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Bryant Burkey Brot h w it h Resazuri ne and Lact ate

Merck Microbiology Manual 12t h Edition 215

The medium is used to enumerate the spores of lactic acidfermenting Clostridia spores in silage, milk and dairy products.During m ilk ing process low num bers of butyri c acid f ermentingbacteria (BAB) origin at ing f rom silage are int roduced i nto theraw m il k. When the contami nated mil k is used for cheeseproduction , cheese brin es become contami nated wi th heatresistant Clostridi a spores. Durin g the ri pening of salt br in ed,semi- and hard cheeses (for example, Gouda, Edammer,Emm entaler, Gruyere, and Parmesan) "l ate blow in g gasogenicClostridi a ferment lactate into bu tyr ic acid, acetic acid and gas(CO2 and H2 ). The gas swell s the cheese and i s respon sibl e for adefect termed "late blowing" or butyric swelling. The blown upcheese has moreov er a bad t aste. The main species causin g t hi s

butyric swelling defect is Co. tyrobutyricum. Other Clostridiabelonging to the butyric acid fermenting bacteria (BAB) are Cl.butyricum or Cl. sporogenes. The causative Clostridia spp. areanaerobi c Gram- positiv e mi croorgani sms form in g heat resistantendospores, whi ch surviv e pasteurisation but n ot UHT orsteri l isat ion of m il k.

M ode of Act ionVegetativ e cells are kil led by a heat tr eatment (75 C for 10 m in .).Resazurin is a redox indicator and monitors the oxygen level.The nutr i ent composit i on of the basal medium, part icularly thehigh quality of the peptones creates the conditions for a rapidgrowt h of l actate fermentin g Clostridi a spp. Sodium acetatepromot es the spore germi nati on, wh ich i s activ ated by the heat

treatment of the sample. Lactateis the substrate for the Clostridiaspp. producin g gas. A strong gas producti on i s visualised by t heraising of t he paraff in pl ug.

Typical Composition (g/litre)Peptone from casein 15.0; yeast extract 5.0; meat extract 7.5;sodiu m acetate 5.0; cysteine HCI 0.5; r esazurin 0.0025; calciu mlactate 5.0.

PreparationDissolve 38 g in 1000 ml of demin. water

Au tocl ave (15 mi n. at 121C).

Cool to 45- 50 C and di spense in bottl es or t ubes.

pH: 5.9 0.1 at 25 C.

The prepared non-boiled broth is pink and the boiled broth iscolourl ess. A pi nk col our i ndi cates the presence of ox ygen.

Expermi ment al Procedure and Evaluat ionThe MPN method is employed in the examination on lactatefermenting Clostridia spp. Tubes of medium are boiled whereappropr iat e (100C for 10 mi n.) to regenerate anaerobiosis andcooled dow n to 25 - 30 C. Colour less tu bes are inocul ated wi thsample or sample dilut ions and overlaid wi th 2cm of steri le(121C for 2 0 mi n.) melt ed (58-60 C) paraf fi n. The tubes areheat treated (75C for 1 0 mi n.) to kil l v egetati ve microo rgani smsand cooled down to 37C to solidi fy t he paraff in .

The inoculated medium is incubated at 37 C for up t o 7 days.The tubes are evalu ated every 48 h. Tubes wi th grow th and gasformation indicated by a raised paraffin plug are considered

positiv e. The M PN index i s used to calculate the number ofClostr idia.

Further biochemical i denti fi cation v erfies the presence of Cl.tyrobutyr icum.

LiteratureBergere, J.L. 1979 Development de l'ensilage. Ses consequences sur la qualitedu l ait et des produit s laiters. Revue laiteri e Francaise.

Bergere, J.L. et al. 1968 Les Clostri dium do group bu tyr ique dans les produi tslaiters. Ann. Institut Pasteur Li l le 19, 41-54 .

Bry ant, M.P. & Burk ey, L.A.: 1953 Cult ural meth ods and some characteristi csof some more numerous gropus of bacteria wi th bov ine rum en. J DairyScience 36 , 205-217.

Cerf , O. & Bergere, J.L. 1968 La num eration des spores de Clostri dium et sonapplication au lait et aux produi ts laiters. Numeration des differents groupsde Clostridium. Le Lait 48 , 501-509.

Franknet, J & de Carheil, M . 1983 Les tests de contr ole des germ es buty ri ques.La technique laiterie 977 , 15-28.

Ordering Informati on

Product M erck Cat . No. Pack size

Bryant Burkey Broth w ithResazuri ne and Lactate

1.01617.0500 500 g

Paraff in 1.07158.1000 1 kg

good growthgasformation (+)

no growt h

M edium f or the select ive enr ichment of lact ate ferment ing Clostr idia spp. (Cl. t ryobutyr i cum) w hich are responsible for" late blowing" in br ine salted semi-hard cheese

Bryant Burkey Broth wit h Resazurine and Lactate

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216 Merck Microbiology Manual 12t h Edition

Bryant Burkey Brot h w it h Resazuri ne and Lact ate

Quality control

Test st rains Growth Gas For m at i on

Clostridium tyrobutyricum W 7 good / very good +

Clostridium tyrobutyricum DSMZ 663 good / very good +

Clostridium perfringens ATCC 10543 good / very good +

Escherichia col i ATCC 25922 good / very good - (poor)

Staphylococcus aureus ATCC 25923 good / very good -

Pseudomonas aeruginosa ATCC 27853 none -