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.
Preparation of the Surgeon
While an assistant is preparing the animal, the surgeon should prepare to carry out surgery. Appropriate protective clothing should be used to avoid contaminating the surgical site. Since operating areas should be kept warmer than animal holding rooms, to help reduce the risk of animals becoming hypothermic. During anaesthesia, it may be more comfortable to change into a scrub suit. This will also avoid long sleeved clothing becoming wet during hand washing. Wearing a head cover and mask is strongly recommended, but the most important step is to perform effective disinfection of your hands by scrubbing up and then putting on a sterile gown and sterile surgical gloves.
Having taken off any watches or jewellery, wash your hands with a disinfectant soap. Methodically work around each finger and down over your hands and forearms and allow sufficient contact time for the disinfectant to act. The process of hand washing aims to remove surface dirt and grease from your skin and allow sufficient contact time with a disinfectant soap to kill or inhibit bacteria in the outer layer of the skin. The only time a scrubbing brush should be used is to clean under your nails. The remainder of the process is just careful and repeated hand washing. During this process, keep the level of your hands above that of your elbows so that water drains away from what should become the cleanest areas.
After completing this process, do not touch any items such as the sink or taps with your hands. Taps and soap dispensers should be elbow operated, but if these are not available, an assistant can operate them so that repeated contamination of your hands is avoided. An assistant opens the pack which contains a sterile gown and cloth or paper towels that are used to dry your hands. Work from the fingers and hand down to the elbows, then discard the cloth again, being careful not to touch any non-sterile surface.
The gown is then picked up and opened after stepping away from the gowning table so that the outer surface of the gown is not contaminated. The surgeon places their arms into the sleeves of the gown and an assistant helps to pull it into a comfortable position. At this stage, the surgeon’s hands remain within the sleeves of the gown. It is then possible to put on a pair of sterile gloves using a technique called closed gloving.
Start by placing one glove, thumb side down, on your wrist. With the fingers of the glove pointing up your arm. Grasp the cuff of the glove using your thumb through the sleeve of your gown. Using your other hand, still within the sleeve of the gown, turn the cuff of the glove down over your hand and slide the hand being gloved out of the sleeve of the gown and into the glove. If necessary, adjust the cuff of the glove, then repeat this process for the other hand. Note that the thumb side of the glove is laid onto the surgeon’s wrist. An assistant completes the process of tying the gown.
Having carried out this process, it is vital that you avoid touching any non-sterile surfaces. Standing with your hands clasped in front of your chest helps avoid inadvertent contact with non-sterile items.
Cloth gowns need to be properly packed and autoclaved. A convenient alternative is to use paper disposable gowns. These are supplied pre-sterilised at relatively low cost in a pack that also contains hand towels.
Preparation for Surgery
The surgeon can now prepare the sterilised items needed for surgery. An assistant opens the outer wrapping of the instrument pack and hands this to the surgeon so they can take it holding only the inner sterilised wrapping. When unwrapping the pack, drop the corners of the drapes so that the back of your hand does not touch the surface of the table. The animal can then be draped, once again, being careful not to touch any non-sterile surface. Using a drape prevents sterilised items touching the animal’s fur and becoming contaminated.
Smaller cloth drapes can be made or purchased for use with mice. Drapes can be either plastic, cloth or paper and sterilised either by autoclaving or purchased pre-sterilised. Disposable paper drapes are very convenient for use for batch surgery of rodents, allowing a new sterile drape to be used for each animal.
When using a paper drape, a suitable sized hole can be cut to access the surgical site. Avoid cutting along one of the pre-folded sections as this can prevent the drape conforming to the animal. The sterile field can be extended by using an additional paper or cloth drape.
Surgical Procedure
After the sterile pack has been opened, the instruments can be checked to ensure all the items needed are present and can then be arranged into a convenient order for undertaking surgery.
An assistant opens the outer packing of items such as scalpel blades and sutures, so that the surgeon can take the item, handling only the inner sterile surfaces. When the assistant opens the item, this should be done adjacent to the sterile area, not over it, so that inadvertent contamination of the sterile field is avoided.
Surgery is now undertaken. After use, each instrument should be placed back on a sterile drape.
If you need to touch a non sterile surface, for example, to adjust an anaesthetic vaporiser, and your assistant is not immediately available, this can be done using a large sterile swab, which is then discarded.
In addition to assisting with these procedures, your assistant can record the start and end time of the procedure and note any complications or other relevant information in your laboratory notebook.
Batch Surgery
It is often necessary to carry out a series of procedures in one operating session. After completing the first procedure, the surgeon should lift the sterile drape and dispose of it. The assistant then moves the animal to a recovery area, or directly into a warm incubator. They may also have been able to anaesthetise the next animal and clipped it ready for surgery. This animal can be positioned on the table and a skin preparation undertaken by the assistant.
During this time, the surgeon can use a hot bead steriliser to re-sterilise the tips of the instruments. The instruments must first be cleaned in sterile water. The instruments heat up rapidly in the steriliser but also cool rapidly so can be used safely within a minute or so. Alternatively, a new set of instruments can be used.
If a new set of instruments is to be used, the surgeon should re-glove before handling them. This can be done without the need to re-gown. The surgeon touches only the inner surface of the gloves with his ungloved hand. He can then use his gloved hand to handle the other glove, making sure that his ungloved fingers do not contact the outer surface of either glove.
The surgeon can then re-drape the animal using a new sterile drape and carry out surgery. Ideally, a new instrument kit should be used for each animal and if using a hot bead steriliser, the kit should be changed when there is obvious gross contamination or after 4-5 surgical procedures have been undertaken.
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.
Preparation of the Surgeon
While an assistant is preparing the animal, the surgeon should prepare to carry out surgery. Appropriate protective clothing should be used to avoid contaminating the surgical site. Since operating areas should be kept warmer than animal holding rooms, to help reduce the risk of animals becoming hypothermic. During anaesthesia, it may be more comfortable to change into a scrub suit. This will also avoid long sleeved clothing becoming wet during hand washing. Wearing a head cover and mask is strongly recommended, but the most important step is to perform effective disinfection of your hands by scrubbing up and then putting on a sterile gown and sterile surgical gloves.
Having taken off any watches or jewellery, wash your hands with a disinfectant soap. Methodically work around each finger and down over your hands and forearms and allow sufficient contact time for the disinfectant to act. The process of hand washing aims to remove surface dirt and grease from your skin and allow sufficient contact time with a disinfectant soap to kill or inhibit bacteria in the outer layer of the skin. The only time a scrubbing brush should be used is to clean under your nails. The remainder of the process is just careful and repeated hand washing. During this process, keep the level of your hands above that of your elbows so that water drains away from what should become the cleanest areas.
After completing this process, do not touch any items such as the sink or taps with your hands. Taps and soap dispensers should be elbow operated, but if these are not available, an assistant can operate them so that repeated contamination of your hands is avoided. An assistant opens the pack which contains a sterile gown and cloth or paper towels that are used to dry your hands. Work from the fingers and hand down to the elbows, then discard the cloth again, being careful not to touch any non-sterile surface.
The gown is then picked up and opened after stepping away from the gowning table so that the outer surface of the gown is not contaminated. The surgeon places their arms into the sleeves of the gown and an assistant helps to pull it into a comfortable position. At this stage, the surgeon’s hands remain within the sleeves of the gown. It is then possible to put on a pair of sterile gloves using a technique called closed gloving.
Start by placing one glove, thumb side down, on your wrist. With the fingers of the glove pointing up your arm. Grasp the cuff of the glove using your thumb through the sleeve of your gown. Using your other hand, still within the sleeve of the gown, turn the cuff of the glove down over your hand and slide the hand being gloved out of the sleeve of the gown and into the glove. If necessary, adjust the cuff of the glove, then repeat this process for the other hand. Note that the thumb side of the glove is laid onto the surgeon’s wrist. An assistant completes the process of tying the gown.
Having carried out this process, it is vital that you avoid touching any non-sterile surfaces. Standing with your hands clasped in front of your chest helps avoid inadvertent contact with non-sterile items.
Cloth gowns need to be properly packed and autoclaved. A convenient alternative is to use paper disposable gowns. These are supplied pre-sterilised at relatively low cost in a pack that also contains hand towels.
Preparation for Surgery
The surgeon can now prepare the sterilised items needed for surgery. An assistant opens the outer wrapping of the instrument pack and hands this to the surgeon so they can take it holding only the inner sterilised wrapping. When unwrapping the pack, drop the corners of the drapes so that the back of your hand does not touch the surface of the table. The animal can then be draped, once again, being careful not to touch any non-sterile surface. Using a drape prevents sterilised items touching the animal’s fur and becoming contaminated.
Smaller cloth drapes can be made or purchased for use with mice. Drapes can be either plastic, cloth or paper and sterilised either by autoclaving or purchased pre-sterilised. Disposable paper drapes are very convenient for use for batch surgery of rodents, allowing a new sterile drape to be used for each animal.
When using a paper drape, a suitable sized hole can be cut to access the surgical site. Avoid cutting along one of the pre-folded sections as this can prevent the drape conforming to the animal. The sterile field can be extended by using an additional paper or cloth drape.
Surgical Procedure
After the sterile pack has been opened, the instruments can be checked to ensure all the items needed are present and can then be arranged into a convenient order for undertaking surgery.
An assistant opens the outer packing of items such as scalpel blades and sutures, so that the surgeon can take the item, handling only the inner sterile surfaces. When the assistant opens the item, this should be done adjacent to the sterile area, not over it, so that inadvertent contamination of the sterile field is avoided.
Surgery is now undertaken. After use, each instrument should be placed back on a sterile drape.
If you need to touch a non sterile surface, for example, to adjust an anaesthetic vaporiser, and your assistant is not immediately available, this can be done using a large sterile swab, which is then discarded.
In addition to assisting with these procedures, your assistant can record the start and end time of the procedure and note any complications or other relevant information in your laboratory notebook.
Batch Surgery
It is often necessary to carry out a series of procedures in one operating session. After completing the first procedure, the surgeon should lift the sterile drape and dispose of it. The assistant then moves the animal to a recovery area, or directly into a warm incubator. They may also have been able to anaesthetise the next animal and clipped it ready for surgery. This animal can be positioned on the table and a skin preparation undertaken by the assistant.
During this time, the surgeon can use a hot bead steriliser to re-sterilise the tips of the instruments. The instruments must first be cleaned in sterile water. The instruments heat up rapidly in the steriliser but also cool rapidly so can be used safely within a minute or so. Alternatively, a new set of instruments can be used.
If a new set of instruments is to be used, the surgeon should re-glove before handling them. This can be done without the need to re-gown. The surgeon touches only the inner surface of the gloves with his ungloved hand. He can then use his gloved hand to handle the other glove, making sure that his ungloved fingers do not contact the outer surface of either glove.
The surgeon can then re-drape the animal using a new sterile drape and carry out surgery. Ideally, a new instrument kit should be used for each animal and if using a hot bead steriliser, the kit should be changed when there is obvious gross contamination or after 4-5 surgical procedures have been undertaken.