Difco™ & BBL™ Manual, 2nd EditionM17 Agar • M17 BrothIntended UseM17

Agar is used for isolating and enumerating lactic strepto-cocci in yogurt, cheese starters and other dairy products.M17 Broth is used for isolating lactic streptococci from yogurt, cheese starters and other dairy products.

Summary and ExplanationLactic streptococci are acid-producing bacteria. They are nutritionally fastidious and require complex culture media for optimum growth. One study showed that in a synthetic medium, all strains had an obligate requirement for at least six amino acids and three vitamins.1 These homofermentative lactic streptococci produce large amounts of acid and, in a culture medium without an adequate buffering system, the pH decreases and adversely affects growth. Lowrie and Pearce2developed M16 Medium but it lacked a strong buffering system. Terzaghi and Sandine3 worked with M16 Medium and demonstrated that the rapid drop in pH that accompanies lactic streptococcal growth can adversely affect colony size and phage plaque formation. They modified M16 Medium using disodium-

β-glycerophosphate as a buffer and called it M17. Shankar and Davies4 found that disodium-β-glycerophosphate in M17 Broth suppressed Lactobacillus bulgaricus and selectively isolated Streptococcus thermophilus from yogurt. Similar results were achieved using M17 Broth solidified with agar. The International Dairy Federation recommends M17 Agar for isolating S. thermophilus from yogurt.5 M17 Agar is a standard methods medium for isolating lactic streptococci.6

Principles of the ProcedureM17 Agar and M17 Broth contain peptones and meat derivatives as sources of carbon, nitrogen, vitamins and minerals. Yeast extract supplies B-complex vitamins which stimulate bacterial growth. Disodium-

β-glycerophosphate buffers the medium as acid is produced from fermentation of lactose. Ascorbic acid stimulates growth of lactic streptococci. Magnesium sulfate provides essential ions for growth. Agar is the solidifying agent in M17 Agar

User Quality ControlIdentity SpecificationsDifco™ M17 AgarDehydrated Appearance:Beige to medium tan, free-flowing, homo- geneous.Solution:48.25 g soluble in 950 mL purified water upon boiling. Solution is light-medium to medium amber, very slightly to slightly opalescent.Prepared Appearance:Light to medium amber, very slightly to slightly opalescent, no significant precipitate.Reaction of 48.25 g/950 mL at 25°C:pH 6.9 ± 0.2Difco™ M17 BrothDehydrated Appearance:Beige to medium tan, free-flowing, homo-geneous.Solution:37.25 g soluble in 950 mL purified water upon boiling. Solution is light-medium to medium amber, clear to very slightly opalescent.Prepared Appearance:Light medium to medium amber, clear to very slightly opalescent, no significant precipitate.Reaction of 37.25 g/950 mL at 25°C:pH 6.9 ± 0.2Cultural ResponseDifco™ M17 Agar or M17 BrothPrepare the medium per label directions with the addition of lactose. Inoculate and incubate for 40-48 hours; Lactococcus lactis subsp. cremorisat 30 ± 2°C and the remaining organisms at 35 ± 2°C.OrgAnISM ATCC™InOCULUM CFU rECOVErYLactobacillus delbrueckiisubsp. bulgaricus11842102-103None to poorLactococcus lactissubsp. cremoris19257102-103GoodLactococcus lactissubsp. cremoris9625102-103GoodStreptococcus thermophilus 19258102-103Good

FormulaeDifco™ M17 Agar Approximate Formula* Per 950 mLPancreatic Digest of Casein .........................................5.0 gSoy Peptone ................................................................5.0 gBeef Extract .................................................................5.0 gYeast Extract ...............................................................2.5 gAscorbic Acid ..............................................................0.5 gMagnesium Sulfate .....................................................0.25 gDisodium-β-glycerophosphate ...................................19.0 gAgar .........................................................................11.0 gDifco™ M17 BrothConsists of the same ingredients without the agar.*Adjusted and/or supplemented as required to meet performance criteria.Directions for Preparation from Dehydrated Product1. Suspend the powder in 950 mL of purified water. Difco™ M17 Agar – 48.25 g;Difco™ M17 Broth – 37.25 g.Mix thoroughly.2. Heat with frequent agitation and boil for 1 minute to com-pletely dissolve the powder.3. Autoclave at 121°C for 15 minutes. Cool to 50°C.4. Add 50 mL sterile 10% lactose solution and mix well.5. Test samples of the finished product for performance using stable, typical control cultures