et g., 1957). C l a s s i f i c a t i o n of t h e s e gram-positive, c a t a l a s e - p o s i t i v e

Transcription

1 342 MFTHODOLQGY FOR STAPHYLOCOCCUS AU WJS AND CUIZX'RIDIUM PERF'RINGE:NSK H W WALKER AND C R EU3Y Iowa S t a t e University The staphylococci and micrococcihave been-placed i n t h e family Micrococcaceae i n "Bergey ' s Manual of Determinative Bacteriology" (Breed et g, 1957) C l a s s i f i c a t i o n of t h e s e gram-positive, c a t a l a s e - p o s i t i v e cocci i n t h i s family is s t i l l somewhat c o n t r o v e r s i a l I n 1965, however, the I n t e r n a t i o n a l Subcommittee on Staphylococci and Micrococci proposed a standard t e s t f o r separating t h e s e organisms i n t o t h e twcj genera :,f Staphylococcus and Micrococcus According t o t h i s cormnittee, t h e genus Staphylococcus should contain the p a r a s i t i c, facultative-anaerobic cocci which produce a c i d from glucose under anaerobic conditions, and t h e genus Micrococcus should contain t h e saprophytic, aerobic cocci which produce a c i d from glucose a e r o b i c a l l y, but not a n a e r o b i c a l l y Our i n t e r e s t is mainly with the genus Sta h lococcus which c o n s i s t s of two well accepted species, S aureus and S e$ idermidis =-7-Breed e t a l, 1957) These tw:, s p e c i e s a r e separgtted as follows: S aureus ferments mannitol and i s coagulase p o s i t i v e, while S epidermidis does n o t ferment mannitol and i s coagulase negative S aureug produces a v a r i e t y of e x t r a c e l l u l a r products, among which i s enterotoxin, a h e a t - s t a b l e exotoxin that causes a severe type of foqd poisoning i n man and ~ o m eo t h e r animal s p e c i e s Animal assays are not convenient f o r testing f o r s t r a i n s t h a t produce this toxin; t h e r e f o r e, other simplified means of d e t e c t i n g pathogenic s t r a i n s have been used o r suggested Coagulase production i s probably the most extensively used t e s t f o r c o r r e l a t i o n with e n t e r o t o x i g e n i c i t y (Chapman, 1944; Morrison, 1962); t h i s enzyme causes t h e formation o f a c l o t o r coagulum i n c i t r a t e d o r oxalated blood plasma According t o I v l e r (197), approxbnately 97$ of t h e staphylococci associated w i t h pathological symptoms i n man a r e capable of elaborating this enzyme Other c h a r a c t e r i s t i c s that have been c o r r e l a t e d with a b i l i t y t o produce enterotoxin have been production of hemolysin and a gdlden pigment (Feldman, 1946), mannitol fermentation under anaerobic c m d i t i o n s ( J o s h i and Dale, 1963); g e l a t i n l i q u e f a c t i o n (Stone, 1935): p r d u c t i m of deoxyribonuclease ( I e c h i c a e t a1,*7 1969, 1971; Brandish and ' i l l i s, i97; Jay, 1962) and production of p r o t e i n A, an e x t r a c e l l u l a r substance formed by staphylococci (Forsgren, 197; Jay, 1971) - * Journal Paper No J-7292of t h e Iowa Agriculture and Home Economics 2xpcriment S t a t i o n, Ames, Iowa P r o j e c t No 1896 Presented a t t h e 25th Annual Reciprocal Meat Conference :,f the American Keat Science Association, 1972

2 343 Exceptions t o a l l t h e s e i n d i c a t o r s of enterotoxin production have been observed For example, Bergdoll e t a l (1967) and Thatcher and Simon (1956) have observed staphylococcal i s o l a t e s t h a t were coagulase-negative but prcauced enterotoxin A l l coagulase-positive staphylococci a r e not necessarily pigmented (Muth, 1971) Production of g e l a t i n a s e i s a poor index of fo& poisoning p o t e n t i a l (Chinn, 1936; Clark e t a l, 1961) These and a d d i t i o n a l examples have been extensively reviewed by MFnor and Marth (1972) Evidence i n d i c a t e s a r e l a t i v e l y high c o r r e l a t i o n between enterotoxigenicity and coagulase production, deoxyribonuclease production, and production o f p r o t e i n A No one c h a r a c t e r i s t i c, however, i s i n f a l l i b l e as an index of enterotoxig e n i c i t y Franklin e t a l (1966) evaluated coagulase-mannitol agar base, deoxyribonuclease t e s t medium, t h e p h o s m t a s e t e s t, t h e fluorescein amine r e a c t i o n and t h e fibrinogen t e s t as s u b s t i t u t e s f o r t h e tube coagulase t e s t ; they found none of t h e s e diagnostic t e s t s s u i t a b l e s u b s t i t u t e s f o r t h e tube coagulase t e s t i n the i d e n t i f i c a t i o n of S aureus In t h e absence of a simple undisputed m e t h d of i d e n t i f y i n g Fnterotoxigenic s t r a i n s, t h e coagulation of plasma i s t h e most widely accepted index of t h i s c h a r a c t e r i s t i c and i s used as a c o n f i r m a t o r y t e s t f o r t y p i c a l colonies occurring on various s e l e c t i v e media f o r staphylococci The numerous media developed f o r i s o l a t i o n of staphylococci from other organisms have been c l a s s i f i e d i n t o t h r e e groups on t h e b a s i s of t h e s e l e c t i v e agents used (Jay, 1962) The f i r s t group includes those t h a t use sodium chloride as t h e s e l e c t i v e agent; l e v e l s used may vary from 55% t o l@ Several examples of this type of medium a r e : Chapman-Stone medium, MannitolS a l t agar, Staphylococcus Medium No 11, and Staphylococcus Medium S o 11 f o r t i f i e d with egg yolk The second group c o n s i s t s of media t h a t depend on various chemicals f o r t h e i r s e l e c t i v i t y Media included i n t h i s category a r e : Tellurite-Glycine agar, Azide agar; and Vogel and Johnson a g a r The t h i r d group includes media t h a t incorporate a n t i b i o t i c s, such as neomycin and polymyxin, as s e l e c t i v e agents P r a c t i c a l l y a l l these media and o t h e r s n o t l i s t e d have been used s u c c e s s f u l l y f o r t h e i s o l a t i o n of coagulasep o s i t l v e s t r a i n s of s aureus from c l i n i c a l specimens and n a s a l and s k i n swabs, but t h e y have-not always been adapted s u c c e s s f u l l y f o r use w i t h food analyses Zebovitz e t a l (1955) developed Tellurite-Glycine medium e s p e c i a l l y f o r t h e d e t e c t i o n of staphylococci i n cured meats Black colonies a r e formed on an agar medium containing t e l l u r i t e because staphylococci can a e r o b i c a l l y reduce t h e t e l l u r i t e s a l t t o elemental t e l l u r i u m This medium,, however, proved excessively i n h i b i t o r y when used f o r o t h e r f m d products (Mowe and Nelson, 1962; Donnelly e t, *,a ; Marshall e t, *,a a; Chou and Msrth, 1969) Vogel and Johnson (1$1) mqdified t h e T e l l u r i t e Glycine agar of Zebovitz and co-workers by increasing t h e mannitol content and by adding phenol red f o r a ph i n d i c a t o r - Another modification of t h e T e l l u r i t e - G l y c i n e type medium i s t h e 3gg Yolk-Tellurite-Glycine-Qruvate Agar formulated by Baird-Parker (1962) "his medium and Vogel-Johnson medium a r e two commonly used f o r enumerating staphylococci The Food and Drug Administration i n i t s B a c t e r i o l o g i c a l Analytical Manual (1969) recommended Vogel-Johnson Medium i n t h e d i r e c t

3 344 plating method f o r t h e detection and enumeration of S aureus occurring in food; however, this recommendation was changed t o BaTrd-Parker medium i n an update In 1971 The USDA a l s o uses this medium This usage gives a c e r t a i n ethtus t o t h e medium, but does not constitute endorsement a s a standard method Comparisons made on recovery of staphylococci by using various selective media have shown that Vogel-Johnson medium does not always yield the greatest percentage recovery (Sessams and Mercuri, 1969; Marshall e t a l, 1%5a; Chou and Marth, 1969) The Food Research I n s t i t u t e a t the University of Wiscmsin uses the procedure of Foster e t a l (197) f o r enumerating staphylococci This technique consists of surface-plating samples on Egg Yolk-PyruvateTellurite-Glycine Agar (Baird-Parker medium) and incubating f o r 24-3 h r a t 35C Black colonies surrounded by a c l e a r zone a r e inoculated i n t o Brain Heart Infusion o r Trypticase Soy broths and incubated overnight a t 35C Coagulase a c t i v i t y of these cultures i s then determined I n their book, "MicroorganJsms in foods; t h e i r significance and methods of enumeration," Thatcher and Clark (1968)describe the methods used by t h e Food and D r u g Administration and t h e Food Research I n s t i t u t e but a l s o recommended Baird-Parker, Egg Yolk-Azide, Milk-Salt, or Tellurite-PolyxayxinEgg Yolk agars In several comparative studies, Tellurite-Polymyxin-Egg Yolk agar gave t h e b e s t o v e r a l l recoveries (Sessoms and Mercuri, 1969; Marshall e t al, l965a; Crisley & &, 1965) Chou and Marth (1%9), however, found that d i r e c t p l a t i n g on Mannitol S a l t agar yielded b e t t e r recovery of coagulase positive staphylococci from n a t u r a l l y contaminated feed-grade meat products than d i d Tellurite-Polymyxin-Egg Yolk agar Chapman (1946) developed a medium containing mannitol, gelatin, tryptone, lactose, yeast e x t r a c t and 754 N a C l f o r i s o l a t i o n of coagulase positive staphylococci, which i s known as Staphylococcus Medium No 11 A modification o f t h i s medium, i n w h i c h t h e amount of N a C l WBS reduced t o 558 and smmonium s u l f a t e w a s added t o aid in t h e detection of g e l a t b a s e a c t i v i t y, is known as Chapman-Stone Medium Yellow or orange colonies surrounded by a c l e a r zone on t h i s medium a r e considered suspect These media o r modif'ications t o which egg yolk has been added a l s o have been used extensively for i s o l a t i o n and enumeration of staphylococci Many coagulase positive strains produce opacity as a result of l i p a s e a c t i v i t y w h e n grown Fn media containing egg ;yolk (Gillespie and Alder, 1952) A medium called Polymyxin-Mannitol-Phenolphthalein Diphosphate agar has been reported as f i e l d i n g good recoveries of coagulase-positive staphylococci i n t h e analysis of foods without the presence of f a l s e p? s i t i v e reactions ( W i l l i a m s, 1972) In t h i s method, poured agar p l a t e s +"-esurface inoculated by streaking and incubated a t 3 C f o r 4 h r When t h e p l a t e s are exposed t o t h e vapor from strong ammonia solution, coagulasepositive staphylococci appear pink, surrounded by a clear zone The theory i s t h a t only coagulase-positive staphylococci produce recognizable acid from mannitol and menolphthalein from phenolphthalein diphosphate within 4 hr a t 37C

4 34 5 A 24-hour method f o r t h e detection of coagulase-positive staphylococci i n f i s h and shrimp has been developed by I n s a l a t a e t a l (1972) i n cooperation with t h e Food and Drug Administration These authors s e t out t 3 d e v e h p a rapid screening method t h a t would y i e l d r e s u l t s within 24 h r Requirements t o be f u l f i l l e d were t h a t t h e t e s t must be d i r e c t and a b l e t o d e t e c t more than 1 coagulase-positive staphylococci per gram and be usable i n t h e microbiological f a c i l i t i e s r o u t i n e l y a v a i l a b l e i n t h e shrimp i n d u s t r y An enrichment medium c o n s i s t i n g of a blended mixture o f 27 m l of Trypticase Soy broth with 75$ N a C l added, 1 m l of B r a i n Heart Infusicn w i t h 75$ TiIaCl added and 3 g of cooked meat medium containing 25% NaCl was recommended The highest percentage of d e t e c t i o n of p o s i t i v e samples w i t h i n 24 hr a f t e r incubation a t 4OC i n the enrichment medium was obtained w i t h t h e coagulase s l i d e t e s t, which detected of t h e possible p o s i t i v e samples This t e s t can be u s e f u l f o r c e r t a i n purposes, but i t s v a l i d i t y f o r rapid evaluation of o t h e r foods needs t o be determined Many media f o r recovery and s e v e r a l i d e n t i f i c a t i o n procedures f o r pathogenic staphylococci a r e a v a i l a b l e and advantages and disadvantages have been proposed f o r each by various individuals and l a b o r a t o r i e s The most popular media a t present f o r recovery of suspect enterotoxigenic s t r a i n s seem t o be Vogler and Johnson medium and Egg Yolk-Pyruvate-TelluriteGlycine agar of Baird-Parker Suspect colonies a r e confirmed u s u a l l y by t e s t i n g f o r coagulase a c t i v i t y C r i s l e y e t a l (1965) have suggested t h a t s p e c i f i c media may give t h e b e s t recoveries f o r c e r t a i n types of foods For example, some of t h e i r c m p a r i s o n s showed t h a t Tellurite-Glyclne agar seemed b e s t f o r a n a l y s i s of chicken p i e and T e l l u r i t e - P o l w x i n - E g g Yolk agar f o r r a w egg Some o f t h e disagreement about t h e use of various media might be resolved if c o l l a b o r a t i v e s t u d i e s were i n i t i a t e d t o e s t a b l i s h acceptable o r p r e f e r r e d procedures f o r c e r t a i n foods Clostridium perfringens is a nonmotile, Gram-positive, anaerobic b a c i l l u s with b l u n t ends (Breed e t al, 1957) This organism can form spores, b u t many s t r a i n s form few, i f any, in laboratory media D i s t i n c t i v e c h a r a c t e r i s t i c s, according t o Breed e t a l a r e stormy fermentation of milk and nonmotility Hobbs (1969)has noted t h a t t h e stormy c l o t reaction associated with C perfringens i n m i l k c u l t u r e s v a r i e s g r e a t l y between strains Observ%ions in our l a b o r a t o r y support t h i s statement Reports on t h e i s o l a t i o n o f Clostridium perfringens i n d i c a t e the occurrence of t h i s organism i n a wide v a r i e t y of foods (Canada and Stson&, 1964; Hall and Angelotti, 1965; Hobbs e t al, 1965; Woodburne and K i m, 1966; Nakamura and Kelly, 1968; Mead, 1969) If a food contaminated w i t h C perfringens i s held between a temperature range of approximately 2 Knd 45C o r s l i g h t l y higher f o r a s u f f i c i e n t time, t h i s organism can multiply t o g r e a t numbers Pihen l a r g e numbers of t h e s e c e l l s a r e lngested by an individual, a mild food poisoning r e s u l t s, characterized by diarrhea and abdominal pain accompanied by a l a r g e volume of gas i n t h e i n t e s t i n e ; symptms occur a f t e r an incubation period of between 4 and 22 hr, with an average r>f 12 h r Nausea and vomiting are r a r e ; f e v e r, headache, and other s i g n s Df i n f e c t i m seldom occur (Dische and Elek, 1957; Hauschild and Thatcher, 1967; Ibbbs e t a l, 1953; Robertson, 1963) c -

5 346 I n 197, C, perfrinaens accounted f o r almost 376 of a l l cases and 15% of a l l outhreaka of food poisoning reported t o t h e Center f o r Disease Control I n the same year, Salmonella caused 2% of a l l food poisoning -s2r :; and 13$ of a l l outbreaks Staphylococcal g a s t r o e n t e r i t i s was t h e h i r d most common type of food poisoning, accounting f o r 1984 of a l l patients and nearly 28% of a l l outbreaks When t h e outbreaks were broken down by vehicles of i n f e c t i o n, beef and turkey were most frequently associated w i t h C p e r f r b g e n s food poisoning; fowl, w i t h salmonella; and pork, fowl and beef tended t o be associated with staphylococcal food poisoning Mass feeding establishments, such as r e s t a u r a n t s and school lunches -in w h i c h l a r g e numbers of people are involved, a r e frequently associated with outbreaks perfringens Food poisoning a t t r i b u t e d t o t h i s organism w a s f i r s t reported i n t h e United S t a t e s i n 1945, when McClung described f o u r outbreaks traced t o poultry dishes; l i t t l e a t t e n t i o n, however, was given t o t h i s type of food poisoning u n t i l the r e p o r t of Hobbs and Co-workers in 1953 Since t h a t time, t h i s i l l n e s s has become recognized a s one of t h e most prevalent food-borne poisonings Food samples t o be examined f o r E perfringens should be taken t o the laboratory r e f r i g e r a t e d b u t n o t frozen Freezing may d e s t r o y 97$ or more of -C perfringens vegetative c e l l s ; even r e f r i g e r a t i o n without f r e e z i n g may k i l l 976 (Food and D r u g Administration, 1969) Refrigeration should not be f o r extended periods, b u t i s useful i n preventing excessive growth of t h i s organism or overgrowth by other organisms i n t h e sample before laboratory analyses are completed Several media have been developed and recommended f o r t h e recovery and enume1,ation of C perfringens i n foods The m s t widely used media a r e a c t u a l l y modifitations of t h e medium of Wilson and B l a i r (1925); t h e s e m e d i a depend on t h e a b i l i t y of c l o s t r i d i a t o reduce s u l f i t e t o s u l f i d e, with t h e formation of black colonies a6 t h e r e s u l t of p r e c i p i t a t i o n of i r o n s u l f i d e Mossel (1959) added polymyxin B sulfate t o suppress growth of many strains of Enterobacteriaceae, which can a l s o produce black colonies on t h i s medium Angelotti e t a l (1562) modified Mossel s medium by addlng sodium s u l f a d i a z i n e and c a l l e d it Sulfite-Polymyxin-Sulfadiazine o r SPS a g a r This medium inhibited growth of Proteus spp, Pseudomonas spp, and coliforms Hall (1968) reported t h e r e s u l t s o f a c o l l a b o r a t i v e study f o r the q u a n t i t a t i o n of C perfringens using SPS agar; d e t a i l s of t h e procedure arc: reported by Angeiotti e t al (1962) and Angelotti (1964) This method has been ad3pted as o f f i c i a l, f i r s t action, by t h e Association of O f f i c i a l Analytical Chemists I n t h i s method, s u i t a b l e d i l u t i o n s of a 1:lO blend of tbe food are p l a t e d on SPS agar and incubated anaerobically f o r 24 hr a t 3 5 C Slack colonies a r e counted and recorded as t o t a l c l o s t r i d i a l count A r e p r e s e n t a t i v e number of colonies is picked t o N i t r a t e - M o t i l i t y medium and iiiioglyc9llate broth, which are incubated f o r 24 h r a t 35C Sporulation 7;;roth i s inoculated and observed f o r spore p r o d u c t b n!he presence of ncmimtile, anaerobic, spore forming organisms t h a t produce H2S and reduce C perfringens According t o Hall (1969),t h i s t e s t n i t, r a t e is t y p i c a l f o r I

6 347 has been f i e l d t e s t e d by a number of workers and found s a t i s f a c t o r y f o r t h e e x a d n a t i o n of foods involved in poisoning outbreaks Essentially, t h e same method is preeented i n the Bacteriological Analytical Manual of t h e U S Food and D r u g Administration (1969); however, t h e s u b s t i t u t i o n of TryptoneSulfite-Neowcin agar (TSN) f o r t h e SPS agar is accepted Marshall e t al (196%) developed TSN agar and claimed that incubation o f t h i s medlum%t-6;6c prevented the development of other sulfite-reducing organisms such as 2 bifermentans They reported t h e i r medium as being q u a n t i t a t i v e l y and s e l e c t i v e l y superior t o SPS agar Tolerance t o neomycin, o p t i n a l growth a t 46C and s u l f i t e - r e d u c i n g p r o p e r t i e s were used as a b a s i s f o r developing t h i s medium A medium r e f e r r e d t o as TDY-C was developed by Green and Litsky (1966 ), which t h e y used w i t h a Most Probable Number technique f o r enumeration They proposed t h e use of t h i s method f o r samples i n which t h e number of 2 perfringens i s low Recoveries w i t h t h i s technique were reported as being superior t o those observed w i t h SPS agar Another s e l e c t i v e and d i f f e r e n t i a l medium containing Tryptose, yeast e x t r a c t, f e r r i c ammonium c i t r a t e, sodium metabisulfite, polymyxin, kanamycin and egg yolk emulsion i s r e f e r r e d t o as Shahidi-Ferguson-Perfringens (SF'P) agar (Shahidi and Ferguson, 1971) Sodium m e t a b i s u l f i t e and f e r r i c ammonium c i t r a t e were added f o r demonstration of H2S production and egg yolk f o r detection of l e c i t h i n a s e a c t i v i t y Lecithinase w i l l r e a c t with egg yolk t o produce a wliitish o r opaque h a l o around t h e colony On t h i s medium, 2 Perfringens produces black colonpes surrounded by an opaque zone A comparative study between SFP agar and noninhibitory agar showed l i t t l e Fnhibition of 16 s t r a i n s o f 2 perfringens This medium w a s a l s o considered superior t o SPS a g a r The SPS medium used in t h i s work and t h e work mentioned previously was prepared commercially Quantitation may vary considerably from l o t t o l o t of c o m e r c i a l l y prepared medium (Green and Litsky, 1966; Hauschild e t al, 1967) SPS agar m i g h t have compared more favorably i f it had been f r e s h l y prepared i n the laboratory Workers a t t h e Food and D r u g Administration (Harmon e t a l, 1971) have proposed Tryptose-Sulfite-Cycloserine (TSC) agar f o r enumeration of E perfringens The medium is t h e same a s SFP agar, except 4 pg of D-cycloserine per ml w a s s u b s t i t u t e d f o r kanamycin and polymyxin Comparison o f t h e two media showed almost complete recovery of most of t h e s t r a i n s o f E perfringens t e s t e d while i n h i b i t i n g p r a c t i c a l l y a l l f a c u l t a t i v e anaerobes On t h e other hand, SF'P agar allowed a slightly higher rate of recovery of 2 perfringens, b u t was much less s e l e c t i v e Fecal s t r e p t o c o c c i can grow r e a d i l y on SPS agar and produce small w h i t r s colonies C perfringens and f e c a l s t r e p t o c o c c i can be traced t o t h c s m v source (Cooper and Ramadan, 1955; H a l l and Hanser, 1966), and streptococcal colonies f r e q u e n t l y appear in numbers equal t o o r higher than black c l o s t r i d i a l colonies This occurrence interferes with the i s o l a t i o n of presumptive colonies and r e q u i r e s p u r i f i c a t i o n of i s o l a t e s before confirmatory t e s t s can be made Fuzi and Csukas (1969) observed that s t r a i n s of C perfringens

7 3443 i s o l a t e d from human and animal f e c a l specimens were h i g h l y r e s i s t a n t t o D-cycloserine and that l a w concentrations of t h e a n t i b i o t i c were i n h i b i t o r y t o rimy types of f a c u l t a t i v e anaerobic b a c t e r i a, including s t r e p t o c o c c i Since t h e procedure of Angelotti e t a l (1962) i s probably the method m 3 s t f r e q u e n t l y used a t present i n t h e United S t a t e s f o r examination of foods, evaluation of f a c t o r s t h a t influence recovery and i s o l a t i o n of E perfringens 3x1 t h i s medium were undertaken i n our l a b o r a t o r i e s The purpose of our s t u d i e e w a s t o simplify rmd r e f i n e s e v e r a l procedures by combining t h e anaerobic-pouch technique of Blade1 and Greenberg (1965) w i t h the enumeration and i d e n t i f i c a t i o n procedures recommended by Angelotti e t a l (1962) Also, observations were made on t h e s u b s t i t u t i o n of D-cycloserine f o r p o l p y x i ~ iand s u l f a d i a z i n e f o r degree of i n h i b i t i o n o f C perfringens and for elimination o f s t r e p t o c o c c i colonies and o t h e r i n t e F f e r i n g organisms B y t h e time our s t u d i e s were completed, t h e work of H a r m m e t a l (1971)w a s published i n which t h e y recommended t h e use of D-cycloserine i n SFP medium SET agar w a s t e s t e d i n our l a b o r a t o r i e s, but did not adapt w e l l t o o u r combined techniques because t h e cloudiness produced by egg yolk i n t h e medium i n t e r f e r e d with observation o f colonies i n t h e pouches MAlTRIALS AND METHODS Twenty-two strains cf Clostridium perfringens were used i n t h i s study and included t h e following: FD1; IIobb's s t r a i n s Em, HR12, HR2, H 1 3 (NCTC 124), H4 (NCTC 8247), H9 (NCTC 8798); ATCC 3624; ATCC 1543; F42 and 2l5B obtained from D r C, Duncan, Food Research I n s t i t u t e, Madison, Wisconsin; and 12 strains I s o l a t e d in our laboratory from various p o u l t r y and meat products I n a d d i t i o n, 3 s t r a i n s of other Clostridium spp were used Seventy-five s t r a i n s of i d e n t i f i e d b a c t e r i a from various sources were used and included species of B a c i l l u s, Arthrobacter, S e r r a t i a, Salmonella, Escherichia, Enterobacter, Micrococcus, Staphylococcus, S t r e p t o c o c c u s Pseudomonas, and Proteus Also, 172 u n i d e n t i f i e d c u l t u r e s i s o l a t e d from r e f r i g e r a t e d and frozen chicken and from meat emulsion f o r f r a n k f u r t e r s were used A l l aerobes and f a c u l t a t i v e anaerobes were precultured in Brain H e a r t I n f u s i m broth (Difco); the c l o s t r i d i a were grown in f l u i d T h i o g l y c ~ l l a t e medium (Difco) a t 37C f o r 24 h r before t e s t i n g For comparative purposes, the basal ingredients 3f SPS agar c o n s i s t i n g of the following were used: Try-ptone, 15 3; Yeast e x t r a c t, 1 5 g; and i r o n c i t r a t e, 5 g These m a t e r i a l s were d i s s d v e d i n 1 l i t e r 3f d i s t i l l e d water The a g a r concentration was increased from 15% t o 2@ t o c o n t r d l i q u e f a c t i o n of t h e medium f r e q u e n t l y observed a f t e r i n c u b a t i m a t 37C for 24 h r Sodium m e t a b i s u l f i t e (1g per 1 of medium) was added t o t h e medium PefDre s t e r i l i z a t i o n Sodium m e t a b i s u l f i t e was s t a b l e t o autoclairing and could be added t o t h e medium before s t e r i l i z a t i o n Yolyqyxin B s u l f a t e (Sigma Chemical Co, S t Inuis, Mo), s u l f a d i a z i n? and D-cycloserine (Sigma) were t e s t e d f o r sel(2ctive i n h i b i t i m of' b a c t e r i a l growth The l e v e l s and combinations of antibiot,ics arc s h m n in Yable 1 Polymyxin w a s added t o the medium p r i o r t o s t e r i l i z a t i m ; su1i'ediazini was s t e r i l i z e d by f i l t r a t i o n and incorporated i n t o t h e m e d i u m j u s t ( S i p ),

8 349 TABLE 1 CONCENTRATIONS AND COMBINATIONS OF ANTIBIOTICS ESTED IN THE BASAL MEDIUM FOR SELECTION OF C PERFRINGENS - Antibiotic Level of a n t i b i o t i c i n t h e medium Polymyxin B s u l f a t e 3 u n i t s per ml Sulfadiazine ( sodium s a l t ) 12 mg per ml P1ym;yxin B s u l f a t e plus s u l f a d i a z i n e 3 units per m l D - cyc loa erine 8 pg p e r D-cycloserine 4 Polymyxin B s u l f a t e plus D-cycloserine 3 units per m l 8 pg per td Polymyxh B s u l f a t e p l u s D-cycloserine 3 units p e r ml 4 pg per m l 12 rag per m l pg tri~ per m l b e f w e p l a t i n g as recommended by Angelotti e t a l (1962) D-cycloserine was dissolved in d i s t i l l e d water (1g per 125 ml), dispensed i n screw cap tubes, and steamed f o r 2 min f o r s t e r i l i z a t i o n The a c t i v i t y o f D-cycloserine was s t a b l e f o r a t least four months when frozen and s t o r e d a t -29C The s o l u t i o n was thawed a t room temperature just before p l a t i n g ; 5 o r 1 ml o f t h e s o l u t i o n was added t o 1 l i t e r of b a s a l medium t o o b t a b a f i n a l c o n c e n t r a t i m of 4 o r 8 pg of t h e a n t i b i o t i c p e r ml The Fnhibitory a c t i o n of the a n t i b i o t i c s was t e s t e d semiquantitatively 3n a l l t h e c u l t u r e s l i s t e d p r e v i m s l y by using t h e r e p l i c a - p l a t i n g procedure of Hartman and P a t t e e (1968) Growth on t h e b a s a l medium w a s compared w i t h growth on t h e medium with a n t i b i o t i c s added The media were poured i n t o square p l a s t i c p l a t e s and allowed t o s o l i d i f y and d r y before inoculation After surface inoculation w i t h 24 h r broth cultures, the p l a t e s were allowed t o d r y f o r 1 t o 15 min a t room temperature An overlay of t h e assay medium was poured on t h e inoculated p l a t e s when anaerobic incubation was t o follow T r i p l i c a t e sets of plates were incubated a e r o b i c a l l y a t 37C f o r 24 hr; another set of t r i p l i c a t e p l e t e s was incubated under nitrogen i n an anaergbic incubator f o r t h e same time and temperature Numerical values were assigned t o d i f f e r e n t l e v e l s of growth on p l a t e s : f o r no growth; 1 f o r scant growth; 2 f o r medium growth; and 3 f o r abundant growth In addition, t h e e f f e c t of polymyxin and D-cycloserine Qn recovery of C - perfringens was t e s t e d by a n anaerobic p l a t e count using t h e pouch method of B h d e l and Greenberg (1965) Pouches were made of 75 Maraflex 55(3): 75 gauge Saran-coated Mylar-polyester w i t h 2 m i l polyethylene Oxygen

9 35 permeability of t h i s film i s 32 pl/in2/24 hr a t 1 F and 9oqb R H The film WRS provided by the American Can Co Neenah, Wisconsin 54956, Some adaptations of this plating procedure were made t o simplify and expedite handling The medium was dispensed i n t o the pouches a t melting temperatures since the film withstands boiling temperature f o r a t l e a s t 1 min; an a u t m t i c syringe with a 3-inch hypodermic needle w a s used t o deliver the medium i n t o t h e pouches The f i l l e d pouches were stored in a small incubator a t 5 C t o keep the agar liquefied u n t i l they were inoculated Three r e p l i c a t e s of 24-hr broth cultures of each of t h e 22 s t r a i n s of' C perfringens were plated In t h e basal medium containing 3 units of ~ o l y l q y x i nper m l and I n t h e basal medium containing 4 l g of D-cycloserine per m l The pouches were incubated aerobically a t 37 C f o r 24 h r since t h e pouches provided adequate anaerobic conditions Analysis of variance was performed on the data obtained by semiquantitative assays f o r evaluation of inhibitory action of the a n t i b i o t i c s cm b a c t e r i a l growth Paired + tests were made on the difference i n numbers of C perfringens recovered by p l a t e count t o evaluate the resistance of thig organism t o polylsyxin and D-cycloserine RESULTS AND DISCUSSION The analysis of variance on t h e semiquantitative assays, as might be expected, showed a significant difference between aerobic and anaerobic growth The average growth l e v e l s on t h e basal medium w i t h incubation at 37 C under aerobic conditions were compared per group of cultures t e s t e d ( f i g u r e 1) Anaerobic incubation t o t a l l y inhibited growth of Arthrobacter spp, Micrococcus lysodeikticus, and the 88 organisms isolated from frozen and refrigerated meat Growth of the strains of S e r r a t i a Escherichia, Proteus, Pseudomonas aerunrinosa, Bacillus and t h e m i s o l a t e s from frankf'urters was g r e a t l y reduced under low oxygen tension; some scant growth, however, took place w i t h mmt of these cultures Anaerobic conditions had limited or no e f f e c t on growth of Salmonella, Enterobacter and Streptococcus spp and enhanced the growth of the s t r a i n s of Clostridium t e s t e d A l l the strains from refrigerated meat and the four species of psychrop h i l i c Pseudomonas were also incubated a t 3 C Fn t h e basal medium under aerobic conditions t o confirm t h e i r v i a b i l i t y The higher temperature o f -lnciibation (37 C ) g r e a t l y inhibited growth of organisms isolated from frozen and refrigerated products The pouch method described by Blade1 and Greenberg (1965) offered r e l i a b l e and consistent anaerobic conditions f o r the cultivation of C perfringens The use of an automatic syringe t o dispense the medium-into the pouches and subsequent storage of the pouches a t 5 C u n t i l inoculation decreased the time necessary for'anaerobic plating t o a l e v e l approximting that f o r aerobic plating procedures

10 1 Comparison of aerobic and anaerobic incubation a t 37 C on growth of various groups of b a c t e r i a i n the basal SPS mdium (See Materials and Methods f o r means of evaluating l e v e l of growth) AVE Scant GROWTH LEVEL Medium 2 I TU I Abundant o I meat i so I a t e s p' se'u'd o m o n a d s b ' 4II, n-k'f'u, te s o I af es m Strafhq ococci q :::

11 352 For s u l f i t e - r e d u c i n g b a c t e r i a, most anaerobic procedures have t h e disadvantage t h a t, once t h e p l a t e s are exposed t o a i r, t h e s u l f i d e oxidizes, w i i f i subsequent fading and disappearance of t h e black p r e c i p i t a t e c h a r a c t e r i s t i c ~f t h e colonies; t h e r e f o r e, counts and i d e n t i f i c a t i o n of presumptive colonies of C perfringens from food samples have t o follow almost immediately Pouches, however, can be saved under r e f r i g e r a t i o n without d i s c e r n i b l e changes occurring; colonies in pouches s t o r e d f o r one month were s t i l l viable, w i t h no fading of the t y p i c a l zone of i r o n s u l f i d e Table 2 presents the average growth l e v e l per group of c u l t u r e s i n a l l 37 C under anaerobic conditions Sulfad i a z i n e i n h i b i t e d growth of all organisms except that of Pseudomonas and Bacillus spp Polymyxin and sulfadiazine, independently o r in combination w i t h each other, showed l i t t l e i n h i b i t i o n o f growth of Salmonella, Enterobacter, Streptococcus and t h e 84 i s o l a t e s from f r a n k f u t e r s These findings a r e i n agreement w i t h r e s u l t s reported by Fuzi and Csukas (l%9) D-cycloserine at a l e v e l of 8 pg/ml was e f f e c t i v e i n c o n t r o l l i n g growth of most o f t h e c u l t u r e s under anaerobic conditions; s t r a i n s of S e r r a t i a marcescens and Enterobacter aerogenes were capable of only s c a n t growth Growth p a t t e r n s were similar i n t h e presence of 4 pg/ml of D-cycloserine The combined a c t i o n of polymyxin and D-cycloserine did not seem t 3 improve i n h i b i t o r y activity the media t e s t e d when incubated a t Polymyxin and D-cycloserine showed no d e t e c t a b l e e f f e c t on growth of Clostridium spp when semiquantitative assays were used However, when counts were made on the basal medium by using t h e p l a s t i c pouches w i t h and without a n t i b i o t i c s, presence of 3 u n i t s of polymyxh and 4 pg of D-cycloserine per ml i n t h e recovery medium showed i n h i b i t i o n of perfringens a t t h e 995$ level when perired + t e s t s were made on the d i f f e r e n c e s i n counts D-cycloserine a t t h e two l e v e l s t e s t e d i n h i b i t e d t h e f e c a l s t r e p t o c o c c i and had a broader spectrum f o r s e l e c t i v e i n h i b i t i o n ( t a b l e 2 ) than d i d polymyxin and s u l f a d i a z i n e D-cycloserine a t the 4 pg l e v e l had no g r e a t e r i n h i b i t o r y a c t i v i t y f o r C perfringens than t h e 3 u n i t s o f polymyxin used i n SPS agar This l e v e l was considered most s u i t a b l e by us and w a s the same l e v e l recormended by Harmon e t a l (1971) f o r use in SF P medium C CONCLUSIOMS AND CDMMENTS S u b s t i t u t i o n of 4 p g of D-cycloserine per m l of SPS medium f o r polymyxin and sulfadiazine, coupled with good anaerobic conditions, provided c o n t r o l of t h e growth of f e c a l s t r e p t o c o c c i and most o f t h e comon contaminants i n food without excessive I n h i b i t i o n of t h e 22 s t r a i n s o f C perfringens t e s t e d L%e pouch method of Blade1 and Greenberg (1965) provided s a t i s f a c t o r y anaerobic conditions f o r enumeration of 2 p e r f r h g e n s The use o r pouches a l s o permitted s t o r a g e of p l a t e s a f t e r incubation u n t i l confirmatory tests mid be made This method also permitted f l e x i b i l i t y i n scheduling platrcounts and use of aerobic incubators f o r anaerobic p l a t i n g S b p l i f i c a t i m and m d i f i c a t i o n OS c e r t a i n procedures i n t h e o r i g i n a l method enabled the Labcratory worker t o do m m e analyses i n less time and made t h e pouch methsd l e s s cumbersome

12 1 a 8 r ( O O d 3 53 O d 8 8 d O O d P i t es C i r u? t- '? ln 8 d O O d O d t- u? t- rldddd?a??u?? r ( r ( O d?c9 v? 8! rl!! I ii r 5 s : b a b E 3 U?a? r l d O d " " dcul-icud d 4 3 d h r l k k u o :-$ $"ui : 3a3 (v (v E dl " 8 T? d Orl 8 ri 5: 9 9 r 1 O O d O Q) 2!! Q) $ a m P-l 3 I - l : 8 5 B a a 2 V UM

13 354 A review of the various media and techniques f o r the isolation, identification, and enumeration of staphylococci and E perfringens emphasizes the need f o r collaborative t e s t s f o r determining t h e acceptability o f c e r t a i n $rocedures Also, the need f o r experience in the use and knowledge of t h e variability of these methods becomes apparent These and other possible imperfections do not necessarily detract from t h e value of t h e t e s t s but emphasize the need f o r i n t e l l i g e n t i n t e r p r e t a t i o n and use of the data gained by t h e various available techniques Journal Paper No of t h e Iuwa Agriculture and Home Economics Experbent Station, Ames, Iowa; Project No 1896 This investigation was supported in part by PHS Grant No FDOdc44 W e wish t o thank P h y l l i s Rohrbaugh, Christine Pearson, and Robert Champion f o r excellent technical assistance and Roger Mracheck f o r helping w i t h t h e s t a t i s t i c a l analyses

14 355 Angelotti, R 1964 Methods f o r i s o l a t i o n and enumeration of Clostridium e r f r i n g e n s I n "Examination of Foods f o r Enteropathogenic and I n d i c a t m E a c t e r i a I' ( L e w i s, K H and R Angelotti, eds ) U S Department of Health, Education and Welfare, Public Health Service Publ 1142 p 5 Angelotti, R, H E H a l l, M J Foter and K H Lewis 1962 Q u a n t i t a t i o n of Clostridium perfringens i n foods Appl Microbiol lo:l93 Baird-Parker, A C 1962 An improved d i a g n o s t i c and s e l e c t i v e medium for i s o l a t i n g coagulase-posit i v e staphylococci J Appl B a c t e r i o l 25 :1 2 Bergdoll, M S, K F Weiss and M J Muster 1967 The production of staphylococcal enterotoxin by a coagulase-negative microorganism B a c t e r i o l Proc : ~ Bladel, B and R A Greenberg 1965 Pouch method f o r t h e i s o l a t i m and enumeration of c l o s t r i d i a Appl Microbiol 13:281 Brandish, J M and J T Willis 197 Observations on t h e coagulase and deoxyribonuclease tests f o r staphylococci J Med Lab Technol Breed, R S, E G D Murray and N R Smith 1957 Bergey's Manual of Determinative bacteriology Seventh e d i t i o n W i l l i a m s and Wilkins Co, Baltimore 194 pp Canada, J C and D H Strong 1964 l i v e r s J Food S c i 29:862 Clostridium perfringens i n bovine Center f o r Disease Control 197 Foodborne Outbreaks Annual Summary US Department o f Health, Education and Welfare, Atlanta, Georgia 3333 Chapman, G H 1944 A suggestion f o r t h e r a p i d presumptive examination of foods suspected of having caused staphylococcal food poisoning Food Res 9: A s i n g l e c u l t u r e medium f o r s e l e c t i v e i s o l a t i o n g f ' Chapman, G H plasma-coagulating staphylococci and f x improved t e s t i n g of' chr3mqy:nr s i s, plasma coagulation, mannitol fermentation, and t h e Stone r e a c t i m J B a c t e r i o l 51:49 Chinn, B D 1936 Observations on r e a c t i o n s of staphylococci of' t h e f m d poisoning types i n g e l a t i n Food Res 1:513 Chou, C C and E H Marth 1969 A comparison Df d i r e c t p l a t i n g and enrichment methods f o r d e t e c t i o n and ennumeration of coagulase-positive staphylococci i n frozen feeds of animal o r i g i n J Milk Food TechnDl 323%

15 C h r k, W S, Jr, T D Moore and F E Nelson 1961 Characterization of c o a ~ u l a s e - p o s i t i v e staphylococci i s o l a t e d from raw milk Appl MicrDbi i'mper, K E and F M Ramadan 1955 Studies i n t h e d i f f e r e n t i a t i o n between human and a n a l p o l l u t i o n by means of f a e c a l s t r e p t o c o c c i J Gen Microbiol 12:l8 Crisley, F D, J T Peeler and R Angelotti 1965 Comparative evaluation of f i v e s e l e c t i v e and d i f f e r e n t i a l media f o r t h e d e t e c t i m and enumeration o f coagulase-positive sta-phylococci i n foods Appl Microbiol 13 :14 Dische, F E and S D Elek welchii Lsncet 2: Experimental food poisoning by C Donnelly, C B, L A Black and K H L e w i s 1964 Occurrence of coagulasep o s i t i v e staphylococci i n Cheddar cheese Appl Microbiol 12:311 Feldman, H, A 1946 Incidence of c e r t a i n b i o l o g i c a l c h a r a c t e r i s t i c s ammg Amer J Public Health 36 :35 fox3 poisoning "staphylococci " Food and D r u g Administration 1969 Bacteriological Analytical Manual 2nd e d i t i o n US D e p a r t m e n t of Health, Education and Welfare, Public Health Eervice, Washbgton, DC 224 Forsgren, A 197 Significance o f p r o t e i n A production by staphylococci I n f e c t ~mmun 2:672 Faster, E M, R H Deibel, C L Duncan, J M Goepfert and 8 Sugiyama 197 Food poisoning In: J E Blair, E H Lennette and J P Truant (Ed ), Manual of C l i n i c a l Microbiology Amer SOC Microbiol Bethesda, Maryland p 624 Franklin, M K, E F Baer and M M Gilden 1966 R e l i a b i l i t y of s e v e r a l diagnostic and determinative t e s t s in the i d e n t i f i c a t i o n of coagulasep o s i t i v e staphylococci J Assoc O f f Anal Chem 49:282 Fuzi, M and 2 C s u k a s 1969 New s e l e c t i v e medium f o r t h e i s o l a t i o n of Clostridium perfringens Acta Microbiol Acad S c i Hung 16:273 G i l l e s p i e, W A and V G Alder 1952 Production of opacity i n e g g - p l k media by coagulase-positive staphylococci J Pathol B a c t e r i o l 64 :187 Green, J H and W L i t s l q y 1966 A new medium and "mimic" KPN method f o r Clostridium perfringens i s o l a t i o n and enumeration J Food S c i 31 :61 Hall, H E 1968 Collaborative study of a q u a n t i t a t i v e method f o r Cloctridium p e r f r i w e n s in foods J Assoc O f f Anal Chem 51:1338 Hall, H E 1469 Current developments i n d e t e c t i o n 3f microorganisms i n J Milk Food Technol 32:426 foodsclostridium perfrinp;ens

16 357 H a l l, H E and R AngeLotti 1965 Clostridium perfringens i n meat and meat products Appl Microbiol H a l l, H E and G H IIanser 1966 Examination of feces from food handlers f o r salmonellae, s h i g e l l a e, enteropathogenic Eschericia c o l i, and Clostridium perfringens Appl Microbiol 14 : 9 8 Harmon, S M, D A Kautter and J T Peeler f o r enumration of Clostridium perfr-ens 1971 Improved medium Appl Microbiol 22:688 Hartman, P A and P A P a t t e e 1968 Improved c a p i l l a r y action r e p l i c a t i n g apparatus Appl Microbiol 16 :151 Hauschild, AHW, J E Erdman, R HillsheFmer and F S Thatcher 1967 Variations in recovery of Clostridium perfringens on c o m e r c i a l s u l f i t e polylqyxin-sulfadiazine (SPS ) agar J Food S c i 32 :469 Hauschild, A H W and F S Thatcher 1967 Experimental food poisoning with heat susceptible Clostridium perfrlngens type A J Food S c i 32 :467 Hobbs, B C 1969 Clostridium perfringens and Bacillus cereus i n f e c t i o n s in: H Riemann (ed ) Academic Food-Borne Infections and Intaxications Press, N e w York p 131 Hobbs, G, D C C a n n, B B Wilson and J M Shewan 1965 The incidence o f organisms of t h e genus Clostridium i n vacuum packed f i s h in t h e United Kingdom J Appl Bacteriol 28:265 Hobbs, B, C, M E Smith, C L Oakley and G H Warrack welchii food poisoning J Hygiene 51: Clostridium I n s a l a t a, N F, C W Mahnke, W G Dunlop and C W Beazley 1972 A 24-hour method f o r t h e detection of coagulase-positive staphylococci i n f i s h and shrimp Food Technol 26:78 I n t e r n a t i o n a l subcommittee on staphylococci and micrococci 1965 Recommendations of Subcommittee I n t B u l l Bacteriol Nomencl Taxon 15:iog I v l e r, D 197 Staphylococcus In: J E B l a i r, E H Lennetti and J P Truant (eds ) Manual of C l i n i c a l Microbiology Amer SOC Microbiol Bethesda, Md p 61 Jay, J M 1962 Further s t u d i e s on staphylococci in meats 111 Occurrence and c h a r a c t e r i s t i c s of coagulase-positive s t r a i n s from a v a r i e t y of nonfrozen market c u t s Appl Microbiol 1:247 Jay, J M 1971 Use of a p l a t i n g method t o estimate e x t r a c e l l u l a r protein production by staphylococci I n f e c t Irmrmn 3 :544

17 Joshl, N and D Dale 1963 Some microbiological aspectg of staphylococcal mastitis Can J Camp Med 27:61 Iachica, RVF, K F Weies and R H Deibel 1969 Relationships among cosgulaee enterotaxin and heat -stable deoxyribonuclease productim by Staphylococcu6 aureus Appl Mlcrobiol 18 :126 + Lachica, RVF, P D Hoeprich and C Genigeorgis 1971 Nuclease production and lysostaphin s u s c e p t i b i l i t y of S t a h loccu, aureus and other catalase-positive cocci A p p l Microbiol Marshall, R T, C D Neighbors, and J E Edmondson l965a I s o l a t i o n of staphylococci fram d r i e d m i l k J Milk Food Technol 28:117 Marshall, R S, J F Steenbergen and L S McClung Rapid technique f o r t h e enumeration of Clostridium perfringens Appl Mlcrobiol 13 :559 McClung, L S 1945 Human food poisoning due t o growth of Clostridium erf ringens ( C welchii 1 i n f r e s h l y cooked chickens : preliminary note Bacteriol-5:229 : 1969 Growth and sporulation of Clostridium welchii i n b r e a s t Mead, G C and l e g muscle of poultry J Appl Bacteriol 32:86 Minor, T E and E H Marth 1972 Staphylococcus aureus and staphylococcal food intoxication A review I V Staphylococci in meat, bakery products, and other f w d s J M i l k Food Technol 35:228 Moore, T D and F E Nelson 1962 The enumeration of Staphylococcus aureus on s e v e r a l t e l l u r i t e - g l y c i n e media J Milk Food Technol 25: Enumeration of sulfite-reducing c l o s t r i d i a occurring Mossel, D A A i n foods J Sci Food A g r 19:662 Morrison, R B 1962 The coagulase t e s t in the i d e n t i f i c a t i o n of pathogenic staphylococci J Appl Bacteriol 25 :432 Muth, W L 1971 Evidence f o r a new extrachromosomal element i n a high coagulase-producing s t r a i n of Staphylococcus aureus B a c t e r i o l Proc 1971:45 Nakamura, M and K D Kelly 1968 Clostridium perfringens in dehydrated soups and sources J Food S c i 33:424 Robert:;m, J S 1963 An outbreak of Clostridium welchii food poisoning Mon B u n M i n i s t Health, Public Health Iab S e r v T B r i t ) 22:144 -I A R and A J Mercuri 1969 Efficiency of f i v e s e l e c t i v e nedia f o r recovering coagulase-positive staphylococci from mixed cultures P o d t r y S c i 4 8 ~ L)t T O U,

18 359 Shahidi, S A and A R Ferguson 1971 New q u a n t i t a t i v e, q u a l i t a t i v e, and confirmatory media f o r rapid a a a l y s i s of food f o r Clostridium perfringens Appl Microbiol 21: 5 Stone, R V, S r 1935 A c u l t u r a l method f o r c l a s s i f y i n g staphylococci as of the "food poisoning" type Proc SOC Exptl Biol Med 33:185 Thatcher, F S and W A Simon 1956 Compazative a p p r a i s a l of t h e properties of "staphylococci" i s o l a t e d from c l i n i c a l s i t e s and from dairy products C a n J Microbiol 2:73 Thatcher, F S and D S Clark (eds ) 1968 Microorganisms i n foods : t h e i r significance and methods of enumeration Univ of Toronto Press Vogel, R and M Johnson 1961 A modification of the t e l l u r i t e glycine medium f o r use i n t h e i d e n t i f i c a t i o n of Staphylococcus aureus Public Health Lab l8:131 Williams, MLB 1972 A note on t h e development of polymyxin-mannitolphenolphthalein diphosphate agar f o r s e l e c t i v e enumeration of coagulase p o s i t i v e staphylococci in foods J Appl Bacteriol 35:139 Wilson, W J and E MM B l a i r 1925 C o r r e h t i o n o f t h e sulphite reduction t e s t with other t e s t s In t h e b a c t e r i o l o g i c a l examination of water J Hyg 24:111 Woodburn, M and C H Kim 1966 Sunrival of Clostridium during baking and holding turkey s t u f f i n g Appl Zebovitz, E, J B Evans and C F Niven, Jr 1955 T e l l u r i t e glycine agar: a s e l e c t i v e p l a t i n g medium f o r q u a n t i t a t i v e detection of coagulasep o s i t i v e sta@aylococci J Bacteriol 7:686

19 36 BRUCE TOMPKI[N: I want t o thank t h e s e f o u r gentlemen f o r t h e i r excellent presentations on four i n t c r e s t i n g and very timely s u b j e c t s Are t h e r e any comments o r questions from t h e f l o o r? W L SULZBACHER: I have a couple of questions f o r Dr Genigeorgis in your f i r s t s i d e how do you know when you were using those large masses, l i k e c e l l s, t h a t you weren't carrying over t o x i n w i t h t h e c e l l s i n t o t h e medium? d All t h e c e l l s were f i r s t washed twice before inoculation However, even i f t o x i n were c a r r i e d over with t h e inoculum the amount of t o x i n produced l a t e r was s o l a r g e that t h e carryover amount w m l d have been i n s i g n i f i c a n t You know what I mean? When you g e t 1 and 2 micrograms you don't c a r r y such an amount by t r a n s f e r r i n g the cells C GENIGEORGIS: W L SULZBACHER: Another point I want t o make here i s the v a r i a b i l i t y of Aw values which you obtained a t d i f f e r e n t temperatures I t h i n k t h i s i n d i c a t e s t h a t there i s something t h e matter w i t h your instrumentation I f you look back i n t h e theory o f Aw you f i n d t h e temperatures should cancel w t C GENIGEORGIS: It should be t h e opposite W L SULZBACHER: A r e you using the l i t h i u m chloride type c e l l s from Iiygrodynamics, Inc? They a r e s u b j e c t t o a good d e a l of e r r o r You have t o watch them very c a r e f u l l y C GEfiIGEORGIS: Actually when you t r y t o measure water a c t i v i t y w i t h pure chemicals from t h e tables they supply, t h e r e i s j u s t t h e opposite r e l a t i o n s h i p A s you increase the temperature, t h e water a c t i v i t y i s increased But i n t h i s ease it was j u s t t h e opposite Well, I don't know, what t h e theory is behind measuring t h e water a c t i v i t y of meat, b u t t h e r e WLS a s i g n i f i c a n t d i f f e r e n c e From 97 i t went down t o 91 T h i s i s more than t h e v a r i a t i o n they allow f o r the sensors; s o, a t t h i s point I cannot enswer w i t h loo$ c e r t a i c t y W L SUUBACHER: That's a very l a r g e e r r o r I think I mentioned something about t h i s once before a t this meeting, t h a t using t h e s e kinds of techniques a r e not very dependable It i s a n unfortunate s i t u a t i q n C GBUGEORGIS: That's why nob3dy studied i t I3 TOMPKIN: I wouldn't w r i t e it off as an e r r o r a t t h i s p o i n t I But, where we use t h e s e same instruments snc! ;Ihc-rcw e have had problems w i t h sensing elemefits, w e ' l l o f t e n g e t an a b n m m l l y high Aw o r 1CC$ r e l a t i v e humidlty He's f i n d i n g a lower reading Lhink it needs double checking I! M HILL: I ' l l add t o t h e confusion j u s t a l i t t l e b i t We j u s t received 1-5 of these senses with a higher Aw range and w e checked tht-rrr out using standard s a l t s o l u t i o n s They were, with t h e exception of :le sensor, almost right on t h e b u t t o n We were r e l a t i v e l y impressed w i t h rwcii:1v

20 361 w h a t we received We checked tlem once s i n c e wc have been using them and we have been using them almoat d a i l y now f o r about two months We f'ound tkat t h e one that was o f f i n i t i a l l y i s s t i l l off', but w e can c o r r e c t for it e a s i l y, and w e have two o t h e r s that have subsequently become s l i g h t l y of'f One t h i n g w e noticed very quickly If you are using t h e s e elements it i s very important that you keep the sensors and your jars in the incubator a t 1 o r 9, o r whatever temperature you a r e running, and take your measurements then a t that temperature If you t a k e t h e jars out of t h e incubator, s e t them on a bench t o p and t u r n on t h e instrument, you can watch t h e needle on t h e instrument diet1 drop l i k e a bomb Maint a i n i n g a constant temperature i s one very important thing, i f you want t o g e t i n t o water a c t i v i t y work using t h e s e devices C GENIGEORGIS: What determines whether you incubate t h e jar? W M H I L L : Well, again w e were using a dry sausage and we found that we could incubate six hours and g e t f a i r l y s t a b l e readings A s a normal operation w e w i l l incubate If w e can g e t these samples in a t 9:OO in t h e morning, w e ' l l mike a reading a t 4:OO Then w e ' l l put on another batch and make a reading a t 8 : O O o r 9 : O O on t h e following morning The time v a r i e s, b u t I t h i n k you need 5 t o 6 hours, depending perhaps on your temperature T N BWMER: We l o u d t h a t i f you put i n s u l a t i o n around t h e jars, s o t h a t only t h e contact points a r e exposed then w e reduce m a t e r i a l l y t h e e r r o r t h a t we g e t from reading t o reading D r Genigeorgis, have you done any s t u d i e s with products where you have t r i e d t o c o r r e l a t e moisture content, salt, and a l l these other f a c t o r s with water a c t i v i t y and growth? C GENIGEORGIS: I n all of our studies with meats we a l s o measure6 the water content It seemed t h a t t h e water content d i d n ' t give a c l e a r cut p i c t u r e But, I would l i k e t o add something Recently t h e people from MIT d i d s t u d i e s with baby formulas based on pork and they t r i e d t o define the water a c t i v i t y permitting not only staph growth, but a l s o growth from some o t h e r b a c t e r i a They reported d a t a i n d i c a t i n g thc a b i l i t y of staphylococcal growth down t o 75 t o 84 T h i s p a r t i c u l a r baby product was brought t o lower water a c t i v i t i e s by adding g l y c e r o l When they took t h e same product which t h e y dehydrated by freeze drying and then rehydrated t o d e s i r a b l e water a c t i v i t i e s, then the m i n i m u m water a c t i v i t y supporting staphylococcal growth was 9O Their conclusion was t h a t it i s not only water a c t i v i t y alone, but a l s o t h e a v a i l a b l e moisture Thus, t h e water content should p l a y some r o l e and t h i s i s where they suggested that we should r e v i s e some of our O M i d e a s This i s t r u e f o r other b a c t e r i a a l s o T IT BLUMER: I ' d l i k e t o mention one more p o i n t When we als9 t e s t e d t h e sens3rs with s a l t s o l u t i o n s we found they were very accurate, except one in which I knocked t h e j a r over Send it back t o t h e factory i f that happens,' d o n ' t f o o l with i t

21 J D FOX: I have more of a comment and a word of' caution than a question Last F a l l, I imagine it was right before Iabor Day, a l o c a l chair *;tore was having a s a l e on luncheon meats I was i n t h e s t c r e on R Xursday evening about 8:3 and they were j u s t stocking this m e a t At tlme t h e r e was approximately 2 Ib of luncheon meat on an unrefrigerateu table i n t h e middle of t h e aisle, and t h i s was t o s t a y t h e r e u n t i l Saturday n i g h t A f t e r discussion w i t h the s t o r e manager I found that t h i s was being done through a n e n t i r e merchandising d i s t r i c t a t t h e d i r e c t i o n of t h e meat merchandising manager f o r t h e d i s t r i c t They that a f t e r discussing t h e problem with them t h a t assured me, tho-, t h i s meat wouldn't spoil, leaving it out t h e r e f o r t b e e days I t h i n k that industry m y need t o reconsider t h e knowledge of the people who handle t h e i r food on down t h e l i n e I ' m afraid industry would be i n about the Sam f i x as that c a t t h e other day No matter who t h e cause i s, t h e industry i s going t o g e t t h e blame i f someone g e t s s i c k B TOMPKIN: One t h i n g t h a t has been going through m y mind i s that about 1 5 years ago we were very concerned about t h e public h e a l t h aspects of vacuum packaged luncheon meats Based upon what I heard today, Dr Genigeorgis, you seem t o i n d i c a t e that vacuum packaging w i l l, in f a c t, reduce enterotoxin production by staphylococci Therefore, vacuum packaging would present l e s s of a public h e a l t h hazard than was formerly believed Do you agree? GENIGEORGIS: I would agree with that because when you campare a e r o b i o s i s versus anaerobiosis the amount o f enterotoxin produced i s always very d i f f e r e n t A tremendous amount of toxin i s produced under aerobic conditions and you g e t a very small amount under anaerobic conditions This a p p l i e s a l s o t o staphylococcal growth, not only the yield of toxin but a l s o growth is e f f e c t e d C D V VADEHRA: D r Walker, in t h e method you outlined j u s t a minute ago, it s e e m so comgdicated and complex I wonder i f i t i s s u i t a b l e f o r a small i n d u s t r i a l packing p l a n t which r e a l l y cannot a f f o r d a s o p h i s t i c a t e d method of checking What do you suggest f o r t h e small plants? 11 W WALICER: Well, that i s a good question f o r t h e small operator who perhaps cannot a f f o r d t o have a laboratory I would say t h i s pouch method, mybe some others do not agree with me, does not r e q u i r e t h e elaborate equipment f o r anaerobic incubators This cuts down on t h e expense and I don't know how much simpler you can g e t Perhaps, w h a t w e need i s a r e a l rapid t e s t within 24 hours A simple t e s t t h a t could be used i n p l a n t s Such a t e s t has j u s t been described f o r S a w e u s by I n s a l a t a and co-workers They have developed a 24 hour t e s t t o detcc! levels of 1 staph o r w e r within 24 hours This was reported i n thv last issue of Food Technology i f you would l i k e t o read more about i t G J COCOMA: D r Bryan, do y m have any information on t h e numbers c h s t r i d i a that were involved in t h e meat t h a t caused t h e outbreaks Do you l a o w t h e minimum numbers of c l o s t r i d i a t h a t w i l l cause i l l n e s s i n nc-t products? Jf