Sterilizing microbiological media

Bio-Validation of Laboratory Autoclaves Used for Sterilizing Microbiological Media... 

Regarding the components of bulk fermentation processes, the strain of the organism used to manufacture the drug substance for the clinical study should be compared with the strain to be used in commercial production. Strain identification includes microbiological, cultural, and biochemical characteristics. A comparison of the media composition and method of sterilization, sterilization parameters, and the pH of the medium after sterilization should be done. All fermentation stages, parameters, and conditions should be described in detail (i.e., temperature, pH) and documented. 

Microbiologic culture studies are useful fc>r bacterial identification, especially when an ocular infection foils to respond to treatment. Cultures are often obtained from the eyelids, the conjimctiva, expressed material from the lacrimal sac, and the cornea. Because preserved ophthalmic anesthetics have a bacteriostatic effect, cultures should be obtained if possible before anesthetic instillation. In the case of corneal sampling, it is necessary to provide topical anesthesia for patient comfort. The anesthetic of choice is 0.5% proparacaine because it causes the least bacterial growth inhibition. To enhance the bacterial yield, sterile preservative-free anesthetic may be used. Samples obtained may be inoculated directly onto soUd media plates (e.g., blood agar). Amies without charcoal transport medium (e g., BBL CultureSwab Plus) appears to be an acceptable alternative to direct plating and has the added benefit of convenience. 

The conduct of aseptic processing validation ordinarily requires the use of a microbiological growth medium in lieu of the product. The PDA/PhRMA joint task force of Validation of Sterile Bulk Processes has outlined some process simulation methodologies which do not... 

Twice 5 ampules, taken at random, are incubated at 22° and 37°C in four different culture media (broth, glucose broth, ascites broth, and an anaerobic medium) at an interval of some weeks. When no growth occurs the lot is considered to be microbiologically sterile. 

To control sterility or cleanness in hospital environment microbiology methods, laborious and time consuming (24-120 h), are used at present. To accelerate and simplify the sterility or cleanness control on different surfaces we applied bioluminescent assay of total bacterial contamination (TBC). Since the most surfaces analyzed in hospital contained low number of bacteria, below the detection limit of ATP-reagent used, incubation of the samples in nutritive medium followed by filtration through special luminometric microcuvettes Filtravette was applied. 

Heat is frequently used to sterilise or pasteurise the tubes before addition of the inoculum, but causes problems if the vitamin is not heat stable. In this case, membrane filtration may be used, and the vitamin preparation added aseptically to tubes of sterile medium. These procedures again imply a considerable level of microbiological expertise and competence. 

The availability of a variety of preformulated dehydrated media has dramatically shortened the time required for preparation. To prepare these media, frequently only distilled water is added and then the medium is sterilized, usually by autoclaving. Most suppliers handle a variety of specialized media and, depending on demand, can prepare others as needed. Even when specialized media caimot be obtained, individual ingredients are normally available. Major suppliers of microbiological media include Difco, BBL (Baltimore Biological Laboratories), and Oxoid. For price conscious individuals, house-brand formulations of routine media are also available from most major suppliers at competitive prices. 

Fig. 4.6 Cyclic voltammograms of a mediator-producing bacterial suspension fed glucose at time 0 (black line), and after 30 min (red line) and 120 min (green line). Arrows indicate peaks while dashed lines that cross between oxidation and reduction peaks intersect the x-axis at the formal potential of the redox couple. The peak at -170 mV disappeared when bacteria were washed by centrifugation, but the peak at 180 mV never disappeared. There were no peaks observed from sterile medium. [From Rabaey et al. (2004), reprinted with permission of the American Society for Microbiology.]...

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