TRYPTIC SOY BROTH with FERROUS SULFATE (7739)

Intended Use Tryptic Soy Broth with Ferrous Sulfate is used in the pre-enrichment of Salmonella for shell and liquid 1 whole eggs as described in FDA/Bacteriological Analytical Manual. Product Summary and Explanation Tryptic Soy Broth is a general purpose medium used for the cultivation of a wide variety of microorganisms, 2,3,4 and conforms to Harmonized USP/EP/JP Requirements. Tryptic Soy Broth, is commonly referred to as Soybean-Casein Digest Medium, and abbreviated as TSB. Tryptic Soy Broth complies with established 5,6 standards in the food industry. Tryptic Soy Broth with Ferrous Sulfate is used in the FDA/Bacteriological Analytical Manual (BAM) Method 1 for the Identification and Isolation of Salmonella in Eggs. TSB with Ferrous Sulfate is used in the Pre1 Enrichment Step for Shell Eggs and Liquid Whole Eggs (Homogenized). Egg-associated illness caused by 7 Salmonella is a serious public health problem. Salmonella enteritidis has been implicated in egg-associated illness, and can cause abdominal cramps, fever, and diarrhea beginning 12 to 72 hours after consuming a 7 8 contaminated food or beverage. Salmonella enteritidis can be found inside eggs that appear normal. Eggs 8 in the shell become contaminated on the farm, primarily because of an infection in the laying hens. Principles of the Procedure Enzymatic Digest of Casein and Enzymatic Digest of Soybean Meal are nitrogen sources in Tryptic Soy Broth with Ferrous Sulfate. Dextrose is the carbon energy source that facilitates organism growth in this medium. Sodium Chloride maintains osmotic balance and Dipotassium Phosphate is a buffering agent. Ferrous Sulfate is added to assist in the recovery of injured target Salmonella strains. Formula / Liter Enzymatic Digest of Casein ................................................. 17.0 g Enzymatic Digest of Soybean Meal ....................................... 3.0 g Sodium Chloride .................................................................... 5.0 g Dipotassium Phosphate ......................................................... 2.5 g Dextrose................................................................................. 2.5 g Ferrous Sulfate .................................................................. 0.035 g Final pH: 7.3 0.2 at 25C
Formula may be adjusted and/or supplemented as required to meet performance specifications.

Precautions 1. For Laboratory Use. 2. IRRITANT. Irritating to eyes, respiratory system, and skin. Directions 1. Suspend 30 g of the medium in one liter of purified water. 2. Autoclave at 121C for 15 minutes. Quality Control Specifications Dehydrated Appearance: Powder is homogeneous, free flowing, and light beige. Prepared Appearance: Prepared medium is clear to brilliantly clear, no to light precipitate, and yellow to amber.

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Cultural Response: Cultural response in Tryptic Soy Broth with Ferrous Sulfate follows the BAM Protocol for the isolation of Salmonella enteritidis. Cultures were enriched for 24 hours in TSB with Ferrous Sulfate and transferred to Tetrathionate Broth (7241) and Rappaport-Vassiliadis R10 Broth (7510). Cultures were incubated for 24 hours at the appropriate temperature and atmosphere, and transferred to XLD Agar (7166). The inoculated XLD Agar was incubated at the appropriate atmosphere and temperature, and examined for growth at 18 24 hours. Microorganism Salmnoella enteric subsp. Enterica serovar Enteridis ATCC 13076 Escherichia coli ATCC 25922 Approx. Inoculum (CFU) 10 - 300 Expected Growth Growth on XLD; confluent growth, red colonies w/ black centers; isolated colonies yellow w/ black centers. Suppressed to inhibited; if recovered on XLD, yellow colonies bile precipitate.

10 - 300

Test Procedure For complete details of the procedure, refer to the FDA Bacteriological Analytical Manual, Identification and 1 Isolation of Salmonella in Eggs. FDA/BAM Method The following methods are based on the analysis of a 25 gram analytical unit at a 1:9 sample/broth ratio. Depending on the extent of compositing, add enough broth to maintain this 1:9 ratio unless otherwise indicated. For samples not analyzed on an exact weight basis, refer to the specific method for instructions. Pre-Enrichment for Shell Eggs Remove material from shell surface. Disinfect eggs with 3:1 solution consisting of 3 parts of 70% alcohol to 1 part iodine/potassium iodide solution. Submerge eggs in disinfection solution for at least 10 seconds. Eggs with chipped, cracked or broken shells should not be included in the sample. Each sample should consist of 20 eggs cracked aseptically into a Whirl-Pak bag, for a total of 50 samples per poultry house. Hold samples at room temperature for 96 2 hours. After 96 hours, remove 25 mL portion from each sample of pooled eggs. Pre-enrich 25 mL test portion in 225 mL Tryptic Soy Broth with Ferrous Sulfate and mix well. Let stand for 60 minutes at room temperature. Mix well and determine pH. Adjust pH, if necessary, to 6.8 1 02. Incubate 24 2 hours at 35C. Proceed with incubation step in the FDA/BAM Manual. Pre-Enrichment for Liquid Whole Eggs (Homogenized) Combine 15, 25 mL test portions into a 375 mL composite contained in a 6 L flask. Hold composites at room temperature for 96 2 hours. After 96 hours, add 3,375 mL Tryptic Soy Broth with Ferrous Sulfate and mix well. Let stand for 60 minutes at room temperature. Mix well and determine pH. Adjust pH, if necessary, to 6.8 02. Incubate 24 2 hours 1 at 35C. Proceed with incubation step in the FDA/BAM Manual. Results 1 Refer to confirmatory results in the FDA Bacteriological Analytical Manual (BAM). Storage Store sealed bottle containing the dehydrated medium at 2 - 30C. Once opened and recapped, place container in a low humidity environment at the same storage temperature. Protect from moisture and light by keeping container tightly closed.
1

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Expiration Refer to expiration date stamped on the container. The dehydrated medium should be discarded if not free flowing, or if the appearance has changed from the original pale to light beige. Expiry applies to medium in its intact container when stored as directed. Limitation of the Procedure Due to nutritional variation, some strains may grow poorly or fail to grow on this medium. Packaging Tryptic Soy Broth with Ferrous Sulfate

Code No. 7739A 7739B 7739C

500 g 2 kg 10 kg

References
1. http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManual/BAM/default.htm 2. United States Pharmacopeial Convention. 2007. The United States pharmacopeia, 31st ed., Amended Chapters 61, 62, 111. The United States Pharmacopeial Convention, Rockville, MD. 3. Directorate for the Quality of Medicines of the Council of Europe (EDQM). 2007. The European Pharmacopoeia, Amended Chapters 2.6.12, 2.6.13, 5.1.4, Council of Europe, 67075 Strasbourg Cedex, France. 4. Japanese Pharmacopoeia. 2007. Society of Japanese Pharmacopoeia. Amended Chapters 35.1, 35.2, 7. The Minister of Health, Labor, and Welfare. 5. Vanderzant, C., and D. F. Splittstoesser (eds.). 1992. Compendium of methods for the microbiological examination of foods, 3 rd ed. American Public Health Association, Washington, D.C. 6. Cunnif, P. 1995. Official methods of analysis AOAC International, 16th ed. AOAC International, Arlington, VA. 7. http://www.cdc.gov/ncidod/dbmd/diseaseinfo/salmet_g.htm 8. http://www.fda/gov/NewsEvents/newsroom/PressAnnouncements/ucm218461.htm

Technical Information
Contact Acumedia Manufacturers, Inc. for Technical Service or questions involving dehydrated culture media preparation or performance at (517)372-9200 or fax us at (517)372-2006.

PI 7164, Rev New, October 2010