STERILE TRANSFER TECHNIQUES

The ability to transfer colonies from one container to another, without contamination, is crucial to success in the microbiology laboratory and serves as the basis for any subsequent work. The need for transferring cultures varies from raising large populations of yeast and/or bacteria to serve as inocula, to having to identify a troublesome organism. Routine transfer of cultures to fresh media is yet another reason. 

Because staffing is usually limited, the microbiologist will need to transfer cultures and make dilutions without assistance. This requires the ability to manipulate a transfer loop (or pipettes) with one hand and tubes or plates with the other. The following techniques are described for right-handed individuals. 

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Figure 25 Sterilizing two scalpels speeds up agar transfer technique.

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. 

Using sterile techniques, transfer the appropriate bacterial cells from slant to a test tube or flask containing 10 mL of sterilized LB medium and 40 fiL of the ampicillin solution. Incubate the culture with vigorous shaking at 37°C for 12 to 15 hours or overnight. 

Inoculation Inoculation is the seeding of a culture vessel with the microbial material (inoculum). The inoculum is introduced with a metal wire or loop which is rapidly sterilized just before its use by heating it in a flame. Transfers of liquid culture are often made by using a sterilized pipette. The inoculation is usually done in a laminar flow hood to minimize the risk of contamination. It is important to know proper pipetting techniques for inoculating or sampling during cultivation. 

For aerobic fermentations, air needs to be supplied continuously. Typical aeration rates for aerobic fermentation are 0.5 - 1.0 vvm (air volume per liquid volume per minute). This requires an enormous amount of air. Therefore, not only the medium but also the air must be free of microbial contaminants. All of the sterilization techniques discussed for medium can also be employed for air. However, sterilization of air by means of heat is economically impractical and is also ineffective due to the low heat-transfer efficiency of air compared with those of liquids. The most effective technique for air sterilization is filtration using fibrous or membrane filters. 

NOTE Sterile technique is essential in the following transfers, incubations, and centrifugations to avoid contamination with other bacteria. 

Transfer 5 to 10 ml of this culture to 100 ml of fresh SPI medium in a 500-ml sidearm flask so that the final cell suspension has an absorbance at 500 nm of about 0.1 when viewed against an SPI medium blank. (NOTE If you do not use a Klett colorimeter and a sidearm flask, remove 1-ml samples for absorbance readings in a spectrophotometer. Use sterile technique for all samplings. Do not return samples from the cuvettes to the cell suspension.) Incubate this culture with shaking at 37°C. 

The following procedures require sterile technique that is, use of sterilized glassware, pipettes, Petri plates, and other equipment. Do not use equipment that has lost a covering cap and consequently has been exposed to the air for long periods. If you are unfamiliar with sterile techniques for transferring bacterial cultures, consult your instructor for details of the techniques required in this experiment. 

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 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. 

After the culture is grown, the flask (fitted with a hose and tank coupling device) is used to inoculate the seed fermenter. However, some transfer the culture from the seed flask to a sterile metal container (in the laboratory) which has a special attachment for the seed fermenter. This technique is usually abandoned in time. Ingenuity for the minimum transfers in the simplest manner will usually give the best results. 

Apart from continuous sterilizers, pumps are a minor concern in the fermentation department. A simple way to transfer inoculum from a large laboratory flask to a seed fermenter, without removing the back pressure on the vessel, is to use a peristaltic pump. Connect the sterile adapter (which is attached to the flask) to the seed fermenter by sterile technique. Install the gum rubber tubing in the pump, open the hose clamp and start the pump. 

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