a zone of hemolysis. Gram stains should Cat. No. 227300 Dehydrated – 500 g 227200 Dehydrated – 10 kg be made and examined to check the macroscopic findings. ™ (Other organisms which may cause hemolysis include Listeria, Difco Tryptic Soy Blood Agar Base EH various corynebacteria, hemolytic staphylococci, Escherichia BAM COMPF Cat. No. 228300 Dehydrated – 500 g coli and Pseudomonas.) In reporting, approximate quantitation 228200 Dehydrated – 10 kg of the number of colonies of hemolytic streptococci may be BBL™ Trypticase™ Soy Agar, Modified (TSA II) helpful to the clinician. BAM COMPF • Pneumococci usually appear as very flat, smooth, translu- Cat. No. 212305 Dehydrated – 500 g 298031 Dehydrated, LitrePak™ – 20 x 1.0 L cent, grayish and sometimes mucoid colonies surrounded 221082 Prepared Pour Tubes, 20 mL – Pkg. of 10 by a narrow zone of “green” (alpha) hemolysis. 297941 Prepared Pour Tubes, 20 mL – Ctn. of 100 • Staphylococci appear as opaque, white to gold-yellow BBL™ Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) colonies with or without zones of beta hemolysis. BS10 CMPH MCM7 • Listeria produce small zones of beta hemolysis. They United States and Canada Cat. No. 221239 Prepared Plates – Pkg. of 20* may be distinguished by their rod shape in stains, and 221261 Prepared Plates – Ctn. of 100* by motility at room temperature. Europe • Haemophilus influenzae produces nonhemolytic, small Cat. No. 254053 Prepared Plates – Pkg. of 20* grayish, translucent colonies with a “mousy” odor on 254087 Prepared Plates – Ctn. of 120* Trypticase Soy Agar (TSA II) with 5% Rabbit Blood. Japan • Other organisms representing minimal flora and Cat. No. 251239 Prepared Plates – Pkg. of 20* 251261 Prepared Plates – Ctn. of 100* clinically significant isolates can also be expected to 251240 Prepared Plates – Ctn. of 200* grow on this nonselective formulation. BBL™ Trypticase Soy Agar with 10% Sheep Blood (TSA II) CAMP Test Cat. No. 221162 Prepared Plates – Pkg. of 20* A positive CAMP reaction is indicated by an arrowhead or 221260 Prepared Plates – Ctn. of 100* triangular shaped area of increased hemolysis which forms around BBL™ Trypticase™ Soy Agar with 5% Sheep Blood (TSA II)// ™ the end of the streptococcal streak line closest to the S. aureus Trypticase Soy Agar with 5% Sheep Blood (TSA II) Cat. No. 221292 Prepared I Plate™ Dishes – Pkg. of 20* growth. The streptococcal growth must be within the wide ™ ™ zone of partial hemolysis that surrounds the S. aureus growth. A BBL Trypticase Soy Agar with 5% Sheep Blood (TSA II)//Group A Selective Strep Agar with 5% negative reaction may appear as a bullet-shaped zone of slightly Sheep Blood (ssA™) increased hemolysis or as no increased hemolysis. Cat. No. 221783 Prepared Bi-Plate Dishes – Pkg. of 20* Bacitracin-negative, CAMP-positive, beta-hemolytic strepto- BBL™ Trypticase™ Soy Agar with 5% Sheep Blood cocci may be reported as presumptive group B streptococci. (TSA II)//Chocolate II Agar CAMP-positive group A species may be differentiated from BS10 CMPH MCM7 United States and Canada group B streptococci by hemolysis, bacitracin susceptibility, Cat. No. 221302 Prepared I Plate™ Dishes – Pkg. of 20* and hippurate hydrolysis. Group B streptococci tend to 221303 Prepared I Plate™ Dishes – Ctn. of 100* produce larger colonies and have less pronounced zones of Europe beta hemolysis than other beta-hemolytic strains, and some Cat. No. 251302 Prepared I Plate™ Dishes – Pkg. of 20* group B strains are nonhemolytic.9 Japan Cat. No. 251302 Prepared I Plate™ Dishes – Pkg. of 20* ™ References 251303 Prepared I Plate Dishes – Ctn. of 100* ™ ™ 1. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC BBL Trypticase Soy Agar with 5% Sheep Blood International, Gaithersburg, Md. (TSA II)//Levine EMB Agar 2. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C. BS10 CMPH MCM7 3. Vera and Power. 1980. In Lennette, Balows, Hausler and Truant (ed.), Manual of clinical microbi- Cat. No. 221286 Prepared I Plate™ Dishes – Pkg. of 20* ology, 3rd ed. American Society for Microbiology, Washington, D.C. ™ 4. Bernheimer, Linder and Avigad. 1979. Infect. Immun. 23:838. 221289 Prepared I Plate Dishes – Ctn. of 100* 5. Krumweide and Kuttner. 1938. J. Exp. Med. 67:429. 6. Vera. 1971. Health Lab Sci. 8:176. 7. Finegold and Martin. 1982. Bailey & Scott’s diagnostic microbiology, 6th ed. The C.V. Mosby Company, St. Louis, Mo. 8. Nash and Krenz. 1991. In Balows, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 9. Ruoff, Whiley and Beighton. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 10. Darling. 1975. J. Clin. Microbiol. 1:171. 11. Facklam and Washington. 1991. In Balows, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C.

586 TSA II with Ampicillin

BBL™ Trypticase™ Soy Agar with 5% Sheep Blood BBL™ Trypticase™ Soy Agar with 5% Horse Blood (TSA II) (TSA II)//MacConkey II Agar United States and Canada BS10 CMPH MCM7 Cat. No. 221372 Prepared Plates – Pkg. of 20* United States and Canada Europe Cat. No. 221290 Prepared I Plate™ Dishes – Pkg. of 20* Cat. No. 212099 Prepared Plates – Pkg. of 20* 221291 Prepared I Plate™ Dishes – Ctn. of 100* BBL™ Trypticase™ Soy Agar with 5% Rabbit Blood (TSA II) Europe Cat. No. 251290 Prepared I Plate™ Dishes – Pkg. of 20* Cat. No. 221356 Prepared Plates – Pkg. of 20* ™ ™ Japan BBL Trypticase Soy Agar (TSA II) with Defibrinated Cat. No. 251290 Prepared I Plate™ Dishes – Ctn. of 20* Sheep Blood Slant 251572 Prepared I Plate™ Dishes – Ctn. of 100* Cat. No. 220830 Prepared Slants – Pkg. of 10* 220831 Prepared Slants – Ctn. of 100* BBL™ Trypticase™ Soy Agar with 5% Sheep Blood (TSA II)//MacConkey II Agar with MUG *Store at 2-8°C. Cat. No. 221949 Prepared I Plate™ Dishes – Pkg. of 20* BBL™ Trypticase™ Soy Agar with 5% Sheep Blood (TSA II)//Chocolate II Agar//MacConkey II Agar Cat. No. 297140 Prepared Y Plate™ Dishes – Pkg. of 20* 299580 Prepared Y Plate™ Dishes – Ctn. of 100*

Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) with Ampicillin Intended Use Expected Results Trypticase Soy Agar with 5% Sheep Blood (TSA II) with Ampi- Colonies of Aeromonas may be round, raised, with an entire cillin is used for the isolation of Aeromonas spp. edge, smooth surface and may be surrounded by a zone of beta hemolysis. Gram staining, biochemical tests and other Summary and Explanation procedures should be performed to confirm findings. Aeromonas spp. are widely distributed in nature, primarily occurring in natural fresh and salt waters, where they infect Limitations of the Procedure T animals, amphibians, reptiles and fish.1 They have been recov- 1. Some diagnostic tests may be performed with the primary ered from a variety of specimens; gastroenteritis, however, is plate. However, a pure culture is recommended for identi- the most common infection associated with this organism.2 fication purposes. Consult appropriate references for detailed information and recommended procedures.1-3 Aeromonas spp. grow on standard media used for the isolation 2. Ampicillin-susceptible species of Aeromonas have been of gram-negative bacilli (e.g., blood agar and MacConkey Agar), reported.1, 4 but some strains are reported to be inhibited on selective media 2 used to isolate Salmonella, Shigella and Campylobacter. References Isolation is facilitated, however, on 5% sheep blood agar 1. Altwegg. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology, containing ampicillin as a selective agent.1 7th ed. American Society for Microbiology, Washington, D.C. 2. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby- Year Book, Inc., St. Louis, Mo. 3. Holt, Krieg, Sneath, Staley and Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology, Principles of the Procedure 9th ed. Williams & Wilkins, Baltimore, Md. The combination of casein and soy peptones in the Trypticase 4. Carnahan, Chakraborty, Fanning, Verma, Ali, Janda and Joseph. 1991. J. Clin. Microbiol. 29:1206. Soy Agar base renders the medium highly nutritious by Availability supplying organic nitrogen, particularly amino acids and BBL™ Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) larger-chained peptides. The sodium chloride maintains osmotic with Ampicillin equilibrium. Defibrinated sheep blood supplies nutrients and, Cat. No. 297346 Prepared Plates – Pkg. of 20* simultaneously, it allows detection of hemolytic reactions. *Store at 2-8°C. Ampicillin is active against gram-negative as well as gram-positive organisms, although most Aeromonas spp. are resistant to it. Agar is the solidifying agent. Procedure Use standard procedures to obtain isolated colonies from specimens. Incubate the plates in an inverted position (agar side up) at 35 ± 2°C in an aerobic atmosphere for 18-48 hours.

587 Section III T TSA II with Gentamicin Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) with Gentamicin

Intended Use fastidious organisms and to detect hemolytic reactions while Trypticase Soy Agar with 5% Sheep Blood (TSA II) with Gen- also inhibiting the growth of Haemophilus haemolyticus, a tamicin is used for the isolation of Streptococcus pneumoniae bacterium commonly found in nose and throat specimens from clinical specimens. that is indistinguishable from beta-hemolytic streptococci.1 Gentamicin is an aminoglycoside antibiotic that inhibits the Summary and Explanation growth of gram-negative bacteria. Agar is the solidifying agent. Streptococcus pneumoniae is the leading cause of bacterial pneumonia and a cause of meningitis, endocarditis, otitis Procedure media and sinusitis.1 Use standard procedures to obtain isolated colonies from specimens. Incubate the plates in an inverted position (agar side To improve the recovery of S. pneumoniae from clinical up) at 35°C in a CO -enriched atmosphere for 18-48 hours. specimens, gentamicin has been incorporated into a general 2 growth medium enriched with sheep blood. Dilworth et al. Expected Results and Sondag et al. reported that supplementing sheep blood Staphylococci and gram-negative bacteria are inhibited. Circular, agar with gentamicin increased the recovery of S. pneumoniae flat, translucent colonies surrounded by zones of alpha hemolysis from respiratory secretions by inhibiting the growth of bacteria may be presumptively identified as Streptococcus pneumoniae. that masked the presence of pneumococcal colonies.2, 3 However, when the colonies are young, they may be dome- Trypticase Soy Agar with 5% Sheep Blood (TSA II) with shaped and may be confused with viridans streptococci, which Gentamicin improves the recovery of Streptococcus will also grow on this medium. Gram staining, biochemical pneumoniae from clinical specimens by inhibiting the growth tests and serological procedures should be performed to of bacteria that could mask its presence, including staphylo- confirm findings. cocci and gram-negative bacilli. The concentration of gentamicin has been reduced to 2.5 mg/L in this medium References 1. Ruoff, Whiley and Beighton. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual because higher concentrations of gentamicin may inhibit of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 2. Dilworth, Stewart, Gwaltney, Hendley and Sande. 1975. J. Clin. Microbiol. 2:453. the growth of some strains of S. pneumoniae. 3. Sondag, Morgens, Hoppe and Marr. 1977. J. Clin. Microbiol. 5:397. Principles of the Procedure Availability The combination of casein and soy peptones renders the BBL™ Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) medium highly nutritious by supplying organic nitrogen, with Gentamicin particularly amino acids and longer-chained peptides. Sodium Cat. No. 297457 Prepared Plates – Pkg of 20* chloride maintains osmotic equilibrium. Defibrinated sheep *Store at 2-8°C. blood supplies nutrients necessary to support the growth of

Bacto™ Tryptic Soy Broth/Trypticase™ Soy Broth (Soybean-Casein Digest Medium) • Trypticase™ Soy Broth with 6.5% Sodium Chloride • Trypticase™ Soy Broth with 5% Fildes Enrichment • Bacto™ Tryptic Soy Broth without Dextrose

Intended Use Trypticase Soy Broth with 6.5% Sodium Chloride is used to Tryptic (Trypticase) Soy Broth conforms with specifications differentiate Enterococcus spp. from the Streptococcus bovis of The United States Pharmacopeia (USP). group of streptococci. Tryptic (Trypticase) Soy Broth (Soybean-Casein Digest Medium) Trypticase Soy Broth with 5% Fildes Enrichment is used for is a general purpose medium used in qualitative procedures for the cultivation of fastidious organisms; e.g., Haemophilus the cultivation of fastidious and nonfastidious microorganisms influenzae. from a variety of clinical and nonclinical specimens.

588 Tryptic Soy Broth, cont.

Identity Specifications Identity Specifications Bacto™ Tryptic Soy Broth BBL™ Trypticase™ Soy Broth Dehydrated Appearance: Light beige, free-flowing, homoge- Dehydrated Appearance: Fine, homogeneous, free of extrane- neous. ous material. Solution: 3.0% solution, soluble in purified Solution: 3.0% solution, soluble in purified water upon warming. Solution is light water upon warming. Solution is light, amber, clear. tan to yellow, clear to slightly hazy. Prepared Appearance: Light amber, clear. Prepared Appearance: Light, tan to yellow, clear to slightly Reaction of 3.0% hazy. Solution at 25°C: pH 7.3 ± 0.2 Reaction of 3.0% Solution at 25°C: pH 7.3 ± 0.2 Bacto™ Tryptic Soy Broth without Dextrose Dehydrated Appearance: Light beige, free-flowing, homoge- Cultural Response neous. BBL™ Trypticase™ Soy Broth Solution: 2.75% solution, soluble in purified water upon warming. Solution is light Prepare the medium per label directions. Inoculate and incubate aero- ° amber, clear to very slightly opalescent. bically (unless otherwise indicated) at 20-25 C for organisms marked (*) and at 33-37°C for the remaining organisms. Incubate for up to 5 Prepared Appearance: Light amber, clear to very slightly opal- days (incubate E. coli and S. aureus for 2 days). escent. Reaction of 2.75% ORGANISM ATCC™ INOCULUM CFU RESULT Solution at 25°C: pH 7.3 ± 0.2 Aspergillus niger* 16404 < 102 Growth Bacillus subtilis* 6633 < 102 Growth Cultural Response Bacteroides fragilis (anaerobic) 25285 5×102-103 Growth Bacto™ Tryptic Soy Broth Candida albicans* 10231 < 102 Growth Prepare the medium per label directions. Inoculate and incubate at Candida albicans* 2091 < 102 Growth 30-35°C for 18-48 hours (up to 72 hours, if necessary). To test for Escherichia coli 25922 < 102 Growth growth promotion according to the USP/EP, inoculate using organisms Micrococcus luteus* 9341 < 102 Growth marked with (*) and incubate at 20-25°C for 3 days and 7 days for Neisseria meningitidis (3-5% CO ) 700344 < 102 Growth bacteria and fungi, respectively (incubate B. subtilis at 20-25°C and 2 × 2 3 30-35°C). Pseudomonas aeruginosa 27853 5 10 -10 Growth T Staphylococcus aureus 25923 < 102 Growth INOCULUM USP/EP Streptococcus pneumoniae 6305 < 102 Growth ORGANISM ATCC™ CFU RECOVERY GROWTH Streptococcus pneumoniae 2 Neisseria meningitidis 13090 10-10 Fair to good N/A (anaerobic) 6305 < 102 Growth 2 Staphylococcus epidermidis 12228 10-10 Good N/A NOTE: A. niger ATCC 16404, B. subtilis ATCC 6633 and C. albicans ATCC 10231 are the USP/EP Streptococcus recommended organisms for growth promotion testing. pneumoniae 6305 10-102 Good N/A Streptococcus pyogenes 19615 10-102 Good N/A Aspergillus niger* 16404 10-102 N/A Growth Bacillus subtilis* (20-25°C) 6633 10-102 N/A Growth Bacillus subtilis* (30-35°C) 6633 10-102 N/A Growth Candida albicans* 10231 10-102 N/A Growth Bacto™ Tryptic Soy Broth without Dextrose Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-48 hours.

ORGANISM ATCC™ INOCULUM CFU RECOVERY Neisseria meningitidis 13090 30-300 Fair to good Staphylococcus epidermidis 12228 30-300 Good Streptococcus pneumoniae 6305 30-300 Good Streptococcus pyogenes 19615 30-300 Good

Uninoculated Staphylococcus Tube epidermidis ATCC™ 12228

589 Section III T Tryptic Soy Broth, cont.

Tryptic Soy Broth without Dextrose, a low carbohydrate Tryptic Soy Broth and Trypticase Soy Broth are provided formulation of Tryptic Soy Broth, is used for cultivating as prepared media in a variety of bottle styles. In addition, fastidious and nonfastidious microorganisms. Tryptic Soy Broth is provided as a Sterile Pack Bottle; i.e., the bottle has been terminally sterilized inside of autoclavable Summary and Explanation double-bags. All varieties of bottled TSB conform with Tryptic (Trypticase) Soy Broth (TSB) is a nutritious medium requirements for Ready-To-Use Media as described in the USP. that will support the growth of a wide variety of microorganisms, including common aerobic, facultative and anaerobic bacteria and Formulae fungi.1-4 Because of its capacity for growth promotion, this Bacto™ Tryptic Soy Broth formulation is included in the USP as a sterility test medium Approximate Formula* Per Liter and for use in performing microbial limit testing.5 Pancreatic Digest of Casein ...... 17.0 g Enzymatic Digest of Soybean Meal ...... 3.0 g TSB was chosen by the USDA Animal and Plant Health Inspection Sodium Chloride ...... 5.0 g 6 Dipotassium Phosphate ...... 2.5 g Service for detecting viable bacteria in live vaccines. It is used in Dextrose ...... 2.5 g the coliphage detection procedure, a methodology in Standard BBL™ Trypticase™ Soy Broth 7 Methods for the Examination of Water and Wastewater. Approximate Formula* Per Liter TSB is recommended for testing bacterial contaminants in Pancreatic Digest of Casein ...... 17.0 g cosmetics8 and complies with established standards in the food Papaic Digest of Soybean Meal ...... 3.0 g 9-11 Sodium Chloride ...... 5.0 g industry. Dipotassium Phosphate ...... 2.5 g TSB is also recommended, because of growth promotion, for Dextrose ...... 2.5 g ™ use as the inoculum broth for disc diffusion and agar dilution Bacto Tryptic Soy Broth without Dextrose Approximate Formula* Per Liter antimicrobial susceptibility testing as standardized by the Pancreatic Digest of Casein ...... 17.0 g National Committeee for Clinical Laboratory Standards Enzymatic Digest of Soybean Meal ...... 3.0 g (NCCLS).12,13 Sodium Chloride ...... 5.0 g Dipotassium Phosphate ...... 2.5 g Trypticase Soy Broth with 6.5% Sodium Chloride is used to *Adjusted and/or supplemented as required to meet performance criteria. differentiate the enterococcal species from the S. bovis group of streptococci by the 6.5% NaCl tolerance test.14 Directions for Preparation from Dehydrated Product Trypticase Soy Broth supplemented with 5% Fildes Enrich- 1. Suspend the powder in 1 L of purified water: ment provides growth factors necessary for the cultivation of Bacto™ Tryptic Soy Broth – 30 g; fastidious organisms.15 BBL™ Trypticase™ Soy Broth – 30 g; Tryptic Soy Broth without Dextrose, a modification of TSB, is Bacto™ Tryptic Soy Broth without Dextrose – 27.5 g. a basal medium to which carbohydrates may be added for use Mix thoroughly. in fermentation studies. Phenol red and other indicators may 2. Warm gently until solution is complete. also be added. 3. Autoclave at 121°C for 15 minutes. 4. Test samples of the finished product for performance using Principles of the Procedure stable, typical control cultures. Enzymatic digests of casein and soybean meal provide amino acids and other complex nitrogenous substances. Dextrose is an energy Procedure source. Sodium chloride maintains the osmotic equilibrium. Dibasic Swab specimens may be inserted into the medium after inocu- potassium phosphate acts as a buffer to control pH. lation of appropriate plated media. For liquid specimens, use The addition of 6.5% sodium chloride to Trypticase Soy Broth a sterile inoculating loop to transfer a loopful of the specimen to the broth medium. Specimens known or suspected to permits the differentiation of salt-tolerant enterococci, which contain obligate anaerobes should be inoculated near the are resistant to the high salt content, from the salt-intolerant bottom of the tube. S. bovis group and other streptococcal species. At this concentration, the sodium chloride is a selective agent that Incubate the tubes and bottles with loosened caps at 35 ± 2°C interferes with membrane permeability and osmotic and aerobically with or without supplementation with carbon electrokinetic equilibria.1 dioxide. Tubed and bottled media intended for the cultivation of anaerobes should be incubated under anaerobic conditions. Fildes Enrichment is a peptic digest of sheep blood that sup- An efficient and easy way to obtain suitable anaerobic condi- plies the X (hemin) and V (nicotinamide adenine dinucleotide, tions is through the use of BBL™ GasPak™ or GasPak EZ NAD) factors necessary for the growth of H. influenzae. anaerobic systems or equivalent alternative system. Dextrose is omitted from the formula for Tryptic Soy Broth Examine for growth after 18-24 hours and 42-48 hours of without Dextrose to permit use of the medium in fermenta- incubation. tion studies. The carbohydrate concentration used most frequently in fermentation reactions is 0.5% or 1%. 590 TSB with 0.15% Agar

For use in sterility testing, consult the USP for procedural BBL™ Trypticase™ Soy Broth (Soybean-Casein Digest details and specifications for volume of medium relative to Medium) container size.5 AOAC BAM BS10 CCAM CMPH COMPF EP ISO MCM7 NCCLS SMD SMWW USDA USP For use in the preparation of standardized inocula for antimi- Cat. No. 211768 Dehydrated – 500 g 12,13 296264 Sterile, Dehydrated – 500 g crobial susceptibility testing, refer to the NCCLS standards. 211771 Dehydrated – 5 lb (2.3 kg) 211772 Dehydrated – 25 lb (11.3 kg) Expected Results 298131 Dehydrated LitrePak™ – 20 × 1.0 L Growth in broth media is indicated by the presence of turbid- 297294 Prepared Tubes, 0.5 mL (K Tubes) – Ctn. of 100 295634 Prepared Tubes, 1 mL (K Tubes) – Ctn. of 100 ity compared to an uninoculated control. Broth cultures should 221815 Prepared Tubes, 2 mL (K Tubes) – Ctn. of 100 be held for at least a week before discarding as negative. 297979 Prepared Tubes, 3 mL (K Tubes) – Ctn. of 100 297482 Prepared Tubes, 4 mL (D Tubes) – Ctn. of 100 221715 Prepared Tubes, 5 mL (K Tubes) – Pkg. of 10 References 221716 Prepared Tubes, 5 mL (K Tubes) – Ctn. of 100 1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams & Wilkins, Baltimore, Md. 297342 Prepared Tubes, 5 mL (D Tubes) – Ctn. of 100 2. Marshall (ed.). 1993. Standard methods for the examination of dairy products, 16th ed. American 221092 Prepared Tubes, 8 mL (K Tubes) – Pkg. of 10 Public Health Association, Washington, D.C. 221093 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100 3. Forbes, Sahm and Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, Inc. St. Louis, Mo. 297354 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100 4. Fredette and Forget. 1961. The sensitivity of several media to small inocula. Extract from a paper 221823 Prepared Tubes, 15 mL (A Tubes) – Ctn. of 100 presented at the Canadian Society of Microbiology Annual Meeting, June 12-15. Kingston, Ontario, 297811 Prepared Tubes, 21 mL (A Tubes) – Pkg. of 10 Canada. 5. United States Pharmacopeial Convention, Inc. 2001. The United States pharmacopeia 25/The 297380 Prepared Bottle – 30 mL national formulary 20 – 2002. United States Pharmacopeial Convention, Inc., Rockville, Md. 299107 Prepared Bottles, 100 mL (serum bottle) – Pkg. of 10 6. Federal Register. 1992. Fed. Regist. 21:113.26. 299416 Prepared Bottles, 100 mL 7. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, D.C. (septum screw cap) – Pkg. of 10 8. Curry, Joyce and McEwen. 1993. CTFA microbiology guidelines. The Cosmetic, Toiletry and 299113 Prepared Bottles, 500 mL – Pkg. of 10 Fragrance Association, Inc., Washington, D.C. 9. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC BBL™ Trypticase™ Soy Broth with 6.5% Sodium Chloride International, Gaithersburg, Md. 10. Horwitz (ed.). 2000. Official methods of analysis AOAC International, 17th ed., vol. 1. AOAC Cat. No. 221351 Prepared Tubes (K Tubes) – Ctn. of 100 International, Gaithersburg, Md. 11. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of BBL™ Trypticase™ Soy Broth with Fildes Enrichment foods, 4th ed. American Public Health Association, Washington, D.C. 12. National Committee for Clinical Laboratory Standards. 2000. Approved standard: M2-A7. Per- Cat. No. 221403 Prepared Tubes (K Tubes) – Pkg. of 10* formance standards for antimicrobial disk susceptibility tests, 7th ed., NCCLS, Wayne, Pa. 221404 Prepared Tubes (K Tubes) – Ctn. of 100* 13. National Committee for Clinical Laboratory Standards. 2000. Approved standard: M7-A5. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Bacto™ Tryptic Soy Broth without Dextrose NCCLS, Wayne, Pa. 14. Facklam, Sahm and Teixeira. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual Cat. No. 286220 Dehydrated – 500 g of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 286210 Dehydrated – 10 kg T 15. Fildes. 1920. Br. J. Exp. Pathol. 1:129. *Store at 2-8°C. Availability Bacto™ Tryptic Soy Broth (Soybean-Casein Digest Medium) AOAC BAM BS10 CCAM CMPH COMPF EP ISO MCM7 NCCLS SMD SMWW USDA USP Cat. No. 211824 Dehydrated – 100 g 211825 Dehydrated – 500 g 211822 Dehydrated – 2 kg 211823 Dehydrated – 10 kg 290631 Prepared Bottles, 100 mL (septum screw cap) – Pkg. of 10 290612 Prepared Bottles, 90 mL (wide mouth) – Pkg. of 10 290613 Prepared Bottles, 100 mL (wide mouth) – Pkg. of 10 257213 Sterile Pack Bottles (double bagged), 100 mL – Pkg. of 10

Trypticase™ Soy Broth with 0.15% Agar

Intended Use enhances the cultivation of some microorganisms, particularly Trypticase Soy Broth (TSB) with 0.15% Agar is a general- anaerobes from root canal and other clinical specimens.1 purpose medium for cultivation of fastidious and nonfastidious microorganisms, especially anaerobic bacteria. Principles of the Procedure TSB contains enzymatic digests of casein and soybean meal to Summary and Explanation provide nitrogenous substances. Dextrose is a source of TSB is a nutritious medium that supports the growth of a wide energy, and sodium chloride provides osmotic equilibrium. variety of microorganisms, including common aerobic, anaerobic and facultative bacteria and fungi.1-3 The addition of agar

591