Biological Safety Manual Page 1 5/26/2005

Transcription

1 Bilgical Safety Manual Page 1 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania IV. Practices and Prcedures A. Administrative Cntrls 1. Bihazard Warning Signs and Psting Each labratry must have a rm sign that prvides safety infrmatin t visitrs and service persnnel. Rm signs must cntain designatins fr all labratry hazards in use within the labratry (carcingens, acutely txic agents, reprductive hazards, bihazards, radiactive materials, lasers and magnetic fields). Cmplete the frm in Appendix I f the University f Pennsylvania Chemical Hygiene Plan t request a rm sign. All areas and labratries that cntain bihazardus agents must be psted with the bihazard warning sign (Figure 1). The backgrund must be red/range in clr with a black universal bihazard symbl and black lettering. Figure 1 All areas and labratries that cntain bihazardus r txic agents must be psted with signs stating, "EATING, DRINKING, SMOKING AND APPLYING COSMETICS ARE PROHIBITED IN THIS AREA." All equipment (centrifuges, water baths, crygenic freezers, incubatrs, etc.) that cmes in cntact with bihazardus materials must be labeled with the universal bihazard symbl. 2. Bisafety Levels The essential elements f the fur Bisafety levels fr activities invlving infectius micrrganisms are summarized in Table 1. The levels are designated in ascending rder, by degree f prtectin prvided t persnnel, the envirnment and the cmmunity. In general, Risk Grup 1 agents are handled at Bisafety Level 1, Risk Grup 2 agents at Bisafety Level 2 and s n. There are NO Bisafety Level 4 labratries at Penn. Agents requiring this level f cntainment may nt be brught t Penn s campus.

2 Bilgical Safety Manual Page 2 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania 3. Vertebrate Animal Bisafety Levels There are fur animal Bisafety levels fr experiments n animals infected with agents that prduce r may prduce human infectin. They are summarized in Table 2. As with Bisafety Levels, increasing levels f prtectin t persnnel and the envirnment are prvided as the rder ascends. There are NO Animal Bisafety Level 4 facilities at Penn. Animal wrk requiring this level f cntainment may nt ccur n Penn s campus. 4. Medical Surveillance A medical surveillance prgram will be prvided thrugh Occupatinal Medicine at HUP fr thse persnnel having substantial direct animal cntact. A medical surveillance prgram will be prvided thrugh Occupatinal Medicine fr thse persnnel wh are ccupatinally at-risk f expsure t bldbrne pathgens. The prgram will include free hepatitis B vaccine, pst-expsure evaluatin and fllw-up. Fr a mre detailed explanatin f this prgram, cnsult the University's Expsure Cntrl Plan. Designated persnnel (Principal Investigatrs / Area Supervisrs) are respnsible t cnduct a risk assessment fr their area t determine the risk fr nscmial r ccupatinal transmissin f M. tuberculsis and implement an apprpriate TB infectin cntrl plan. See Appendix D f the University f Pennsylvania Expsure Cntrl Plan, describing a Tuberculsis (TB) Infectin Cntrl Plan. Vaccines fr which the benefits (levels f antibdy cnsidered t be prtective) clearly exceed the risk (lcal r systemic reactins) will be ffered t all clearly identified at-risk persnnel, because immunprphylaxis may prvide an additinal level f prtectin. It is the Principal Investigatr s respnsibility t ensure that labratry persnnel wh wrk in Bisafety Level 2 r Bisafety Level 3 facilities receive the apprpriate immunizatins r tests fr the agents handled r ptentially present in the labratry (e.g. hepatitis B vaccine, Rabies vaccine, TB skin test, Vaccinia (smallpx) vaccine, etc.) and peridic testing as recmmended fr the agent(s) being handled. Baseline serum samples may be cllected as apprpriate. Additinal serum specimens may be peridically cllected, depending n the agents handled r the functin f the labratry. Fr mre infrmatin, cntact EHRS at (215) B. Engineering Cntrls

3 Bilgical Safety Manual Page 3 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania 1. Bilgical Safety Cabinets BSCs are designed t cntain aersls generated during wrk with infectius material thrugh the use f laminar airflw and high efficiency particulate air (HEPA) filtratin. All persnnel must develp prficient lab technique befre wrking with infectius materials in a BSC. Three types f BSCs (Class I, II and III) are used in micrbilgical labratries. The Class I BSC is suitable fr wrk invlving lw t mderate risk agents, where there is a need fr cntainment, but nt fr prduct prtectin. It prvides prtectin t persnnel and the envirnment frm cntaminants within the cabinet. The Class I BSC des nt prtect the prduct frm "dirty" rm air. It is similar in air mvement t a chemical fume hd, but has a HEPA filter in the exhaust system t prtect the envirnment. In many cases Class I BSCs are used specifically t enclse equipment (e.g., centrifuges, harvesting equipment r small fermenters), r prcedures (e.g. cage dumping, aerating cultures r hmgenizing tissues) with a ptential t generate aersls that may flw back int the rm. The Class II BSC prtects the material being manipulated inside the cabinet (e.g., cell cultures, micrbilgical stcks) frm external cntaminatin as well as meeting requirements t prtect persnnel and the envirnment. There are fur types f Class II BSCs: Type A1, Type A2, Type B1 and Type B2. The majr differences between the three types may be fund in the percent f air that is exhausted r recirculated, and the manner in which exhaust air is remved frm the wrk area (EHRS des nt supprt exhausting f Type A cabinets t the utside thrugh a thimble cnnectin t a building s exhaust system.)

4 Bilgical Safety Manual Page 4 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania BSC Class II, A1 II, A2 II, B1 II, B2 Cmparisn f Class II Bisafety Cabinet Characteristics Face Velcity Airflw Pattern Nnvlatile Txic Chemicals and Radinuclides % recirculated t the cabinet wrk area thrugh HEPA; 30% balance can be exhausted thrugh HEPA back int the rm r t the utside thrugh a thimble unit Same as II, A, but plenums are under negative pressure t rm; exhaust air is thimbleducted t the utside thrugh a HEPA filter Exhaust cabinet air must pass thrugh a dedicated duct t the utside thrugh a HEPA filter N recirculatin; ttal exhaust t the utside thrugh hardduct and a HEPA filter YES YES YES YES Applicatins Vlatile Txic Chemicals and Radinuclides NO YES [minute amunts (1)] YES [minute amunts (1)] YES (small amunts) (1) In n circumstances shuld the chemical cncentratin

5 Bilgical Safety Manual Page 5 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania apprach the lwer explsin limits f the cmpund. The gas-tight Class III BSC, r glve bx, prvides the highest attainable level f prtectin t persnnel, the envirnment and the prduct. It is the nly cabinetry that prvides a ttal physical barrier between the prduct and persnnel. It is fr use with high-risk bilgical agents and is used when abslute cntainment f highly infectius r hazardus material is required. It is imprtant t nte that laminar flw clean benches must nt be utilized fr wrk with bihazardus r chemically hazardus agents. Clean benches prvide prduct prtectin by ensuring that the prduct is expsed nly t HEPA-filtered air. They d nt prvide prtectin t persnnel r the ambient envirnment. The Class I BSC A. frnt pening B. sash C. exhaust HEPA D. exhaust plenum The Class II, Type A BSC A. frnt pening B. sash C. exhaust HEPA filter D. rear plenum E. supply HEPA filter F. blwer

6 Bilgical Safety Manual Page 6 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania The Class III BSC A. glve prts with O-ring fr attaching arm-length glves t cabinet B. sash C. exhaust HEPA filter D. supply HEPA filter E. duble-ended autclave r pass-thrugh bx a. Selectin and Placement f Bisafety Cabinets in the Labratry Certain cnsideratins must be met t ensure maximum effectiveness f these primary barriers. Cntact EHRS prir t purchase f a bisafety cabinet t ensure yu have selected an apprpriate unit fr the prpsed usage. Cntact yur building administratr and EHRS t ensure prper installatin and placement f a bisafety cabinet in yur labratry space.

7 Bilgical Safety Manual Page 7 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Adequate clearance shuld be prvided behind and n each side f the cabinet t allw easy access fr maintenance, and t ensure that the air return t the labratry is nt hindered. The ideal lcatin fr the bilgical safety cabinet is remte frm the entry (i.e., the rear f the labratry away frm traffic), since peple walking parallel t the face f a BSC can disrupt the prtective laminar flw air curtain. The air curtain created at the frnt f the cabinet is quite fragile, amunting t a nminal inward and dwnward velcity f 1 mph. A BSC shuld be lcated away frm pen windws, air supply registers, r labratry equipment (e.g., centrifuges, vacuum pumps) that creates turbulence. Similarly, a BSC shuld nt be lcated clse t a chemical fume hd. 2. Other Safety Equipment Safety equipment includes items fr persnal prtectin such as glves, cats, gwns, she cvers, bts, respiratrs, face shields, safety glasses r gggles. Persnal prtective equipment (PPE) must be used in cmbinatin with BSCs and ther devices that cntain bihazardus agents, animals r materials. When it is impractical t wrk in BSCs, PPE frms the primary barrier between persnnel and infectius materials. Examples include certain animal studies, animal necrpsy, agent prductin activities and activities relating t maintenance, service r supprt f the labratry facility. Other safety equipment such as safety centrifuge cups and safety blenders are enclsed cntainers designed t prevent aersls frm being released during centrifugatin r hmgenizatin f infectius material. Cntainment cntrls such as BSCs, safety centrifuge cups and blenders must be used fr handling infectius agents that can be transmitted thrugh the aersl rute f expsure.

8 Bilgical Safety Manual Page 8 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Fr assistance in the selectin f a BSC r ther safety equipment, cntact EHRS by phne ( ) r . Fr mre infrmatin n PPE, a descriptin f effective use f BSCs and infrmatin n ther safety equipment may be fund in the Recmmended Wrk Practices sectin belw. C. Recmmended Wrk Practices 1. Pipettes and Pipetting Aids Pipettes are used fr vlumetric measurements and transfer f fluids that may cntain infectius, txic, crrsive r radiactive agents. Labratry-assciated infectins have ccurred frm ral aspiratin f infectius materials, muth transfer via a cntaminated finger and inhalatin f aersls. Expsure t aersls may ccur when liquid frm a pipette is drpped nt the wrk surface, when cultures are mixed by pipetting, r when the last drp f an inculum is blwn ut. A pipette may becme a hazardus piece f equipment if imprperly used. The safe pipetting techniques that fllw are required t minimize the ptential fr expsure t bilgically hazardus materials: Never muth pipette. Always use a pipetting aid. If wrking with bihazardus r txic fluid, cnfine pipetting peratins t a bilgical safety cabinet. Always use cttn-plugged pipettes when pipetting bihazardus r txic materials, even when safety pipetting aids are used. D nt prepare bihazardus materials by bubbling expiratry air thrugh a liquid with a pipette. D nt frcibly expel bihazardus material ut f a pipette. Never mix bihazardus r txic material by suctin and expulsin thrugh a pipette. When pipetting, avid accidental release f infectius drplets. Place a disinfectant saked twel n the wrk surface and autclave the twel after use. Use "t deliver" pipettes rather than thse requiring "blwut". D nt discharge material frm a pipette at a height. Whenever pssible allw the discharge t run dwn the cntainer wall. Place cntaminated, reusable pipettes hrizntally in a pan cntaining enugh liquid disinfectant t cmpletely cver them. D nt place pipettes vertically int

9 Bilgical Safety Manual Page 9 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania a cylinder. Autclave the pan and pipettes as a unit befre prcessing them as dirty glassware fr reuse (see sectin D, Decntaminatin). Discard cntaminated dispsable pipettes in an apprpriate sharps cntainer. Autclave the cntainer when it is 2/3 t 3/4 full and dispse f as infectius waste. Place pans r sharps cntainers fr cntaminated pipettes inside the bilgical safety cabinet t minimize mvement in and ut f the BSC. 2. Syringes and Needles Syringes and hypdermic needles are dangerus instruments. The use f needles and syringes shuld be restricted t prcedures fr which there is n alternative. Blunt cannulas shuld be used as alternatives t needles wherever pssible (i.e., prcedures such as ral r intranasal animal inculatins). Needles and syringes shuld never be used as a substitute fr pipettes. When needles and syringes must be used, the fllwing prcedures are recmmended: Use dispsable safety-engineered needle-lcking syringe units whenever pssible. When using syringes and needles with bihazardus r ptentially infectius agents, wrk in a bilgical safety cabinet whenever pssible. Wear glves. Fill the syringe carefully t minimize air bubbles. Expel air, liquid and bubbles frm the syringe vertically int a cttn pledget mistened with disinfectant. D nt use a syringe t mix infectius fluid frcefully. D nt cntaminate the needle hub when filling the syringe in rder t avid transfer f infectius material t fingers. Wrap the needle and stpper in a cttn pledget mistened with disinfectant when remving a needle frm a rubber-stppered bttle. Bending, recapping, clipping r remval f needles frm syringes is prhibited. If yu must recap r remve a cntaminated needle frm a syringe, use a mechanical device (e.g. frceps) r the ne-handed scp methd. The use f needle-nipping devices is prhibited (needle-nipping devices must be discarded as infectius waste).

10 Bilgical Safety Manual Page 10 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Use a separate pan f disinfectant fr reusable syringes and needles. D nt place them in pans cntaining pipettes r ther glassware in rder t eliminate srting later. Used dispsable needles and syringes must be placed in apprpriate sharps dispsal cntainers and discarded as infectius waste. The Occupatinal Safety and Health Administratin (OSHA) revised the Occupatinal Expsure t Bldbrne Pathgens Standard (29 CFR Part ) in 2001 t include new effrts t help reduce needlestick injuries amng healthcare wrkers and thers wh handle medical sharps. OSHA nw requires the University t invlve nn-managerial emplyees in selecting safer medical sharps devices. Evaluative data will be made available in the University f Pennsylvania Expsure Cntrl Plan. If yu use sharps and are interested in evaluating safer medical devices, refer t the Safety Engineered Sharps Evaluatin Prgram. 3. Safe and Effective Use f Bilgical Safety Cabinets In general: Make sure yur BSC is certified prir t use, when it is installed r after it is mved, and annually thereafter. (Fr infrmatin n cabinet certificatin cntact EHRS by phne ( ) r . Understand hw yur cabinet wrks. The NIH / CDC dcument, Primary Cntainment fr Bihazards: Selectin, Installatin and Use f Bilgical Safety Cabinets, prvides thrugh infrmatin. Als cnsult the manufacturer s peratinal manual. Mnitr alarms, pressure gauges r flw indicatrs fr any majr fluctuatin r changes pssibly indicating a prblem with the unit. Immediately ntify EHRS f cabinets that are nt perating prperly. DO NOT attempt t adjust the speed cntrl r alarm settings. D nt disrupt the prtective airflw pattern f the BSC. Make sure lab drs are clsed befre starting wrk in the BSC. Plan yur wrk and prceed cnscientiusly. Minimize the strage f materials in and arund the BSC. Hard ducted (Type B2, Ttal Exhaust) cabinets shuld be left running at all times. Cabinets that are nt vented t the utside may be turned ff when nt in use, hwever, be sure t allw the BSC t run fr at least 10 minutes befre starting wrk.

11 Bilgical Safety Manual Page 11 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Operatinal directins Limit traffic in the area when the cabinet is in use. If there is an UV light incrprated within the cabinet, d nt leave it n while wrking in the cabinet r when ccupants are in the labratry. Befre using, wipe wrk surface with 70% alchl. Wipe ff each item yu need fr yur prcedures and place in cabinet. DO NOT place bjects ver the frnt air intake grille. Keep all materials at least 4 inches inside the sash. DO NOT place items n tp f the unit r blck the rear exhaust grille. Segregate cntaminated and clean items. Wrk frm "clean t dirty." Place a pan with disinfectant and/r a sharps cntainer inside the BSC fr pipette discards. DO NOT use vertical pipette discard canisters n the flr utside cabinet. It is nt necessary t flame items. This creates turbulence in airflw and may cmprmise sterility; heat buildup may damage the filters. Mve arms slwly when remving r intrducing new items int the BSC. If yu use a piece f equipment that creates air turbulence in the BSC (such as a centrifuge, blender) place equipment in the back 1/3 f the cabinet; stp ther wrk while equipment is perating. Prtect the building vacuum system frm bihazards by placing an in-line HEPA cartridge filter between the vacuum trap system and the surce valve in the cabinet. Clean up all spills in the cabinet immediately. Allw cabinet t run fr 10 minutes befre resuming wrk. When wrk is cmpleted-remve all materials and wipe all interir surfaces with 70% alchl. Remve lab cat and wash hands thrughly befre leaving labratry. 4. Crystats Frzen sectins f unfixed human tissue r animal tissue infected with an etilgic agent pse a risk because accidents can ccur. Freezing tissue des nt necessarily inactivate infectius agents. Freezing prpellants under pressure shuld nt be used fr frzen sectins as they may cause spattering f drplets f infectius material. Glves shuld be wrn during preparatin f frzen sectins.

12 Bilgical Safety Manual Page 12 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania When wrking with bihazardus material in a crystat, the fllwing is recmmended: Cnsider the cntents f the crystat t be cntaminated and decntaminate it frequently with 70% ethanl. Cnsider trimmings and sectins f tissue that accumulate in the crystat t be ptentially infectius and remve them during decntaminatin. Defrst and decntaminate the crystat with a tuberculcidal hspital disinfectant nce a week and immediately after tissue knwn t cntain bldbrne pathgens, M. tuberculsis r ther infectius agents is cut. Handle micrtme knives with extreme care. Stainless steel mesh glves shuld be wrn when changing knife blades. Cnsider slutins used fr staining ptentially infected frzen tissue sectins t be cntaminated. 5. Centrifuge Equipment Hazards assciated with centrifuging include mechanical failure and the creatin f aersls. T minimize the risk f mechanical failure, centrifuges must be maintained and used accrding t the manufacturer's instructins. Users shuld be prperly trained and perating instructins that include safety precautins shuld be prminently psted n the unit. Aersls are created by practices such as filling centrifuge tubes, remving plugs r caps frm tubes after centrifugatin, remving supernatant, and resuspending sedimented pellets. The greatest aersl hazard is created if a tube breaks during centrifugatin. T minimize the generatin f aersls when centrifuging bihazardus material, fllw the prcedures belw: Use sealed tubes and safety buckets that seal with O-rings. Befre use, inspect tubes, O-rings and buckets fr cracks, chips, ersins, bits f brken glass, etc. D nt use aluminum fil t cap centrifuge tubes because it may detach r rupture during centrifugatin. Fill and pen centrifuge tubes, rtrs and accessries in a BSC. Avid verfilling f centrifuge tubes s that clsures d nt becme wet. After tubes are filled and sealed, wipe them dwn with disinfectant. Add disinfectant t the space between the tube and the bucket t disinfect material in the event f breakage during centrifugatin. Always balance buckets, tubes and rtrs prperly befre centrifugatin.

13 Bilgical Safety Manual Page 13 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania D nt decant r pur ff supernatant. Use a vacuum system with apprpriate in-line reservirs and filters. Wrk in a BSC when re-suspending sedimented material. Use a swirling rtary mtin rather than shaking. If shaking is necessary, wait a few minutes t permit the aersl t settle befre pening the tube. Small lw-speed centrifuges may be placed in a BSC during use t cntain aersls. High-speed centrifuges pse additinal hazards. Take precautins t filter the exhaust air frm vacuum lines; avid metal fatigue resulting in disintegratin f rtrs; and use prper cleaning techniques and centrifuge cmpnents. Fllw manufacturers' recmmendatins meticulusly t avid metal fatigue, distrtin and crrsin. Avid the use f cellulid (cellulse nitrate) tubes with bihazardus materials. Cellulid centrifuge tubes are highly flammable and prne t shrinkage with age. They distrt n biling and can be highly explsive in an autclave. If cellulid tubes must be used, an apprpriate chemical disinfectant must be used t decntaminate them. 6. Persnal Prtective Equipment OSHA standard 29 CFR requires wrkplace assessment fr ptential hazards and mandates that emplyers prvide apprpriate PPE fr emplyees. PPE is used t prtect persnnel frm cntact with hazardus materials and infectius agents. Apprpriate clthing may als prtect the experiment frm cntaminatin. PPE must be prvided withut cst t persnnel. Supervisrs are respnsible t perfrm the assessments and t select and train emplyees in the use f rutine items such as lab cats, prtective glves, safety glasses, face shields, etc. Fr assistance in selectin f PPE cntact EHRS. The fllwing PPE is recmmended fr regular use: Face Prtectin Gggles r safety glasses with slid side shields in cmbinatin with masks, r chin length face shields r ther splatter guards are required fr anticipated splashes, sprays r splatters f infectius r ther hazardus materials t the face. Infrmatin n safety eyewear and the availability f lw cst prescriptin safety eyewear is available at the EHRS website.

14 Bilgical Safety Manual Page 14 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Labratry Clthing This categry includes: labratry cats, smcks, scrub suits, and gwns. Lng sleeved garments shuld be used t minimize the cntaminatin f skin r street clthes and t reduce shedding f micrrganisms frm the arms. In a circumstance where it is anticipated that splashes may ccur, the garment must be resistant t liquid penetratin t prtect clthing frm cntaminatin. If the garment is nt dispsable, it must be capable f withstanding sterilizatin, in the event it becmes cntaminated. Additinal criteria fr selecting clthing are: cmfrt, appearance, clsure types and lcatin, antistatic prperties and durability. Prtective clthing must be remved and left in the labratry befre leaving fr nn-labratry areas. If required, dispsables shuld be prvided fr visitrs, maintenance and service wrkers. All prtective clthing shuld be either discarded in the labratry r laundered by the facility. Persnnel must nt launder labratry clthing at hme. Glves Glves must be selected based n the hazards invlved and the activity t be cnducted. Glves must be wrn when wrking with bihazards, txics and ther physically hazardus agents. Temperature resistant glves must be wrn when handling ht material r dry ice. Delicate wrk requiring a high degree f precisin dictates the use f thin walled glves. Prtectin frm cntact with txic r crrsive chemicals may als be required. In additin, fr sme wrkers, expsures t latex may result in allergic reactins. Reprts f such reactins have increased in recent years, prmpting EHRS t recmmend the use f nitrile glves ver latex glves. Fr assistance in glve selectin, cntact EHRS. When wrking with hazardus materials, the glve shuld verlap the lwer sleeve and the cuff f the labratry garment. A lng sleeved glve r dispsable arm-shield may be wrn fr further prtectin f the garment. In sme instances duble glving may be apprpriate. If a spill ccurs, hands will be prtected after the cntaminated uter glves are remved. Glves must be dispsed f when trn r cntaminated, remved when wrk with infectius materials is cmpleted and nt wrn utside the labratry. Dispsable glves must nt be washed r reused. Glves must be remved crrectly in rder t avid cntaminatin. Fllw the prcedure belw t remve glves safely*:

15 Bilgical Safety Manual Page 15 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Pull ne glve near yur wrist twards yur fingertips until the glve flds ver. Carefully grab the fld and pull twards yur fingertips. As yu pull yu are turning the inside f the glve utwards. * 2005 University f Maryland Pull the fld until the glve is almst ff. T avid cntaminatin f yur envirnment, cntinue t hld the remved glve. Cmpletely remve yur hand frm the glve. Slide a finger frm yur glve-free hand under the remaining glve. Cntinue t slide yur finger twards yur fingertips until almst half f yur finger is under the glve. Turn yu finger 180 degrees and pull the glve utwards and twards yur fingertips. As yu d this, the first glve will be encased in the secnd glve. The inside f the secnd glve will als be turned utwards. Grab the glves firmly, by the uncntaminated surface (the side that was riginally tuching yur hand). Release yur grasp f the first glve yu remved. Pull yur secnd hand free frm its glve. Dispse f the glves prperly.

16 Bilgical Safety Manual Page 16 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Respiratrs In certain instances additinal PPE may be required. Respiratr selectin is based n the hazard and the prtectin factr required. If yur wrk requires the use f a respiratr, yu must participate in the University's respiratry prtectin prgram. Persnnel wh require respiratry prtectin must be medically evaluated by Occupatinal Medicine prir t respiratr use. After evaluatin, EHRS must assist the user in respiratr selectin and usage training. Cntact EHRS t initiate the prcess. 7. Aersl Prducing Devices The use f devices such as ultrasnic disrupters, grinders and hmgenizers t disrupt bihazardus materials results in cnsiderable aersl prductin and shuld be perfrmed in a BSC whenever pssible. Special care and barrier prtectin (splash shields, gggles, bench napkins, glves, etc.) are imprtant nt nly during the agitatin/disruptin prcess but als when handling the finished prduct. Allw yur vessel t sit fr a shrt time t allw yur prduct t settle befre pening. Review the peratins manual fr the device yu are using, paying special attentin t thse areas f the device that are susceptible t cntaminatin by yur prduct and decntaminate apprpriately after use, especially when wrking with ptentially infectius materials. Safety blenders are designed t prevent leakage frm the bttm f the blender jar, prvide a cling jacket t avid bilgical inactivatin and t withstand sterilizatin by autclaving. If blender rtrs are nt leak prf, test them with sterile saline r dye slutin prir t use with bihazardus material. The use f glass blender jars is nt recmmended because f the breakage ptential. If they must be used, cver the glass jar with a plyprpylene jar t prevent spraying f glass and cntents in the event it breaks. Use safety blenders in a BSC t prevent the accidental release f aersl during the blending prcess. During use, place a twel mistened with disinfectant ver the tp f the blender. Befre pening the blender jar, allw the unit t rest fr at least ne minute t allw the aersl t settle. Decntaminate the device prmptly after use. Lyphilizers may be used t freeze-dry bihazardus material. Depending n each lyphilizer design, infectius aersl prductin may ccur when bihazardus material is laded r remved frm the lyphilizer unit. If pssible, lad sample material in a BSC. Be sure the vacuum pump exhaust is HEPA-filtered t remve any hazardus agents r, alternatively, vent the pump int a BSC. After lyphilizatin is cmpleted, disinfect all surfaces f the unit that have been expsed t the agent. If the lyphilizer is equipped with a remvable chamber, clse it ff and mve it t a BSC fr unlading and decntaminatin. Handle cultures as infrequently as pssible and use vapr traps wherever pssible.

17 Bilgical Safety Manual Page 17 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Open all glass ampules cntaining liquid r lyphilized culture material in a BSC t cntain the aersl prduced. Glves must be wrn. T pen, nick the neck f the ampule with a file, wrap it in disinfectant saked twel, hld the ampule upright and snap it pen at the nick. Recnstitute the cntents f the ampule by slwly adding liquid t avid aerslizatin f the dried material. Mix the cntents withut bubbling and withdraw it int a fresh cntainer. Discard the twel and ampule tp and bttm as infectius waste. Glass ampules used t stre bihazardus material in liquid nitrgen have explded causing eye injuries. The use f plyprpylene tubes eliminates this hazard. These tubes are available dust-free r pre-sterilized and are fitted with plyethylene caps with silicne washers. Heat sealable plyprpylene tubes are als available. 8. Lp Sterilizers and Bunsen Burners Sterilizatin f inculating lps r needles in an pen flame generates smallparticle aersls that may cntain viable micrrganisms. The use f a shielded electric incineratr minimizes aersl prductin during lp sterilizatin. Alternatively, dispsable plastic lps and needles may be used fr culture wrk where electric incineratrs r gas flames are nt available. The lps are semiquantitative and can be used fr cunting bacteria. The use f gas burners in BSCs is nt recmmended. These burners can prduce turbulence that disturbs the prtective airflw patterns f the cabinet. In many bisafety cabinets, a prtin f the ttal air vlume is recirculated in the wrk area allwing flammable vaprs r gases t accumulate thereby creating a fire hazard. Additinally, the heat prduced by the cntinuus flame may damage the HEPA filter. If a gas burner must be used, select a tuch-plate burner with a pilt light. In additin, apprpriate hard piping frm the huse gas line must be used and an easily accessible emergency shut-ff valve (specifically identified as such) must be placed n the utside f the bisafety cabinet. 9. Laundry All persnal prtective clthing must be cleaned, laundered and dispsed f by the emplyer at n cst t emplyees. Apparel cntaminated with bld r ther ptentially infectius materials shuld be handled as little as pssible and decntaminated, preferably by autclaving, befre being sent t the laundry fr cleaning. Emplyees wh handle cntaminated laundry must wear apprpriate PPE.

18 Bilgical Safety Manual Page 18 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania 10. Husekeeping Gd husekeeping in labratries is essential t reduce risks and prtect the integrity f bilgical experiments. Rutine husekeeping must be relied upn t prvide wrk areas free f significant surces f cntaminatin. Husekeeping prcedures shuld be based n the highest degree f risk t which persnnel and experimental integrity may be subjected. Labratry persnnel are respnsible t clean labratry benches, equipment and areas that require specialized technical knwledge. Labratry staff is respnsible t: Secure bihazardus materials at the cnclusin f wrk. Keep the labratry neat and free f clutter - surfaces shuld be clean and free f infrequently used chemicals, bilgicals, glassware and equipment. Access t sinks, eyewashes, emergency shwers and fire extinguishers must nt be blcked. Decntaminate and discard infectius waste d nt allw it t accumulate in the labratry. Dispse f ld and unused chemicals prmptly and prperly. T have yur waste chemicals remved, cmplete an EHRS Chemical Waste Pickup frm. Prvide a wrkplace that is free f physical hazards - aisles and crridrs shuld be free f tripping hazards. Attentin shuld be paid t electrical safety, especially as it relates t the use f extensin crds, prper grunding f equipment, avidance f verladed electrical circuits and avidance f the creatin f electrical hazards in wet areas. Remve unnecessary items n flrs, under benches r in crners. Prperly secure all cmpressed gas cylinders. Never use fume hds r bisafety cabinets fr strage. Practical custdial cncerns include: Dry sweeping and dusting that may lead t the frmatin f aersls is nt permitted. The use f a wet r dry industrial type vacuum cleaner is prhibited t prtect persnnel as well as the integrity f the experiment. They are ptent aersl generatrs and, unless equipped with high efficiency particulate air (HEPA) filters, must nt be used in the bilgical research labratry. Wet and dry units with HEPA filters n the exhaust are available frm a number f manufacturers.

19 Bilgical Safety Manual Page 19 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania 11. Bihazard Spill Cleanup Prcedures The fllwing prcedures are prvided as a guideline t bihazardus spill cleanup. a. Bisafety Level 2 Spill Prtcl NOTE: If spill als invlves radiactive materials, cntact EHRS at Instruct injured persnnel t g t Occupatinal Medicine, (first flr, Silverstein), HUP, immediately. Ask a c-wrker t call ahead t alert Occupatinal Medicine ( ). If transprtatin assistance is required, call Penn Plice at 511 r After hurs, injured persnnel shuld g directly t HUP Emergency Rm. Small spills: Wipe up spill with a disinfectant-saked paper twel & clean the surface with a suitable disinfectant. Larger spills: Within a Bilgical Safety Cabinet (BSC) BSC must run during cleanup t cntain aersls & HEPAfilter exhaust air. Dn apprpriate persnal prtective gear befre initiating cleanup. Initiate clean up as sn as pssible using a germicidal disinfectant (phenlic r idphr). Alchl is nt recmmended. Large quantities may create the risk f fire. If the spill is cntained n a bench diaper, remve the cntaminated bench diaper & discard as infectius waste. If the spill is n the wrk area surface, cver spilled material with disinfectant-saked twels. Allw 20 minutes cntact time then remve the cntaminated twels & discard as infectius waste. Wipe dwn the interir f the cabinet & any splatter n items within the cabinet with a disinfectant-saked twel.

20 Bilgical Safety Manual Page 20 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Wipe dwn nn-autclavable materials with disinfectant. Allw 20 minutes f cntact time with disinfectant befre any items are remved frm cabinet. Place items designated as cntaminated used sharps in an apprpriate infectius waste sharps cntainer using tngs/frceps. Place ther cntaminated dispsable materials used in the cleanup prcess in an autclave bag. Prcess as infectius waste. Place cntaminated re-usable items in bihazard bags, autclavable pans with lids r wrap them in newspaper. Sterilize, preferably by autclaving, then clean fr re-use. If the cabinet has a catch basin beneath the wrk surface & the spill resulted in liquids flwing int this area, mre extensive decntaminatin is required. 1. Ensure the drain valve under the cabinet is clsed. 2. Pur disinfectant nt the wrk surface & thrugh the frnt and rear grilles int the drain pan. Allw minutes cntact time. 3. Absrb spilled fluid-disinfectant frm wrk surface with paper twels & discard in bihazard bag. 4. Prepare t empty drain pan. Place disinfectant slutin in a cllectin vessel. Attach flexible tubing t the drain valve. The tube shuld be f sufficient length t allw the pen end t be submerged in the cllectin vessel t minimize aersl generatin. 5. Open the drain valve & empty the drain pan int the cllectin vessel cntaining disinfectant. Flush the drain pan with water & remve the flexible tubing. Manage cntaminated materials as if they are infectius. 6. Remve prtective clthing used during cleanup & place in a bihazard bag fr autclaving. Wash hands when glves are remved. 7. Ntify Principal Investigatr r supervisr and EHRS ( ). Cnsult with EHRS t determine whether frmaldehyde decntaminatin f the cabinet and filters is necessary, especially if a

21 Bilgical Safety Manual Page 21 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania high-risk agent r a majr spill f a mderate-risk agent ccurred. 8. Run BSC at least 10 minutes after cleanup, befre resuming activity in the cabinet. Outside the Cabinet, Inside the Labratry If a spill ccurs in a Bisafety Level 2 facility, utside the BSC, ntify ther individuals in the labratry t evacuate. Exit the labratry t the hallway, clsing the dr behind yu. Remve any cntaminated clthing (turn cntaminated prtin inward) & place it in an autclave bag. Wash all expsed skin. Place signs n dr(s) t the labratry warning individuals wh may want t enter that a spill ccurred & access is denied. Allw aersls t settle fr 30 minutes befre re-entering the labratry. Assemble supplies (disinfectant, sharps cntainers, twels, tngs, autclave bags, etc.) befre entering the labratry. Dn apprpriate persnal prtective equipment (i.e. dispsable gwn, prtective eyewear, glves, she cverings & respiratry prtectin [if needed]). Clean up spill with a suitable disinfectant as fllws: 1. Surrund spill area with disinfectant r diking material that is saked in disinfectant. 2. Place paper twels saked in a disinfectant ver the entire spill area. 3. Allw 20-minute cntact time with the disinfectant t ensure adequate germicidal actin. 4. Wipe dwn nn-autclavable materials with germicidal disinfectant.

22 Bilgical Safety Manual Page 22 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania 5. Place items designated as cntaminated used sharps in an apprpriate infectius waste sharps cntainer. Place ther dispsable materials used in the cleanup prcess in an autclave bag. Prcess as infectius waste. 6. Place cntaminated re-usable items in bihazard bags, autclavable pans with lids r wrap them in newspaper. Sterilize, preferably by autclaving, then clean fr re-use. Remve prtective clthing used during cleanup then place in a bihazard bag fr autclaving. Wash hands when glves are remved. Ntify Principal Investigatr r supervisr & EHRS ( ) Inside a Centrifuge The ptential fr multiple infectins frm a single centrifuge accident is great. Aersls are created when fluid escapes frm the rtr r cup while the centrifuge is perating at high speed. All pening f centrifuges must be perfrmed slwly. Unsealed buckets: If a centrifuge tube breaks while the centrifuge is running, turn ff mtr. Allw the machine t be at rest fr 30 minutes befre pening. If breakage is discvered after the machine has stpped, re-clse the lid immediately & allw the unit t be at rest fr 30 minutes. Unplug centrifuge befre initiating clean up. Dn strng, thick rubber glves & ther PPE befre prceeding with clean up. Fld centrifuge bwl with a germicidal disinfectant. Place paper twels saked in a disinfectant ver the entire spill area. Allw 20 minutes cntact time.

23 Bilgical Safety Manual Page 23 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Use mechanical means (such as frceps) t remve brken tubes & glass fragments. Place them in a sharps cntainer fr autclaving & dispsal as infectius waste. Remve buckets, trunnins & rtr then place in disinfectant fr 24 hurs r autclave. Unbrken, capped tubes may be placed in disinfectant & recvered after 20 minutes cntact time r autclaved. Use mechanical means t remve remaining disinfectant saked materials frm centrifuge bwl & discard as infectius waste. Place paper twels saked in a disinfectant in the centrifuge bwl & allw it t sak vernight, wipe dwn again with disinfectant, wash with water & dry. Discard disinfectant saked materials as infectius waste. Remve prtective clthing used during cleanup & place in a bihazard bag fr autclaving. Wash hands whenever glves are remved. Sealed buckets (safety cups): If breakage is suspected, remve the sealed bucket t a bilgical safety cabinet befre pening. If breakage ccurred, replace the cap n the safety cup lsely and autclave. Ntify Principal Investigatr r supervisr & EHRS ( ). Outside the Labratry; during Transprt (n Penn's Campus) The majr emphasis shuld be n preventing spills during transprt. All transprt f infectius materials must be in a rigid, securely sealed, watertight primary cntainer, which is cntained within a secnd rigid, leak prf sealed cntainer. Sufficient absrbent shuld be added t the secnd cntainer t take up cntents in case f leakage frm the primary cntainer. The uter cntainer must be labeled with the universal bihazard symbl. If a spill ccurrs during transprt, dn glves and initiate cleanup immediately as fllws:

24 Bilgical Safety Manual Page 24 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Surrund spill area with disinfectant r diking material that is saked in disinfectant. Place paper twels saked in a disinfectant ver the entire spill area. Allw a minimum 20 minutes cntact time with the disinfectant t ensure adequate germicidal actin. Place cntaminated used sharps in an apprpriate infectius waste sharps cntainer. Place ther materials used in the cleanup prcess (including cntaminated glves) in an autclave bag and prcess as infectius waste. Repeat decntaminatin f spill area after cntaminated materials are remved. Wash hands as sn as pssible. Cntact EHRS ( ) if assistance is needed. b. Bisafety Level 3 Spill Prtcl NOTE: All labratry persnnel (faculty, staff, students) wrking with a Risk Grup 3 agent in a Bisafety Level 3 facility must be trained in the use f respiratry equipment by the EHRS prir t beginning wrk. If spill invlves radiactive materials, cntact EHRS immediately at Instruct injured persnnel t g t Occupatinal Medicine, (first flr, Silverstein), HUP, immediately. Ask a c-wrker t call ahead t alert Occupatinal Medicine ( ). If transprtatin assistance is required, call Penn Plice at 511 r After hurs, injured persnnel shuld g directly t HUP Emergency Rm. Within a Bilgical Safety Cabinet (BSC) BSC must run during cleanup t cntain aersls & HEPAfilter exhaust air. Dn apprpriate persnal prtective gear befre initiating cleanup (dispsable back-clsing gwn, duble glves).

25 Bilgical Safety Manual Page 25 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Initiate clean up as sn as pssible using a germicidal disinfectant (phenlic r idphr). Alchl is nt recmmended. Large quantities may create the risk f fire. If the spill is cntained n a bench diaper, remve the cntaminated bench diaper & discard as infectius waste. If the spill is n the wrk area surface, cver spilled material with disinfectant-saked twels. Allw 20 minutes cntact time then remve the cntaminated twels & discard as infectius waste. Wipe dwn the interir f the cabinet & any splatter n items within the cabinet with a disinfectant-saked twel. Wipe dwn nn-autclavable materials with disinfectant. Allw 20 minutes f cntact time with disinfectant befre any items are remved frm cabinet. Place items designated as cntaminated used sharps in an apprpriate infectius waste sharps cntainer using tngs/frceps. Place ther cntaminated dispsable materials used in the cleanup prcess in an autclave bag. Prcess as infectius waste. Place cntaminated re-usable items in bihazard bags, autclavable pans with lids r wrap them in newspaper. Sterilize, preferably by autclaving, then clean fr re-use. If the cabinet has a catch basin beneath the wrk surface & the spill resulted in liquids flwing int this area, mre extensive decntaminatin is required. 1. Ensure the drain valve under the cabinet is clsed. 2. Pur disinfectant nt the wrk surface & thrugh the frnt and rear grilles int the drain pan. Allw minutes cntact time. 3. Absrb spilled fluid-disinfectant frm wrk surface with paper twels & discard in bihazard bag. 4. Prepare t empty drain pan. Place disinfectant slutin in a cllectin vessel. Attach flexible tubing t the drain valve. The tube shuld be f sufficient length t allw the pen end t be submerged in the cllectin vessel t minimize aersl generatin.

26 Bilgical Safety Manual Page 26 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania 5. Open the drain valve & empty the drain pan int the cllectin vessel cntaining disinfectant. Flush the drain pan with water & remve the flexible tubing. Manage cntaminated materials as if they are infectius. Remve prtective clthing used during cleanup & place in a bihazard bag fr autclaving. Wash hands when glves are remved. Ntify Principal Investigatr r supervisr and EHRS ( ). Cnsult with EHRS t determine whether frmaldehyde decntaminatin f the cabinet and filters is necessary, especially if a high-risk agent r a majr spill f a mderate-risk agent ccurred. Run BSC at least 10 minutes after cleanup, befre resuming activity in the cabinet. Outside the Cabinet, Inside the Labratry Ntify ther individuals in the labratry t evacuate the labratry immediately. Hld yur breath and exit the labratry t the anterm. Remve cntaminated clthing (turn cntaminated prtin inward; place int autclave bag). Wash hands after glves are remved. Wash all expsed skin with germicidal sap. If eyes were splashed, flush at eyewash statin fr 15 minutes then reprt t Occupatinal Medicine. Ntify Principal Investigatr r supervisr and EHRS ( ). EHRS will cnsult with the Principal Investigatr t determine the apprpriate methd f decntaminatin and spill cleanup (persnnel spill respnse r frmaldehyde decntaminatin f the entire facility). Place a sign n the dr t the BL3 lab t warn individuals f the spill and advise them keep ut f the lab. If persnnel spill respnse is required, d the fllwing:

27 Bilgical Safety Manual Page 27 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania Allw aersls t settle fr a minimum f 30 minutes befre re-entering the labratry. Assemble supplies (disinfectant, sharps cntainers, twels, tngs, autclave bags and prtective gear [dispsable Tyvek suit/back-clsing gwn, prtective eyewear, glves, she cverings, respiratr], etc.) befre initiating spill cleanup. Dn apprpriate persnal prtective equipment (PPE). Duble glving is recmmended. Clean up spill with a suitable disinfectant as fllws: 1. Surrund spill area with disinfectant r diking material that is saked in disinfectant. 2. Place paper twels saked in a disinfectant ver the entire spill area. 3. Allw a minimum 20 minutes cntact time with the disinfectant t ensure adequate germicidal actin. 4. Wipe dwn nn-autclavable materials with germicidal disinfectant, allwing 20-minute cntact time. 5. Place items designated as cntaminated used sharps in an apprpriate infectius waste sharps cntainer using tngs/frceps. Place ther cntaminated dispsable materials used in the cleanup prcess in an autclave bag. Prcess as infectius waste. 6. Place cntaminated autclavable re-usable items in bihazard bags, autclavable pans with lids r wrap them in newspaper. Sterilize, preferably by autclaving, then clean fr re-use. 7. Repeat decntaminatin f spill area (flr and wrk surfaces) after cntaminated materials are remved. Remve uter glves befre exiting labratry t the anterm. Remve prtective clthing used during cleanup in the fllwing rder: she cverings, gwn/suit, respiratr, and

28 Bilgical Safety Manual Page 28 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania glves last. If reusable, wipe dwn respiratr with disinfectant. Place dispsable PPE in a bihazard bag fr autclaving. Wash hands with germicidal sap after glves are remved; shwer recmmended. Inside a Centrifuge The ptential fr multiple infectins frm a single centrifuge accident is great. Aersls are created when fluid escapes frm the rtr r cup while the centrifuge is perating at high speed. All pening f centrifuges must be perfrmed slwly. If a centrifuge tube breaks while the centrifuge is running, turn ff the mtr, ntify thers in the lab, hld yur breath, and evacuate. Allw the centrifuge t be at rest fr 30 minutes befre attempting t pen. Ntify Principal Investigatr r supervisr and EHRS ( ). EHRS will cnsult with the Principal Investigatr t determine the mst apprpriate methd f decntaminatin and spill cleanup. Place a sign n the dr t the BL3 lab, t warn individuals f the spill and t keep ut f the lab. If persnnel spill respnse is required, d the fllwing: Assemble supplies (disinfectant, sharps cntainers, twels, tngs, autclave bags and prtective gear [dispsable Tyvek suit/back-clsing gwn, prtective eyewear, glves, she cverings, respiratr], etc.) befre initiating spill cleanup. Dn apprpriate persnal prtective equipment (PPE). Duble glving is recmmended. Re-enter BL3 lab. Unplug centrifuge and slwly pen centrifuge. If safety cup(s) is intact, remve unit t bilgical safety cabinet fr further decntaminatin.

29 Bilgical Safety Manual Page 29 5/26/2005 Envirnmental Health & Radiatin Safety University f Pennsylvania If integrity f safety cup(s) is breached, decntaminate all expsed surfaces befre remving cup(s) t bilgical safety cabinet fr further decntaminatin. Outside the Labratry; during Transprt (n Penn's Campus) The majr emphasis shuld be n preventing spills during transprt. Please nte that transprtatin f a large number f cultures at the same time is discuraged. If a cart is used t transprt the material, it must have side rails. All transprt f infectius materials must be in a rigid, securely sealed, watertight primary cntainer, which is cntained within a secnd rigid, sealed, leak-prf cntainer. Sufficient absrbent shuld be added t the secnd cntainer t take up cntents in case f leakage. The uter cntainer must be labeled with the universal bihazard symbl. The cntainer shuld be placed n the bttm shelf f the cart. If a spill ccurrs during transprt, dn glves and initiate cleanup immediately as fllws: Place absrbent twels, preferably saked in a disinfectant, n the spilled material. Cntact EHRS ( ). Remain nearby until EHRS arrives t assist in the spill cleanup. D. Decntaminatin Decntaminatin is a term used t describe a prcess r treatment that renders a medical device, instrument, r envirnmental surface safe t handle. A decntaminatin prcedure can range frm sterilizatin t simple cleaning with sap and water. Sterilizatin, disinfectin and antisepsis are all frms f decntaminatin. Sterilizatin is the use f a physical r chemical prcedure t destry all micrbial life, including highly resistant bacterial endspres. Disinfectin eliminates virtually all pathgenic nn-spre-frming micrrganisms but nt necessarily all micrbial frms n inanimate bjects (wrk surfaces, equipment, etc.). Effectiveness is influenced by the kinds and numbers f rganisms, the amunt f rganic matter, the bject t be disinfected and chemical expsure time, temperature and cncentratin. Antisepsis is the applicatin f a liquid antimicrbial chemical t skin r living tissue t inhibit r destry micrrganisms. It includes swabbing an injectin site n a persn r animal and hand washing with germicidal slutins. Althugh sme chemicals may be utilized as either a disinfectant r an antiseptic, adequacy fr ne applicatin des nt