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Safety Operating Procedure (SOP)

Biosafety Level 2 Laboratory

4nd Edition

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

Updated in June 2012 (1st

draft in June 2008)

All Lab users must read and understand this SOP before conduct

work in the lab.

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Chapter 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 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. Chen Chih Hui Gabriel, Safety and Health Officer, Faculty of Engineering. In case of emergencies Ms. Tang Fenglin can be contacted at 6516 1941, or at engtf@nus.edu.sg.

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Chapter 2 First Aid

Acid and alkali in

eyes

Irrigate thoroughly with solution from eyewash bottle or, if this is not available, cold water from the tap.

Obtain medical attention immediately.

Extensive splashing with acid or alkali

Douse with water (use a shower) and remove affected clothing. Obtain immediate medical attention at the

Emergency Department, NUH.

Thermal burs

Apply cold water to affected area, dry carefully, cover with dry dressing and seek medical assistance.

Severely burnt persons should be kept warm until medical assistance arrives.

Cuts and wounds

Wash the wound under running water. Dress simple wounds with iodine and gauze/plaster. Deep

lacerated wounds need further medical attention after application of pressure dressing to stop bleeding. If

there is detachment of body parts like parts of fingers, place them in a container packed with ice and bring

them along with the patient to the Emergency Department, NUH

Electric shock

Put on rubber gloves to switch off electrical supply. If breathing of the electrocuted person has stopped or

is feeble, obtain immediate medical attention and, if possible, apply artificial respiration.

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Chapter 3 Laboratory Safety

The cell culture facility is an environment that poses physical, chemical and biological dangers to all

users. Nonetheless, it is possible to make this a safer workplace by simply observing certain safety

precautions. The main principle behind these precautions is the minimization of exposure of users to these

hazards.

Housekeeping

Work areas should be kept clean and free from obstructions. There should be a clear demarcation between

‘wet’ and ‘dry’ areas, where paperwork is done.

Smoking is not allowed in the laboratory.

Walkways and passages should be kept clear of any equipment or boxes, as this may pose a fire hazard or

cause accidents

Never use any chemical found in an unlabelled container. Unlabelled containers and chemical wastes

should be disposed of promptly by appropriate procedures.

Spilt chemicals should be cleaned up immediately and disposed of properly. All spillage must be reported

to the lab officers or supervisors.

Cleaning up should follow the completion of any operation or take place at the end of each day.

First Aid Kits, Emergency Exits and Safety Equipment

Every laboratory must be equipped with emergency safety equipment such as eye washer and safety

shower. The equipment should be inspected and maintained regularly. All workers must be familiar with

emergency and first aid procedures. First aid kits should be inspected and replenished frequently.

Every laboratory should have an appropriate number of fire extinguishers. Every worker must know the

location of the nearest fire extinguisher.

Access to emergency exits and equipment should never be blocked

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Personal Protection and Hygiene

Laboratory coats should be worn in the laboratory. Gloves should be worn as needed. In addition, safety

spectacles, dust masks, etc should be worn where appropriate.

Hand must be washed well with detergent and water before leaving the laboratory area.

Working Alone or After Office Hours

Where the work involves potential hazard, personnel should not work alone or after office hours in the

laboratory.

It is prudent to avoid working in a laboratory alone. If this must be done, arrangements should be made

between individuals working in separate laboratories outside of conventional hours to cross-check

periodically. Alternatively, security guards may be asked to check on a laboratory worker. Experiments

known to be hazardous should not be undertaken by a worker who is alone in a laboratory.

The supervisor of the laboratory has the responsibility for determining whether the work requires special

safety precautions, such as having two persons in the same room or in close proximity during a particular

operation.

Experimental Techniques

Be familiar with the potential hazards of the techniques being used, and the appropriate response. At the

end of the experiment, conduct a check to make sure all equipment is cleaned and put away and gas

supply, vacuum, electrical apparatus etc turned off. Hot plates and water baths which are still hot should

be clearly indicated as such.

Chemicals and Reagents

Guidelines on Chemical Safety are found in the next Chapter.

All Chemicals and reagents must be clearly labeled and any special hazards or storage condition indicated.

Store chemicals appropriately. Treat all chemicals as potentially hazardous.

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Spills

If the spill is minor and presents no danger, clean up spill immediately. If the spill is large or hazardous,

evacuate all personnel from the area and inform the relevant people. A spill kit for spill removal is

available in the lab.

Disposal of Waste

Ascertain the correct procedure for disposal of waste materials, particularly biological materials and

chemicals. Organic solvents and corrosive chemicals should not be poured down the sink, but placed into

the appropriate waste bottles, which should then be disposed of in the correct manner.

Sharp objects, such as broken glassware, needles and blades, must be disposed of in a sharp container and

not in the general waste. Do not attempt to pick up broken glassware with bare hands. Use a brush and

pan.

Biological waste must be autoclaved prior disposal. A autoclave dedicated for waste is in the BSL2 lab.

Chemical waste disposal procedures can be found in the MSDS and in the Risk Assessment forms for

chemicals. All are available as hardcopy in the lab

Electrical Equipment

Examine all electrical cords periodically for signs of wear and damage. If damaged electrical cords are

discovered, unplug the equipment and send it off for repair.

Properly ground all electrical equipment. If sparks are noticed while plugging or unplugging equipment or

if the cord feels hot, do not use the equipment until it can be serviced by an electrician.

Do not plug too many items into a single outlet. Cords that enable you to plug more than one item in at a

time should not be used. Multi-plug strips can be used if they are protected with a circuit breaker and if

they are not overloaded.

Use only carbon dioxide, halon or dry chemical fire extinguishers for electrical fires.

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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.

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.

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

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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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Chapter 4 Chemical Safety

BSL2 lab is a common facility lab in Division of Bioengineering, it is strongly recommended that all

users store chemicals in their respective own lab. Here, a general guideline is listed here.

GENERAL PRINCIPLES AND PRECAUTIONS

Handling of Hazardous Chemicals

It is essential for laboratory workers to be familiar with the potential hazards of chemicals used in

laboratory experiments and to follow recommended procedures for their use, handling, storage, and

disposal.

Laboratory managers/officers have the responsibility for establishing waste-disposal procedures for

routine and emergency situations and for communicating these procedures to laboratory workers.

Workers must observe them to avoid hazards or damage to the environment.

Appropriate waste disposal bags for chemically hazardous and biohazard materials should be used. This is

to distinguish them from normal wastes.

Material Safety Data Sheets

Material Safety Data Sheets (MSDS) always accompany all hazardous chemicals that are shipped. These

data sheets contain detailed safety information. MSDS should be filed in the laboratory in a central

location as a reference guide. Workers should consult MSDS concerning the relevant chemicals before

commencing their experiments.

Risk Assessment Documents for most hazardous chemicals

A Risk Assessment Document is available for the most hazardous chemicals in Lab. The Documents

are provided as hardcopy and softcopy on the Lab homepage. Lab users ordering a new hazardous

chemical are required to prepare a Risk Assessment document to update the existing documents.

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Labeling and Storage of Chemicals

Be certain that all chemicals are correctly and clearly labeled. Post warning signs if chemicals are

inflammable, highly toxic, and carcinogenic or other special problems exist.

If the label on the container does not give safety information, obtain the information from some reference

sources: your supervisor; a handbook such as the CRC Handbook of Laboratory Safety, Merck Index; or

MSDS.

Centralized storage of bulk quantities of flammable liquids provides the best method of controlling fire

hazards. The flammable liquids must be stored in fire-resistant cupboard.

Bunsen burners should not be used near flammable liquids.

Chemical which are light-sensitive should be stored in dark bottles.

Chemicals that have high chronic toxicity, including those classified as potential carcinogens, should be

stored in ventilated storage areas in unbreakable, chemically resistant containers. Storage vessels

containing such substances should carry a label:

CAUTION: HIGH CHRONIC TOXICITY or CANCER SUSPECT AGENT.

Carcinogenic Materials

The use of carcinogenic materials should be avoided if possible. If they are required, extra precautions are

necessary in handling the materials. Follow the prescribed procedures for use and waste disposal.

Carcinogens must be stored in locked cabinets for poisons, according to temperature sensitivity.

Operations should be performed in fume cupboards, gloves should be worn and immediately after use,

hands should be washed with cold water.

Contact is dangerous through lungs, mouth and skin absorption. Known carcinogens usually have

warning labels. If in doubt, refer to MSDS.

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Incompatible Chemicals

Some chemicals are incompatible with others. The incompatible chemicals should be kept segregated. A

more complete listing may be found in the National Fire Protection Association (NFPA) information at

http://www.nfpa.org/home.html and in various laboratory and chemical reference manuals.

Transport of Chemicals

When chemicals are carried by hand, they should be placed in a box or acid-carrying bucket to prevent

breakage and spillage. For transport of chemicals between labs at E3A 7 floor: Transport only in closed

containers or bottles; Cover microtipter plates; do not walk around on the floor with pipettes filled with

liquid/chemicals (remove Pipetting tip). Chemicals should be carried up the stairs or transported on

freight elevators to avoid exposure to other persons on passenger elevators. However, it is unwise to carry

solid carbon dioxide in any closed elevator.

Lab Coats, Face Masks, Gloves and Eye Protection

Laboratory coats and appropriate type of gloves must be worn when handling chemicals. In addition,

safety glasses or goggles must be worn when handling corrosive chemicals. Use face shields when

handling large quantity of corrosive chemicals.

Personnel should not wear lab coats in canteens or other eating area. They should leave their lab coats in

their laboratory before proceeding to canteen.

Gloves should be selected on the basis of the substance(s) being handled, the particular hazard involved,

and their suitability for manipulations in the operation being conducted. Remove contaminated gloves

before touching doorknobs or equipment handles.

Personnel should not wear gloves in the common area or lift. They should leave the gloves either in their

laboratory or in the pockets of their lab coats. This is to prevent contamination of door knobs or lift

buttons from the dirty gloves.

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Many carcinogens either in solid form or in solution can permeate glove material. It is advisable therefore

to work with two pairs, replacing the outer pair as soon as any contamination is observed.

Wear a facemask when handling chemicals that are harmful when inhaled. Warn other workers in the

laboratory when such chemicals are used. Lab coats and gloves are not allowed in the Pantry area.

Mouth Pipetting

Do not pipette chemicals or start a siphon by mouth; a pipette bulb or an aspirator should be used.

Tasting and Smelling of Chemicals

Most chemicals used in laboratories are poisonous when taken internally; never taste and avoid smelling

any reagent or product. Exceptions to this rule will be rare and clear.

Food and Beverage Consumption

Eating and drinking is strictly prohibited in the laboratory. Contamination of food and drink is a potential

route for exposure to toxic chemicals.

Food and drink should be stored, handled, or consumed in the pantry area only. Under no

circumstances store drinks/food in the lab fridges, freezers or lockers.

Cylinders of Compressed Gases

Cylinders that contain compressed gases should not be subjected to rough handling or abuse. Such misuse

can seriously weaken the cylinder, rendering it unfit for further use or causing a highly dangerous fracture.

To protect the valve during transportation, the cover cap should be screwed on tightly by hand and remain

on until the cylinder is securely chained up in place and ready for use. Cylinders should never be rolled or

dragged. The preferred transport, even for short distances, is by cart with the cylinder strapped in place

with a chain.

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Fume Cupboard and Biohazard Hood

Perform experiments that involve the use of chemicals that release gases, vapors or aerosols in fume

cupboard or biohazard hood.

Weighing of powdered chemicals which are harmful when inhaled, e.g. acrylamide should be carried out

in a fume hood.

Fume cupboard and biohazard hood should be inspected periodically by authorized personnel for proper

and optimum working conditions.

Equipment Maintenance

Good equipment and maintenance is important for safe, efficient operations. Equipment should be

inspected and maintained regularly. Servicing schedules should be related to expected usage and the

reliability of the equipment. Equipment awaiting repair or maintenance should be removed from service

or clearly labeled “Out of Order”

Equipment must be cleaned periodically to prevent accumulation of dust which might affect the

performance of the equipment.

Safety Training

Appropriate safety training on handling of various types of hazardous chemicals and radioisotopes must

be given to individuals when they commence their work in the laboratory. Supervisors in the laboratory

are responsible for ensuring that the workers undergo safety training. Such training is organized regularly

by the Office of Safety, Health and Environment, NUS.

Resources on Chemical Safety

1. Hazards in the Chemical Laboratory, 5th

edition, 1992, Edited by S.G Luxon, Cambridge, The Royal

Society of Chemistry, QD63.5Haz, Science Library and Central library, NUS

2. Working Safely in the Chemistry Laboratory. Harry G. Hajian Sr and Robert

L. Pecsok, American Chemical Society, Washington, DC, 1994, QD63.5Haj, Science Library, NUS.

3. Chemical Safety Matters, International Union of Pure and Applied Chemistry, International

Programme on Chemical Safety, 1992, Cambridge University Press, QD63.5Che, Science Library, NUS

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4. Safety Sense: A Laboratory Standard, Cold Spring Laboratory Press, 1999, QD63.5Saf, Science

Library, NUS.

5. Dealing with Chemical Safety (Video recording), 1993, T55.3Haz.De, SVC1529, Science Library,

NUS.

6. Chemical Safety for laboratory Workers (Video recording), 1986, TP149Che, SVC928, Science

Library, NUS

7. World Wide Web Sites:

Office of Radiation, Chemical & Biological Safety (ORCBS), Michigan State University. The

site contains useful safety information on radiation, chemical, biological safety and hazardous

waste. It also contains links to searchable MSDS databases. http://www.orcbs.msu.edu/

Office of Chemical, Biological, and Radiation Safety, University of Missouri, Kansas City.

http://www.umkc.edu/cbrs/

Hazardous Chemical Database, the University of AKRON, Department of Chemistry. This site

contains a searchable database for safety information on hazardous chemicals.

http://ull.chemistry.uakron.edu/

Material Safety Data Sheets, PDC Cornell University. The site contained searchable MSDS

database http://msds.pdc.cornell.edu/

The Physical and Theoretical Chemistry Laboratory, Oxford University. The site contains

information on Chemical Safety, especially industrial and scientific gases.

http://physchem.ox.ac.uk/MSDS/

Chemical Safety, Stanford University. The site provides a searchable database for chemicals.

http://www.stanford.edu/dept/EHS/prod/index.html

National OCCUPATIONAL Health & Safety Commission, Australia. The site provides

searchable chemical databases, including the hazardous Substances Database and Australian

Exposure Standards Database. http://www.safetyline.wa.gov.au/

NIOSH, National Institute for Occupational Safety and Health

http://www.cdc.gov/niosh/homepage.html

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NIEHS, National Institute of Environmental Health Sciences. Research on Environment-related

diseases http://www.niehs.nih.gov/

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Chapter 5 Biological Safety

Introduction

In establishing safe practices in a biological laboratory, it is paramount that appropriate and sufficient

concern be given to the more esoteric and poorly understood risks, such as those arising from genetic

manipulation, handling and culture of tissues and microorganisms, etc. It should always be assumed that

any microorganisms and biological agents (or materials containing biological agents whose epidemiology

and aetiology are unknown or incompletely understood) handled in the laboratory are capable of causing

disease. Great care should therefore be taken in handling cultures, slides and all materials that contain or

have been in contact with living microorganisms and questionable biological agents. Any hazardous

biological agent must be properly handled or disposed of so as not to constitute a health risk. It should be

borne in mind that any accidents involving these biological agents may result in infection. Remember the

main routes of entry of infection to the body are: by inhalation, by ingestion, through cuts and abrasions

and by infecting the eyes. The majority of exposures often result from more subtle sources such as the

production of aerosols during routine laboratory procedures. Safety procedure should be directed towards

the prevention of infection.

In general, biosafety level 2 or 3 practices are required for all research involving human blood, blood

components, blood products, human body fluid, or tissues and organs as these may contain human

pathogens and other potentially hazardous human materials. Although primary human cell culture are not

included in the above, it must be strongly emphasized that primary human cell cultures are not entirely

hazard free and that prudent laboratory practices required that standard microbiological practices and

biosafety level 2or 3 be employed for all such procedures as well.

More information on Biological safety can be found in the WHO Biosafety Manual (copy in the lab and

on the Lab Webpage).

Standard Biological Practices

1. In handling biological specimens appropriate barrier protection (e.g. gloves, eye and face

protection, lab coats) should be used at all times.

2. Cover any abrasion, cut or open wound with adhesive plaster before beginning work.

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3. The working bench should be free of any personal items

4. Report to a staff member immediately when a laboratory accident occurs, whether it is a spilt

culture or burn from a Bunsen flame.

5. Wear a laboratory coat at all times in the laboratory. Do not wear this protective clothing outside

the laboratory. Laboratory coats should not be kept in the lockers where personal clothing is

stored but placed on convenient hooks, preferably in the laboratory where they are worn. All lab

coats should be washed regularly and when contaminated, should be disinfected before washing.

6. Do not eat, drink or smoke in the laboratory.

7. Wash hands frequently and always before leaving the laboratory.

8. Disinfect work surfaces and any equipment immediately after spillage of biological samples. A

spilt culture should be flooded with a suitable disinfectant solution and left for 15-30 minutes

before clearing up.

9. Biohazardous waste (like contaminated labware, biological waste etc.) must not be discarded into

normal disposal bins.

10. Exercise care in all procedures and manipulations to minimize aerosol formation. Aerosols can be

generated while using pipettes, wire loops and even during the removal of a screw cap or a rubber

bung from a culture tube. Only sealed containers are to be used when centrifuging microbial

cultures.

11. Wearing of safety goggles is necessary for all work that generates aerosols that are biohazardous

in nature. Only those wearing glasses are exempted.

12. Inoculating needles and wire loops must be sterilized before and after use, by heating in a Bunsen

flame until red hot along the entire length of the wire. Splattering of material from the wire

should be avoided by very gradual introduction into the Bunsen flame. The flame should be

turned off when not in use.

13. Test tube cultures should always be kept in test tube racks. Never place the test tubes horizontally

on the bench top.

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14. Never pipette cultures by mouth.

15. Rapid and forceful ejection of the contents of a blow-out pipette can produce an aerosol. In

general, slow and unhurried movements are to be preferred in microbiological work, but with

minimum delays between operations.

16. Microscope slides and cover slips must be discarded into jars of disinfection solution, separating

the cover slips first from the slides.

17. Homogenizers and blenders must not be used in conjunction with bacterial cultures without

adequate precautions against the spread of air-borned contamination. Use a biological safety

cabinet and cover the top of the blender with a towel moistened with disinfectant. Before opening

a blend bowl, wait for at least 1 minute to allow the aerosol to settle.

18. Autoclave or otherwise disinfect contaminated items such as glassware, animal cages and

laboratory equipment before washing, re-using, or discarding

19. Autoclaves should be regularly maintained and checked to ensure that they operate at the required

temperature and pressures.

20. Used needles, disposable syringes, scalpel blades, pipettes, and other sharp items are to be placed

in puncture resistant containers marked with a biohazard symbol for disposal.

21. Used needles should not be re-caped, bent, broken or manipulated by hand. Discard these items

intact to prevent accidental skin puncture.

22. Nothing containing living micro-organisms or tissue cultures should be directly poured down the

sink.

23. Used Petri dishes and other culture vessel (tubes, bottles, etc) should be put into the appropriate

discard container for sterilization. Equipment which contained living microorganisms should be

sterilized as soon as possible.

24. Living cultures must not be removed from the laboratory without permission.

25. The contents of discard bins should not be searched.

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26. Biological material when transported between buildings must be in a double container system. If

any materials is dropped or spilt whilst being carried between buildings, the accident must be

reported and the spillage must be treated immediately.

27. Laboratory refrigerators, cold rooms and other equipment must not be used to store any food for

human consumption. Food can only be stored, handled and consumed using designated

refrigerators in specified rooms like pantry and tea-rooms.

28. Materials to be autoclaved should be placed in leak proof containers and properly labeled with

autoclave tape. Glassware should be autoclaved separately from disposable waste, rinsed

following autoclaving, and then sent through the normal wash cycle. Containers should be

available in each lab for the disposal of liquids and solids which are contaminated with

microorganisms or mammalian cells. Autoclavable bags on holders are good for discarding most

solid waste and small volumes of liquid waste. For large volumes of liquid waste, closed flasks or

autoclavable bottles can be used. Disposable waste that has been autoclaved may be combined

with normal waste.

29. All vessels containing viable biological materials should be properly labeled to provide

information to users in case of breakage and spillage. Label information should include: organism

present, special features if any, and name of the investigator responsible for it.

30. The location of first aid kits, eye irrigation bottles, emergency showers and fire extinguishers

should be made known to all laboratory workers

Use of Biohazard Safety Cabinets

1. The appropriate type of biohazard safety cabinets should be used according to the class of

biosafety level required for the biological agents used (See following Tables)

2. Experiments involving all agents (especially mutant species) that are of Biosafety Level 2 and

above should never be performed on open bench top.

3. All used pipettes, glassware, etc. should be discarded into containers placed within the cabinet

and with appropriate disinfectants.

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Biosafety in Biological and Biomedical Laboratories

Table 1 Classification of Biosafety Levels for Infectious Agents

Biosafety level

(BSL)

Agents

Practices

Safety Equipment (Primary Barriers)

Facilities (Secondary-Barriers)

1. Not known to cause disease in healthy adult humans

Standard Microbiological Practices

None required Open bench top sink required

2. Associated with human disease, hazard= autoinoculation, ingestion, mucous membrane exposure

BSL-1 practice plus: Limited access; Biohazard warning signs; “Sharps” precautions; Biosafety manual defining any needed waste decontamination or medical surveillance policies.

Primary barriers= Class I or II BSC or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; laboratory coats; gloves; face protection as needed.

BSL-1 plus: Autoclave.

3. Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

BSL-2 practice plus: Controlled access; Decontamination of all waste: Decontamination of lab clothing before laundering; Baseline serum.

Primary barriers=Class I or II BCSs or other physical containment devices used for all manipulations of agents; protective lab clothing; gloves; respiratory protection as needed

BSL-2 plus: Physical separation from access corridors; Self-closing, double-door access; Exhausted air not recirculated; Negative airflow into laboratory

4.

Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission

BSL-3 practices plus: Clothing change before entering; Shower on exit; all material decontaminated on exit from facility.

Primary barriers= All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure personnel suit.

BSL-3 plus: Separate building or isolated zone; dedicated supply/exhaust, vacuum, and Decon systems; requirements outlined in the reference1

Table 2 Types of Biohazard Safety Cabinets

Face Velocity (lfpm)

Airflow Pattern Radionuclides/Toxic Chemicals

Biosafety Level (s)

Product Protection

Class I*, open

75 In at front; out rear and top through HEPA**

No 2, 3 No

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front filter Class II: Type A

75 70% recirculated through HEPA; exhaust through HEPA

No 2, 3 Yes

Type B1 100 30% recirculated through HEPA; exhaust via HEPA and hard ducted

Yes (Low Levels/volatility)

2, 3 Yes

Type B2 100 No recirculation; total exhaust via HEPA and hard ducted

Yes 2, 3 Yes

Type B3 100

Same as IIA, but plus under negative pressure to room and exhaust air is ducted

Yes 2, 3 Yes

Class III NA Supply air inlets and exhaust through 2 HEPA filters

Yes 3, 4 Yes

*Glove panels may be added and will increase face velocity to 150 lfpm; gloves may be added with an inlet air pressure release that will allow work with chemicals/radionuclides

** HEPA: High Efficiency Particulate Airfilter

lfpm: linear flow per minute

Appropriate References and Websites

1. Biosafety in Microbiological and Biomedical Laboratories. CDC, NIH. 3rd

edition. 1993.

2. http://www.niehs.nih.gov/odhsb/biosafe/bio.htm

3. http://www.niehs.nih.gov/odhsb/biosafe/bsc/bsc.htm

4. http://www.niehs.nih.gov/odhsb/biosafe/bsc/section1.htm

5. Laboratory manual, Microbiology Dept, NUS

6. Safety manual, Faculty of Engineering, NUS

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Some References on Sharps Injuries and Control of Occupationally Acquired Bloodborne Infections

1. Guidelines for prevention of transmission of HIV and HBV to health care and public safety

workers. US Dept of Health and Human Services. 1989.

2. A code of practice for the safe use and disposal of sharps. British Medical Association, 1990

3. Exposure control plan for bloodborne pathogens. American College of Occupational and

Environmental Medical, 1992.

4. Selecting, evaluation and using sharps disposal containers. NIOSH 1998

24

Chapter 6 Electrical Safety

This chapter on electrical safety gives general guidelines on safe practices related more to laboratory

which have power driven machinery. It covers general electrical safety practices for all electrical or

electronic hardware.

Electrical Safety Practices The following practices are to be followed by all: Individual

Eye protection is required during any electronic or electrical hardware repair, installation and/or open

front operation.

Electrical safety shoes, long sleeve non-polyester, low flammability shirts and insulating gloves will be

worn when operating or testing 600 volt or higher equipment.

Protective apron will be worn over polyester or other highly flammable clothing during soldering

operations.

Laboratory

All new equipment and electronic laboratory equipment must be inspected for electrical hazards before

using.

1. All electrical equipment must be grounded (earthed) through power cords, frame grounding and/or

grounding through wiring in conduit system

2. All external metal casing of electrical apparatus, cables and conduits must be grounded (earth) to

prevent any rise of voltage if some fault arises. It is always advisable to check that the new equipment is

properly earthed before putting it into use, and the Office of Estate and Development will carry out such

checks on request.

3. Try to avoid installing electrical equipment into areas that would require work to be carried out in close

proximity to water supplies or other earthed metal work where there may be a risk of putting one hand on

the earthed metal and the other on ‘live’ conductors.

4. Wiring must not be overloaded, otherwise it will overheat and the insulation will be damaged.

Insulation of wiring that has been in use for a number of years tends to become brittle. Where alterations

or additions are required, the installed cable must always be checked by a competent electrician and

replaced completely if there are indications of failure of the insulation.

25

5. Laboratory equipment should be kept of electrical panel-boards with the following clearances: 100-cm

(36 inches) for 120/208 volts and 115-cm (42 inches) for 277/480 volts and up to 600-volt equipment.

6. Operation of panel-board circuit breakers by laboratory personnel is prohibited except in case of

personal emergency. Contact the Office of Estate and Development for assistance.

7. When work is to be performed on electrical equipment, care must be taken to make sure the electrical

source is turned off, rendered inoperative, tagged and locked. Working on live parts of 50 volts or more

should not be done except in an emergency and with proper procedure and/or qualified personnel with

appropriate safety equipment.

8. The circulation space in the laboratories must be kept clear of wire and cables to prevent any hazards

occurring. E.g. mishaps could happen as a result of tripping over a loose cable stretching across the room.

9. The Electrophoresis equipment uses high potential! Read manual carefully before operation. Always

make sure the power is switched of when handling/open the equipment!

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Chapter 7 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.

1. 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 possibility although slight, that

chemical-, or heat- and high-pressure-resistant pathogens might survive these processes and

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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.

8. Only one of the two doors of the airlock window should be opened at any point in time.

9. To use it:

10. Open the airlock door from within the cell culture area;

11. Place the biohazard bag within the airlock;

12. Close the airlock from within the cell culture area;

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

14. Remove the biohazad bag and close the airlock door.

15. All biohazard bags should be enclosed in an additional biohazard bag before being autoclaved.

16. Discard all autoclaved waste into the collector bins provided for final disposal.

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Chapter 8 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.

9. Injuries:

10. 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.

11. 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.

12. Cuts are dangerous because they tear the skin and make the interior of the body vulnerable to

toxins and other dangerous agents.

13. If a cut is bleeding, apply pressure to it with a bandage, or with gloved hands, to staunch the flow

of blood.

14. 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

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environment.

15. 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

engcecf@nus.edu.sg, or your supervisor immediately.

16. 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.

17. 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.

18. 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 engcecf@nus.edu.sg, or your supervisor should be informed and the injured user

should seek medical assistance immediately.

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

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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 engcecf@nus.edu.sg, or your

supervisor.

13. If you are alone, remove all contaminated clothing and rinse yourself thoroughly under the

emergency shower. Use soap if possible.

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 engcecf@nus.edu.sg, 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.

31

Chapter 9 Equipment Operation Safety

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 56C 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.

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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.

Gas Cylinders

The contents of gas cylinders must be clearly labeled. Empty cylinders should be separated from full ones

and clearly labeled as such.

Gas cylinders should be placed in a stand or strapped into an approved rack. Cylinders must only be

transported in a cylinder trolley, and with a cylinder cap. Ensure that the regulator used is appropriate for

the gas and the pressure required.

Readily combustible substances, such as oil or grease, must not be permitted to come into contact with

oxygen cylinder valves, regulators, gauges or fittings. Gas cylinders, in particular oxygen cylinders

should be placed as faar away as possible from flammable solvents and materials. Suspected leaks can be

checked by applying a dilute soap solution. Bubbles will occur where there is a leak.

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Liquid Nitrogen

Cryogenic fluids are usually stored in special insulated containers. When transferring liquid nitrogen from

one container to another, the operator must be adequately shielded, with a face mask or goggles, heavy-

duty gloves and protective clothing.

Store and use in well-ventilated areas. Keep away from sparks and flames, which may cause explosions.

Cryogenics present the hazard of asphyxiation due to displacement of oxygen, or embitterment of metals

due to extreme cold.

When liquid nitrogen is split on the skin, it will roll of due to its movement on a cushion of gas. If,

however, it is trapped by garments, it may produce burns. Such clothing should be discarded until the

liquid has vaporized.

Low pressure Equipment

When using low-pressure equipment, such as freeze-dryers, safety glasses or goggles must always be

worn. Vessels under low pressure should be shielded by thick cardboard or cloth, and air should be

introduced gradually.

Centrifuges Operation: Study operation manual before operating centrifuge. Select correct rotor

and change rotor if necessary. Balance tubes to be placed in rotor and place pair of balanced tubes on

opposite side of rotor. Close the lid of the rotor if applicable. Close the lid of the centrifuge. Program

RPM, temperature (if applicable) and time. Check if the correct rotor is recognized by the centrifuge.

Start the centrifuge. Wait until full RPM is reached and check for vibrations. Stop centrifuge manually or

wait until time program is ended.

Safe operation and precautions:

1. Securely anchor tabletop centrifuges and place in a location where the vibration will not

cause bottle to fall off the bench.

2. Keep the centrifuge lid closed while operating and do not leave the centrifuge until you are

certain it is running safely without vibration.

34

3. If the centrifuge starts vibrating, stop and check the load balances.

4. Regularly clean rotors and buckets with a non-corrosive cleaning solution.

5. Use sealed safety cups while centrifuging hazardous materials.

Ultraviolet Lamps

1. Wear ultraviolet absorbing protective safety glasses while working with ultraviolet light.

2. Protect skin from potential burns due to ultraviolet light.

3. Shield any experiment in which ultraviolet light is used to prevent escape of the direct beam

or scattered radiation.

Autoclave

Operation: Study operation manual before operating the autoclave. Switch on the autoclave. Check

the pressure gauge. Make sure that the level of the exhaust water lies between the high and low mark.

Ensure that there is sufficient volume of the sterilizing water. Label the items to be autoclaved with

autoclave tape and place items into the steel mesh basket. Press START. Once the sterilization cycle

is over, remove the items and check for activation of the autoclave tape. Place items in convection

oven for drying.

Safety Operation and Precautions: Autoclaves are pressurized sterilizing chambers and are used to sterilize glassware, instruments, gloves,

and liquids in bottles, biological waste, and other materials by steam under pressure. Autoclaves are

typically at pressures a little under two atmospheres, at temperatures of up to 135°C. The stored energy in

the steam can cause serious injury.

1. Users must be trained in the correct operation of the autoclave.

2. The autoclave MUST be depressurized before it is opened. Check pressure gauge!

35

3. Use non-sealed Pyrex containers which are designed for the temperatures and pressures of the

autoclave, as liquids placed in sealed bottles or in ordinary glass bottles may rupture.

4. Be aware that if the unit is set to exhaust rapidly, as might be done for instrumental sterilization,

boiling may take place in bottles of liquids, with a consequent loss of liquids into the autoclave.

5. Do not run flammable liquids or chemicals which could become unstable at the temperatures

reached in the autoclave through the sterilizing cycle.

6. Clean and check autoclave regularly.

7. Two Autoclaves are available at the BSL2 lab and are dedicated for media and waste.

Biological Safety Cabinet

1. To assure sterility inside the cabinet and establish proper air flow for containment, the blower

should be turned on at least ten minutes before infectious materials are to be put into the biosafety

cabinet.

2. Biosafety cabinets must be certified prior to use. A qualified outside contractor must certify these

cabinets annually. Check the certification sticker on the front of the unit to verify your biosafety

cabinet’s condition.

3. The biosafety cabinet air flow (“Magnehelic”) gauge should be checked (reading is equal to

approximately 0.5 inches) to assure proper operation of the cabinet before placing any materials

into it. Readings indicate relative pressure drop across the HEPA filter. Higher readings may,

therefore, indicate filter clogging. Zero readings may indicate loss of filter integrity. In either of

these cases, notify the Laboratory Manager or PI.

4. NEVER place anything over the front or rear grill of a biosafety cabinet.

5. Disrupting the airflow into the front grill allows contaminated air from inside the cabinet to blow

into the lab or directly at the person sitting at the cabinet. It also allows non-sterile air from the

36

room to blow into the biosafety cabinet over the experiments.

6. Materials should be placed in the cabinet so as not to block air flow into the rear grill. Leave a

few inches for air to flow around objects. Any disruption of the air flow in the cabinet decreases

its effectiveness.

7. Before manipulating infectious materials, make sure that you have everything you need in the

cabinet. The fewer times you pull your hands out of the cabinet, the less disruption of the air flow.

8. Work should be performed in the center of the work surface of the cabinet whenever possible.

Work outward progressing from clean to dirty (contaminated). However, infectious agents should

not be placed directly adjacent to or directly on the intake grills.

9. After manipulating infectious agents, make sure all containers are tightly closed.

10. All waste and disposable items generated by work in the cabinet should be left (describe where it

should be stored) until properly decontaminated or contained for transport to the autoclave.

11. After the cabinet has been emptied, wipe inner surfaces with (name of disinfectant), followed by

70% ethanol. Do not shut down the blower. (these instructions must be written to accommodate

your lab practices).

12. The bleach in the vacuum traps must be changed after one week of use or when the flask is half

full. To discard trap liquid, first treat with fresh bleach for 20 minutes, and then empty it into the

sink.

13. The vacuum filters must be replaced if clogged or if liquid makes contact with the filter. Used

filters should be placed in the waste to be autoclaved.

Note: Though ClassII B cabinets are hard-ducted (so that all air is removed from the room). ClassII A

cabinets recirculate about 70& of the air inside themselves and exhaust the remainder to the lab. Any use

of volatile solvents, such as absolute ethanol, should be kept to a minimum or done elsewhere.

37

Dangerously high levels of volatile vapors can accumulate inside the cabinet and pose a threat of

fire or explosion.

Incubator

1. Incubators are normally set at 37o

C.

2. Temperature should be checked each day by all users.

3. Operation manuals are located beside the incubator.

4. If an alarm is sounding, check the panel for the identifying blinking light. 1) If there is no obvious

reason for the alarm, contact the Lab Manager or PI. 2) The “CO2 Low” (or High) message

indicates a deviation from 5% CO2. Check the hose from the wall to the unit. 3) The “tank farm”

must be checked for empty tanks once/week. Always keep one CO2 gas cylinder working and the

other spare one is full for changing.

5. Decontaminate incubators at least every week.

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

June 2012