Aseptic transfer techniques

The ability to transfer microorganisms from one container to another without contamination is crucial to success in the microbiology laboratory. These techniques serve as the basis for subsequent work such as starter culture preparation or maintaining viable cultures in long-term storage. Transfer loops are normally used to transfer to the surface of agar (Petri plates and slants), whereas transfer needles are used to prepare stab cultures. Both implements are sterilized by heating in an open flame until red hot (Fig. 13.1). 

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The USP describes two general methods for conducting the test the direct transfer, or direct inoculation, method and the membrane filtration method. As the name indicates, the direct inoculation method involves the aseptic transfer of a sample of test product solution into the sterility test growth medium. To use this method, it must first be demonstrated that the product solution itself does not inhibit the growth of typical indicator microorganisms specified in the USP method. It should be self-evident why it is important to perform testing to negate the chance of product inhibition of possible microbial contaminants, as this is the purpose of the sterility test. The direct inoculation method, while not theoretically complex, requires the utmost technical precision and aseptic manipulation techniques for proper execution. As a consequence of the repetitive motions involved, it is prone to human error. 

Appendix B outlines the basics of laboratory media preparation and skills involved in handling microorganisms without contamination. For those who need to brush up on technique, the fundamentals of routine aseptic transfer are covered in detail. 

Membrane filtration is the technique reeommended by most pharmacopoeias and involves filtration of fluids through a sterile membrane filter (pore size = 0.45 im), any microorganism present being retained on the surfaee of the filter. After washing in situ, the filter is divided aseptically and portions transferred to suitable culture media which are then incubated at the appropriate temperature for the required period of time. Water-soluble solids can be dissolved in a suitable diluent and processed in this way. 

Aseptic Assembly. The first interventions performed are those that prepare the equipment for the aseptic process. This entails the removal of sterilized materials and equipment items from the autoclave and transfer to the location where the aseptic processing activities will be performed. This is ordinarily followed by the assembly/preparation of the equipment for the process. Aseptic assembly in which sterilized parts are removed from protective materials, installed and adjusted in preparation for the aseptic process are perhaps the most potentially invasive of all of the activities which must be performed. The operator must be meticulous in their execution of these tasks to prevent the inadvertent contamination of product contact surfaces. Strict adherence to the principles of aseptic technique described earlier is essential. These interventions are a necessary part of every aseptic activity, and it is common to identify the first containers filled as they may be more indicative of potential problems with the aseptic assembly. For this reason, the validation program should include process simulations that include containers filled immediately after the set-up of the equipment. 

The sample size has a considerable effect on the separation characteristics of a certain system. Therefore, the sample diameter on the plate should be kept as small as possible (<3mm). Inefficient separations and artificial results are caused by too-large spots. The standard TLC technique for applying a sample onto the plate is to use micropipets or glass capillaries for single use. The volume is typically 5 pi. A certain drawback of this technique is the fact that the sample is withdrawn aseptically with a syringe from the vial and needs to be transferred for spotting. 

The membrane filtration technique is technically more elaborate and requires that the radiopharmaceutical under test, after aseptic dilution, is passed through a membrane filter with a pore size of 0.45 m, which has been moistened with a sterile nutrient diluent. After filtration, the membrane is either transferred to a suitable culture medium or aseptically cut into two equal parts and one half placed in each of two suitable media. Incubation at the appropriate temperature is required for at least 7 days. 

Each vial contains the freeze-dried, sterile components under nitrogen atmosphere in a multidose vial. Depending on the intended use, the vial is reconstituted with sterile saline or with sterile sodium Tc-pertechnetate injection solution. All transfers and vial stopper entries must be done using aseptic techniques. Following reconstitution, the vial is agitated to dissolve the lyophilized material. 

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