Standard Operating Procedure Osmium Tetroxide

Standard Operating Procedure

Osmium Tetroxide

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Type of SOP: ☐ Process ☒Hazardous Chemical ☐ Hazardous Class

Purpose

Osmium tetroxide is commonly used as a stain in electron microscopy. It is used in both transmission electron microscopy and scanning electron microscopy. Osmium tetroxide operates as a secondary fixative that binds to lipids, and its Osmium heavy metal component adds to the density and contrast of electron microscopy images. It typically involves glutaraldehyde fixation followed by osmium tetroxide staining. This is frequently combined with plastic embedding of the fixed sample to generate thin sections for more effective electron penetration.

Physical & Chemical Properties/Definition of Chemical Group

CAS#: 20816-12-0

Class: Toxic

Molecular Formula: OsO₄

Form (physical state): Solid

Color: Pale yellow

Potential Hazards/Toxicity

Toxic Effects: Oxidizing material. Highly toxic. Contact with combustible materials may cause fire. May cause sensitization by inhalation or skin contact.

Acute Effects: The acute toxicity of osmium tetroxide is high, and it is a severe irritant of the eyes and respiratory tract.
Exposure to osmium tetroxide vapor can damage the cornea of the eye. Irritation is generally the initial symptom of exposure to low concentrations of osmium tetroxide vapor, and lacrimation, a gritty feeling in the eyes, the eyes can temporarily cloud, and the appearance of rings around lights may also be noted. In most cases, recovery occurs in a few days. Concentrations of vapor that do not cause immediate irritation can have an insidious cumulative effect; symptoms may not be noted until several hours after exposure. Contact of the eyes with concentrated solutions of this substance can cause severe damage and possible blindness.
Inhalation can cause headache, coughing, dizziness, lung damage, difficult breathing and may be fatal.
Contact of the vapor with skin can cause dermatitis, and direct contact with the solid can lead to severe irritation and burns. Exposure to osmium tetroxide via inhalation, skin contact, or ingestion can lead to systemic toxic effects involving liver and kidney damage. Osmium tetroxide is regarded as a substance with poor warning properties.
Chronic Effects: Chronic phenol poisoning is characterized by vomiting, difficult swallowing,

excessive salivation, diarrhea, anorexia, headache, fainting, vertigo, mental disturbances, and possibly skin eruptions. Prolonged cutaneous exposure may result in deposition of dark pigment in the skin.

Exposure Limits: Cal-OSHA PEL= 0.0002 ppm (0.002 mg/m³)

Personal Protective Equipment (PPE)

Respirator Protection

Where risk assessment shows air-purifying respirators are appropriate use a full-face particle respirator type N99 (US) or type P2 (EN 143) respirator cartridges as a backup to engineering controls. If the respirator is the sole means of protection, use a full-face supplied air respirator. Use respirators and components tested and approved under appropriate government standards such as NIOSH (US) or CEN (EU).

Respirators should be used only under any of the following circumstances:

• 
  As a last line of defense (i.e., after engineering and administrative controls have been exhausted).
  
• 
  When Permissible Exposure Limit (PEL) has exceeded or when there is a possibility that PEL will be exceeded.
  
• 
  Regulations require the use of a respirator.
  
• 
  An employer requires the use of a respirator.
  
• 
  There is potential for harmful exposure due to an atmospheric contaminant (in the absence of PEL)
  
• 
  As PPE in the event of a chemical spill clean-up process
  

Lab personnel intending to use/wear a respirator mask must be trained and fit-tested by EH&S. This is a regulatory requirement. (https://www.ehs.ucla.edu/ep/ih/resp)

Hand Protection

Double-gloving is recommended when working with pure osmium tetroxide or concentrated solutions. Change gloves frequently and when contaminated, punctured or torn. Wash hands immediately after removing gloves.

NOTE: Consult with your preferred glove manufacturer to ensure that the gloves you plan on using are compatible with osmium tetroxide.

Refer to glove selection chart from the links below:

http://www.ansellpro.com/download/Ansell_8thEditionChemicalResistanceGuide.pdf

OR

http://www.allsafetyproducts.com/glove-selection-chart-chemical-breakthrough-ratings.html

OR

http://www.showabestglove.com/site/default.aspx

OR

http://www.mapaglove.com/

Eye Protection

Chemical goggles (safety glasses alone are not adequate protection because of osmium tetroxide’s severe effects on the eyes).

Skin and Body Protection

Lab coats should be worn. These laboratory coats must be appropriately sized for the individual and be buttoned to their full length. Laboratory coat sleeves must be of a sufficient length to prevent skin exposure while wearing gloves. Full length pants and close-toed shoes must be worn at all times by all individuals that are occupying the laboratory area. The area of skin between the shoe and ankle should not be exposed.

Hygiene Measures

Avoid contact with skin, eyes and clothing. Wash hands before breaks and immediately after handling the product.

Engineering Controls

Osmium tetroxide solutions must be prepared and handled in a certified chemical hood.

Choose a hood with minimal equipment or obstructions to ensure good containment of vapors.
Working surfaces should be protected with plastic backed absorbent pads to insure containment of any spills.
Post the hood with a warning sign to alert others to the hazards, see warning sign at the end of this SOP.
Ensure that the safety shower and eyewash are operational and access is unblocked.
First Aid Procedures

If inhaled

Move person into fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Consult a physician.

In case of skin contact

Flush with plenty of water for at least 15 minutes while removing contaminated clothing. Take victim immediately to hospital.

In case of eye contact

Flush eyes with plenty of water for at least 15 minutes lifting upper and lower eyelids and removing contact lenses. Consult a physician. Continue rinsing eyes during transport to the hospital.

If swallowed

Never give anything by mouth to an unconscious person. Get medical aid immediately. Do NOT induce vomiting. If conscious and alert, rinse mouth with water.

Special Handling and Storage Requirements

Keep locked up. Keep away from heat. Keep away from sources of ignition. Keep away from combustible material. Do not ingest. Do not breathe dust. Wear suitable protective clothing In case of insufficient ventilation, wear suitable respiratory equipment. If ingested, seek medical advice immediately and show the container or the label. Avoid contact with skin and eyes.

Storage: Keep container tightly closed. Keep in a cool, well-ventilated place. Highly toxic or infectious materials should be stored in a separate locked safety storage cabinet or room.
Because of osmium tetroxide’s high acute toxicity and powerful oxidizing ability, osmium tetroxide must be handled in the laboratory using prudent practices. In particular, all work with osmium tetroxide must be conducted in a fume hood to prevent exposure by inhalation, and personal protective equipment (see section) must be worn at all times to prevent eye and skin contact.
Osmium tetroxide should be purchased as a liquid to avoid particulate exposure from the powdered form. The solutions should be stored in labeled tightly sealed containers, and these should be placed in secondary containment. Secondary containment should be used anytime the material is transported to another lab location.

When osmium tetroxide is freshly prepared and active, it is colorless to pale yellow in color. When the material reacts and causes oxidation, it turns black. This is helpful to know especially in the event of a splash or spill (see below) or inadvertent dermal exposure (black dots on skin).

Pure osmium tetroxide and concentrated solutions should be stored in a location that is secure to unauthorized access.
A refrigerator containing osmium tetroxide must be labeled with a caution sign noting the presence of osmium tetroxide and its hazards.
Store pure osmium tetroxide and its concentrated solutions in appropriate, sealed glass containers within unbreakable secondary containment (i.e., a bottle or vial within a sealed compatible plastic jar or metal can with lid). Label all containers, including secondary containment, with the chemical name and hazard warning.
Handling and Solution Preparation:

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  When moving pure osmium tetroxide to a chemical hood, do not remove it from the secondary containment until it is in the hood.
  
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  Prepare the smallest amount of solution necessary for the procedure, typically 50 mL or less. Prepare the solution volumetrically rather than gravimetrically. If a balance must be used, weighing must take place in the chemical hood.
  
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  Pure osmium tetroxide or its concentrated solutions must be opened only in a chemical hood that has been certified within the last 12 months. Just before use, the operation of the chemical hood must be verified by the use of an installed chemical hood monitoring device, a smoke test using a smoke generating tube, or a mechanical or electronic device that indicates air flow. During use, the sash must be lowered to operating height.
  
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  All lab ware that has contacted osmium tetroxide must be decontaminated by rinsing or dipping in corn oil or aqueous solutions of sodium sulfide or sodium sulfite before removing from the hood.
  
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  Immediately after work with osmium tetroxide, decontaminate any spills with kitty litter soaked with corn oil. Discard kitty litter as hazardous waste. Or use aqueous solutions of sodium sulfide or sodium sulfite.
  

Spill and Accident Procedure

Chemical Spill Dial 911 and x59797

Spill – Assess the extent of danger. Help contaminated or injured persons. Evacuate the spill area. Avoid breathing vapors. If possible, confine the spill to a small area using a spill kit or absorbent material. Keep others from entering contaminated area (e.g., use caution tape, barriers, etc.).

Small (<1 L) – If you have training, you may assist in the clean-up effort. Use appropriate personal protective equipment and clean-up material for chemical spilled. Double bag spill waste in clear plastic bags, label and take to the next chemical waste pick-up.

Large (>1 L) – Dial 911 (or 310-825-1491 from cell phone) and EH&S at x59797 for assistance.

Chemical Spill on Body or Clothes – Remove clothing and rinse body thoroughly in emergency shower for at least 15 minutes. Seek medical attention. Notify supervisor and EH&S at x59797 immediately.

Chemical Splash Into Eyes – Immediately rinse eyeball and inner surface of eyelid with water from the emergency eyewash station for 15 minutes by forcibly holding the eye open. Seek medical attention. Notify supervisor and EH&S at x59797 immediately.

Medical Emergency Dial 911 or x52111

Life Threatening Emergency, After Hours, Weekends And Holidays – Dial 911 (or 310-825-1491 from cell phone) or contact the Ronald Reagan UCLA Medical Center (emergency room) directly at x52111 (located at 757 Westwood Plaza, enter from Gayley Avenue). Note: All serious injuries must be reported to EH&S at x59797 within 8 hours.

Non-Life Threatening Emergency – Go to the Occupational Health Facility (OHF), x56771, CHS room 67-120 (This is on the 6^th floor, 7^th corridor, room 120. Enter through the School of Dentistry on Tiverton Drive and proceed to the “O” elevator to the 6th floor.)Hours: M - F, 7:30 a.m. to 4:30 p.m. At all other times report to Ronald Reagan UCLA Medical Center (emergency room) at x52111. Note: All serious injuries must be reported to EH&S at x59797 within 8 hours.

Needle stick/puncture exposure (as applicable to chemical handling procedure) – Wash the affected area with antiseptic soap and warm water for 15 minutes. For mucous membrane exposure, flush the affected area for 15 minutes using an eyewash station. Page the needle stick nurse by dialing 231 from a campus phone, enter 93333 when prompted and then enter your extension. Hours: M – F, 8:00 a.m. to 4:00 p.m. At all other times report to Ronald Reagan UCLA Medical Center (emergency room) at x52111. Note: All needle stick/puncture exposures must be reported to EH&S at x59797 within 8 hours.

Decontamination/Waste Disposal Procedure

To reduce hazards involved in discarding osmium tetroxide, the following neutralization procedure should be employed:

1. 
   Perform neutralization in a chemical hood.
   
2. 
   A 2% solution of osmium tetroxide can be fully neutralized by twice its volume of vegetable oil (corn oil is preferred because of its high percentage of unsaturated bonds). For every 10 mL of 2% osmium textroxide solution, 20 mL of corn oil is required. Pour the corn oil into the osmium tetroxide solution.
   
3. 
   Wait for the oil to completely turn black.
   
4. 
   To test if osmium tetroxide is fully neutralized, hold a piece of filter paper soaked in corn oil over the solution. Blackening indicates that osmium tetroxide is still present and more corn oil should be added.
   
5. 
   Aqueous solutions contaminated with osmium tetroxide can be fully neutralized by adding sodium sulfide or sodium sulfite to reduce osmium tetroxide to less hazardous forms.
   
6. 
   Dispose of neutralized solutions as hazardous waste.
   

To dispose of waste:

SOLUTIONS: DO NOT dispose of leftover or spent solutions of osmium tetroxide by emptying into the laboratory sink. Collect solutions and solid osmium tetroxide in a labeled leak proof waste container for pickup and proper disposal by EH&S Hazardous Waste Pickup service. A completed waste label must be attached to the container.

SOLIDS: Collect pipette tips, gloves, ampoules, etc in a rigid leak proof container. Affix a completed waste label to the container. Due to the toxicity of osmium tetroxide, empty containers must also be collected and disposed as hazardous waste.

General hazardous waste disposal guidelines:

Label Waste

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  Affix an on-line hazardous waste tag on all waste containers using the WASTe Online Tag Program https://ehs.ucop.edu/waste as soon as the first drop of waste is added to the container
  

Store Waste

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  Store hazardous waste in closed containers, in secondary containment and in a designated location
  
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  Double-bag dry waste using transparent bags https://www.ehs.ucla.edu/hazwaste/management/containers
  
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  Waste must be under the control of the person generating & disposing of it
  

Dispose of Waste

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  Dispose of regularly generated chemical waste within 90 days
  
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  Call EH&S at x61887 for questions
  
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  Empty Containers
  

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  Dispose as hazardous waste if it once held extremely hazardous waste (irrespective of the container size) https://www.ehs.ucla.edu/hazwaste/types/extremely-hazardous
  
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  Consult waste pick-up schedule
  

https://www.ehs.ucla.edu/hazwaste/management/pick-ups

Prepare for transport to pick-up location

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  Check on-line waste tag
  
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  Write date of pick-up on the waste tag
  
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  Use secondary containment
  

Safety Data Sheet (SDS) Location

Online SDS can be accessed at http://msds.ehs.ucla.edu.

Protocol/Procedure

Amounts covered by this SOP:

1g from 10 g stock bottle to prepare 2% solution

Conditions covered by this SOP:

-20°C - 4°C

*Hazardous skin and eye irritant. Extremely hazardous in case of ingestion or inhalation, which can cause death following prolonged exposure. Wear standard PPE when handling. Advisable to wear dust respirator/mask or handle in the fume hood.

1. 
   Prepare the fixative solution (25mM phosphate buffer pH 7; 3% glutaraldehyde). Then place tissue in scintillation vial filled with fixative solution. Incubate overnight at 4oC to fix.
   
2. 
   Then prepare 2% osmium tetroxide solution by dissolving 1g in 50mL ddH2O. Once the osmium tetroxide tablet is fully dissolved, the solution can be stored at 4oC or -20oC for up to a month (should be yellowish if still good, purple if gone bad). Dilute the osmium tetroxide solution to 1% 25mM phosphate buffer pH 7. Following fixation step, pour out fix and incubate tissue in 1% osmium tetroxide solution overnight until the osmium color is black.
   
3. 
   Now rinse 3X with PBS solution making sure osmium solution and first wash are discarded in osmium waste.
   
4. 
   Now soak the tissue in series of alcohol washes for 15min each to dehydrate or store long-term in 100% alcohol.
   
   1. 
      50% ethanol 15min
      
   2. 
      70% ethanol 15min
      
   3. 
      95% ethanol 15min
      
   4. 
      100% ethanol 2X15min
      
5. 
   Dry & mount for SEM analysis
   

NOTE

Any deviation from this SOP requires approval from PI.

Documentation of Training (signature of all users is required)

• 
  Prior to conducting any work with osmium tetroxide, designated personnel must provide training to his/her laboratory personnel specific to the hazards involved in working with this substance, work area decontamination, and emergency procedures.
  

• 
  The Principal Investigator must provide his/her laboratory personnel with a copy of this SOP and a copy of the SDS provided by the manufacturer.
  

• 
  The Principal Investigator must ensure that his/her laboratory personnel have attended appropriate laboratory safety training or refresher training within the last one year.
  

Principal Investigator SOP Approval

Print name __________________________Signature___________________________   

Approval Date:

I have read and understand the content of this SOP:

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Osmium Tetroxide Date: 9/16/2015
UCLA- EH&S SA/JW