A comparison of the MicroCount Digital System to plate count and membrane filtration methods for the enumeration of microorganisms in water for pharmaceutical purposes.

The enumeration of microorganisms in water for pharmaceutical purposes using the MicroCount Digital System (Millipore Corporation, Bedford, MA) was compared to the USP-recommended Pour Plate and Membrane Filtration Count methods. A study, using a pure culture of Buckholderia cepacia, ATCC#25416, showed that the accuracy, precision, reproducibility and linearity of the MicroCount ATP Bioluminescence System was equivalent to or better than the traditional methods. When the MicroCount System was used to monitor purified water and water for injection taps in a pharmaceutical plant over a month, comparable counts to the traditional methods were obtained within 24 hours compared to 48 to 72 hours with the other methods. The effectiveness of the memory device used for the isolation of colonies for characterization was demonstrated by comparing the number and pattern of the positive wells in the MicroCount plates with the isolation of colonies on the microbial count agar plates. The recovery on agar plates, although slightly higher, was not statistically different to the MicroCount plates. The predominated microorganisms isolated using all three methods were Ralstonia pickettii, Bacillus sphaericus, Stenotrophomonas maltophia, and a Staphylococcus species.

The application of response surface methodology to sterilization process development and validation of a water cascade autoclave.

The application of Response Surface Methodology (RSM) to the performance validation of a water cascade sterilizer is described. The methodology was successfully used to 1) identify the cold and hot zones within maximum load configurations through three dimensional thermal mapping, 2) demonstrate the consistency of consecutive sterilization cycles in companion sterilizers, and 3) set target sterilizing values (F0) to achieve a high statistical assurance that any location will be above a minimum F0 (sterility assurance) and below a maximum F0 (for stability considerations).

Statistical analysis of environmental monitoring data: does a worst case time for monitoring clean rooms exist?

To determine the relationship between the sampling time of the environmental monitoring, i.e., viable counts, in aseptic filling areas and the microbial count and frequency of alerts for air, surface and personnel microbial monitoring, statistical analyses were conducted on 1) the frequency of alerts versus the time of day for routine environmental sampling conducted in calendar year 1994, and 2) environmental monitoring data collected at 30-minute intervals during routine aseptic filling operations over two separate days in four different clean rooms with multiple shifts and equipment set-ups at a parenteral manufacturing facility. Statistical analyses showed, except for one floor location that had significantly higher number of counts but no alert or action level samplings in the first two hours of operation, there was no relationship between the number of counts and the time of sampling. Further studies over a 30-day period at the floor location showed no relationship between time of sampling and microbial counts. The conclusion reached in the study was that there is no worst case time for environmental monitoring at that facility and that sampling any time during the aseptic filling operation will give a satisfactory measure of the microbial cleanliness in the clean room during the set-up and aseptic filling operation.

The use of tri(n-butyl)phosphate detergent mixtures to inactivate hepatitis viruses and human immunodeficiency virus in plasma and plasma's subsequent fractionation.

The treatment of plasma with organic solvent/detergent mixtures at the time of plasma collection or pooling could reduce the exposure of technical staff to infectious viruses and enhance the viral safety of the final product. Treatment of plasma for 4 hours with 2-percent tri(n-butyl)phosphate (TNBP) at 37 degrees C, with 1-percent TNBP and 1-percent polyoxyethylensorbitan monooleate (Tween 80) at 30 degrees C, or with 1-percent TNBP and 1-percent polyoxyethylene ethers, (Triton X-45) at 30 degrees C resulted in the rapid and complete inactivation of greater than or equal to 10(4) tissue culture-infectious doses (TCID50) of vesicular stomatitis and Sindbis viruses, which are used as surrogates. Treatment of plasma with TNBP and TNBP and Tween-80 was shown to inactivate greater than or equal to 10(4) TCID50 of human immunodeficiency virus. TNBP treatment of plasma contaminated with 10(6) chimpanzee-infectious doses (CID50) of hepatitis B virus and 10(5) CID50 of non-A,non-B hepatitis virus prevented the transmission of hepatitis to chimpanzees. Immediately after treatment of plasma with 2-percent TNBP, the recovery of factors VIII, IX, and V and antithrombin III was 80, 90, 40, and 100 percent, respectively. Recovery of all factors was greater than or equal to 90 percent after treatment with TNBP and detergent mixtures. Treated plasma was fractionated by standard techniques into antihemophilic factor and prothrombin complex concentrates, immune globulin, and albumin. Prior treatment with TNBP or TNBP and detergent did not affect the separations of desired proteins. Therefore, it appears possible to inactivate viruses in plasma before the execution of standard fractionation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbial populations associated with the surface of the brown algaAscophyllum nodosum.

The microorganisms on the surface of the brown algaAscophyllum nodosum, collected from an intertidal area in Nahant, Massachusetts, were examined using scanning electron microscopy. Differences in the microbial populations on the holdfast, internodal regions of the stipe, and the apical tips were apparent. The populations ranged from a lawn of end-attached bacteria above the holdfast to microcolonies of yeast cells near the apical tips. The greatest diversity of microorganisms was noted in the internodal region representing the fourth year of growth where a dense lawn of end-attached bacteria was overlaid by filamentous bacteria, pennate diatoms, and filamentous blue-green algae. A simple procedure was developed to estimate the number of bacteria on the surface of the seaweed using the scanning electron microscope. The observed distribution of epiphytes may be explained in terms of the age of the algal surface, differences in light intensity, and the differential secretion of tannin by various parts ofAscophyllum.

Morphology and ultrastructure of a Penicillium sp. grown on n-hexadecane or peptone.

The morphology and ultrastructure of a Penicillium sp. grown on n-hexadecane or on peptone in shake-culture were compared using transmission and scanning electron microscopy. The fungus grew as hollow mycelial balls surrounding individual hydrocarbon droplets on n-hexadecane and as solid mycelial balls on peptone. A dense layer of fungal mycelium that showed irregular forms, fusion, and increase in hyphal size formed at the hydrocarbon-water interface. Inclusions were present in the hexadecane-grown fungus that were absent when the Penicillium sp. was grown on peptone. Problems of fixation made it difficult to differentiate detailed changes in the cytoplasm when the fungus was examined with the transmission electron microscope.

Persistence and biodegradation of spilled residual fuel oil on an estuarine beach.

The enrichment of hydrocarbon-degrading bacteria and the persistence of petroleum hydrocarbons on an estuarine beach after a spill of residual fuel oil on 11 April 1973 in Upper Narragansett Bay, R.I. was investigated. A rapid enrichment occurred during days 4 to 16 after the oil spill and a significant population of hydrocarbon-degrading bacteria was maintained in the beach sand for at least a year. The concentration of petroleum hydrocarbons in the mid-tide area declined rapidly during the bacterial enrichment period, remained fairly constant throughout the summer, and then declined to a low concentration after 1 year. An increased concentration of branched and cyclic aliphatic hydrocarbons in the low-tide sediment 128 days after the spill suggested a migration of hydrocarbons during the summer. Hydrocarbon biodegradation was apparent during the winter months at a rate of less than 1 mug of hydrocarbon per g of dry sediment per day.