Standard Operating Procedure (SOP) Biosafety Level 2 Laboratory 1st Edition

E3A, #07-03/04 Division of Bioengineering NUS

June 2008

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Section 1 Introduction to the cell culture facility

1. The E3A#07-03/04 facility is a tissue culture laboratory of Biosafety Level 2 (BSL-2). This means that only work that falls under Biosafety Levels 1 or 2 can be performed in this facility. Figure 1 is a diagram of the facility, as well as the various equipment installed therein (Table 1). 2. Accordingly, the main doors to the facility are labeled with the universal symbol for biohazards (Fig. 2) and with the Office of Safety, Health and Environment (OSHE) laboratory signpost. 3. Biosafety Levels are a means to determine the level of danger that the work done in a facility poses to its users, other users present, as well as the environment. 4. Biosafety Level 1, as defined by the WHO Laboratory Biosafety Manual (Third Edition) and the CDC Biosafety in Microbiological and Biomedical Laboratories (5th Edition), describes any facility wherein work involving well-characterised materials that are not known to cause disease in humans, and which present little danger to other laboratory users or the environment, are carried out. 5. In contrast, work that is described as Biosafety Level 2 involve the use of agents that are associated with disease in humans, that pose a possible threat to others as well as the environment, but for which known and effective treatments are available. 6. In summary, users should be aware that other users of this facility may be handling materials that may cause illness upon exposure, and should take appropriate precautions, some of which are described herein, when working in it. 7. Owing to the special dangers to health posed by these materials, they are described as biohazadous, or biohazards. 8. Since this facility is designed for work at Biosafety Level 2, it is augmented with equipment for containment of aerosols generated during the course of experimental work, as well as equipment for the proper treatment of biohazardous waste prior to its disposal. 9. A list of the equipment in the facility is given in Table 1. A detailed set of 1

operating instructions will be included in Section 5 of this Standard Operating Procedure. 10. It should be noted that proper use of these equipment must be supplemented with the use of appropriate microbiological techniques. 11. This facility is under the purview of Mr. Francis Cheng, Safety and Health Officer, Faculty of Engineering. In case of emergencies Mr. Francis Cheng can be contacted at 6516 8599 or hp 90063314, or at [email protected].

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Figure 1. Floor plan and equipment footprint of the cell culture facility.

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S/N Item

Quantity Function

01

Water purification system

01

02

Thermostated water bath

01

03

Centrifuge

01

04

Fridge (-20, 4 degree)

01

To supply ultra-pure and RO (reverse osmosis) water. To provide a water bath for heating. For the centrifugation of cells. For the storage of chemicals/ medium. For the sterilization of clean

05

Autoclave

02

glassware

and

biohazard

wastes. For the disposal of glass 06

Glass disposal bin

01

slides and shards of broken glass.

07

Flammable cabinet

02

08

Weighing balance

01

09

Pipette aid

01

10

Sharps disposal box

01

11

Spill kit

01

12

First Aid Kit

01

13

Container for liquid nitrogen

01

4

For storage of flammable chemicals For

the

measurement

of

sample mass. For the use of pipettes. For the disposal of syringe needles. For

the

management

of

chemical spills. For

the

management

of

minor injuries. For the maintenance of a cell bank.

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Inverted,phase-contrast microscope 01

15

CO2 incubators

05

16

CO2 gas cylinders

02

For observation of cells and samples. For the maintenance of cell cultures. To supply carbon dioxide to the incubators. For

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Biosafety cabinets

03

the

containment

of

aerosols generated by sample handling.

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Eye wash

01

20

Fire extinguisher

01

21

pH meter

01

22

Shaker

01

23

Fine vortex

01

24

Heat sealer

01

For the washing the eyes in case of contamination. For the management of fires. For the measurement of fluid pH.

Table 1. List of equipment installed in the cell culture facility.

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Figure 2. Biohazard label.

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Section 2 Safety

1. The cell culture facility is an environment that poses physical, chemical and biological dangers to all users. 2. Nonetheless, it is possible to make this a safer workplace by simply observing certain safety precautions. 3. The main principle behind these precautions is the minimization of exposure of users to these hazards. 4. The first part of this section describes general safety precautions that are applicable to all laboratories, while the second part describes precautions and practices that should be observed when working with in a facility designated as BSL-2.

General safety precautions: 1. All users should wear proper personal protective equipment including a laboratory coat, gloves and goggles. These items would minimize contact of samples with the skin and the eyes. Perspex goggles, especially for those who do not wear spectacles, would also serve to protect the user’s eyes from accidental exposure to ultra- violet irradiation. 2. Never touch door handles or telephone receivers with gloved hands! These are surfaces that are commonly handled without gloves and come into contact with the unprotected face. As such, contamination of these surfaces pose a danger to unwary users. Gloves must always be assumed to be contaminated with biological material. As such handling door handles and telephone receivers is always assumed to contaminate these items. 3. Clothing should extend to the ankles to protect the lower limbs. This is to ensure that any sample or liquid spill will not come into direct contact with the skin of the lower limbs.

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4. No cloth or open-toed footwear should be used in this laboratory. Similarly, footwear serves as a barrier to direct contact of contaminants with the skin. However, to be effective the footwear should completely encase the feet. Sandals and slippers should never be used in this facility. Shoes made of cloth are also not effective barriers, as liquid spills could easily soak through the material. Leather or polyester shoes are most appropriate. 5. Be sure to wash your hands thoroughly before leaving the facility. Always assume that the hands have come into contact with samples, even when handling them with gloves. In order to protect the users, as well as to prevent the exit of contaminants, everybody exiting the cell culture facility must first wash their hands using the proper hand washing technique shown in Figure 3. 6. Never consume any food or drink inside the facility. Since the samples handled in the facility are potentially dangerous, ingestion of food and drink will increase the danger of toxins entering one’s system. As with all laboratories, food and beverage consumption is strictly forbidden in the cell culture facility. It should also be noted that the application of cosmetic products in the facility poses similar threats and should be avoided.

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From the Department of Health, Republic of the Philippines Figure 3. Proper technique for hand-washing.

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Safety precautions for working at BSL-2: 1. Always use aseptic technique when handling biological samples. Proper aseptic technique ensures that contamination of clean surfaces is minimized if not eliminated altogether. The use of this technique not only protects your sample from contamination, it also confines and prevents your sample from contaminating the environment. 2. Using the biosafety cabinets for work that could potentially generate aerosols, such as fluid dispensing using a pipette, would minimise the contamination of the environment with these aerosols. However, for this to be effective, proper use of the biosafety cabinet must be ensured. This will be discussed in detail in Section 3. 3. It is advisable to have yourself immunized against Hepatitis B before starting work in the facility. Since BSL-2 work often entails the use of materials derived from human sources the possibility of infection by contaminating hepatitis B viruses in these samples exists. 4. Any incidents involving a sample spill or an injury must be reported as soon as possible to facilitate treatment. This is for reasons of safety, not punishment. Contamination of the environment poses are real hazard to other users who might not be aware of the contamination, or who might not be competent in managing such incidents. Getting help immediately would ensure that the contamination is contained and treated. Also, any injury would be attended to immediately. 5. All biohazard wastes must be double-bagged and autoclaved prior to disposal by SembCorp. Each biohazard bag must not be taken out of the cell culture facility prior to autoclaving. Transfer to the autoclaves must be effected by way of the transfer chamber (See Fig. 1). 6. All deposits into the cell bank must be documented using the forms provided in Appendix 1. This is to allow an accurate inventory of all cellular materials used in the facility.

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Section 3 Waste disposal

1. Like all laboratories, this cell culture facility will be generating considerable waste material that must be disposed of. 2. However, owing to the biohazardous nature of the materials used, special methods of disposal must also be employed for these biological wastes, in addition to the regular means of disposing non-biohazardous materials. 3. These special methods serve the following purposes: •

To minimize or remove the hazardous nature of these materials;

•

To minimize the risk of user exposure to still-hazardous wastes;

•

To minimize the risk of contaminating the environment with these materials.

4. This following description of waste disposal procedures begins with general practices for common waste disposal, followed by those specific to dealing with biohazardous wastes generated from BSL-2 work.

Basic disposal protocol - General: 1. All solid non-biohazardous wastes must be discarded into the regular waste bin provided. 2. Do not discard biohazardous wastes into the regular bins or the sink. 3. Syringe needles must only be discarded in the sharps disposal bins. 4. Broken glass should not be handled manually, but should be collected using a broom and dust-pan, and then disposed of in the glass disposal box. Basic disposal protocol - Biological: 1. All biohazardous wastes should be treated chemically or by autoclaving in order to minimize their hazardous nature prior to disposal. 2. Users should bear in mind, however, that although chemical treatment and autoclaving are very effective methods of destroying pathogens, there is a 11

possibility although slight, that chemical-, or heat- and high-pressure-resistant pathogens might survive these processes and still pose a threat. 3. Treat all liquid biohazards overnight with 10% (v/v) bleach before disposal into a labeled collector bottle. Do not discard into the sink. 4. All solid biohazardous wastes must be disposed of into the biohazard bins only. Do not discard into the regular waste bins. 5. All biohazard bins must be lined with biohazard bags when in use. 6. Biohazardous wastes, even when in biohazard bags, should never be brought out of the facility until properly treated, such as by autoclaving. 7. To transfer the autoclave bag from the cell culture area to the autoclave room, use the airlock window as shown in Figure . 8. Only one of the two doors of the airlock window should be opened at any point in time. 9. To use it: •

Open the airlock door from within the cell culture area;

•

Place the biohazard bag within the airlock;

•

Close the airlock from within the cell culture area;

•

Go to the autoclave room and open the airlock from within it;

•

Remove the biohazad bag and close the airlock door.

10. All biohazard bags should be enclosed in an additional biohazard bag before being autoclaved. 11. Discard all autoclaved waste into the collector bins provided for final disposal.

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Section 4 Accident management

1. Accidents can and do occur in laboratories. 2. As with all such incidences, the foremost concern is to ensure that safety to all users is preserved. 3. Once all users have been brought to safety, one can then begin to deal with the effects of the accident. 4. Accidents tend to generate situations of danger owing either to exposure to poisons or to physically harmful objects. As before, these can be categorized into common dangers, and biohazardous dangers. 5. Managing these dangers requires that we first determine the nature of these dangers, and whether remaining in the site of the accident would pose a health threat. 6. If immediate evacuation is not necessary, we can then assist anyone that might have been hurt as a result of the accident. Various means of treating injuries will be dealt with later in this section. 7. Once all users have been brought to safety, the next concern would be to prevent the threat from spreading from the site of the accident. This might necessitate the containment of spills or leaks. 8. Once the spread of the spilled biohazardous material is stopped or retarded, one may proceed to remove them and treat the contaminated surfaces.

Injuries: 1. A blunt blow to parts of the body might result in mild internal bleeding leading to a bluish-black discolouration, or a bruise, just beneath the skin. 2. In the event of a bruise apply ice cubes wrapped in a piece of cloth to the injured area. If possible, apply pressure and raise the injured part of the body to reduce swelling and bruising. 13

3. Cuts are dangerous because they tear the skin and make the interior of the body vulnerable to toxins and other dangerous agents. 4. If a cut is bleeding, apply pressure to it with a bandage, or with gloved hands, to staunch the flow of blood. 5. Then, the wound should be cleaned very thoroughly by rinsing with soap and water. Finally, a bandage or sticking plaster should be used to protect the wound from exposure to the environment. 6. In the event that the cut was caused by an object known to contain or to be contaminated by a biohazardous material, immediate medical attention should be sought. If possible, seek the help of the Safety and Health Officer, Mr. Francis Cheng, at 6516 8599 or hp 90063314, or at [email protected], or your supervisor immediately. 7. Needles are commonly used the laboratory and it is possible that a user might be accidentally stabbed with one. A needle-stick injury like this is similar to a cut and poses the same dangers. 8. Unlike a cut, a needle-stick injury may not bleed profusely and so, might not seem as severe. However, in such cases, the skin has also been penetrated and it should always be assumed that the interior of the body has been exposed to dangerous materials. As such, needle-stick injuries should be treated the same way as cuts. 9. It should be noted however, that in the interest of safety, any object that has caused a cut, or a needle that has stabbed a user should always be assumed to have been contaminated with a biohazard. As such, the Safety and Health Officer, Mr. Francis Cheng, at 6516 8599 or hp 90063314, or at [email protected], or your supervisor should be informed and the injured user should seek medical assistance immediately.

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Containment protocol: Containing solid spills Containing liquid spills

Basic decontamination protocol: 1. Make sure to put up clear warning signs to others to keep clear of the contaminated area until decontamination is completed. 2. In case of a solid sample spill, double-glove your hands and dispose of the contaminant, along with the outer-most pair of gloves into the biohazard bin. 3. Disinfect the contaminated surface with 10% bleach for 30 mins. 4. Wipe away the bleach, then disinfect with 70% ethanol for 30 mins. 5. Dispose of all wiping towels, along with the second pair of gloves, in the biohazard bin. 6. In case of liquid spills, prevent spread of the liquid first and foremost. 7. With double-gloved hands, use either a spill kit sponge pad or wad of paper towels to surround the liquid. 8. Proceed to soak up the main body of the spill with paper towels or sprinkle with crystalising agent (such as Red Z from the spill kit) before disposing, along with the outermost pair of gloves, into the biohazard bin. 9. Disinfect the contaminated surface with 10% bleach for 30 mins. 10. Wipe away the bleach, then disinfect with 70% ethanol for 30 mins. 11. Dispose of all sponge pads and wiping towels, along with the second pair of gloves, in the biohazard bin. 12. In case of bodily contact with a contaminant, seek help immediately from the Safety and Health Officer, Mr. Francis Cheng, at 6516 8599 or hp 90063314, or at [email protected], or your supervisor. 13. If you are alone, remove all contaminated clothing and rinse yourself thoroughly under the emergency shower. Use soap if possible. 15

14. In case of a minor contamination, wash the area thoroughly with soap and rinse in the sink. 15. Report all incidents of contamination to the Safety and Health Officer, Mr. Francis Cheng, at 6516 8599 or hp 90063314, or at [email protected], or your supervisor. 16. It should be noted that the reasons for reporting these incidences is solely for the purpose of ensuring the safety of the users, and not for the purpose of punishment.

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Section 5 Equipment operation

The following are simple descriptions of how each piece of equipment in the cell culture facility should be used. However, the main purpose of this section is to highlight certain important precautions that must be taken when using these instruments. For more detailed descriptions of each equipment, users should refer to the individual equipment manuals.

Water purification system The water purification system in the cell culture facility is able dispense ultra-pure water and water treated by reverse osmosis (RO). RO water should be used for the final rinsing of glassware during washing and can be dispensed by turning the knob at the side of the instrument. Be sure to hold the container beneath the tube while dispensing to avoid spillage. To prepare the ultra-pure water for dispensing, press “operate/stop” on the control panel of the instrument and wait until the resistance of the water reaches 18.2 MΩ. To dispense the ultra-pure water, shift the dispenser bar all the way to the right. Be sure to have the container ready beneath the nozzle to avoid spillage.

Thermostated water bath The thermostated water bath is switched on by pressing the ‘On’ button. The temperature of the water is set by depressing the ‘Set’ button and adjusting the temperature dial. The temperature of the water bath is maintained at 37°C for the purpose of warming liquids used for cell culture. Should you need to use the water bath at higher temperatures – such as 56°C for heat-inactivation of serum – be sure to place a note warning other users of the change in temperature where it will be easily seen. Be sure to re-set the temperature to 37°C once you are finished. 17

Inverted, phase-contrast microscope The microscope in the cell culture facility is equipped with a light source that illuminates samples on the stage from above. Accordingly, the microscope objective lenses are positioned below the stage. This is the typical configuration of an inverted microscope. To operate the microscope, switch on the light source by flipping the switch. Position the sample above the stage aperture and focus the image using the coarse and fine focusing knobs. Make sure the appropriate phase rings are in place in the slot of the light source column. Adjust the light intensity using the intensity knob. Before placing your samples on the stage, it is advisable to wipe the platform with a C-fold towel soaked with 70% ethanol. The stage must be similarly disinfected with 70% ethanol once you are done. Never spray diluted alcohol directly onto any part of the microscope as this might result in penetration of the alcohol into the objective lenses, or the chassis of the micrscope. Resultant evaporation of the alcohol would leave moisture trapped within the objective lenses, or on the reflective mirrors in the chassis, which would facilitate mold growth on these surfaces. Should this occur, the microscope will have to be decommissioned and stripped of its chassis or objective lenses, in order to have the mold removed. This is a time-consuming and expensive process and should be avoided.

Simplified operation procedures for other equipment will be posted beside each of the equipment.

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Appendix 1. Cryostock Inventory

SHELF NO.: BOX NO.:

NAME OF USER:

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Please note that any special precautions pertinent to the specific work carried out in this facility should be documented and displayed beside the facility/equipment.

BSL2 Lab management team June 2008

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