Retention of water-borne bacteria by membrane filters. Part I: Bacterial challenge tests on 0.2 and 0.22 micron rated filters

The results of bacterial challenge tests conducted on several 0.2 and 0.22 micron rated "sterilizing grade" filter cartridge types with bacteria from a natural water source are presented. Eight different 0.2/0.22 micron rated "sterilizing grade" filter types from four different filter manufacturers, claimed to be capable of retaining Brevundimonas diminuta at a challenge level of 10(7) CFU/cm2, were tested. The filters tested included nylon 6.6 and polyamide filters from two manufacturers, modified or hydrophilic PVDF filters from two manufacturers, modified or asymmetric PES filters from three manufacturers, and cellulose acetate filters from a single manufacturer. Consistent bacterial penetration was observed, over the 18-24 h challenge period, for all twenty-five integral 0.2 and 0.22 micron rated filter cartridges tested, at challenge levels of about 10(1)-10(4) CFU/cm2, indicating that natural waterborne bacteria were more penetrative than B. diminuta. The observed penetration was thus qualitatively independent of filter media type or manufacturer. These results add to the growing body of evidence that shows 0.2 and 0.22 micron rated filters may not remove all microorganisms under all conditions. These results further establish that bacterial penetration of 0.2/0.22 micron rated filters is not limited just to (1) specific membrane types, or (2) extended duration challenges (> 24 h), or (3) extremely high challenge levels, or (4) bacteria that can only exist in a penetrative state in an artificial laboratory setting.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part III: bacterial challenge tests on 0.2/0.22 and 0.1 micron rated filter cartridges with Hydrogenophaga (formerly Pseudomonas) pseudoflava.

We have previously reported on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. This article reports on the retention efficiencies of a large panel of 0.2/0.22 microm and 0.1 microm rated filter cartridges for H. pseudoflava (ATCC 700892) versus the retention capabilities of the same filters for Brevundimonas diminuta (ATCC 19146). A total of thirty-two 0.2/0.22 microm rated filter cartridges, spanning nine different "sterilizing grade" filter types from four different filter manufacturers, were challenged with H. pseudoflava at challenge levels exceeding 10(7) cfu/cm2. H. pseudoflava was shown to penetrate every 0.2/0.22 microm rated filter tested, with log titer reduction (LTR) values ranging from 3.5 to 7.7 logs. H. pseudoflava was shown to be more penetrative than B. diminuta under the same challenge conditions. B. diminuta was fully retained by nineteen of the twenty 0.2/0.22 microm rated filters that were challenged with both organisms. In the case of 0.1 microm rated filters, eighteen filter cartridges, spanning five different filter types from three manufacturers were tested. H. pseudoflava was consistently retained by four out of the five filter types tested, with LTR values in excess of 11.5 to 12.2 logs. The 0.1 microm rated filter type that was penetrated by H. pseudoflava has been previously demonstrated to be not fully retentive for naturally occurring bacteria. The data show that H. pseudoflava penetrates 0.2/0.22 microm rated filters just as readily as B. diminuta penetrates 0.45 microm rated filters. In addition, titer reductions provided by 0.2/0.22 microm rated filters for H. pseudoflava are comparable to those reported for A. laidlawii mycoplasma, albeit under different conditions. This study demonstrates that H. pseudoflava meets all criteria for use as a standard organism for qualifying the microbial removal performance of 0.1 microm rated filters for enhanced sterility assurance.     

Retention of water-borne bacteria by membrane filters. Part III: Bacterial challenge tests on 0.1 micron rated filters

Clear performance differences were observed between different 0.1 micron rated filters in terms of their microbial removal efficiency when challenged with naturally occurring waterborne bacteria from a water source. Penetration occurred with three 0.1 micron rated "sterilizing grade" filter types tested, from three different filter manufacturers, that did not have a specific high titer reduction claim for Acholeplasma laidlawii. Bacteria shown to penetrate these 0.1 micron rated filters were quite similar to those recovered downstream of 0.2.0.22 micron rated filters (described in Part II). All of the isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. In contrast, four different 0.1 micron rated "sterilizing grade" filter types from two different manufacturers, which had been qualified with both B. diminuta and A. laidlawii, consistently produced sterile effluents under similar test conditions. This study thus highlights the need for an industry or regulatory standard method of defining the microbial removal performance of 0.1 micron rated filters, and supports the use of functionally qualified 0.1 micron rated filters as sterilizing grade filters in pharmaceutical operations for enhanced sterility assurance.     

Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes

In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii. R. pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R. pickettii contamination in filter-sterilized parenteral fluids. This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R. pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation. Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved. Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation. The SEM analyses of R. pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours. In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge. These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms. Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B. diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent. Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R. pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B. diminuta, in the drug solution. This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part II: preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for use as a standard challenge organism to qualify 0.1 micron rated filters.

In this article, we report on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. Filter-cloned H. pseudoflava (ATCC 700892) was easily cultured in a low nutrient broth (R2A broth) under standard laboratory conditions, reaching high titers of 10(8)-10(9) cfu/mL within 48-65 hours of incubation at 25+/-5 degrees C. Under these conditions, H. pseudoflava is a rod-shaped bacterium, averaging 0.25+/-0.03 microm by 1.65+/-0.35 microm, and appears to be smaller than Brevundimonas diminuta in width (0.31+/-0.03 microm), but somewhat longer in length (0.88+/-0.19 microm), which may partly explain the observed penetration. In total, thirty-five 0.2/0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from two different filter manufacturers were challenged with H. pseudoflava. In all cases, H. pseudoflava was shown to consistently penetrate every 0.2/0.22 microm rated filter disc tested. These tests also spanned three different challenge durations, including short-term challenges (30-40 minutes), and two different challenge fluids. The use of serial (double) 0.22 mm rated filters, which is a common industry practice to reduce the prefiltration bioburden to the final "sterilizing" filter, was also shown to be inadequate to fully retain H. pseudoflava under the challenge condition used. In contrast, two different 0.1 microm rated filter types functionally qualified with a specified high titer reduction claim for Acholeplasma laidlawii, were shown to consistently and fully retain H. pseudoflava, and retention by these two filter types was shown to be robust and independent of the challenge duration.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part IV: Retention of hydrogenophaga pseudoflava (ATCC 700892) and Ralstonia pickettii (ATCC 700591) by 0.2 and 0.22 micron rated filters

Ralstonia pickettii has emerged as a bioburden microorganism of considerable importance in pharmaceutical processes utilizing conventional 0.2 or 0.22 micron rated "sterilizing grade" filters. In this article, we re-evaluated and studied the retention efficiencies of 0.2 micron rated nylon 6.6 and 0.22 microns rated modified polyvinylidene fluoride (PVDF) filters for Hydrogenophaga pseudoflava (ATCC 700892) and R. pickettii (ATCC 700591). Out of a total of forty-four 0.2/0.22 micron rated filters discs tested in this study (spanning different challenge fluids, different challenge conditions, and different filter types), H. pseudoflava penetration was observed for every filter disc tested. Log titer reduction (LTR) values ranged from 0.3 to 2.0 logs for 20-48 hour challenges conducted in Water for Injection (WFI), and 3.8-7.1 logs for 6-hour challenges conducted in Minimal Media Davis (MMD). For 0.2 micron nylon 6.6 filter discs, penetration by R. pickettii was observed only in WFI challenges and was dependent on the culture and challenge conditions used. Penetration by R. pickettii was also restricted to only those membrane discs that were very close to the filter manufacturer's production integrity test (the Quantitative Bubble Point, QBP, test) limit. Where R. pickettii penetration was observed, LTR values were significantly higher than those observed for H. pseudoflava with the same filter discs. This study: 1) supports the use of H. pseudoflava as a worst-case challenge model for R. pickettii in process- and product-specific bacterial retention testing; 2) provides experimental evidence, for the first time, for the need to include filter membrane lots that have a physical integrity test value at or near the filter manufacturer's production (lower) limit in these tests; and 3) demonstrates how a standardized membrane integrity test (such as the QBP test) can be used select such "worst-case" membranes and to verify the inclusion of such "worst-case" membranes in these tests, thus serving as the link between the membrane disc used in bacterial retention validation testing and the production process filter.     

Changes in the cell size of Brevundimonas diminuta using different growth agitation rates

Brevundimonas diminuta (ATCC 19146) is a standard organism for validation of sterilizing-grade membrane filters. Cell size is critical for the determination of retention characteristics of 0.2 micron rated membrane filters. In this study, cell size changes of B. diminuta cultured under different physiologic states and variable agitations at 50, 100 and 200 rpm were measured by a particle size analyzer and scanning electron microscope (SEM). The smallest cells were obtained at initial stationary phase in saline lactose broth (SLB) as a shaking culture at 50 rpm. Cells grown under agitation at 50, 100 and 200 rpm showed an increase of specific growth rate (mu), about 2.9, 3.6 and 3.6 fold, respectively, compared to the non-agitated cells in SLB media. These results suggested that the cell size decreased proportionally with increase of the specific growth rate (mu) in SLB. These size changes were associated with penetration through a 0.2 micron rated cellulose acetate filter. A scale-down filtration system was developed and performed bacterial challenge test and bubble point test with cells cultured in SLB. Cells grown under agitation conditions in SLB were not retained by 0.2 micron rated membrane filter.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part I: characterization of water isolates for potential use as standard challenge organisms to qualify 0.1 micron rated filters.

Although 0.1 microm rated filters intended for pharmaceutical sterilization applications have been commercially available for at least 15 years, there is no industry-wide standard for qualifying the microbial removal performance of these filters. In this article, we report on the bacterial challenge methodology used to screen four bacterial species for potential utility as a standard challenge organism to qualify 0.1 microm rated filters. These isolates were, in their natural state, demonstrated to penetrate 0.2/0.22 microm rated filters in prior studies. In the screening challenges described in this study, three out of these four candidates tested demonstrated consistent penetration of one 0.22 microm rated filter type tested (when cultured in a low nutrient medium under standard laboratory conditions). These included 6204-22 (FAME ID Acidovorax avenae citrulli), 6266-15 (FAME ID Comamonas acidovorans), and 6266-34 (FAME ID Hydrogenophaga pseudoflava). Of these, H. pseudoflava (6266-34) was chosen for additional experiments with other 0.2 microm rated filter membranes. In total, seventeen 0.2 and 0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from three different filter manufacturers were tested. H. pseudoflava penetration was observed for every filter tested. Under the same challenge conditions, H. pseudoflava was consistently retained by a 0.1 microm rated hydrophilic PVDF (polyvinylidenefluoride) filter with a specified high titer reduction claim for Acholeplasma laidlawii. In order to ensure selection of the most stable penetrative phenotype (i.e., select for nonrevertants), H. pseudoflava was subjected to three rounds of "filter cloning," and these results are described herein. The advantages of using H. pseudoflava for qualifying the microbial removal performance of 0.1 microm rated filters are also discussed.     

Microbial retention characteristics of sterilizing-grade membrane filters with alginate substituted for oil-based products

For oil-based products, FDA recommends substitution of the oil with a compound which has similar viscosity and physical characteristics. In this study, a substitute for oil-based products was screened by measuring the viscosity and filterability, and examined for the presence of cell clumps in the various test fluids using an optical microscopy. The viscosity of the test fluids measured in the range of about 60-75 cP. Brevundimonas diminuta (formerly Pseudomonas diminuta), a standard challenge test organism for validation of 0.2 micron rated membrane filters, formed clumps in oils (corn, olive, sesame, and soybean) and polyethylene glycol (PEG, Molecular Weight (MW) = 400 and 1,000). During the viability test, cells suspended in 80% glycerol showed a ten-fold mortality rate after an exposure for 6 hours, but there was no significant change in viability in alginate (low, medium, and high viscosity) for 24 hours. These results suggested that alginate is better suited as a substitute for oil-based products than 80% glycerol. Since high viscosity fluids take longer to filter, the glycerol mortality rate would influence the challenge test negatively. A scaled-down filtration system has been developed for the described trials, and the bacterial challenge and bubble point tests have been performed in 1.6% alginate (66.7 cP), which was the choice of carrier fluid.     

The importance of accurate microorganism identification in microbial challenge tests of membrane filters--part I

Microbial challenge testing is a common procedure to determine the retention efficiency, performance, and validity of a sterilizing-grade filter. The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146), routinely used to verify a 0.2 μm rated sterilizing-grade filter. Process validation procedures most often also utilize B. diminuta (ATCC 19146), but instead of the standard procedures and fluids, process, and product parameters are employed to determine whether these parameters influence the retentivity of the filter or changes to the challenge organism, which might result in the penetration of the filter. In certain instances, the native bioburden within the drug manufacturing process is used to perform such process validation challenge tests. Filter penetrations can happen and cause concern; therefore, it is essential to identify the organism species with accuracy to avoid unnecessary confusion. This paper and its follow-up will describe such imprecision and the resulting misconceptions. It will clarify past determinations and put perspective on the findings. LAY ABSTRACT: Sterilizing-grade filters are used to remove microorganisms from biopharmaceutical solutions. To determine the retention performance of such filters, bacteria challenge tests are utilized, often with a standard challenge organism (Brevundimonas diminuta), in instances with native bioburden. The accuracy of the microorganism identification is of importance to avoid flawed results and misinterpretation of the filter's performance.     

Bacterial endotoxin retention by inline intravenous filters

Filters used in i.v. administration sets were tested for their ability to retain bacterial endotoxins for up to 96 hours of continuous infusion. Inline filters composed of cellulose ester, polyacrylate, polypropylene, polyethylene, or Posidyne Nylon 66 were used during continuous infusion of 5% dextrose injection at 83 mL/hr. One milliliter of inoculum containing 10(8) Escherichia coli was injected through a port upstream from the filter. A bacterial filter was used to monitor the sterility of effluent from the inline filters. The effluent was tested with limulus amebocyte lysate (LAL) that could detect endotoxin concentrations greater than 50 pg/mL. A control solution was monitored for viability of the bacteria throughout the course of the study, and positive endotoxin controls were used to confirm the sensitivity of the LAL. Samples of effluent were tested at 0, 4, 19, 24, 48, 72, and 96 hours. Effluent from all filters was sterile throughout the study. LAL assay indicated that only the effluent from filters containing Posidyne Nylon 66 was free of endotoxins for 96 hours. Effluent from the other filters contained endotoxins immediately after injection of the E. coli. Of the inline filters tested, only the one composed of Posidyne Nylon 66 was able to retain E. coli endotoxin for 96 hours. Further study is needed with E. coli and other microorganisms that are likely contaminants of i.v. infusions.     

Investigation of antibacterial activity of rosemary essential oil against Propionibacterium acnes with atomic force microscopy

In the present study, the antibacterial activity of rosemary (Rosmarinus officinalis L. Labiatae) essential oil against Propionibacterium acnes (P. acnes) was observed with atomic force microscopy (AFM). The MIC (minimal inhibitory concentration) value of rosemary essential oil against P.acnes was 0.56 mg/mL. Significant changes in morphology and size of P. acnes were observed by atomic force microscopy (AFM) in response to essential oil treatment. The essential oil first attached to the surface of P. acnes at low concentration, the width and height of the bacterial body became larger, whereas the length did not change considerably. With increasing concentration of the essential oil, the bacterial bodies were severely damaged. The length, width and height were all reduced, when the concentration was increased up to 64xMIC, the length, width and height were reduced by 42.56%, 92.00% and 41.58%, respectively. Furthermore, treated bacteria lost their native shape, the cell wall desquamated, and the cytoplasm leaked out of the bacterial body, finally leading to bacterial death. With the increasing time at MIC, the bacteria length was reduced at 8 h, the width and height gradually became smaller, the shape of the cell became distorted, and finally led to cell wall damage and bacterial death at 8 h. In conclusion, the AFM investigation of morphology and size of P. ACNES treated with rosemary essential oil represents a powerful technique, which can generally be applied to reveal the biological changing mechanisms of bacteria induced by antibacterial agents at the nanometer level.     

Characterization of filter extractables by proton NMR spectroscopy: studies on intact filters with process buffers

Studies were conducted to characterize potential extractables from sterilizing grade filters. The focus of this report is the 0.22 micron Durapore (hydrophilic modified PVDF) filter which is used throughout our recovery processes. The objectives of this study are (1) to identify potential filter extractables from the hydrophilic PVDF filters; (2) to show that NMR spectroscopy may be used to detect filter extractables in the presence of product and excipients; and (3) to establish levels of filter extractables obtained by extraction with a variety of buffers. The data show that the primary source of filter extractables is the hydrophilic modification of the PVDF membrane surface. Extractables from the modified hydrophilic PVDF filter include propylene glycol (PG) and soluble oligomers of the hydroxypropyl acrylate and cross-linker. Propylene glycol, arising from the hydrolysis of the hydroxypropyl acrylate, appears to be the primary extractable in buffers above pH 11. Since the 1H-NMR method can easily detect the methyl proton signals of PG, an NMR assay was developed to detect PG in the presence of buffer excipients and final product. Propylene glycol can be used as a marker for the extractables from Durapore hydrophilic PVDF filters. Although numerous buffers were used to generate extractables from the PVDF filter, significant extractables (PG and soluble oligomers) were found only in high pH extraction buffers. As a result of this finding, only a limited number of new buffers or new PVDF filters will require testing for future validation studies. Process validation studies have shown that neither PG nor soluble oligomers are at levels that impact the quality or safety of the product.     

Sepsis in rabbits following administration of contaminated infusions through filters of various pore sizes

The effects of three final filter pore sizes on sepsis and survival times in rabbits infused with contaiminated intravenous fluids were compared. Intravenous fluids were administered for up to 12 hours to five groups of rabbits. Groups 2, 3 and 4 received fluids, contaminated with Gram-negative rods, that were filtered with 0.22-, 1.0- and 5.0-micrometers filters, respectively. Rabbits in Groups 1 and 5 received uncontaminated and contaminated fluids, respectively, neither of which was filtered. Cultures were taken of blood and of fluid below the filters, and rabbit survival times were recorded. At 6, 8 and 12 hours, Group 2 survival time was not significantly different from that of the negative control; the survival times for Groups 3, 4 and 5 were significantly less than for the negative control. Fluid and blood cultures of Group 2 were negative or statistically indistinguishable from those of Group 1. Cultures for Groups 3, 4 and 5 were positive. The study suggests that 1.0- and 5.0-micrometers inline final filters have no beneficial effect on survival following infusion of contaminated fluids, but that 0.22-micrometer inline final filters increase survival time.     

Effects of sterilizing-grade filters on the physico-chemical properties of onion-like vesicles

Spherulites are new promising multilamellar vesicles that we study in a drug delivery context. The sterilization of spherulites suspensions is a necessary step before biological tests and later, before pharmaceutical applications (for example, parenteral or local injections). Among all sterilizing operations, the filtration through 0.22 microm sterilizing-grade filters (of the type Millex (? 4 mm) by Millipore) is easy and rapid, and we decided to study it as a mean to obtain sterile suspensions. The spherulites diameter is usually comprised between 0.2 and 0.5 microm but bigger vesicles occur and reach ? 1 microm. The effects of such filters on vesicles' size and lipids' concentration were then compromised. After examination of this challenging operation, results proved that the sterilizing filtration had no effect on these two parameters whatever the formulation chosen. Then, the possible release of amaranth, an encapsulated hydrophilic dye was followed. With the formulations and in spite of a filter diameter inferior to that of the vesicles, the encapsulation yields were not significantly different before and after the filtration and no leakage could be detected. Finally, the spherulites' functionality after sterilizing filtration was studied under the chemical angle: vesicles containing an amphiphilic reactive anchor (CholE3ONH2) were still able to bind covalently a peptidic molecular recognition pattern. The ligation was quantified by fluorimetry as high as for non-filtrated suspensions. Thus, though spherulites can present a diameter superior to that of the sterilizing filters, their passage through them do not alter the physico-chemical properties of these vesicles.     

Physical and microchemical alterations of chrysotile and amosite asbestos in the hamster lung

The physical and microchemical alterations of chrysotile and amosite asbestos (Union International Contre le Cancer standard samples) in the hamster lung and in vitro following acid treatments were studied by scanning electron microscopy (SEM) and x-ray energy-dispersive spectrometry (XEDS). Following intratracheal instillation, the ratio of short chrysotile fibers (less than 5 microns in length) decreased initially from 38% to 13% in the hamster lung, but increased again to 56% 2 years after the instillation. The majority of these new short chrysotile fibers had diameters less than 0.05 micron. Contrary to this, short amosite fibers (less than 5 microns in length) decreased from 41% initially to 4% 2 yr after instillating into the hamster lung. The diameters of amosite fibers appeared much less altered than that of chrysotile during the same time period. After 2 yr in the hamster lung, 33% of chrysotile and 68% of amosite found were asbestos bodies. The Si/Mg ratios of chrysotile fibers with diameters less than 0.2 micron were significantly higher than those with diameters between 0.2 and 0.6 micron in all groups: this relationship was reversed in all amosite groups. The Si/Mg ratios of the instillated and acid-treated chrysotile fibers were both higher than that of the same-sized control fibers. Acid treatments of chrysotile fibers in asbestos bodies from the hamster lung further altered their Si/Mg ratio. The Si/Mg ratios of the instillated amosite fibers were lower than that of the same-sized control fibers, but the difference between them disappeared following acid treatments. The hamster lung disposed of both chrysotile and amosite fibers smaller than 5 microns efficiently. Chrysotile and its asbestos bodies appeared to lose Mg ions and to fragment continuously in the hamster lung, and also in vitro with acid treatments. Amosite appeared also to fragment but lose more silicon than magnesium ions, at a much slower rate than that of chrysotile, presumably from the difference in their basic structures.     

In line final filters for removing particles from amphotericin B infusions.

The feasibility of using membrane filters to remove particles from intravenous infusions of amphotericin B in dextrose 5% (a colloidal solution) was studied. Six types of commercial membrane filters, ranging in pore size from 0.45-1.0 mum, were used. Because of the effect of pH on the particle size of colloidal solutions, each filter was tested at solution pH 4.7, 5.6 and 6.5. Samples of filtrate were analyzed spectrophotometrically for amphotericin content. All filters of pore size 0.22 mum removed amphotericin B from solution and were inappropriate for use with this product. Solutions at pH 4.7 were turbid, filtered slowly and were generally unacceptable for clinical use. At pH 5.6, only filters with pore sizes of 1.0 mum or greater showed acceptable results. At pH 6.5, filters with pore sizes of 0.45 mum or greater gave acceptable results; the use of a filter with a pore size of not less than 1.0 mum would provide a margin for error to compensate for variations in the colloidal particle size of amphotericin B.     

Nutritional Effects on the Growth, Cell Size, and Resistance to Stress of Acholeplasma laidlawii

Due to their lack of a cell wall, mycoplasmas are useful models for the study of biological membranes. However, they are much less appreciated by cell culture laboratories due to their unfortunate tendency to contaminate cell culture lines (1-5). One of the primary protections of a cell line is through filtration of cell culture media. It is necessary to use 0.1 μm-rated filters because mycoplasmas can often penetrate the more commonly used 0.2 (or 0.22) μm-rated filters. In the battle against mycoplasma contamination, it is helpful to delineate factors that may affect mycoplasma replication, cell size, and resistance to contamination control. The addition of cholesterol and unsaturated long chain fatty acids to growth media enhance replication of Acholeplasma laidlawii (6-10). The absence of glucose, presence of unsaturated fatty acids, increased incubation period, and exposure to stress promote the production of small mycoplasma cells (6-12). Exposure to a high salt concentration, hydrogen peroxide, and UV light, which are commonly used for contamination control, can enhance the production of small resistant cells (13-15).     

DNA solution(R) in cigarette filters reduces polycyclic aromatic hydrocarbon (PAH) levels in mainstream tobacco smoke.

Tobacco consumption represents a major health hazard to humans and, despite anti-smoking campaigns, the number of smokers remains high; thus the reduction of toxic compounds from tobacco smoke may reduce the health hazards of smoking. In the last 25 years cigarette manufacturers have introduced a variety of filter designs to reduce toxic and carcinogenic substances in tobacco smoke (normal filters, NF). However, large quantities of harmful constituents are inefficiently retained by commonly used cigarette filters. Following a patented method we modified commercial cigarette filters (modified filter, MF) by injecting a DNA solution into the filter tips; we then evaluated the reduced polycyclic aromatic hydrocarbon (PAH) levels in mainstream tobacco smoke of MF relative to NF. The PAH measured were: fluoranthene (FLUO), pyrene (PY), benzo(a)anthracene (B(a)A), chrysene (CRY), benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(k)fluoranthene (B(k)F), benzo(g,h,i)perylene (BGP), dibenzo(a,h)anthracene (DBA). The levels of PAH in cigarette smoke after MF were significantly reduced (P<0.001) compared to NF, using a variety of cigarette brands in a smoking machine (44.5%+/-8.4 % and 41.8%+/-5% for total and carcinogenic PAH, respectively, means+/-SE). Using B(a)P(TEF) values the reduction in PAH concentrations were similar for all cigarette brands with the exception of Camel, where the reduction was lower considering B(a)P(TEF) values. Amongst carcinogenic PAH, B(a)A, B(b)F and B(k)F) were reduced by 50-58%, CRY, B(a)P and DBA by about 40%. In conclusion MF filters treated with DNA have the potential of decreasing the exposure to PAH in cigarette smoke. Since, unlike some previously proposed biological filters MF do not retain additional nicotine, the main addictive compound of tobacco smoke, these filters may not induce increased smoking to compensate for the reduction in the nicotine delivery to smokers.     

GTX(4) imposters: characterization of fluorescent compounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1, symbionts of saxitoxin-producing Alexandrium spp.

Saxitoxins, the etiological agent of paralytic shellfish poisoning, are synthesized by dinoflagellates and cyanobacteria. Several reports indicate that bacteria are capable of saxitoxin synthesis. Two bacterial strains were isolated from saxitoxin-producing dinoflagellates, Alexandrium tamarense and A. lusitanicum (=Alexandrium minutum), and grown under a variety of culture conditions including those previously reported to induce saxitoxin synthesis in bacteria. Five fluorescent compounds were accumulated by the bacteria that had HPLC-FLD retention times similar to a reference standard of GTX(4), one of the saxitoxin congeners. However, we were unable to detect GTX(1), the epimeric partner of GTX(4), in the bacterial samples. The GTX(4) standard was hydrolyzed by NaOH/heat treatment but four of the bacterial compounds were stable. Unlike GTX(4), none of the five bacterial compounds were detectable by HPLC-FLD following electrochemical oxidation. The fluorescence emission spectrum of each of the five bacterial compounds was unique and readily discernable from the spectrum of GTX(4). None of the samples containing the putative GTX(4) toxin yielded positive results when analyzed by a 3H-saxitoxin receptor-binding assay for saxitoxin-like activity. We cannot rule out the possibility that these bacteria produce saxitoxins, however, our data clearly demonstrate that they accumulate at least five different fluorescent compounds that could be easily mistaken for GTX(4). We conclude that these five fluorescent compounds are GTX(4) imposters and that fluorescence scanning and chemical/heat stability should, at a minimum, be incorporated into HPLC-FLD protocols for identification of saxitoxins.