Maheshri:SOPs

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Maheshri Lab Standard Operating Procedures (SOPs)

The Occupational Safety and Health Administration (OHSA) requires that all experiments performed in the laboratory that involve chemical and/or other safety concerns be done in accordance with a Standard Operating Procedure (SOP) if all safety concerns are not covered in the standard SOPs listed in the Department's Chemical Hygiene Plan. Since the construction of the SOP is the best opportunity to consider all aspects of safety before an experiment is performed, SOPs are useful and do not just serve to meet the regulations. It is everyone’s responsibility to get familiarized with all the SOPs listed here.

Operating Procedures for Equipment and Hazardous Materials

Chemical (Fume) Hood

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: The chemical fume hood is designed to provide a safe environment for working with noxious and/or volatile chemicals. It is not designed to provide an environment for conducting biological experiments.

Potential Safety Issues: Chemical exposure from improper operation.

Startup Procedure:

1. Ensure that air is flowing through the hood prior to use.
2. Turn the light on in the hood using the light switch found on the left side of the hood.
3. Lift the hood’s sash up 9-12 inches to allow room to work in the hood.

Shutdown Procedure:

1. Ensure that all chemicals are put away and the hood is clean.
2. Lower the hood’s sash to about 1-2 inches above the floor of the hood.
3. Turn on the hood’s light using the light switch found on the left side of the hood.

In the Event of a Power or Airflow Loss: Discontinue use of the hood and shut it down.

Monitoring Required: The chemical hood must be inspected and validated annually. The chemical hood is also inspected weekly by the student safety coordinator.

Chemical Solvent Cabinet

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: The chemical solvent cabinet is used to store toxic, corrosive, and hazardous chemicals.

Potential Safety Issues: Chemical exposure, unintended chemical reactions due to improper chemical storage.

Startup Procedure:

1. Open the cabinet doors.
2. Carefully take out of the cabinet the chemical you intend to use.
3. Close the cabinet doors.

Shutdown Procedure:

1. Open the cabinet doors.
2. Place the chemicals used back in an appropriate area of the cabinet (organic acid, inorganic acid, base, or solvent) based on the identities and hazards of the chemicals used.
3. Firmly close the cabinet doors.

In the Event of an Evacuation: If it can be safely done, place all chemicals back in the appropriate region of the chemical cabinet and firmly close the cabinet doors.

Monitoring Required: The chemical storage cabinet is inspected weekly by the student safety coordinator.

Ethidium Bromide (EtBr) and agarose gel imaging

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Ethidium Bromide (EtBr) is used in staining to visualize DNA bound in agarose gels.

Potential Safety Issues: Exposure to EtBr (mutagenic). Exposure to UV radiation.

Startup Procedure:

1. Use gloves when adding EtBr to agarose gel. EtBr contaminated pipette tips must be disposed into designated waste container in the fume hood.
2. After electrophoresis, place the gel in the gel imager on top of a sheet of plastic wrap. Use spatula (instead of bare hands) to move the gel.
3. In order to turn on the UV lamp while keeping the imager door open, use a pipette tip to hold the safety trigger.
4. Wear face-shield or UV protective goggles when cutting the agarose gel under direct UV exposure.

Shutdown Procedure:

1. Turn off the UV lamp in the imager. Remove the pipette tip from the safety trigger.
2. Remove the gel and dispose it into the five gallon black EtBr gel solid waste container located on the ground near the -80 freezer.
3. Dispose the plastic wrap into regular biological waste and razor blade into the bench-top red sharps container.
4. Remove your EtBr-contaminated gloves and thoroughly wash your hands. The gloves can be disposed into regular biological waste.

In the Event of EtBr Exposure: Immediately and thoroughly wash the exposed area with soap and water.

Monitoring Required: The gel electrophoresis boxes and UV imager are inspected weekly by the student safety coordinator for cleanliness. The EtBr contaminated pipette tips are routinely transferred from the fume hood to the five gallon white solid waste container located on the ground near the -80 freezer.

Phenol and chloroform (P/C)

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Phenol and chloroform (P/C) are used to extract proteins during DNA preps.

Potential Safety Issues: Exposure to phenol (toxic, corrosive and mutagenic) and chloroform (harmful).

Startup Procedure:

1. Use gloves when working with P/C. Work should be done in the chemical fume hood.

Shutdown Procedure:

1. Dispose the liquid P/C waste into the designated waste container in the fume hood.
2. Dispose tips and tubes contaminated by P/C into the designated waste container in the fume hood.

In the Event of P/C Exposure: Immediately and thoroughly wash the exposed area with soap and water.

Monitoring Required: P/C contaminated solid waste is routinely transferred from the fume hood to the five gallon white EtBr + P/C solid waste container located on the ground near the -80 freezer.

Incubator Shakers

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Incubator shakers are used to grow and culture live microbial cell cultures.

Potential Safety Issues: Exposure to Biosafety Level 1 microorganisms.

Startup Procedure:

1. Program the incubator to shake at the desired rpm. Set the timer if needed.
2. Turn on the shaking function of the incubator.
3. Only Biosafety Level 1 microorganisms should be placed in incubators.

Shutdown Procedure:

1. Turn off the shaking function of the incubator.
2. Remove all microbial cultures from the incubator.
3. After experiment, suspend all microbial cultures in bleach to a final concentration of 10% by volume for at least 15 minutes.
4. Dispose of bleached microbial cultures by washing them down the drain with several volumes of water.

In the Event of Power Loss: Shut down the incubator and remove all microbial cultures from the incubator chamber.

Monitoring Required: The incubators are inspected weekly by the student safety coordinator for cleanliness.

Refrigerators and Freezers

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Refrigerators and freezers are used to store and preserve Biosafetly Level 1 microbial cultures and other experimental samples.

Potential Safety Issues: Exposure to Biosafety Level 1 microorganisms, cryogenic hazard.

Startup Procedure:

1. Set the refrigerator or freezer to a desired temperature.
2. Turn on the refrigerator or freezer.
3. Allow the refrigerator or freezer to come to equilibrium at the desired temperature.
4. Securely place samples to be stored or preserved in the refrigerator or freezer. Wear gloves when operating the refrigerator or freezer.

Shutdown (or defrosting) Procedure:

1. Remove all items from the refrigerator or freezer. Wear gloves when operating the refrigerator or freezer.
2. Place paper towels around the refrigerator or freezer.
3. Turn off the refrigerator or freezer.
4. Ensure that the water that will leak out of the refrigerator or freezer as it warms up is captured by the paper towels.

In the Event of Power Loss: Remove all items from the refrigerator or freezer and place them in an alternate but equivalent refrigerator or freezer in another laboratory (such as the Prather lab).

Monitoring Required: The refrigerators and freezers are inspected weekly by the student safety coordinator for cleanliness. Frost deposit is removed during inspections.

Centrifuges

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Centrifuges are used to separate chemicals based on their densities.

Potential Safety Issues: Mechanical hazard from rapidly moving centrifuge parts.

Startup Procedure:

1. Turn on power to the centrifuge.
2. Set the centrifuge to spin at the desired speed, temperature, and duration. Do not spin samples and sample containers at a centrifugal force higher than they are rated to withstand.
3. Place samples in the centrifuge in a balanced manner.
4. Before starting, make sure that the rotor tie-down device is securely fastened.
5. Close the centrifugation chamber and start the centrifuge.

Shutdown Procedure:

1. Ensure that the centrifuge has come to a complete stop.
2. Open the centrifugation chamber and remove all samples.
3. Close the centrifugation chamber.

In the Event of an Power Failure: Shut down all centrifuges. Do not attempt to retrieve the sample from the centrifuge for at least one hour.

Monitoring Required: Centrifuges are inspected weekly by the student safety coordinator for cleanliness.

Hot Plates, Oven and Water Baths

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Hot plates, oven and water baths are used to incubate experimental samples at elevated temperatures.

Potential Safety Issues: Thermal hazard from elevated temperatures. Chemical and biological exposure hazard from sample container caps bursting or failing due to internal pressure buildup during heating.

Startup Procedure:

1. Set the equipment to the desired temperature setpoint.
2. Turn on heating and allow the equipment to reach the setpoint.
3. Put on a pair of insulating gloves and place the experimental samples to be heated into the equipment and incubate them for the desired amount of time.

Shutdown Procedure:

1. While wearing a pair of insulating gloves, remove the experimental samples from the equipment.
2. Turn off the equipment and allow it to cool.

In the Event of a Power Failure: Put on a pair of insulating gloves and remove all samples from the equipment.

Monitoring Required: Hot plates, oven and water baths are inspected weekly by the student safety coordinator for cleanliness.

Thermocycler

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Thermocyclers are used to carry out polymerase chain reactions (PCR reactions) or to incubate experimental samples at elevated temperatures.

Potential Safety Issues: Thermal hazard from elevated temperatures. Chemical and biological exposure hazard from sample container caps bursting or failing due to internal pressure buildup during heating.

Startup Procedure:

1. Turn on the thermocycler and set up the desired thermal cycling program.
2. Allow the thermocycler cover to heat up to equilibrium.
3. Place the experimental samples to be PCRed into the thermocycler.
4. Start the thermocycling program.

Shutdown Procedure:

1. Ensure that the thermocycling program is complete.
2. Remove the experimental samples from the thermocycler.
3. Allow the thermocycler to cool down.

In the Event of a Power Failure: Put on a pair of insulating gloves and remove all samples from the thermocycler.

Monitoring Required: Thermocyclers are inspected weekly by the student safety coordinator for cleanliness.

Microwave oven

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: The miocrowave oven is used to prepare agarose gel and heat up chemical solutions.

Potential Safety Issues: Chemical exposure hazard from agarose gel containing EtBr. Thermal hazard from elevated sample temperatures. Possible exposure to excessive microwave energy.

Startup Procedure:

1. Place paper towels on the turntable and place the samples on the paper towel.
2. Set the power level and time and start the program.
3. Stay near the oven while it is in use to check if there is overflow of boiling liquid.

Shutdown Procedure:

1. Ensure that the program is either complete or paused.
2. Wait a few seconds before opening the oven door to avoid steam burns.
3. Always wear a pair of insulating gloves or use the hot-hands to remove the experimental samples from the microwave oven.

In the Event of a Power Failure: Put on a pair of insulating gloves and remove all samples from the microwave oven.

Monitoring Required: Microwave oven are inspected weekly by the student safety coordinator for cleanliness. The removable turntable should be clear of chemical samples.

Vortex mixers

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Vortex mixers are used to achieve mix homogeneity in the samples

Potential Safety Issues: Exposure to Biosafety Level 1 microorganisms and hazardous chemicals.

Startup Procedure:

1. Ensure the pop-off cup or three-inch platform are firmly attached to the motor.
2. Cap the tubes before mixing.
3. Adjust mixing speed to prevent overflow.

Shutdown Procedure:

1. Clean up any spills after use

Monitoring Required: The vortex mixers are inspected weekly by the student safety coordinator for cleanliness.

Varioskan Flash microplate reader

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: Cell-based spectral scanning multimode reader for photometric and fluorometric measurements.

Potential Safety Issues: Exposure to Biosafety Level 1 microorganisms.

Startup Procedure:

1. Install the appropriate microplate adapter (96 or 384 wells). The adaptor should be taken off if the microplate is covered by plastic lid.

Shutdown Procedure:

1. Remove the microplate and clean up any spills after use.

In the Event of Power Loss: Shut down the microplate reader. Do not attempt to retrieve the sample from the microplate reader for at least one hour.

Monitoring Required: The microplate reader is inspected weekly by the student safety coordinator for cleanliness.

Electroporation apparatus

Author: Tsz-Leung To

Effective date: November 01, 2007

Description: The electroporation unit is used for transforming bacteria, yeast and other microorganisms with DNA materials.

Potential Safety Issues: Electrical hazards generated by high voltages. Mechanical hazards caused by arcing. Exposure to Biosafety Level 1 microorganisms.

Startup Procedure:

1. Wear goggles and insulating gloves.
2. Ensure that the samples are suspended in high resistance media (> 600 ohms).
3. Select the appropriate program for electroporation.
4. Place the cell suspension in an electroporation cuvette, insert the cuvette into the slide of the shocking chamber and push the slide until the cuvette makes firm contact with the chamber electrodes.
5. Press the yellow “Pulse” button to deliver the pulse.

Shutdown Procedure:

1. Remove the cuvette and turn the unit off.
2. If desired, disconnect the sample chamber. Never remove the sample chamber until the leads are disconnected.

Monitoring Required: The electroporation apparatus is inspected weekly by the student safety coordinator for cleanliness.

Fluorescence microscope

Author: Christopher Zopf Effective data: November 01, 2007

Description: Zeiss Axio Observer Microscope with high-intensity, white light source for multiple bandwidth fluorescent microscopy.

Potential Safety Issues: Exposure to Biosafety Level I microorganisms. Exposure to high-intensity light. Risk of rupturing rubber hosing during high-pressure microfluidic experiments.

Startup Procedure:

1. Turn on the white light source (black box connected to the fiber optic cable). DO NOT remove the fiber optic cable from the microscope while the light source is on as the high-intensity light can damage the eye. Also, do not bend the cable sharply as it will damage the fibers.
2. Turn on the Ludl control box. After the “BUSY” LEDs turn off, turn on the microscope (ensure the flat, white voltage source is on).
3. Place either the slide or well-plate stage mount on the stage. To use the 63x or 100x objective, place a drop of immersion oil on the objective before loading the sample onto the stage.
4. Load samples mounted on glass slides into the slide stage mount (coverglass side down), or alternatively, load the CellASIC microfludic well plate onto the well-plate stage mount. If using the microfluidic device, follow the protocol provided by CellASIC for sample loading and imaging. Ensure all hose connections are tight and do not exceed 20 psi.

Shutdown Procedure:

1. If using glass slides, discard in the sharps waste container. If using the microfluidic device, turn off pressurized lines and then the vacuum line before disconnecting the device, discard the well-plate in the biological waste container.
2. Clean the oil objective if used. Wipe excess oil from the edges using LENS PAPER being CAREFUL NOT TO APPLY PRESSURE TO THE LENS. The lens may be cleaned with a cotton-tipped wooden stick dipped in an 80:20 mixture of Petroleum Ether and Isopropyl Alcohol. Discard the cotton in the solid waste container.
3. Clean any spills on or around the microscope.
4. Turn off the control box, the microscope and the light source. Replace the microscope cover to protect the optics from dust.

In the Event of Power Loss: Turn off all devices and power sources.

Monitoring Required: The microscope is inspected weekly by the student safety coordinator for cleanliness.

Maheshri Lab Emergency Operating Procedures

Author: Tsz-Leung To

Effective date: November 01, 2007

In the event of an emergency (fire alarm goes off or lab members forced to evacuate lab), the following should only be done if they can be done both rapidly and safely.

• Turn off all heat and ignition sources (Bunsen burners, gas lines, hot plates).
• Cap and contain any open chemical bottles that are being used at the time. Ideally, large chemical bottles should be placed back in the solvent cabinet or in the chemical hood.
• Take your laboratory notebooks and other critical data and carry them with you out of the building.
• Assist other members of the lab in evacuating (especially any injured or incapacitated members) and inform them of the emergency.
• If members of the lab are injured as a result of the emergency, evacuate them along with the lab’s first aid kit (located in the office). Carefully apply first aid to any injured labmates.

You should not concern yourself with the following during emergency:

• Any room temperature or sub-room temperature biological reactions.
• The thermocycler.
• Pipettes, tips, tubes, glassware, and other non-chemical laboratory items.

Evacuate the building using the stairwells – never use an elevator during an emergency.

If a minor emergency such as a small spill or fire occurs in the lab, you may try to contain the emergency (though you are not required to do so).

If the emergency cannot be contained within our lab (a major fire or a toxic spill or smoke that leaks into a public area like the hallway), campus police (dial 100) should be notified. If any member of the lab sustains significant injury, dial 100 for an ambulance and attempt to apply first aid. The lab’s first aid kit is located in the lab on the shelf of the center bench closest to the main door.

Waste Management Operating Procedures

Autoclaving Biological Waste

Author: Tsz-Leung To

Effective date: November 01, 2007

Waste from the biohazard waste bins must be autoclaved before it can be disposed. Biological waste must be autoclaved for extended periods (~90-180 minutes) to ensure its sterility. Because treating biological waste requires extensive use of the autoclave, it is more polite to the other labs that use the autoclave to only treat the waste in the evenings or on weekends.

1. Seal off the large, clear biological waste bags with cable ties.
2. Fill out and tie an “Autoclaved Biological Waste” white tag around the neck of the bag.
3. Take the biological waste bags to the autoclave room (56-415).
4. Fill out the “autoclave log sheet” in the autoclave room (the one that asks for the autoclaved waste bag tag numbers).
5. Set the autoclave to dry and make sure the temperature is set to 121°C. Set the sterilization and drying times according to the table below. Remember to set the drying time to 20 minutes. Otherwise, your waste will be quite wet and smelly when you take it out of the autoclave.
6. Let the autoclave run. Be sure to let the autoclave depressurize before removing your trash bags. Note that it is ok to let the autoclave run overnight, as long as you pick up your waste the next morning.
7. Remove the waste bags from the autoclave and discard them in the lab’ regular trash bins.

Number of Bags	   Sterilization Time (min)	Drying Time (min)
     1			90				20
     2			105				20
     3			120				20
     4			130				20
     5			140				20
     6			145				20
     7 or More		150				20

Disposing of Biological Sharps Bins

Author: Tsz-Leung To

Effective date: November 01, 2007

The biological sharps bins are picked up and disposed of by EHS every Thursday morning.

1. Push any pipettes or other waste down into the container, such that nothing is sticking out above the rim of the bin.
2. Close the bin by pulling the bin’s cover all the way over the top of the bin.
3. Use two cable ties to seal the bin’s cover. Holes for cable ties are found on one side of the cover.
4. In Wednesday evening place the filled, sealed biosharps bin(s) out in the hallway. It’s easy to forget to do this when Wednesday evening rolls around, so make a little reminder note to yourself to do this.
5. On Thursday, EHS will come by to remove the filled bin(s). They will only come by if you sent them an email on Tuesday (or earlier). When they remove the filled bins they will replace them with empty ones. EHS can come by anytime between 7:30am and 5:00pm to pick up the filled bins.
6. Move the new empty bins into the lab and place them in the lab appropriately.