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U.S. Department of Health and Human Services

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Evaluation and Definition of Potentially Hazardous Foods - Appendix B. Data from Industry and Trade Organizations - Challenge Studies

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Challenge Studies - "Potentially Hazardous Foods"
ProductMicroorganismRepInoculation MethodLevelEndpointTIncubation totherEnumeration methodsPass/fail criteriaOther
Italian Salad DressingS. Enteritidisno 3log/g>1035°C5 mincoveredFDA's BAMno growth/ pass 
S. Typhimuriumno 3log/g>1035°C5 mincovered 
67 Salad DressingsSalmonella spp (3 ck)  500cfu/gnot detected   XLD agar, enrich in lactose broth/FDA BA protocolno Salmonella within 72 hpass
3 Barbecue SaucesSalmonella spp (3 ck)  500cfu/gnot detected   XLD agar, enrich in lactose broth/FDA BA protocolno Salmonella within 72 hpass
Pumpkin PieS. aureus3on and in
filling
106, 107, 108,
 109/g (1 ml)
103, 103, 106, 106/no toxin*Room T6 d (3 samp)bagged, open air, or boxBHI agar/oxoid staph testno toxin production/pass*> 1ng/g
Pumpkin PieS. aureus3in pie filling140/g (0.05 ml) 140cps<100 cps/g25°C3 d (2 samp) Baird - Parker /coagulase +no growth /g/pass 
Lemon Meringue PieS. aureus (2 cultures)/ L. monocytogenes (2 cultures)3rows in filling, intersection,
and merengue.
3,000/1,500/
sample
<10 / sampleRoom5 d (3 samp) Baird - Parker and Mox agarno growth/passTPC, Salmonella -
Hush Puppy MixB. cereus2 ~3.5log/g~3log35°C8 h ICMSF and FDA's BAMno growth 
S. aureus2 ~4log~5log35°C4 h  
~6.5log8 h 
S. senftenberg2 ~2.3log~2.5log35°C8 h  
~6log19 h 
Hush Puppy Mix w/OnionsS. aureus2 ~2.3log~2.3log, ~3.3log35°C4 h, 8 h ICMSF and FDA's BAMno growth/pass at 4 h 
ProductMicroorganismRepInoculation MethodLevelEndpointTIncubation totherEnumeration methodsPass/fail criteriaOther
Apple - Cheese DanishS. aureus ck, (5)2in four places360/g320, 395, 10/g25°C24 h, 4d, 11din sterile bagsBaird - Parkerno growth at 14 - 18h/pass 
L. monocytogenes2in four places435/g235, 160, 10/g25°C24 h, 4d, 11din sterile bagsMox 
E. coli ck (2)2in four places1900/g470, 10,10/g25°C24 h, 4d, 11din sterile bagsE. coli petrifilm 
S. enteritidis/typhimurium ck(5)2in four places145/g15,10,10/g25°C24 h, 4d, 11din sterile bagsXLD 
Blueberry - Cheese DanishS. aureus ck, (5)2in four places360/g340, 215, 10/g25°C24 h, 4d, 11din sterile bagsBaird - Parkerno growth at 14 - 18h/pass 
L. monocytogenes ,2in four places435/g190, 235, 50/g25°C24 h, 4d, 11din sterile bagsMox 
E. coli ck (2)2in four places1900/g300, 35, 10/g25°C24 h, 4d, 11din sterile bagsE. coli petrifilm 
S. enteritidis/typhimurium ck(5)2in four places145/g70,10,10/g25°C24 h, 4d, 11din sterile bagsXLD 
Cherry - Cheese DanishS. aureus ck, (5)2 2 2 2in four places360/g170, 170, 170/g25°C 25°C 25°C24 h, 4d, 11din sterile bagsBaird - Parkerno growth at 14 - 18h/pass 
L. monocytogenes ,2in four places435/g275, 25, 30/g25°C24 h, 4d, 11din sterile bagsMox 
E. coli ck (2)2in four places1900/g155, 30,10/g25°C24 h, 4d, 11din sterile bagsE. coli petrifilm 
S. enteritidis/typhimurium ck(5)2in four places145/g25,10,10/g25°C24 h, 4d, 11din sterile bagsXLD 
EclairesSalmonella spp ck (4) in filling90/g<100, <100, <10026,12,7°C4 din sterile bagsXLDno growth or toxin at 4.5 days/fail 
S. aureus ck (5)in filling2.3 x 102/g<5log, 2log, <10026,12, 7°C4 din sterile bagsBaird - Parker 
L. monocytogenes ckin filling4.8 x 102/g<4log, 4log, 4log26,12, 7°C4 din sterile bagsMox 
RTE Chocolate Pie and Donut FillingS. aureus ck (5)  8 x 2lognot detected10, 32°C21 din sterile bagsBaird - Parkerno growth or toxin at 1.5 x shelf - life/pass at 2 weeks 
L. monocytogenes (2)  1.5 x 3log~20010, 32°C21 din sterile bagsMox 
B. cereus (2)  3 x 2lognot detected32°C21 din sterile bagsMYP 
S. enteritidis/typhimurium (6)  ~2.5 x 2lognot detected10, 32°C21 din sterile bags/anaXLD 
C. sporogenes (2)  2lognot detected32°C21 dSFP 
Fettuccine Alfredo with ChickenC botulinum ck  4log cfu/mlno toxin12, 21°C60 d, 5 d  no toxin for 5 d at 21°C or 60 d at 12°C pass 
Chopped LettuceS. Enteritidis, Cholerasuis, and Typhimurium,2 ~3log~5log24°C24 hsterile containersXLD? 
L. monocytogenes ck (3) ~3log~4log24°C24 hsterile containersMOX 
Chopped OnionsS. Enteritidis, Cholerasuis, and Typhimurium,2 ~3log~2log24°C24 hsterile containersXLD? 
L. monocytogenes ck (3) ~3log~4log24°C24 hsterile containersMOX 
TomatoesSalmonella spp  50~1log,~7log30°C4h, 24 h  ?Distr. At 5°C
Poultry/Bacon/ Cheese - Filled BreadC. botulinum prot. A + B
C. botulinum non - prot. B
 added to
ground
product/
anaerob
100 - 500 spores/gtoxin at aw .98 at 26°C/14d no toxin at aw .89 - 9426/18/13°C14 d/1 wk/8 wk mouse bioassayno toxin for 14d at 26°C, 1 wk at 18°C, and 14 wk at 13/pass or fail aw dependant
Chicken/ Cheese - Filled BreadC. botulinum prot. A + B
C. botulinum non - prot. B
 added to
ground
product/
anaero
100 - 500 spores/gtoxin at aw .98 at 26°C/14d no toxin at aw .89 - 9426/18/13°C14 d/1 wk/8 wk mouse bioassayno toxin for 14d at 26°C, 1 wk at 18°C, and 14 wk at 13/pass or fail aw dependant
ProductMicroorganismRepInoculation MethodLevelEndpointTIncubation totherEnumeration methodsPass/fail criteriaOther
Cheese - Filled BreadC. botulinum prot. A + B
C. botulinum non - prot. B
 added to
ground
product/
anaero
100 - 500 spores/gno toxin at aw .89 - .9826/18/13°C14 d/1 wk/8 wk mouse bioassayno toxin for 14d at 26°C, 1 wk at 18°C, and 14 wk at 13/ 
Ham - Cheese Filled BreadC. botulinum prot. A + B
C. botulinum non - prot. B
 added to
ground
product/
anaero
100 - 500 spores/gtoxin at aw .98 at 26°C/14d, no toxin at aw .89 - 9426/18/13°C14 d/1 wk/8 wk mouse bioassayno toxin for 14d at 26°C, 1 wk at 18°C, and 14 wk at 13/pass or fail aw dependant
Precooked Ground Beef/Cheese/Ham - Filled BreadC. botulinum prot. A + B
C. botulinum non - prot. B
 added to
ground
product/
anaero
100 - 500 spores/gtoxin at aw .98 at 26°C/14d, no toxin at aw .89 - 9426/18/13°C14 d/1 wk/8 wk mouse bioassayno toxin for 14d at 26°C, 1 wk at 18°C, and 14 wk at 13/pass or fail aw dependant
Cheese - Filled BreadC. botulinum prot. A + B
C. botulinum non - prot. B
 added to
ground
product/
anaero
100 - 500 spores/gtoxin at aw .97 at 26°C/14d, no toxin at aw .89 - 9426/18/13°C14 d/1 wk/8 wk mouse bioassayno toxin for 14d at 26°C, 1 wk at 18°C, and 14 wk at 13/pass or fail aw dependant
Process Cheese Slices (Monterey)L. monocytogenes surface inoculated1000cfu/g3.3log30°C(24, 48, 72,) 96h selective mediano growthfailed (pass at 24 only)
S.aureus  7log 
Salmonella 1log 
E. coli O157:H7 2.7log 
Process Cheese Slices (Swiss)L. monocytogenes surface inoculated1000cfu/g2.8log30°C(24, 48, 72,) 96h selective mediano growthpass
S.aureus  3.2log 
Salmonella 1log 
E. coli O157:H7 2.5log 
Process Cheese Slices (American)L. monocytogenes surface inoculated1000cfu/g 30°C(24, 48, 72,) 96h selective mediano growthno data
S.aureus    
Salmonella   
E. coli O157:H7   
Cottage Cheese Hot PackC. botulinum prot. A + B
C. botulinum non - prot. B
 added to
ground
product/
anaero
100 - 500 spores/g 26°C108 h mouse bioassayno toxinpass
S. aureus  4log5log (no toxin)140°F to 45°F30h Baird - Paker agar or SET - EIA kit for toxinno toxinpass
  5log (no toxin)140°F to 45°F30h 
  plus 98°F8h 
  5log (no toxin)140°F to 45°F4h 
  2log (no toxin)45°F60d 
ProductMicroorganismRepInoculation MethodLevelEndpointTIncubation totherEnumeration methodsPass/fail criteriaOther
18 Process Cheese Slices (Cream)C. botulinum (mixed strains) added to
heated
cheese/
agitation into tubes
1000 spores/g 86°F4 wk, 8wk mouse bioassayno toxindepending on formulation (salt?)
14 Process Cheese SauceC. botulinum (mixed strains) added to
heated
cheese/
agitation into tubes
1000 spores/g 45, 55, 65, 86°F4 wk, 8wk mouse bioassayfail 
Process Cheese Sauce (with Spices)C. botulinum (mixed strains)  20 or 70 spores/g 86°F13 mo (13 sampling times) mouse bioassayno toxinpass
Process Cheese SpreadC. botulinum (mixed strains) added uniformly to cheese1000 spores/g 86°F  mouse bioassayno toxin1 out of 5 fail at 6 mo
Process Cheese SpreadC. botulinum (mixed strains) added uniformly to cheese1000 spores/g 45, 55, 65, and 86°F25 wk mouse bioassayno toxin1 out of 4 fail at 65°F/12 wk
Cheese Blend (Pizza)S. aureus (3) added & mixed630/g3,700/g No toxin80°F10 hr FDA's BAMno hazardous level / no toxinalso used pathogen model 4.0
B. cereus (3) 850/g10/g No toxin