Aseptic techniques

The details of a good aseptic technique cannot be written in a book. They must be demonstrated and practised assiduously under the guidance of a skilled operator. It is, however, largely a matter of common sense, and a few do s and dont s are listed below. 

If all the above precautions are taken, then the only remaining source of contamination is the person working with the cells. Personnel carry microorganisms on the clothes and on their person, especially on their hair. It is a good idea to wear a clean laboratory coat restricted to use in the cell culture laboratory and never to move from an animal house into a cell culture laboratory without changing laboratory coats. People with long hair should tie it back and wear a clean head covering. It is also recommended that face masks are used especially if there is any doubt about a person s health. 

Laminar flow cabinets serve two purposes to protect the samples and to protect the worker and the environment. For most tissue culture applications sample protection is sufficient, but increasingly often we are becoming aware of hazards associated with biological samples and the cabinet to choose combines both aspects of protection. Such are the vertical laminar flow cabinets available, for example, from Flow Laboratories (Gelaire) or M.D.H. (Appendix 3). 

Horizontal-flow cabinets are satisfactory for aseptic manipulations such as media preparation where potentially hazardous aerosols are not generated. In these the filtered air enters through the back of the cabinet and leaves through the front. A horizontal-flow cabinet is a miniaseptic room which is much easier and cheaper to maintain than a walk-in aseptic room. In addition, it has the advantage that the worker is not confined within a small room which may become extremely hot and stuffy and, secondly, that the worker s head is separated from the cultures by a perspex screen thus further reducing the chance of contamination. 

The use of a laminar flow system would appear to make it impossible for bacteria to pass from the worker to the culture and in principle they provide ideal protection for both cultures and workers. However, the very act of putting the hands into the air stream disturbs the laminar flow and causes eddies. For this reason laminar flow cabinets should only be used in addition to standard aseptic technique although it is unnecessary to work near a bunsen flame. 

---

Most of these procedures are proprietary. Formulation development is also becoming more complex for preparation and deHvery of new vaccines. The classical vaccines are mostly prepared as injectable solutions. Aseptic techniques are required in the design and operation of the faciHties. 

Aseptic technique Manipulating sterile instruments or culture media in such a way as to maintain sterihty. 

A solution of 10 grams of d-glucoheptonic acid lactone in 50 ml of distilled water is warmed on a steam bath for about 2 hours to hydrolyze the lactone to the acid. The mixture is cooled and 100 ml of 95% ethanol are added. To the solution of glucoheptonic acid are added about 37 grams of erythromycin and the volume of the reaction mixture is brought to 200 ml by the addition of 95% ethanol. The reaction mixture is stirred for about 2 hours and is filtered through a porcelain filter candle of porosity 02. To provide a steriie product, aseptic technique is used throughout the remainder of the procedure. 

As a result of extensive development and testing by thermoprocessing or aseptic techniques, the use of flexible, laminated aluminum pouches and formed aluminum containers for shelf-stable foods is nearing commercial reality. The increasing use of aluminum for food packaging has been made possible by successfully combining it with specialized plastics, papers, adhesives, and coatings. In many applications, aesthetic as well as protective characteristics are also provided. 

Always observe aseptic technique when handling syringes and needles. 

Access to the epidural route is made through the use of a percutaneous epidural catheter. The placement of the catheter requires strict aseptic technique by a skilled physician. The epidural catheter is placed into the space... 

Aseptic manipulations should be performed in the sterile air of a laminar airflow unit. Speed, accuracy and simplicity of movement, in accordance with a complete understanding of what is required, are essential features of a good aseptic technique. 

People are the principal source of contamination in clean room operations. All personnel involved throughout the development and production of a parenteral product must be aware of the factors that influence the overall quality of a product as well as the factors on which they directly impinge. It is of particular importance that production personnel be properly trained so that human error is minimized. They should be made aware of the use of the products with which they are involved and the importance of following all procedures, especially proper aseptic techniques. Procedures must be set up to verify that the product is being manufactured as intended. After manufacture of a batch, production tickets must be carefully checked, sterilization charts examined, and labels verified for correctness and count. 

The interpretation of sterility results is divided into two stages by the USP relative to the type of sterility failure if one occurs. If sterility failure of the test samples occurred because of improper aseptic technique or as a fault of the test itself, stage 1 may be repeated with the same sample size. Sample size is doubled in a stage 2 testing, which is performed if microbial growth is observed in stage 1 and there is no reason to believe that the test was invalid. The only absolute method to guarantee the sterility of a batch would be to test every vial or ampoule. 

A separately packaged sterile diluent and sterile dropper assembly is provided with the sterile powder and requires aseptic technique to reconstitute. The pharmacist should only use the diluent supplied by the manufacturer since it has been developed to maintain the optimum potency and preservation of the reconstituted solution. The storage conditions and expiration dating for the final solution should be emphasized to the patient. 

The blood is collected using an aseptic technique into sterile containers. It can then be allowed to clot with subsequent recovery of the antibody-containing antisera by centrifugation. Alternatively, the blood may be collected in the presence of heparin, or another suitable anticoagulant, with subsequent removal of the suspended cellular elements, again by centrifugation. In this case, the resultant antibody-containing solution is termed plasma . 

The identity of each blood donor should be recorded, and all donor blood bags must be labelled carefully. Traceability of individual blood donors/donations is essential, in case the donor or product is subsequently found to harbour blood-borne pathogens. The risk of contamination of blood during collection/processing is minimized by using closed systems and strict aseptic technique. 

A concentrated Ca-D-pantothenate solution (1 mg/ml) is prepared in distilled water and dilutions made as needed. Refrigerated solutions are stable for 6 months. Pantothenate is added at 5, 10, 20, 40, 60, 80, and 100 mpg/ml final concentrations the control flask consists of basal medium alone for estimation of carry-over error—i.e., the pantothenate activity of the inoculum. The details of aseptic technique have been discussed elsewhere (H18, H19). Growth is measured in optical density units with a Welch Densichron, equipped with a red-sensitive probe to minimize blank readings due to the color of the medium. 

A prime practical consideration in the use of the IP route for acute testing should be the utilization of aseptic techniques to preclude bacterial or viral contamination. If these are not exercised, the resulting infected and compromised animals cannot be expected to produce either valid or reproducible indications or actual chemical toxicity. 

Aseptic techniques are used to avoid the possibility of infection of the animals or ceU cultures. These include the preparation of the vaccines and spleens under aseptic conditions in a class 100 clean room equipped with a laminar airfiow hood, sterilization of instruments, and treatment of work surfaces with disinfectant before and after use, washing of the investigator s hands with an antiseptic surgical scrub preparation, and wearing of sterile gloves, face mask, and eyeglasses. 

As is the case with all other pharmaceutical substances, all aspects of antisera production must be undertaken by means conducive to the principles of GMP. Most regulatory authorities publish guidelines which outline acceptable standards/procedures for the production of such blood-derived products. Donor animals must be healthy and screened for the presence of (particularly blood-borne) pathogens. They must be housed in appropriate animal facilities, and withdrawal of blood must be undertaken by aseptic technique. Subsequent downstream processing must be undertaken according to the principles of GMP, as laid down in Chapter 3. 

Before incubation of the vials, powder should be reconstituted with adapted media (TSB or thioglycolate broth) using aseptic technique under laminar flow. The reconstitution volume is according to the volume described in the original formula. Strict environmental monitoring should be followed through this step. 

Follow up aseptic technique rules. Wear the proper garment, sterile gloves, and mask. 

Observe and report any deviation from the proper aseptic techniques in cleanroom. See attachment no. 1700.80(L). 

To prevent contamination aseptic technique should be strictly applied during handling of agar strips all handling should be under LAP. 

Gowning sterilization cycle Operator aseptic technique ... 

Aseptic techniques used should be reviewed periodically to ensure that departures from aseptic practices do not develop. Personnel should undergo periodic aseptic technique training (SOP [provide number]), particularly when problems are detected (during the course of routine environmental and negative control monitoring) or when operators perform the test infrequently. 

Perform the entire operation in a laminar flow hood with aseptic technique. 

Validation of aseptic technique for ml vials. Media fill batch No. 

Pour an additional 10 ml (approximately) of sterile 0.1% Tween solution into each filter using aseptic technique. This rinse is intended to wash the interior walls of the filtration units and carry any bacteria adhering to these surfaces onto the surface of the membrane filter. 

---