基本无菌化技术

 

INTRODUCTION

The regulations promulgated to implement the amended Animal Welfare Act require that all survival surgery be performed using aseptic procedures. This includes the use of surgical gloves, masks, sterile instruments and aseptic technique.

In this chapter, the Principles of Aseptic Technique will be discussed with the emphasis on the practical application of these principles in the laboratory set ting. In centralized experimental surgeries , a well-trained staff should be available to advise those who use such facilities and oversee its operation to ensure the maintenance of an aseptic environment for survival surgery. When survival surgery is conducted outside such an environment, it is th e principal investigator's responsibility to ensure that appropriate aseptic conditions and practices are maintained. This chapter will provide the necessary information to carry out this responsibility.

Prior to discussing the specific principle s of aseptic surgery a brief review of pertinent terminology is necessary.

TERMINOLOGY

Antimicrobial - An agent or action that kills or inhibits the growth of micro-organisms.

Antiseptic - A chemical agent that is applied topically to inhibit the growth of micro-organisms.

Asepsis - Prevention of microbial contamination of living tissues or sterile materials by excluding, removing or killing micro-organisms.

Autoclave - A steam ste rilizer consisting of a metal chamber constructed to withstand the pressure that is required to raise the temperature of steam to the level required for sterilization. Early models were termed "autoclaves" because they were fitted with a self-closing door .

Bactericide - A chemical or physical agent that kills vegetative (non-spore forming) bacteria.

Bacteriostat - An agent that prevents multiplication of bacteria.

Commensals - Non-pathogenic micro-organisms tha t are living and reproducing as human or animal parasites.

Contamination - Introduction of micro-organisms to sterile articles, materials or tissues.

Disinfectant - An agent that is intended to kill or remove pathogenic micro -organisms, with the exception of bacterial spores.

Pasteurization - A process that kills nonspore-forming micro-organisms by hot water or steam at 65-100oC.

Pathogenic - A species that is capable of causing disease micro-or ganism in a susceptible host.

Sanitization - A process that reduces microbial contamination to a low level by the use of cleaning solutions, hot water or chemical disinfectants.

Sterilant - An agent that kills all types of mi cro-organisms.

Sterile - Free from micro-organisms.

Sterilization - The complete destruction of micro-organisms.

Since the pioneering work of such surgeons as Joseph Lister, who introduced the use of carbolic acid ant iseptics in 1865, and William Halstead, who advocated the use of surgical gloves in 1898, surgeons have strived to eliminate surgical infections through the use of aseptic technique. Potential sources of contamination are well defined. They include the pa tient and the surgical environment: the surgeon and support staff, the instruments, sutures, drapes and all other equipment which can have contact with the surgical field.

FACILITIES

The basis for this discussion about facilities wi ll be the recommendations for Aseptic Surgery contained in the Guide for the Care and Use of Laboratory Animals . The Guide states:

"Functional areas for aseptic surgery should include a separate support area, a preparation area, the operating room or rooms and an area for intensive care and supportive treatment of animals. The interior surfaces of this facility should be constructed of materials that are impervious to moisture and easily cleaned. The surgical support area should be d esigned for storing instruments and supplies for washing and sterilizing instruments. Items that are used on a regular basis, such as anesthetic machines and suture materials, can be stored in the operating room."

"There should be a separate surgi cal preparation area for animals. An area equipped with surgical sinks should be close to, but apart from, the operating room. A dressing area should be provided for personnel to change into surgical attire."

The surgical facility should be locate d outside normal facility traffic patterns. This can help to minimize the potential for surgical suite contamination by the movement of personnel and equipment. Personnel access to these areas should be restricted to essential surgical support staff.

Ideally, the operating room ventilation system should provide a net positive pressure with respect to the surrounding facilities. The system should be regularly monitored. Maintenance work should be performed when the surgery is idle. Ventilation filte rs should be inspected and cleaned or replaced at regular intervals. If explosive anesthetics agents are to be used, the Guide recommends that floors should be conductive and electrical outlets should be explosion-proof and located not less than 5 feet off the floor. Dedicated surgical facilities should be used for aseptic surgeries and the storage of essential surgical equipment, not as general storage space.

EQUIPMENT

The equipment in areas used for aseptic surgery should b e easy to clean and portable to simplify sanitization of the area. The operating table should be con structed with a durable surface material impervious to moisture which can be readily cleaned. Plastic or stainless steel is frequently used for this purpo se. Other useful table design features which assist patient positioning include height and tilt adjustments, V-trough configuration and restraint strap cleats. A disadvantage of stainless steel construction is that it predisposes animals to hypothermia. T his can be corrected by the routine use of a heating pad placed under the surgical patient. Reusable, easy to clean vinyl heating pads which recirculate hot water are frequently used for this purpose. Inexpensive short-term alternatives include hot water bottles or heat lamps. Any heat source should be used with caution to prevent patient burns.

Instrument tables provide the surgeon ready access to the surgical instruments and minimize the risk of sterilized instrument contamination by contact wit h non-sterile fields. Commercially available instrument tables, such as Mayo stands, consist of a stainless steel tray supported by a pedestal base with a foot-operated height adjustment device, but any tray arrangement may be used for this purpose. The u nit should be easy to clean and simple to operate. The drapes in an instrument pack frequently include impervious table covers which can minimize instrument contamination and allow the surgeon to reposition the table without breaking aseptic technique dur ing the procedure. Surgical buckets on wheels (kick buckets), which can be readily positioned with the feet, are another recommended piece of equipment. They should be easy to clean and lined with a plastic bag which should be changed at the end of the pr ocedure.

Adequate lighting is essential for performing surgical procedures. A variety of fixtures can be used to provide sufficient light. The commercially available surgical light fixtures may be ceiling or wall-mounted or free standing. Surgical lights are often positioned above the operative area and should be regularly wiped with a moist towel prior to use to minimize potential contamination of the sterile field below. Light fixtures designed with detachable sterilizable handles allow the surg eon to adjust the beam during surgery. Wheeled, height-adjustable intravenous drip stands should be available when conducting major surgery. Care should be taken to assure that the I.V. tubing does not contaminate the sterile fields. Positioning the I.V. tubing along the heating blanket helps warm I.V. solutions before infusion.

Surgical suction is another useful accessory. Sterilized tubing and suction tips are provided for use in the aseptic field. The tubing is connected to a non-sterile suctio n bottle which in turn is connected to a built-in vacuum line. If built-in vacuum lines are not available, portable electric vaccum pumps are commercially available.

Ancillary equipment such as respirators, electrosurgical units and ECG monitors should be portable and included with the light fixtures in a routine equipment cleaning schedule. Specific details on such devices could be obtained from an institutional veterinarian or surgical supervisor.

Surgical instrumentation and pack prepa ration will vary with the type and complexity of surgery to be performed. Consultation with an institutional veterinarian or surgical supervisor could be helpful when selecting the appropriate surgical instruments necessary to perform a proposed procedure . Instrument packs should be double wrapped. Various commercial materials are available for this purpose. Although pack instrument preparation will be discussed later, as many sterilizable items as possible should be included. These might include prepacka ged surgical blades, sponges, saline bowls and miscellaneous catheters.

Personnel

Aseptic technique requires careful attention to a series of steps which begins with patient and instrument preparation and ends at final wound closure . Failure at any one step may result in wound infection which could compromise the animal's health and the experimental data derived from the animal. Aseptic technique designs all actions and motions to protect the sterile field from contamination. The su rgeon and surgical support staff must be adequately trained to perform each step correctly. Acquiring and developing the necessary skills to maintain aseptic technique requires practice. Personnel should receive instruction on the indications for aseptic technique, the sources of potential contamination, patient, instrument and equipment preparation, sterilization systems, gowning and gloving techniques, and intraoperative aseptic management. Once this theoretical knowledge is gained, trainees can rapidly learn by observing the aseptic management techniques of a well-trained surgical support staff. Trainees should practice each step until correct techniques become second nature.

Assistance with employee training may be available from the instituti onal veterinarian, a member of the animal care staff and/or a member of a hospital surgery staff.

STERILIZATION

Sterilization is the process that is intended to kill or remove all types of micro-organisms. There are two principal st erilization methods:

1) Physical (dry heat or saturated steam)

2) Chemical (ethylene oxide gas or chemical liquids).

Factors which determine the method to be used are the type of micro-organisms involved, the nature of the article to be sterilized and the time available for sterilization.

Physical Methods (Steam)

Steam sterilization (frequently referred to as autoclaving) depends on the use of steam above 100oC. Temperatures ranging from 121-134oC at pressure s of 15-30 psi are generally recommended. The biocidal action of moist heat is a denaturation of major cell constituents. Many sterilizers are designed to provide an automatic sterilization cycle. In the first stage of the cycle, air is evacuated and the chamber brought to the pre-set sterilizing temperature, which is maintained for a holding period sufficient to kill all microbial contaminants. Minimum holding times for the sterilization of medical equipment are 15 minutes at 121oC, 10 minutes at 126oC, and 3 minutes at 134oC. The steam is then removed and instrument packs are allowed to dry or liquids cool. The drying stage may be adjusted to suit the load.

The chamber is then pressure by the introduction of filtered air.

The recommende d periods of exposure vary with the nature of the article to be sterilized and the method used to wrap the article. Specific details are available from the references at the end of the chapter.

Steam sterilization has the advantage of rapid penetr ation of wrapped materials with the destruction of all viruses and bacteria, including the most resistant spores. The sterilization of different supplies is more readily controlled than in other types of sterilizers. However oils, grease and powdered subs tances cannot be sterilized by this method. The steam autoclave must be maintained in good repair and operated correctly in order to perform to specifications. Sterilization failure can occur when machines are not regularly serviced.

Steam autocla ve function should be monitored continuously using one or more of several commercially available indicator systems. The color change on a chemical dye impregnated indicator strip placed within the pack can provide a convenient and rapid visual check thatt he appropriate sterilization conditions were reached. Function should also be monitored on a regular basis using commercially available biological indicators. Spore strips of Bacillus stearothermophilus are placed within the wrapped article prior t o sterilization. After sterilization the strip is incubated at 57oC for 48 hours. The absence of growth indicates effective sporicidal autoclave action.

Chemical Methods (Gas)

Ethylene oxide gas is effective against all types of mic ro-organisms. The biocidal action of this gas is considered to be alkylation of nucleic acids. It is non-corrosive and safe for most plastic and polyethylene materials. However, it is not applicable to liquids or to articles in impervious packaging materi al. It cannot be used to sterilize animal diets due to the potential toxic effects of this gas. It can also be a toxic hazard for animals receiving prosthetic implants which have been sterilized by this gas. The operating pressures and temperatures (45-60 oC and 10-12 psi) of ethylene oxide sterilizers are considerably less than for steam units. Articles should be well aerated prior to use to minimize the potential for tissue toxicity. Aeration should be done in a manner which minimizes exposure of personn el.

This can be accomplished through the use of self-aerating sterilizers or separate aeration cabinets.

Ethylene oxide gas is a potential carcinogen and mutagen and represents a potential occupational health hazard for personnel operating sterilizers. Operation of gas sterilizers and aerators should be in strict conformance with manufacturers' recommendations and institutional policies. Personnel exposure should be minimized by appropriate ventilation of exhaust gas. A regular monitoring program for personnel should be in place.

Gas sterilizer function should be monitored continuously using one of several commercially available indicator systems. The color change on a chemical dye-impregnated indicator strip placed within the pack can provide a convenient and rapid visual check that the appropriate sterilization conditions were reached. Function should also be monitored on a regular basis using a commercially available biological indicator such as spore strips of Bacillus subti lus which are placed within the wrapped article prior to sterilization. After sterilization the strip is incubated at 37oC for 24 hours. The absence of growth indicates effective sterilization.

Temperature-sensitive adhesive tape used to secur e packages prior to sterilization only indicates that the package has been exposed to the sterilizer; this tape does not monitor sterilizer function.

Chemical Methods (Liquids)

The use of chemical solutions as a sterilization techni que for surgical equipment is frequently employed, but it should be stressed that most solutions only disinfect and do not guarantee sterility. When the necessity for maintaining sterility is a critical factor, as in the implantation of prosthetic devices , indwelling catheters or vascular access ports, disinfection in chemical solutions is not recommended. Such prostheses should be thoroughly sterilized by either gas or steam. Chemical solutions, however, offer the advantages of safety for delicate and th ermolabile plastics.

Other limitations of chemical solutions should also be appreciated. Equipment must be thoroughly cleaned before immersion, as chemical action is ineffective in the presence of proteins or fats. There are currently no indicator s commercially available to monitor the effectiveness of this sterilization method.

Alcohols are neither sporicidal nor viricidal. They are not stable and lose effectiveness through evaporation. Alcohols cannot be used for instruments that have pl astic or cemented parts.

The chlorine compounds exert their biocidal action by oxidization. The for mulations which require the mixing of acid and base components with water to generate chlorine dioxide, offer the advantages of wide spectrum bioci dal action and a safe alternative to the more hazardous phenols or formaldehydes. The active shelf life of mixed chemicals is reported to be 24-48 hours.

If chemical sterilization of instruments is the method to be used, it can be performed in cov ered trays containing fresh solutions. A two-tray system, one each for even-numbered and odd-numbered days, will ensure that instruments have a full 24-hour contact time.

PREPARATION OF THE ANIMAL

The animals should be prepared in a n area separate from where surgery will be performed. Preparation is facilitated by first inducing anesthesia. The stomach, rectum and urinary bladder can then be evacuated as required at this stage. Hair is then removed from the surgical site using elect ric clippers equipped with a fine blade. A liberal area is clipped to anticipate any enlargement of the initial surgical incision and minimize wound contamination from adjacent unclipped areas. In rodents the need to minimize the loss of heat during surge ry and recovery must be balanced against the need to provide an adequate aseptic field when clipping the animal. Animal hair, particularly rabbit hair, tends to clog clipper blades. This can be minimized by frequent cleaning of the blades and regular lubr ication with a commercial aerosol product between use. A vacuum can be used to clean up after clipping. Depilatory creams may be applied to the surgical site, but they may cause contact dermatitis which may interfere with the healing process.

Init ial skin cleaning can be done prior to moving the animal to the operating area. When the animal is moved to the operating area, it should be positioned on a heating pad on the surgical table. To avoid burns heating pads should be wrapped to prevent direct contact with the animal. Inclined positioning with a tilt table is indicated for some procedures and some species. The surgical approach will dictate actual animal position; however, some guidelines to consider are:

a. The animal's respiratory fu nction should not be compromised by overextension of forelegs stretched towards the head, or by excessive body tilt which causes pressure from the abdominal organs on the diaphragm.

b. Limbs should not be extended beyond their normal range of moti on and restraint straps should be padded as needed to prevent impaired venous return in extremities.

c. After the animal has been secured, any monitoring devices such as ECG electrodes and esophageal stethoscopes should be placed and their functio n tested.

d. Ruminants are frequently positioned on a slight incline with the head dependent, to minimize the potential for aspiration of rumen fluids. After intubation with a cuffed endotracheal tube, a large bore stomach tube is also frequently placed down the esophagus to remove rumen fluids and gas.

The animal is now ready for final preparation of the surgical site. Personnel who perform the presurgical skin preparation should wear a cap and mask when preparing the surgical scrub suppl ies and when opening pre-sterilized sponge and drape packs. Skin preparation solutions may be applied with a sterile sponge held by a pair of sterile forceps or by a hand wearing a sterile glove. A sterile surgical glove is put on one hand, while the othe r hand is used to hold and manipulate non-sterile bottles of surgical scrub solution. A sterile sponge held in the gloved hand is saturated with surgical scrub solution and the surgical area is scrubbed beginning with the central incision site and working progressively in a circular fashion to the margins of the shaved area (see Figure 1 ). The sponge is then discarded and the process repeated, working from the center to the outside to minimize contamination of the surgical site.</ P>

Some of the most frequently used chemical solutions for preoperative surgical skin preparation are: chlorhexidine, iodophors and povidone-iodine surgical scrubs. Recommended contact times vary from 2 to 4 minutes.

Following removal of the s crub solution with a 70 percent alcohol solution using the same technique, an iodine skin solution is painted on the site using the above technique and left to dry.

Drapes serve to isolate the surgical site and minimize wound contamination. Drapes should be positioned without the fabric dragging across a non-sterile surface. There are two basic types of drape systems used: fenestrated and four corner.

Fenestrated drapes have a hole in them which is placed over the surgical site. Frequently used for smaller species, these drapes are utilized for routine elective procedures. The fenestration should be just slightly larger than the intended incision.

The second alternative is the four corner drape system in which a drape is placed at each of the four margins of the surgical site. Four corner drapes are applied one by one in a clockwise or counterclockwise direction. Each drape should be carefully positioned with a 6 to 8 inch edge folded underneath at the incision site (see Figure 2 A to D ). Small adjustments in position can then be made without contaminating the underside of the drape. Drapes can be secured in place with towel clamps at the four corners or aerosol adhesive applied to the margins of the surgic al site prior to draping.

Some surgeons prefer to secure four corner drapes, then apply a fenestrated drape as a second layer of protection (see Figure 2, E and F ). Ideally, the patient and entire surgical table should be draped, and the drape extended to the instrument table. The need to monitor the draped patient should always be considered. The surgeon who has to work alone often has to assess eye and jaw reflexes, mucous membrane or tongue color; therefore the head sho uld not be entirely covered by drape material.

Self-adhesive backed paper drapes and clear plastic drape material with one adhesive surface are also commercially available.

PREPARATION OF A SURGICAL PACK

A well-organized an d consistent surgical pack preparation system can avoid errors and facilitate surgery. Instruments can be cleaned by hand or with an ultrasonic cleaning unit. After cleaning, each instrument should be inspected to ensure that all debris has been removed. After physical cleaning, instruments can be dipped in a commercial protective lubricant solution and allowed to drain dry. Items should be assembled on a tray and arranged in a consistent order. Materials should be placed in sequential order so that items used first are placed on top (see Figure 3 ). Packs should not be too densely packed in the autoclave to allow for adequate steam or gas penetration. Indicator test strips can be placed deep within the pack. Packs should be double wrapped, and the outer wrap should be secured with adhesive indicator tape on which is recorded the date of sterilization. When applicable, the type or contents of pack (e.g., laparotomy, thorocotomy) can also be noted on the tape.

Note the follo wing points when opening a sterilized surgical pack. The sterilization date should be checked; the shelf life of wrapped instruments is generally considered to be up to 6 months. The adhesive indicator tape should be noted for the appropriate color change and the pack description should be checked, when applicable. Packs should be placed on a dry instrument tray and the outer wrapping carefully unfolded by touching only the corners of the outside drape surface. The operator should avoid reaching over thep ack. The packs should not be opened too early. The surgeon working without assistance should open the pack immediately before scrubbing. Any other sterilized supplies which can be opened onto a sterile field should be made ready at this time.

P REPARATION OF THE SURGEON

In a laboratory setting, the extent of surgeon preparation will depend on the facilities and the need for strict attention to aseptic technique. Well-equipped surgical facilities, in which sophisticated survival proce dures are performed, generally require surgeons to wear appropriate surgical clothing and to scrub, gown and glove. Instruction in such procedures should be done on a one-to-one or small group basis in appropriately designed scrub rooms. To augment the ac tual hands-on approach or when necessary a video tape demonstration or pictorial diagrams can be used. Readers are advised to consult the references quoted at the end of the chapter for instructional details.

To minimize wound contamination potent ial, the surgeon should change into surgical scrubs and shoes or wear shoe covers. Head covers and face masks should cover all facial hair. Remove all rings, jewelry and wrist watches before scrubbing. Finger-nails should be trimmed short and cleaned with a disposable nail cleaner. Scrub sinks equipped with leg or foot-operated faucets are ideal. Regular faucets must be turned on, adjusted and not touched again. The hands and forearms are washed for 30 to 60 seconds with a surgical scrub soap. Then a ster ile brush is used to methodically scrub all surfaces of the hands, fingers and forearms down to the elbows. Both arms are rinsed and the process repeated starting with fingertips working down to the elbows. The definition of a "complete surgical scrub" is controversial. However, contact times of 3 to 15 minutes and/or 5 to 20 strokes per surface are frequently recommended.

After rinsing, the hands are held together high and rinse water allowed to drip from the elbows. This minimizes the contaminat ion of hands by water dripping from the non-sterile upper arm areas. The surgeon should avoid touching anything at this stage except to dry the hands with a sterile towel. Next the sterile gown is carefully removed from the pack to avoid touching the outs ide of the gown. It is held away from the body and shaken out. The sleeve hole is located and each arm inserted in turn. Correct gowning requires an assistant to tie the back of the gown at the neck and waist (being careful to touch only the inner gown su rface).

Sterile surgical gloves are packaged with the cuff of each glove turned down. This allows the gloves to be put on without the bare hands ever touching the outside surface of the glove. One glove is picked up by the turned-down cuff and pul led onto the hand with the cuff left turned down (see Figure 4 - 1 and 2 ). Using the gloved hand, pick up the remaining glove by inserting the fingers into the cuff and pulling it onto the opposite hand (see F igure 4 - 3 ). Then the glove cuff is lifted over and onto the gown cuff and the process repeated on the other hand (see Figure 4 - 4,- 5,- 6 ). This technique is known as "open gloving." An alternative and more difficult method is closed gloving, descriptions of which can be found in general surgical texts. Remove the powder on the outer glove surface by wiping the gloved hands with a damp sterile gauze. Arms and hands should be held above the waist at all times. Aseptic techni que is maintained when the gowned and gloved surgical team only touches sterilized equipment within the sterile field.

The surgeon working alone faces logistical problems when attempting rigid aseptic protocol as defined above. A proposed practica l sequence of steps to minimize errors is presented as follows:

1. Assemble all sterilized supplies.

2. Change into scrubs.

3. Set up table, heat pads and gas machines, check equipment.

4. Weigh animal, induce anesthesia. P repare animal by hair clip and shave, catheters placed as required.

5. Position and secure animal on the table.

6. Connect to gas machine, connect accessory monitors. Start I.V. lines as required.

7. Make certain that a stable anes thetic plane is attained.

8. Put on cap, mask. Open sterile instrument and prep packs.

9. Using one sterile glove, prepare surgical site with scrub solutions.

10. Put on new sterile glove and drape patient.

11. Remove glove s. Recheck stable anesthetic state. Open glove and gown packs if not included in instrument pack.

12. Perform surgical scrub.

13. Put on gown and gloves.

14. Start surgery.

SUMMARY

The practice of aseptic tec hnique, when performing survival surgical procedures, minimizes the chances that animal health or experimental data will be compromised by post-surgical infections. Aseptic techniques require that appropriate facilities and equipment be available and that the personnel involved be adequately trained. The key element in maintaining an aseptic environment is well-trained personnel who understand the principles of aseptic technique and utilize this knowledge on an ongoing basis.

REFERENCES

Animal Welfare Act (Title 7 U.S.C. 21 31-2156) as amended by PL 99-198, December 23, 1980.

Lang, C.M. Animal Physiologic Surgery . Springer-Verlag, New York, 1976.

Leonard, E.P. Fundamentals of Small Animal Surgery . W.B.S anders, Philadelphia, 1968.

Knecht, C.D., Allen, A.R., Williams, D.J., et al. Fundamental Techniques in Veterinary Surgery . W.B. Sanders, Philadelphia, 1981.

Gardner, J.F. and Peel, M.M. Introduction to Sterilization and Disinfec tion . Churchill Livingstone, Melbourne, 1986.

McCredie, J.A. and Burns, G.P. (eds.), Basic Surgery . MacMillan Pub. Co., New York, 1986.

Banerjee, K. and Cheremisinoff, P.N. Sterilization Systems . Technomic Publishing Comp any Inc., Lancaster, PA; 1985.

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