Sterility testing positive controls

For each test compound concentration, vehicle, negative or positive controls, make 8.25 mL of serum culture medium containing 70% rat serum, 30% sterile Tyrode s salt buffer with less than 1 mg/500 mL phenol red (free acid indicator) and 35 p,g/mL streptomycin sulfates. 

Repeat this for each test organism. For positive control use sterile DW instead of sanitizer solution. For negative control use sterile strips. Swab each strip thoroughly with a premoistened Ca alginate swab and place in Lecithin broth (or other suitable neutralizer). Vortex for 30 sec, then perform the usual pour plate method. 

Positive Control Inoculated with coli showed growth after 48 h Negative Control Filtrated sterile WFI under the same conditions of the test... 

Sterility testing requires a strict control of microbial contamination challenge from outside the controlled environment, but not of the particulate contamination liberated by the process itself. Hence, a positive-pressure isolator in a controlled environment, using... 

The growth promotion test constitutes the positive control of the sterility test. Accordingly, if a failing growth promotion result is obtained, the sterility test is deemed to be invalid. Each lot of media is tested with preselected types and numbers of organisms to prove that the medium is/was capable of supporting... 

LAL test kit QCL-1000 (Bio-Whittaker UK Ltd., Cat No. 50-647U) this contains sterile pyrogen-free water, E. cdi endotoxin positive control, chromogenic substrate, and limulus amoebocyte lysate reconstitute the reagents as instructed... 

Strains were inoculated into liquid YPD medium and grown overnight at 37°C. The cells were then pelleted and washed three times with distilled water. Approximately lO cells/ ml were inoculated in molten agar media at 40°C and poured into 100-mm-diameter petri plates. The test materials initially dissolved in 1% DMSO were further diluted in distilled water to concentration ranges of 10-fold of their respective MICs. 4-mm-diameter sterile filter discs were impregnated with the test compounds as reported previously [61]. 1% DMSO (solvent) and 100 pg/ml of fluconazole were also applied on the discs to serve as negative and positive controls, respectively. The diameter of zone of inhibition was recorded after 48 h and was compared with that of control. The type of halo produced depicts fungicidal/static characteristic of test entity. 

There should be at least three replicate plates per treatment with at least five test doses plus untreated controls. Duplicate plates are sufficient for the positive and sterility control treatments. The use of twice as many negative control plates as used... 

This effort must extend to the microbiological laboratory as well, in which validation of methods is essential to assure confidence in the results. This will include appropriate testing environments, laboratory sterilization/depyrogenation validation, equipment qualification and calibration, use of standards, and positive and negative controls. 

Sterilization by moist heat is suitable only for water-wetable materials and aqueous solutions. Both temperature and pressure should be used to monitor the process. The temperature recorder should normally be independent of the controller, and there should be an independent temperature indicator, the reading from which is routinely checked against the chart recorder during the sterilization period. For sterilizers fitted with a drain at the bottom of the chamber, it may also be necessary to record the temperature at this position, throughout the sterilization period, here should be regular leak tests on the chamber when a vacuum phase is part of the cycle. 

Willey and Waterbury (1989) used a substantially different method to study chemotaxis in the marine cyanobacterium Synechococcus sp. This experiment involved the use of blind-well chemotaxis chambers (Neuroprobe, Inc., Cabin John, MD) that consisted of an upper (800- il) and lower (200- li1) acrylic chamber separated by a polycarbonate filter (3.0 pm). A cell suspension (165 pi) of the cyanobacterium was placed in the lower chamber over which the polycarbonate filter was placed. An air space was left between the cell suspension and the filter to control the starting time of the experiment. The upper chamber was filled with sterile seawater containing the compound to be tested and then inverted, allowing the cell suspension to contact the polycarbonate filter and the seawater/compound solution. The experiments were run for 65 min, after which time the chambers were inverted to stop the experiment. The number of cells crossing the filter into the seawater chamber was determined by direct cell counts using epifluorescence microscopy. The motile strain of Synechococcus sp. tested in this assay elicited positive chemotaxis to compounds such as ammonia, nitrate, urea, glycine, and P-alanine. Control chambers with the same concentration of chemoattractant in both the upper and lower chambers failed to elicit a chemotactic response. While the compounds tested in this study were relatively simple metabolites, one could... 

Utility systems such as water for injection (WFl). clean steam, clean-in-placc (CIP) solutions and sterile process air must be similarly proven. Also the building system itself has to be validated. Many bioprocess operations which contain potentially hazardous materials are operated in closely-controlled negative pressure enclosures with filtration of exhaust ventilating air. Sterile and particularly parenteral products arc processed in clean rooms which are maintained at positive pressure with filtered incoming air. Validation of building control systems and of personnel changing facilities and systems of work are necessary to meet CMP requirements. Manuals for formal test procedures are required to validate these activities. 

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