Part III: Culture
specimen handling

Receipt of incoming specimens
Specimens should be received in the office area of the laboratory, preferably at a separate specimen delivery counter. Delivery boxes should be opened in the biosafety cabinet and the following procedures applied:

• Wear disposable gloves during receipt and  inspection of incoming specimens
• Inspect the delivery box for signs of  leakage. If mass leakage is evident discard the box by autoclaving or burning
• Disinfect the outside of the delivery box  using cotton wool or paper towels saturated with a suitable disinfectant (eg. 5% phenol)
• Open the delivery box carefully and check  for cracked or broken specimen containers. Autoclave or burn these without processing and  request another specimen
• Check that specimens have been adequately  labelled with individual identification numbers and that these correspond with the numbers  on the accompanying list
• Disinfect the inside of the delivery box,  discard gloves and wash hands after handling specimen containers

Safe handling of specimens
Working within a BSC

• Because of the increased risks of aerosol production during  culture procedures, all manipulations should be carried out within the BSC. The cabinets  are intended to protect the worker from airborne infection. They will not, however,  protect him/her from spillage and the consequences of poor techniques
• BSCs are designed to be operated 24 hours per day and in order to maintain room air balance they  should not be switched off. If electrical power has been interrupted or the cabinet has been switched off (eg. following replacement of filters), the cabinet blower should be operated for a least five minutes before work is started. The work surface, interior walls  and interior window surface should be wiped with an appropriate disinfectant (eg.  methylated spirits). This should be followed by a second wiping with sterile water
• Prepare a  written checklist of materials necessary for tuberculosis culture. This will minimise the number of arm-movement disruptions across the fragile air barrier of the BSC, which may disrupt the air curtain and compromise the partial barrier
• Place only the materials and equipment required for immediate work in the BSC and store extra  supplies (eg. additional culture media) outside the cabinet. Materials and equipment  placed inside the BSC may cause disruption to the airflow resulting in turbulence,  cross-contamination or breach of containment
• Allow a delay  of 60 seconds after placing hands/arms inside the cabinet, before manipulation of  materials. This allows the BSC to stabilise and to remove surface microbial contaminants
• Ensure that  the front grille is not blocked with laboratory notes, discarded plastic wrappers,  pipetting devices, etc
• Perform all  operations at least four inches from the front grille on the work surface. Raise arms slightly to allow room air to be drawn through the front grille
• Place  absorbent paper towelling on the work surface (but not on the front or rear grille openings). This will facilitate routine cleanup and will reduce splatter and aerosol formation during an overt spill. Towelling can then be folded and placed in an autoclavable bag when work is completed
• Place all  materials and aerosol-generating equipment (eg. vortex mixers) as far back in the cabinet as practical, towards the rear edge of the work surface and away from the front grille
• Place bulky  items such as autoclavable bags, pipette trays and collection flasks to one side in the cabinet
• Arrange  materials and equipment to allow work to flow from a clean to a contaminated area across  the work surface. Place materials and supplies in such a way as to limit the movement of dirty items over clean ones
• Avoid the  following common practices which may interfere with the operation of the BSC:
• taping autoclavable disposal bags to the outside of the cabinet
• placing pipette collection containers  upright in the BSC or on the floor outside the cabinet
• The frequent inward/outward movement needed to place objects in these containers disrupts the integrity of the cabinet air barrier and  can compromise both staff and product protection
• Use only horizontal pipette discard trays containing an appropriate disinfectant (eg. 5% phenol)
• Use proper microbiological techniques to avoid splatter and aerosols. This will minimise the potential for staff exposure to infectious materials manipulated within the cabinet. As a  general rule, keeping clean materials at least 12cm away from aerosol-generating activities will minimise the potential for cross-contamination
• Do not hold  opened tubes or bottles in a vertical position and recap or cover them as soon as possible. This will reduce the chance for cross-contamination
• Do not use large open flames in the BSC. This creates turbulence which disrupts the pattern of air supplied to the work surface. Special Bunsen burners for use in BSC?s are  recommended
• Use an appropriate liquid disinfectant (eg. 5% phenol) in a discard pan to decontaminate materials before removal from the BSC. Introduce items into the pan with the minimal splatter and allow sufficient contact time before removal. Alternatively, contaminated items may be placed into an autoclavable disposal bag within the BSC. Water should be added to the bag prior to autoclaving to ensure steam generation during the autoclave  cycle
• Surface-decontaminate  all containers and equipment before removal from the BSC
• At the end of  the work day, surface-decontaminate the work surface of the BSC, the sides and back and the interior of the glass window
• Handle small spills within the BSC immediately by removing the contaminated absorbent paper towelling and placing it into the autoclavable disposable bag. Wipe any splatter onto items within the cabinet or on its interior immediately with a paper towel saturated with a disinfectant solution (eg. 5% phenol)
• Spills large enough to result in liquids flowing through the front or rear grilles require more  extensive decontamination. Surface-decontaminate and remove all items from the BSC. Ensure that the drain valve is closed and pour appropriate disinfectant (eg. 5% phenol) onto the  work surface and through the grille(s) into the drain pan. Allow at least 30 minutes for  decontamination. Empty the drain pan into a collection vessel containing disinfectant by attaching a flexible tube to the drain valve with the open end submerged in the disinfectant within the collection vessel. After decontamination, flush the drain pan with water and remove the drain tube
• Always decontaminate the BSC before HEPA filters are changed or internal repair work is done. The most common decontamination method uses formaldehyde gas and is described in the Management Series

Using the centrifuge

• Select two centrifuge tubes of identical length and thickness. Place the specimen to be centrifuged in one tube and an equal amount of 70% ethanol in the other. Ensure that the tubes are  balanced
• Place the  tubes in paired centrifuge buckets and place the paired buckets in diametrically opposite positions in the centrifuge head
• Close the  centrifuge lid and ensure that the speed control is at zero before switching on the current. (Many centrifuges are fitted with a ?no volt? release to  prevent the machine starting unless this is done)
• Move the speed control slowly until the speed indicator shows the required rpm or g

Precautions

• Make sure  that the rubber buffers are in the buckets, otherwise tubes will break
• Check the  balancing carefully. Improperly balanced tubes will cause ?head wobble?, spin-off accidents and wear out bearings
• Check that the balanced tubes are opposite one another in multi-bucket centrifuges
• Never start  or stop the centrifuge with a jerk
• Observe the  manufacturer instruction about the speed limits for various loads
• Open sealed  centrifuge buckets in the BSC

Using a pressure cooker autoclave

• There must be  sufficient water inside the chamber
• The autoclave  is loaded and the lid is fastened down with the discharge tap open. The safety valve is  then adjusted to the required temperature and the heat is turned on
• When the water boils, the steam will issue from the discharge tap and carry the air from the chamber with it. The steam and air should be allowed to escape freely until all of the air  has been removed. This may be tested by attaching one end of a length of rubber tubing to the discharge tap and inserting the other end into a bucket or similar large container of  water. Steam condenses in the water and the air rises as bubbles to the surface. When all  of the air has been removed from the chamber, bubbling in the bucket will cease. When this  stage has been reached, the air-steam discharge tap is closed and the rubber tubing  removed. The steam pressure then rises in the chamber until the desired pressure, usually  15lb/in², is reached and steam issues from the safety valve
• When the load  has reached the required temperature, the pressure is held for 30 minutes
• At the end of  the sterilising period, the heater is turned off and the autoclave allowed to cool
• The air and  steam discharge tap is opened very slowly after the pressure gauge has reached zero  (atmospheric pressure). If the tap is opened too soon, while the autoclave is still under pressure, any fluid inside will boil explosively and bottles containing liquids may even  burst
• The contents are allowed to cool. Depending on the nature of the materials being sterilised, the  cooling (or ?run-down?) period needed may be several hours

Using an autoclave with air discharge by gravity displacement

• If the autoclave is jacketed, the jacket must first be brought to the operating temperature
• The  chamber is loaded, the door is closed and the steam-valve is opened, allowing steam to enter the top of the chamber. Air and condense flow out through the drain at the bottom
• When the drain thermometer reaches the required temperature a further period must be allowed for the load to reach that temperature. This should be determined initially and periodically  for each autoclave. Unless this is done the load is unlikely to be sterilised
• The  autoclave cycle is then continued for the holding time. When it is completed the steam valves are closed and the autoclave allowed to cool until the temperature dial reads less  than 80EC. Not  until then is the autoclave safe to open
• The  autoclave door should first be ?cracked? or opened very slightly and left in that position for several minutes to allow steam to escape and the load to cool further