TRYPTIC SOY AGAR - For in vitro use only - Plated Media Tubed Media PT80 –Tryptic Soy Agar (TSA) TT80 – TSA Slant PT81 – TSA (SXT) TT80-18 – TSA Pour Plate [18-mL] PT89 – TSA w Yeast Extract TB75 – TSA Blood Slant PB75 – TSA w 5% Sheep Blood PB81 – TSA w 7% Sheep Blood PB69 – TSA w 5% Horse Blood PB80 – TSA w 7% Horse Blood Tryptic Soy Agar (TSA) is a general purpose The CAMP test can also be used to help identify plating medium used for the isolation, cultivation, pathogenic species of Listeria . and maintenance of a variety of fastidious and non- TSA with horse blood is used to isolate more fastidious microorganisms. fastidious organisms. Horse blood contains both X Leavitt et al. demonstrated the versatility of and V factor, which are essential growth factors for TSA by cultivating both aerobic and anaerobic some organisms such as Haemophilus species. microbes using TSA. TSA is recognized and Sheep and human blood are not suitable since they recommended by numerous agencies around the contain specific enzymes that inactivate V Factor. world. Our standard formulation is prepared Although, some laboratories prefer a plated according to the United States Pharmacopeia medium with a higher blood content (7-10%) or (USP) and recommended for various different with horse blood, these mediums should not be applications put forth by the Association of used for determination of hemolytic reactions or Official Analytical Chemists (AOAC), the for the CAMP test. The increased blood content International Dairy Federation (IDF), the United can make hemolytic reactions less distinct and States Department of Agriculture (USDA), and the more difficult to read while defibrinated horse American Public Health Association (APHA). blood, in some instances, has shown to give Tryptic Soy Agar is a highly nutritious base hemolytic reactions different from sheep blood. that meets the growth requirements of many types TSA supplemented with yeast extract is of microorganisms including bacteria, yeasts, and described in the FDA Bacteriological Analytical molds. Many modifications have been to the TSA Manual for the isolation and purification of formulation to increase both its nutritious and Listeria monocytogenes as well as other selective properties. TSA with 5% defibrinated heterotrophic organisms. Gunn, Ohashi, Gaydos, sheep blood is used extensively for the cultivation and Holt developed the selective SXT formulation and recovery of fastidious microbial species and by adding the selective agents sulfamethoxazole for the determination of hemolytic reactions that and trimethoprim. They found that this medium are important differential characteristic especially gave superior isolation of group A and B among the streptococci. This medium is also streptococci from throat specimens by inhibiting suitable for performing the overnight CAMP test; the growth of the normal throat flora. Group B streptococci produce an extracellular Plain TSA can also be used in the substance (CAMP factor) that can act differentiation of Haemophilus species when used synergistically with the beta-toxin produced by in conjunction with X, V, and XV factor disks. some Staphylococcus aureus strains. When placed Differentiation is based on the growth pattern in close proximity to one another the two around the various disks. organisms produce a zone of increased hemolysis. Formulation per Litre of Medium environment at 35°C (plates should be inverted). PT80 & TT80 Tryptic Soy Agar 5. Examine plates and tubes after 18 to 24 hours Pancreatic Digest of Casein ........................... 15.0 g and at 48 hours. Papaic Digest of Soybean Meal ....................... 5.0 g Sodium Chloride .............................................. 5.0 g CAMP Procedure Agar ................................................................ 15.0 g 1. Allow medium to adjust to room temperature pH 7.3 ± 0.2 and ensure that the plate surface is dry prior to inoculation. Additional Ingredients per Liter: 2. Obtain a pure overnight culture of Staphylococcus aureus ATCC 25923 or 33862. PB75 & TB75 TSA with 5% Sheep Blood With an inoculating needle or edge of a loop Defibrinated Sheep Blood .......................... 50.0 mL streak Staphylococcus in a straight line across the center of the plate. PB69 TSB with 5% Horse Blood 3. Streak test organism in a straight line 2 to 3cm Defibrinated Horse Blood .......................... 50.0 mL long and perpendicular to the staphylococci streak. The line should come close (approx PB80 TSA with 7% Horse Blood 3mm) but not touch the staphylococci streak. Defibrinated Horse Blood .......................... 70.0 mL Four test streaks can be performed on each plate although one of the streaks should be a PB81 TSA with 7% Sheep Blood known positive ( Streptococcus agalactiae Defibrinated Sheep Blood .......................... 70.0 mL ATCC 12386). 4. Label the streaks on the bottom of the plate PT81 TSA (SXT) (media side). Defibrinated Sheep Blood .......................... 50.0 mL 5. Incubate plates aerobically in an inverted Sulfa methoxazole ........................................23.75 µg position at 35 °C. Trimethoprim ............................................... 1.25 µg 6. Examine plates after 18 to 24 hours. PT89 TSA with Yeast Extract Yeast Extract .................................................... 6.0 g Interpretation of Results TSA with 5% Defibrinated Sheep Blood is Recommended Procedure commonly used as a primary plating medium. (Please refer to appropriate literature for a more Primary isolation is performed to separate and detailed procedure) isolate organisms present in a specimen. This separation allows for characterization of colony 1. Allow medium to adjust to room temperature types and may indicate the presence of clinically prior to inoculation. significant bacteria. When examining primary 2. Inoculate by performing a four-quadrant streak plates a hand lens or stereoscopic microscope on the plated media to obtain well-isolated should be available for examining very small colonies. For tubed media, streak the surface colonies. The different types of colonial of the medium in a fishtail motion from bottom morphology appearing on the agar plate should be up. noted as well as the number of each morphotype 3. For TSA with blood, several stabs should be present. Hemolysis is a useful differential made into the medium during inoculation to characteristic that is best viewed when a bright better detect beta-hemolysis reactions. light is transmitted from behind the plate. Four 4. Incubate aerobically or in CO 2–rich different types of hemolysis can be described: 1. Alpha-hemolysis ( α) – Partial hemolysis that Quality Control results in a greenish discoloration around the colony After checking for correct pH, colour, depth, 2. Beta-hemolysis ( β) – Complete lysis of red and sterility, the following organisms are used to blood cells resulting in a clear zone around the determine the growth performance of the colony completed medium. 3. Gamma-hemolysis ( γ) – No hemolysis resulting in no change in the medium Organism Expected Results α′ 4. Alpha-prime-hemolysis ( ) – A small zone of TSA complete hydrolysis that is surrounded by an Pseudomonas aeruginosa Growth area of partial hemolysis ATCC 27853 Streptococcus pneumoniae Growth Additional results such as pigment production ATCC 6305 and odor should also be recorded. The CAMP test can be used to presumptively TSA w/ 5% Sheep Blood identify group B streptococci ( S. agalactiae ). A Streptococcus pneumoniae Growth, α-hemolysis positive CAMP reaction is defined by the ATCC 6305 production of a distinct arrowhead zone of Streptococcus pyogenes β complete hemolysis at the point of intersection Growth, -hemolysis, ATCC 19615 − between the test streak and the S. aureus streak. CAMP ( ) β The hemolysis reaction must extend throughout the Streptococcus agalactiae Growth, -hemolysis, + depth of the agar plate. A negative CAMP reaction ATCC 12386 CAMP ( ) is no arrowhead phenomenon or a slight increased Escherichia coli Growth zone of hemolysis but with no arrowhead ATCC 25922 formation. Some organisms such as group A streptococci may show increased hemolysis at the TSA (SXT) zone of intersection. Streptococcus agalactiae Growth Additional tests should be performed on ATCC 12386 isolated colonies from pure culture in order to Streptococcus pyogenes Growth complete identification. ATCC 19615 Streptococcus pneumoniae Inhibition • For TSA with sheep blood, stabbing into the ATCC 6305 medium during inoculation creates an area of Escherichia coli Inhibition reduced oxygen tension that is necessary for ATCC 25922 hemolysis by oxygen-labile hemolysin O TSA w/ Yeast Extract • Before performing the CAMP test, a beta- Listeria monocytogenes Growth hemolytic organism must be presumptively ATCC 19114 identified as a member of the genus Streptococcus by a catalase test and gram TSA w/ Horse Blood stain Haemophilus influenzae Growth • Bacteria, other than group B streptococci, may ATCC 10211 give a positive CAMP reaction, such as Haemophilus haemolyticus Growth Pasteurella haemolytica, Listeria ATCC 33390 monocytogenes, Burkholderia pseudomallei, Corynebacterium renale, Mobiluncus mulieris, Mobiluncus curtsii, and Propionibacterium Vol I. Baltimore: Williams & Wilkins, 1985. Storage and Shelf Life 7. Isenberg HD, Ed. Clinical microbiology procedures handbook. Washington, DC: Our various Tryptic Soy Agar formulations ASM, 1992. should be stored away from direct light at 4 °C to 8. Orth DS. Handbook of cosmetic 8°C. For plated media,