Introduction
Aseptic technique is the method adopted to eliminate all potential sources of contamination of the surgical field. Adopting high standards of asepsis will reduce the risk of post-surgical wound infections developing. This is important for both animal welfare and scientific reasons.
Wound infections can cause pain, distress, and if severe can result in the breakdown of the surgical wound and generalised infections that cause severe illness or death of the animal. Even low grade infections can result in changes to the animal’s physiology that may interact or interfere with specific research procedures.
Adopting good aseptic technique requires careful attention to the preparation of the operating room environment, the animal, the surgeon, and all materials that will come into contact with the surgical field.
The video tutorial below demonstrates all of these aspects of preparing and undertaking aseptic surgery.
Video Tutorial (18:31)
Transcript
Introduction
Before commencing any surgical procedure, it is important to prepare a protocol and a list of equipment that will be required. This can include a simple checklist that can be used to ensure that all the required instruments are prepared and sterilised and that sutures and other items are readily available in the quantities required. A checklist will also help to ensure that the anaesthetic apparatus, heating pads, incubators and other equipment are all prepared for use.
Aseptic surgical procedures can be carried out successfully either in purpose-designed operating theatres or in a modified procedure room. Whichever location is used, provision must be made for an area to prepare the animal, an operating area, and a recovery area. Separate areas are also needed for instrument preparation and for the surgeon to scrub up, gown and glove.
Instruments
Instruments must be sterilised before they are used for aseptic surgery. This can be achieved in a number of ways, but the easiest and most reliable method is by use of an autoclave. Small tabletop models are available that can easily be installed in most research facilities at modest cost.
To prepare the instruments for sterilisation and to ensure they remain sterile prior to use, they need to be cleaned, dried, and then appropriately packed. A cloth or paper drape is wrapped around the instruments. This will provide a sterile working surface after the pack has been opened. The pack is sealed with autoclave tape and is then placed in an outer bag or wrapper. The pack is typically autoclaved for 15 minutes at 134º C.
Successful completion of the sterilisation cycle can be confirmed in a number of ways. Some autoclaves provide a printed record of the sterilisation cycle. The autoclave tape on the pack should change colour and indicator tubes may be used to confirm successful sterilisation. If the sterilised packs are to be stored for future use, the date of sterilisation and shelf life should be recorded.
Ideally, a separate set of sterilised instruments should be used for each animal. But for batch surgery of rodents, an acceptable compromise is to sterilise the instrument tips using a hot bead steriliser. Instruments first need to be cleaned using a sterile brush in sterile water to avoid becoming clogged with organic material. Sterile water must be used, not saline.
The instrument tips are then briefly placed into the hot bead steriliser which rapidly raises their temperature to over 200º C. They are then replaced on a sterile drape ready for use on the next animal. Briefly, immersing instruments in alcohol is not an effective means of sterilisation.
Other Materials
All other materials needed for the surgical procedure that will come into contact with the surgical site must be sterile and must be used within their stated expiry dates.
Many of these items, such as scalpel blades and sutures, can be purchased in sterile packs and opened when required. An assistant opens the outer packing so that the surgeon can take the item, handling only the inner sterile surfaces. When the assistant opens the items, this should be done adjacent to the surgical area, not over it, so that inadvertent contamination of the sterile field is avoided.
Some delicate materials that could not withstand autoclaving, for example catheters can be purchased pre-sterilised or sterilised using other methods such as ethylene oxide.
Preparation of the Animal
After induction of anaesthesia, the surgical site should be clipped to remove the fur. It is important that clippers are used carefully to avoid abrading the skin. The angled end of the clipper blade should remain parallel to the animal’s skin. A vacuum cleaner is used to remove the clipped fur. Clipping of the fur must be done in a separate area to avoid contaminating the operating area.
After removal of the fur, the skin is cleaned using a suitable disinfectant, for example chlorhexidine, either in alcohol or water. Alcohol alone is not suitable for disinfection of the skin. A number of different techniques can be used. For example, the skin can be cleaned using sponges or swabs, wiping from the centre in a circular motion to close to the edge of the clipped area. The sterile sponge used should then be discarded and the process repeated two or three times using a fresh sponge each time. Alternatively, the skin can be wiped from the centre, where the incision will be made, to the edges of the clipped area. The sterile swab used should then be discarded and the process repeated using a fresh swab.
When clipping mice, particular care needs to be taken to avoid damaging the delicate skin. The skin may need to be gently tensed to enable clipping. A clip in the opposite direction provides an even cleaner final result. In both rats and mice, ointment should be applied to the eyes to prevent drying during anaesthesia. A similar technique of wiping from the centre to the edges of the clipped area can be used to clean and disinfect the skin in mice. The process can be repeated up to three times to ensure thorough cleaning. Avoid using too much disinfectant solution as this can increase the risk of the animal becoming hypothermic.
Before commencing any surgical procedure, it is important to prepare a protocol and a list of equipment that will be required. This can include a simple checklist that can be used to ensure that all the required instruments are prepared and sterilised and that sutures and other items are readily available in the quantities required. A checklist will also help to ensure that the anaesthetic apparatus, heating pads, incubators and other equipment are all prepared for use.
Aseptic surgical procedures can be carried out successfully either in purpose-designed operating theatres or in a modified procedure room. Whichever location is used, provision must be made for an area to prepare the animal, an operating area, and a recovery area. Separate areas are also needed for instrument preparation and for the surgeon to scrub up, gown and glove.
Instruments
Instruments must be sterilised before they are used for aseptic surgery. This can be achieved in a number of ways, but the easiest and most reliable method is by use of an autoclave. Small tabletop models are available that can easily be installed in most research facilities at modest cost.
To prepare the instruments for sterilisation and to ensure they remain sterile prior to use, they need to be cleaned, dried, and then appropriately packed. A cloth or paper drape is wrapped around the instruments. This will provide a sterile working surface after the pack has been opened. The pack is sealed with autoclave tape and is then placed in an outer bag or wrapper. The pack is typically autoclaved for 15 minutes at 134º C.
Successful completion of the sterilisation cycle can be confirmed in a number of ways. Some autoclaves provide a printed record of the sterilisation cycle. The autoclave tape on the pack should change colour and indicator tubes may be used to confirm successful sterilisation. If the sterilised packs are to be stored for future use, the date of sterilisation and shelf life should be recorded.
Ideally, a separate set of sterilised instruments should be used for each animal. But for batch surgery of rodents, an acceptable compromise is to sterilise the instrument tips using a hot bead steriliser. Instruments first need to be cleaned using a sterile brush in sterile water to avoid becoming clogged with organic material. Sterile water must be used, not saline.
The instrument tips are then briefly placed into the hot bead steriliser which rapidly raises their temperature to over 200º C. They are then replaced on a sterile drape ready for use on the next animal. Briefly, immersing instruments in alcohol is not an effective means of sterilisation.
Other Materials
All other materials needed for the surgical procedure that will come into contact with the surgical site must be sterile and must be used within their stated expiry dates.
Many of these items, such as scalpel blades and sutures, can be purchased in sterile packs and opened when required. An assistant opens the outer packing so that the surgeon can take the item, handling only the inner sterile surfaces. When the assistant opens the items, this should be done adjacent to the surgical area, not over it, so that inadvertent contamination of the sterile field is avoided.
Some delicate materials that could not withstand autoclaving, for example catheters can be purchased pre-sterilised or sterilised using other methods such as ethylene oxide.
Preparation of the Animal
After induction of anaesthesia, the surgical site should be clipped to remove the fur. It is important that clippers are used carefully to avoid abrading the skin. The angled end of the clipper blade should remain parallel to the animal’s skin. A vacuum cleaner is used to remove the clipped fur. Clipping of the fur must be done in a separate area to avoid contaminating the operating area.
After removal of the fur, the skin is cleaned using a suitable disinfectant, for example chlorhexidine, either in alcohol or water. Alcohol alone is not suitable for disinfection of the skin. A number of different techniques can be used. For example, the skin can be cleaned using sponges or swabs, wiping from the centre in a circular motion to close to the edge of the clipped area. The sterile sponge used should then be discarded and the process repeated two or three times using a fresh sponge each time. Alternatively, the skin can be wiped from the centre, where the incision will be made, to the edges of the clipped area. The sterile swab used should then be discarded and the process repeated using a fresh swab.
When clipping mice, particular care needs to be taken to avoid damaging the delicate skin. The skin may need to be gently tensed to enable clipping. A clip in the opposite direction provides an even cleaner final result. In both rats and mice, ointment should be applied to the eyes to prevent drying during anaesthesia. A similar technique of wiping from the centre to the edges of the clipped area can be used to clean and disinfect the skin in mice. The process can be repeated up to three times to ensure thorough cleaning. Avoid using too much disinfectant solution as this can increase the risk of the animal becoming hypothermic.