Article Figures & Data
Figures
- Figure 1
Schematic of the detection of the autofluorescence of microcolonies. Top: Microorganisms fluoresce under blue light and their location on the membrane captured by the CCD detector. Middle: Images taken at 4-hour intervals and an increase in a fluorescent object size or brightness confirms a growing colony. Bottom: Accuracy of the system can be shown by comparison with human counts at the end of incubation.
- Figure 2
TTR curves for 5 pharmacopeial and 7 environmental organisms, namely Staphylococcus epidermidis, Micrococcus luteus, Kocuria rhizophila, Bacillus pumilus, Brevundimonas diminuta, Candida tropicalis, and Aspergillus fumigatus are incubated and imaged every 4 h for 72 h at 30°C–35°C. The cumulative percentage of emerging colonies is shown on the y axis.
Tables
- Table I
The Media, Test Microorganisms, and Growth Direct® Incubation Temperature and Time at the Different Study Sites
Site and Media Control Microorganism Incubation Temperature and time Company 1 B. subtilis 30°C–35°C for 36 hours TSA P. aeruginosa R2A C. albicans SDA A. brasiliensis TSA + LP80 R. pickettii S. aureus S. epidermidis (in-house) Penicillium species (in-house) P. glucanolyticus (in-house) B. cereus/thuringiensis (in-house) Mixed culture Company 2 B. subtilis 22.5°C–27.5°C for 44 hours TSA + LP80 A. brasiliensis E. coli Company 3 B. subtilis 30°C–35°C for 44 hours TSA + LP80 A. brasiliensis E. coli S. aureus P. aeruginosa C. albicans Mixed culture In-house isolates Company 4 B. subtilis 28°C–32°C for 44 hours TSA + LP80 A. brasiliensis E. coli Company 5 EM:E. coliB. cepacia EM:Water: TSA + LP80HT B. subtilis 30°C–35°C for 44 hours A. brasiliensis R2A Water: E. coli Water: 30°C–35°C for < 7 days B. cepacia Company 6 S.aureus 30°C–35°C for 44 hours TSA + LP80HT C. albicans S. hominis M. luteus C. tuberculostearicum B. subtilis A. brasiliensis E. coli P. aeruginosa Company 7 B. subtilis 30°C–35°C for 44 hours TSA A. brasiliensis R2A E. coli TSA + LP80HT Company 8 B. subtilis 30°C–35°C for 44 hours TSA + LP80 A. brasiliensis R2A E. coli - Table II
Verification of the Equivalency of the Automated Plate Counter and Traditionally Read Plate Counts
Site B. subtilis ATCC 6633 E. coli ATCC 8739 A. brasiliensis ATCC 16404 P. aeruginosa ATCC 9027 S. aureus ATCC 6538 C. albicans ATTC 10231 Site 1 Pass Pass Pass Pass Pass Pass Site 2 Pass Pass Pass Site 3 Pass Pass Pass Pass Pass Pass Site 4 Pass Pass Pass Site 5 Pass Pass Pass Site 6 Pass Pass Pass Pass Pass Pass Site 7 Pass Pass Pass Site 8 Pass Pass Pass Note: Acceptance Specification: Growth Direct® Colony Count ≥85% of the mean colony count by 3 analysts. Organism colonial shape recorded in PQ to verify vision accuracy by site. Grayed cells indicate the organism was not tested during company PQ.
- Table III
Determination of TTR. Acceptance Specification, Recovery ≥85% of the Visual Count for the Maximal Incubation Time
Siteand Media System Incubation Temperature and time Time to Results Comments Site 1 TSA R2A SDA TSA + LP80 EM, water and product: 30°C–35°C for 72 to 120 hours Product: 36 hoursRinse Water 44 hours EM: 60 hours Many species recovered >85% much earlier for EM but minimum times will be driven by worst case recovery organism(s) Site 2 TSA + LP80 EM: 22.5°C–27.5°C for 120 hours EM: 36 hours 100% recovery at 72 hours Microorganisms included standard molds and bacteria and in-house molds and bacteria Site 3 TSA + LP80 EM: 30°C–35°C for 120 hours EM 44 hours TTR is defined by ATCC strains, site-isolates, slow-growers, and heat-stressed organisms. Verified with real samples in routine Site 4R2ATSA + LP80TSA Water: 30°C–35°C for 144 hoursEM: 28°C–32°C for 120 hoursProduct: 30°C–35°C for 120 hours Water: 116 hours EM: 76 hours Product: 52 hours Representative purified water samples from site water loop Real air and surface samples from C/D/CNC areas pH stressed ATCC strains on phosphate buffer (without product matrix) Site 5 R2ATSA + LP80HT EM:25°C–30°C(surface and air monitoring)30°C–35°C(personnel monitoring)for 96 hoursWater:30°C–35°C for 164 hours EM: 56 hours Water: 116 hours Microorganisms for EM TTR: environmental samples from surface, air and personnel monitoring Microorganisms for water TTR: Stenotrophomonas maltophilia (in-house strain) Methylobacterium extorquens (in-house strain) Water samples of different quality Site 6 TSA + LP80HT EM: 25°C–30°C for 120 hours EM: 72 hours Site-isolates from air and surface monitoring Site 7TSA + LP80HTR2ATSA EM30°C–35°C for 168 hours Water 30°C–35°C for 120 hours Product 30°C–35°C for 120 hours EM: 52 hours Water: 64 and 108 hours Product: 36 hours Water TTR varies by facility flora.Bioburden used pH 4.0 stress method Site 8TSA + LP80R2A EM30°C–35°C for 72 hours Water 30°C–35°C for 100 hours EM: 68 hours Water: 100 hours Microorganisms for WFI water TTR were each “stressed” per JP pharmacopeia: Pseudomonas protegens (ATCC 17386)Stenotrophomonas maltophilia (in-house strain) Methylobacterium extorquens (ATCC BAA-2500) Endogenous bacteria were used for purified water (PUW) and pretreatment water systems, using historically highest count sites based on annual trend reports. EM used typical 5 USP organisms plus 3 in-house organisms.
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