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Why does an autoclave need to pressurize steam?

Why does an autoclave need to pressurize steam?



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I've looked this up on other sites and they say that an autoclave needs high pressure in order to raise the boiling point of water so that you can achieve steam of a higher temperature.

If you look at this diagram though, it seems to me as if it would be much simpler to further heat the steam at the ambient pressure of the autoclave room. Does the pressure of the chamber play some role that I am unaware of? Thanks in advance for clearing this up!


An autoclave can sterilize both solids and liquids, whereas an oven with no pressure control is typically not suitable to sterilize liquids.

Not only do you need to heat the chamber to 121°C, but you also need to make sure that the things you are trying to sterilize are not degraded by the treatment.

For dry objects (e.g. glassware) you could heat the chamber up to 121°C at ambient pressure. In that case, the instrument you're using is called a "dry heat sterilizer" rather than an "autoclave" (REF)

So why use an autoclave at all, and not just a dry heat sterilizer? Quite often the things you would put in an autoclave are water-based solutions (for example, LB broth or buffers). If you do not increase the pressure and try to heat to 121°C, the liquid in your bottles will first boil around 100°C until all the aqueous solvent is in the gas state (i.e. your solutions are going to evaporate) and then the temperature will increase to 121°C. However if the pressure is raised to stay at the liquid-gas phase transition, then the gas and the liquid are in equilibrium, therefore you neither lose or gain any volume in your solution.

So in theory, if you autoclave 1L of medium, then you get 1L back at the end. Note that in real life, the temperature and pressure of the autoclave are set based on the diagram for water, but for concentrated solutions these settings may be slightly off, meaning that the volume in the bottle at the end of the cycle is sometimes a little different from what you put in there at the beginning. See this illustrative diagram of how a solution behaves differently from the pure solvent:

(Copyright: Lumen Learning)


it would be much simpler to further heat the steam at the ambient pressure of the autoclave room

You cannot do that, physically. If you put water in a closed (water-vapor-tight) container, then heat it, the vapor "wants" to expand with growing temperature. But as it cannot expand your metal container (autoclave), the pressure goes up instead. It happens automatically, that's how molecules function in a gas (don't ask me to remember the name of that law please).

You could, I guess, try constructing an elastic autoclave where the pressure stays the same and the volume changes. But that would be mechanically challenging - what material are you going to use that is elastic, won't tire with time, won't deform on you during use, won't burn at the heat source, will conduct warmth well, etc.? Also, you will lose your convenient way of ensuring that you are at a target temperature. Since a certain pressure always equals a certain temperature (at constant volume), you can construct a weighted valve to keep a constant pressure in your autoclave - this is cheap, easily doable with 19th century tech, and more accurate and failsafe than creating a cybernetic control loop with a measuring device such as a thermometer or a manometer.

I don't know if there are also biological reasons to prefer it with with a higher pressure - the number of bacterial species which can withstand high temperature is higher or equal to the number of species which can withstand high temperature combined with high pressure - but I doubt that this was a driving factor in the design and adoption of autoclaves.

The standard autoclave is easy to invent, does its job, does it well, and is cheap and unfussy (when compared to a hypothetical room-pressure-wet-autoclave). The pressure is more of a side effect nobody sees the need to remove - on this site, I'm tempted to call it a spandrel.


As you say it is possible to increase the temperature of the steam over the saturation pressure. And with overheated steam, the instrument would reach the desired temperature as is the case in an autoclave.

But the difference is how many time it would take to heat the sterilized instruments. And the difference is actually huge, we are talking of order of magnitude. The reason is that the heat transfer inside an autoclave is evaporation-condensation. But if the autoclave were not pressurized, there would be a first efficient phase of evaporation-condensation followed by an extremely slow natural convection phase.

Let's try to explain it in few lines.

On the difference between the heat transfer by evaporation-condensation and heat transfer by condenstation

Evaporation - condensation is the most effective way to transfer heat. Just consider the fact that uranium is used in nuclear power plant to boil water.

Transfer of heat by natural convection of gas is very slow.

The difference is many order of magnitude, heat transfer by evaporation is typically of the order of 1'000'000 W/m² (1MW/m²). Heat transfer by natural convection it is of the order of 100 W/m².

What is happening when the autocalve steam is saturated.

Liquid water inside the autoclave boils. Its temperature is the saturation temperature that is a function of the autoclave pressure. The vapor moves and condenses on the surface of the instruments that are being sterilized. The temperature of the liquid film that is formed by condensation on the instrument surface is the boiling temperature. The heat transferred by this evaporation-condensation is huge, in few seconds the surface of the sterilized instruments get close to the saturation temperature and in few minute instruments are homogeneously heated.

What would happen if the steam was overheated without pressurization.

Liquid water boils at 100°C and then a heater in the gaz phase overheat this vapor. The steam reach the instrument and condense. But the temperature of the liquid film that is formed on the instrument is still the saturation temperature: 100°C, not more. So in few seconds the instrument surface reaches 100°C. After a few minutes, the instrument reach an homogeneous temperature of 100°C. Then start a second and slow phase: the condensed liquid films dries, then the instrument get heated by the overheated steam through natural convection, and this is very slow. It would take hours for the instrument to reach the steam temperature.


What effect does ultraviolet light exposure have on bacterial growth?

Ionizing radiation (e.g. x-rays, gamma radiation) carries enough energy to remove electrons from molecules in a cell. When electrons are removed from molecules, free radicals are formed that damage the cell leading to DNA damage, mutations, and cell death. Non-ionizing radiation, such as ultraviolet (UV) light, excites electrons in molecules. The excitation of electrons in DNA molecules often results in the formation of extra bonds between adjacent pyrimidines (specifically thymine) in DNA. When two pyrimidines are bound together in this way, it is called a pyrimidine dimer. These dimers often change the shape of the DNA in the cell and cause problems during replication ultimately leading to cell death. Both ionizing and non-ionizing radiation are used to control the growth of microorganisms in clinical settings, the food industry, and in laboratories.

WHAT IS THE EFFECT OF UV LIGHT EXPOSURE ON 3 DIFFERENT SPECIES OF BACTERIA?

1. Each table will be given one of the following bacterial species/stages:

  • Staphylococcus aureus
  • Serratia marcescens
  • Bacillus cereus (vegetative)
  • Bacillus cereus (spores)

2. Working as a table, use a cotton swab to make a lawn on the following 8 plates:

  • no UV exposure (control)
  • 15 seconds
  • 30 seconds
  • 1 min
  • 3 min
  • 5 min
  • 10 min
  • 10 min with the plate fully covered with a lid

3. Put your plates (with the half lids) under UV light for the appropriate exposure time. Make sure to mark on the bottom of your plate which half of the plate was exposed to UV light and incubate them at 37°C.

UV light can burn your skin and eyes. Take proper precautions to protect yourself.


Sterilization: Monitoring

Sterilization procedures should be monitored using biological, mechanical, and chemical indicators. Biological indicators, or spore tests, are the most accepted means of monitoring sterilization because they assess the sterilization process directly by killing known highly resistant microorganisms (e.g., Geobacillus or Bacillus species). However, because spore tests are only done weekly and the results are usually not obtained immediately, mechanical and chemical monitoring should also be done.

Mechanical and chemical indicators do not guarantee sterilization however, they help detect procedural errors (e.g., overloaded sterilizer, incorrect packaging) and equipment malfunctions. Mechanical and chemical monitoring should be done for every sterilizer load.

Mechanical monitoring involves checking the sterilizer gauges, computer displays, or printouts, and documenting in your sterilization records that pressure, temperature, and exposure time have reached the levels recommended by the sterilizer manufacturer. Since these parameters can be observed during the sterilization cycle, this might be the first indication of a problem.

Chemical monitoring uses sensitive chemicals that change color when exposed to high temperatures or combinations of time and temperature. Examples include chemical indicator tapes, strips, or tabs and special markings on packaging materials. Chemical indicator results are obtained immediately following the sterilization cycle and therefore can provide more timely information about the sterilization cycle than a spore test.

A chemical indicator should be used inside every package to verify that the sterilizing agent has penetrated the package and reached the instruments inside. If the internal chemical indicator is not visible from the outside of the package, an external indicator should also be used. Chemical indicators help to differentiate between processed and unprocessed items, eliminating the possibility of using instruments that have not been sterilized.

Do not use instrument packages if mechanical or chemical indicators indicate inadequate processing. Chemical indicators should be inspected immediately when removing packages from the sterilizer if the appropriate color change did not occur, do not use the instruments.

No. The two categories of chemical indicators are single-parameter and multiparameter. A single-parameter chemical indicator provides information about only one sterilization parameter (e.g., time or temperature). Multiparameter chemical indicators are designed to react to two or more parameters (e.g., time and temperature or time, temperature, and the presence of steam) and can provide a more reliable indication that sterilization conditions have been met. Chemical indicators (no matter what class or type) do not verify sterility and do not replace the need for weekly spore testing.

The Food and Drug Administration (FDA) has determined that a risk of infection exists with these devices because of their potential failure to sterilize dental instruments and has required their commercial distribution to cease unless the manufacturer files a premarket approval application. If a bead sterilizer is used, dental health care personnel assume the risk of using a dental device FDA has deemed neither safe nor effective.

An air removal test is designed to detect inadequate air removal in pre-vacuum sterilizers. Air not removed from the sterilizer chamber prevents steam from contacting the items in a load and therefore interferes with sterilization. Follow manufacturer instructions for how to perform the test and frequency of testing. If a sterilizer fails the air removal test, the sterilizer should not be used until it passes inspection by sterilizer repair personnel.

A spore test should be used on each sterilizer at least weekly. Users should follow the manufacturer&rsquos directions for how to place the biological indicator in the sterilizer. A spore test should also be used for every load with an implantable device. Ideally, implantable items should not be used until they test negative.

If the mechanical (e.g., time, temperature, pressure) and chemical (internal or external) indicators suggest that the sterilizer is functioning properly, a single positive spore test result probably does not indicate sterilizer malfunction. Items other than implantable items do not necessarily need to be recalled. However, the sterilizer should be removed from service and sterilization operating procedures reviewed to determine whether operator error could be responsible. Sterilizer operators should repeat the spore test immediately using the same cycle that produced the positive spore test.

If the result of the repeat spore test is negative and operating procedures were correct, then the sterilizer can be returned to service. If the repeat spore test result is positive, do not use the sterilizer until it has been inspected or repaired and rechallenged with spore tests in three consecutive fully loaded chamber sterilization cycles. When possible, items from suspect loads dating back to the last negative spore test should be recalled, rewrapped, and resterilized. Results of biological monitoring and sterilization monitoring reports should be documented.

See Table 12 of the Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008 for the suggested protocol to manage a positive biological indicator in a steam sterilizer.

Common Causes of Sterilization Failure

  • Improper cleaning of instruments
  • Protein and salt debris may insulate organisms from direct contact with the sterilizing agent and interfere with its efficacy.
  • Improper packaging
  • Wrong packaging material for the method of sterilization
  • Excessive packaging material
  • Prevents penetration of the sterilizing agent packaging material may melt.
  • Retards penetration of the sterilizing agent.
  • Improper loading of the sterilizer
  • Overloading
  • No separation between packages or cassettes, even without overloading
  • Increases heat-up time and will retard penetration of the sterilizing agent to the center of the sterilizer load.
  • May prevent or retard thorough contact of the sterilizing agent with all items in the chamber.
  • Improper timing and temperature
  • Incorrect operation of the sterilizer
  • Insufficient time at proper temperature to kill organisms.

Modified from Miller CH and Palenik CJ (2010).

For each sterilization cycle, record the type of sterilizer and cycle used the load identification number the load contents the exposure parameters (e.g., time and temperature) the operator&rsquos name or initials and the results of mechanical, chemical, and biological monitoring.

Records of sterilization monitoring (mechanical, chemical, and biological) should be maintained long enough to comply with state and local regulations. The Centers for Disease Control and Prevention (CDC) does not maintain information on time limits for every state but provides an example of 3 years in its sterilization guidelines, which is the time frame used by the Joint Commission inspection agency.

References

Association for the Advancement of Medical Instrumentation, American National Standards Institute. Comprehensive guide to steam sterilization and sterility assurance in health care facilities. ANSI/AAMI ST79-2010 A1:2010 A2:2011 A3:2012 and A4: 2013. Arlington, VA: Association for the Advancement of Medical Instrumentation, 2010.

Harte JA, Molinari JA. Sterilization Procedures and Monitoring. In: Molinari JA, Harte JA eds. Cottone&rsquos Practical Infection Control in Dentistry, 3rd ed. Baltimore: Lippincott Williams & Wilkins, 2010148&ndash170.

Miller CH, Palenik CJ. Instrument Processing. In: Miller CH, Palenik DJ, eds. Infection Control and Management of Hazardous Materials for the Dental Team, 4th ed. St. Louis: Mosby, 2010135&ndash169.


Conclusion

Sterilizing liquid loads can seem intimidating, but Consolidated Sterilizer Systems controllers come preprogrammed with cycles designed for any application. If you are sterilizing small or precise amounts of liquid, the Air-Over-Pressure cycle will prevent evaporation by cooling the load under pressure. To sterilize other types of liquid loads, check out our previous posts on the Liquids Cycle and Steam-Air-Mix cycles.

For more information please contact us today.


Frequently Asked Questions

Recommended sterilization times by volume: (from AC manual)

Volume Time
<500ml 30 min
500ml-1L 40 min
2-4L 55 min
>4L 60 min

Sterilization time depends on following key variables:

  1. Volume - The greater the volume of liquid, the longer it takes for the product to reach temperature. 10x 200ml heats faster than 1x 2L.
  2. Viscosity - Thicker, more viscous solutions absorb heat more slowly than products such as water.
  3. Material of the container - Different containers with the same volume of liquid will reach temperature at different rates. Metal containers conduct heat more rapidly than glass or PP.
  4. Load volume - The greater the physical size of the load, the longer it will take to reach exposure temperature. 1x 1L heats faster than 1x 10L.
  5. Load density - If the bottles are jammed together, it essentially becomes one large mass. So 10x 200ml, if packed tightly together, will become 1x 2L and you will need to select a 55min liquid cycle. If the bottles are separated enough to allow steam to envelope each bottle in the load, each 200ml bottle will come up at approximately the same time as long as the variables mentioned in 1-3 are consistent and a 30min liquid cycle will suffice.
  6. Location in the autoclave - Bottles that are positioned nearest the heated jacket of the autoclave chamber will tend to reach temperature faster than those in the center of the load.

Since all of the variables mentioned above can affect the come-up time of the product, when processing mixed liquid loads, the total exposure time selected for the load should be based on the largest volume of liquid in a bottle, placed in the most-difficult-to-sterilize location in the chamber.


What is an autoclave?

An autoclave is a machine that is used to eradicate biohazardous waste from the surface of tools or instruments. It was invented by Charles Chamberland in 1884. Autoclaves sterilize or disinfect through physical means by using pressure, temperature and steam. They are often referred to as steam sterilization machines.

(Photo Credit: Labtronixed / Wikimedia Commons)

Microbes on medical or scientific instruments cannot simply be cleaned with water or swept away with a brush or cloth. They require extra care because microorganisms and germs are highly resilient and impervious to these frivolous attempts of cleansing. They can only be eliminated by killing them, which is done by subjecting them to harsh environments.

An autoclave, with the help of a cloud of steam and elevated pressure, maintains a temperature that is too high for any bacteria, virus, fungi or parasite to survive in. However, other than these germs, spores are microscopic varmints that are indifferent to high temperatures. Fortunately, they can be eliminated if these extreme conditions are maintained for a prolonged period of time.

Autoclaves are basically used in any field where tools come in contact with biological matter. This would include tattooing, podiatry, funeral homes and prosthetics.


Who invented autoclaves?

Photo: Scientific autoclave: Inspecting a crystal grown in microgravity inside a cylindrical autoclave. This scientific experiment was carried out onboard the Space Shuttle in October 1995. Photo by courtesy of NASA Marshall Space Flight Center (NASA-MSFC).

  • Ancient Greeks use boiling water to sterilize medical tools.
  • 1679: French engineer Denis Papin (1647&ndash1712) invents the steam pressure cooker&mdashan important step in the development of steam engines.
  • 1860s: French biologist Louis Pasteur (1822&ndash1895) helps to confirm the germ theory of disease. He realizes that heating things to kill germs can prevent diseases and extend the life of foodstuffs (which leads him to the invention of pasteurization).
  • 1879: Pasteur's collaborator Charles Chamberland (1851&ndash1908) invents the modern autoclave. It looks like much like a pressure cooker, with a lid on top sealed tight with clips.
  • 1881: Microbiologist Robert Koch and others criticize Chamberland's high-pressure steam method, which they believe may damage laboratory equipment, and develop an alternative, unpressurized sterilizer instead. This eventually evolves into a machine called the Koch autoclave.
  • 1889: German physician Curt Schimmelbusch builds on the work of Chamberland and Koch to produce a drum-type sterilizer known as the Schimmelbusch autoclave (sterilization drum).

What's the difference between an autoclave and a pressure cooker?

Want to cook your dinner in a fraction of the time? You could use a microwave to zap it with energetic waves. But another popular solution is to seal it in a pressure cooker : a kind of saucepan that cooks things quicker by boiling them at a higher temperature than usual. Although considered old-fashioned by some, pressure cookers are still a convenient and economical way to prepare food. The basic concept&mdashusing pressure to achieve a higher temperature&mdashis the same as what happens in an autoclave.

Photo: A pressure cooker in action. Notice the valve on the top through which steam escapes and the double handle arrangement used to lock the lid. Photo by George Danor, US Office of War Administration, courtesy of US Library of Congress.

We've already seen that high pressure raises the boiling point of water. Suppose we could somehow arrange things so that the air above our saucepan was actually at a much higher pressure than usual. That would make the water boil at a significantly hotter temperature, which would make the potatoes cook more quickly.

This is the basic idea behind pressure cookers. A pressure cooker is a big steel saucepan with a tight-fitting lid. The outer edge of the lid has a thick circle of rubber called a gasket that fits between the bottom of the lid and the top of the pan to make a really tight seal.

When you fill the pan with water and place it on the stove, the water heats up and some of its molecules escape to form steam up above it. With a normal pan, the steam would just drift off into your kitchen and disappear. But with a pressure cooker, the gasket and lid stop the steam escaping so the pressure soon builds up. Although the water inside the pan boils, the higher pressure means it boils at a higher temperature than normal that cooks your food more quickly. A special valve on the top of the lid allows a small amount of steam to escape, keeping the pressure higher than normal but not so high that the cooker explodes. If the pressure inside the pan builds up too much, the valve pops right out, rapidly lowering the pressure to a safe level again.


Where to Find Steam Sterilization Equipment

Hospitals and other facilities looking to acquire an autoclave for medical use are advised to contact Consolidated Sterilizer Systems. We offer medical series steam sterilizers designed to sterilize at temperatures between 250°F and 275°F, as well a stainless steel vessel construction in a variety of sizes and program control options.

To learn more about our medical series steam sterilizers or what Consolidated can do for you, contact us today.


Biological Waste Guide

Biological and regulated medical solid waste shall be disposed of through the Biological Solid Waste Stream established by the Department of Environmental Health and Safety. You play an important role in UConn’s biological waste program if you generate biological waste in a research, teaching, clinical laboratory or clinical area. This guide will help you dispose of your biological waste in an easy and legal manner. Our program is designed to protect the people who handle, transport and dispose of your waste, to protect the environment and minimize UConn's regulatory liability. Some waste generators may attempt to work around this program. These attempts are counter productive because they place other people and the University at risk. The cost associated with one injury or violation can easily exceed annual operational costs. If you have complaints, concerns or suggestions for program improvement, we would rather have you tell us than have you implement unauthorized procedures. Environmental Health and Safety will continually work to improve this program and to control its costs.

Definitions

At The University of Connecticut, biological waste is defined as infectious waste, pathological waste, chemotherapy waste and the receptacles and supplies generated during its handling and/or storage. This definition is in accordance with the definition of biological waste as defined by the Connecticut Department of Energy and Environmental Protection (DEEP). It is further defined as waste that, because of its quantity, character or composition, has been determined to require special handling.

Infectious waste is defined by seven categories of waste:

  1. Cultures and stocks: Agents infectious to humans and associated biologicals, waste from biological production, live and attenuated vaccines and anything used to contain, mix or transfer agents. This includes but is not limited to petri dishes, pipettes, pipette tips, microtiter plates, disposable loops, eppendorfs and toothpicks.
  2. Human blood, blood products and infectious body fluids: This category includes blood that is not contained by a disposable item or is visibly dripping, serum, plasma, and other blood products or non-glass containers filled with such discarded fluids. It further includes any substance which contains visible blood, semen, vaginal secretions, cerebrospinal fluid, synovial fluid, peritoneal fluid and pericardial fluid. Glass containers filled with such discarded fluids shall be considered sharps. Intravenous bags which did not contain blood or blood products shall not be considered a blood product. Dialysates are not considered blood or body fluids.
  3. Sharps: needles, scalpel blades, hypodermic needles, syringes (with or without attached needles) and needles with attached tubing regardless of contact with infectious agents are considered by EPA and DEP to be regulated medical waste. Other sharps: pasteur pipettes, disposable pipettes, razor blades, blood vials, test tubes, pipette tips, broken plastic culture dishes, glass culture dishes and other types of broken and unbroken glass waste (including microscope slides and cover slips) that may have been in contact with infectious material. Items that can puncture or tear autoclave bags.
  4. Research animal waste: contaminated carcasses, body parts and bedding of animals that were intentionally exposed to infectious agents during research or testing. Animal carcasses and body parts not intentionally exposed to infectious agents during research or testing are disposed of by Inserve and are not picked up by the Biosafety section.
  5. Isolation waste: biological waste and discarded material contaminated with body fluids from humans or animals which are isolated because they are known to be infected with a highly communicable disease (biosafety level 4 agent).
  6. Any material collected during or resulting from the cleanup of a spill of infectious or chemotherapy waste.
  7. Any waste mixed with infectious waste that cannot be considered as chemical hazardous waste or radioactive waste.

Potentially Infectious Material is defined by the OHSA Bloodborne Pathogens Standard as:

  1. Human body fluids: semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericadial fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, any body fluid that is visibly contaminated with blood, and all body fluids in situations where it is difficult or impossible to differentiate between body fluids,
  2. Any unfixed tissue or organ (other than intact skin) from a human (living or dead) including cell or tissue cultures and
  3. HIV-containing cell or tissue cultures, organ cultures and HIV- or HBV-containing culture medium or other solutions and blood, organs, or other tissues from experimental animals infected with HIV or HBV.

"Look - a - Like" infectious waste is defined as: laboratory materials that can be used to contain, transfer or mix infectious agents but has been used with non-infectious agents. For example: disposable micropipette tips may have transferred sterile water or broth, but an identical tip in the same laboratory may have transferred an infectious agent. In the trash you could not distinguish between them. These "look- a -like" materials will be handled as infectious waste if the facility routinely generates infectious or potentially infectious biological waste or is engaged in a temporary project that generates infectious or potentially infectious biological waste.

Disposal Procedures

RADIOACTIVE WASTE IS DISPOSED OF THROUGH THE RADIATION SAFETY SECTION (860) 486-3613. HAZARDOUS CHEMICAL WASTE IS DISPOSED OF THROUGH THE CHEMICAL SAFETY SECTION (860) 486-3613.

  1. Sharps waste: All sharps as described by category 3 must be discarded in an approved sharps container. These containers are provided by Environmental Health & Safety. Some sharps containers may melt if autoclaved in which case decontamination of the contents may be accomplished by chemical means. If chemical means are used, the liquid must be drained from the containers before they are sealed and placed in the box-bag units. Alternately, untreated sealed sharps containers may be placed in the box-bag units with other untreated biological waste. A University address label provided by the Biological Safety section must be affixed to each sharps container, treated or untreated, that is placed in the box-bag unit. For chemical decontamination, the disinfectant shall be an EPA registered tuberculocidal agent. An example is standard household bleach diluted to the final concentration of 5250ppm (10%). Fill leak-proof receptacle with the appropriate dilution of disinfectant and let stand over-night. Empty liquid, seal and label receptacle and put in box-bag unit.
  2. Non-sharps: There are three acceptable methods for disposal:
  1. Certain biological waste can be disposed of as non-biohazardous/ non-infectious waste, if approved in writing by Biological Safety. The waste must have been decontaminated by autoclave, chemical disinfection or other appropriate decontamination method. If the treatment of choice is a validated decontamination procedure, the waste will be labeled as "non-biohazardous/non-infectious" and can go as regular trash. See below for validation procedures.
  2. If a non-validated decontamination autoclave is available, autoclave the waste in an autoclave bag, affix autoclave indicator tape and place in an autoclave safe tray. CT DEEP regulation requires that autoclaves be monitored for effective kill. See paragraphs d, e and f (validation procedure). After autoclaving and the bag has cooled, drain off any remaining liquid and place the sealed waste in the box-bag unit for pickup. Do not pour liquefied agar media down the drain. See below for box-bag unit instructions.
  3. If an autoclave is not available the waste may be collected in orange/red autoclave bags, closed with tape and placed in the box-bag unit as untreated biological waste. Environmental Health & Safety will pick up all box-bag units on, at least, a weekly basis.

Do not autoclave containers or other receptacles containing bleach. The combination of bleach and residual cotton and oil (improperly cleaned autoclaves) may result in an explosive combustion within the autoclave.

  1. Liquid waste: The sanitary sewer was designed for the disposal of certain liquid wastes. Use of the sanitary sewer reduces the chance for leaks or spills during transport and reduces disposal costs. Biological liquid waste can be poured down the drain (sanitary sewer), under running water after it has been decontaminated by autoclave or chemical means. Human or animal blood and body fluids do not need to be disinfected before being poured down the drain. The sink should be rinsed well and disinfected if necessary, after the disposal procedure.
  2. Mixed waste: Follow the formula below to determine which waste stream.

Biological + Radiation = Radiation Waste

Biological + Hazardous Chemical = Chemical Waste

Transport and Storage of Biological WasteThe transport of biological waste outside of the laboratory, for decontamination purposes or storage until pick-up, must be in a closed leakproof container that is labeled "biohazard". Labeling may be accomplished by the use of red or orange autoclave bags or biohazard box-bag units. Biological Safety must authorize the transport or transfer of regulated medical waste or biohazardous biological waste through public streets or roadways in order to comply with DOT regulations. Biological waste must not be allowed to accumulate. Material should be decontaminated and disposed of daily or on a regular basis, as needed. If the storage of contaminated material is necessary, it must be done in a rigid container away from general traffic and preferably in a secured area. Treated biological waste, excluding used sharps, may be stored at room temperature until the storage container or box-bag unit is full, but no longer than 48 hours from the date the storage container is first put into service. It may be refrigerated for up to 1 week from the date of generation. Biological waste must be dated when refrigerated for storage. If biological waste becomes putrescent during storage it must be moved offsite within 24 hours for processing and disposal. Sharps containers may be used until 2/3-3/4 full at which time they should be decontaminated, preferably by autoclaving, and disposed of as regulated medical waste. Biological waste generated at regional campuses is picked up directly by University contracted biological waste vendors. Coordinate all biological waste pick-ups at regional campuses by calling Environmental Health and Safety at 860-486-3613.

Labeling of Biomedical Waste

Materials that are put into the supplied box-bag units must be labeled with a University of Connecticut address label. Each individual bag or sharps container must have a separate label. The box-bag unit must be labeled with the generator’s building and room number. It must indicate whether or not the waste in the box is treated or untreated.

When a biological waste pick-up is desired, submit a biowaste pickup/supply delivery form on our web site. A waste inspection and removal approval may be required for some waste. The inspector will seal approved waste and affix an approval label. Normally, the waste will be picked up within 48 hours after the request has been submitted. Non-biohazardous/non-infectious waste (validated decontamination method) will be tagged with labels provided by Biological Safety. Autoclave indicator tape should be used as evidence of decontamination.

Box-Bag Unit Assembly Instructions

Instructions can be found in the presentation Biowaste Management

Validation Procedures for Steam Sterilization Units

Steam treatment units shall subject loads of biological waste to sufficient temperature, pressure, and time to demonstrate a minimum Log 4 kill of Bacillus stearothermophilus spores placed at the center of the waste load, and shall be operated in accordance with the following:

  1. Before placing a steam treatment unit into service, operating parameters such as temperature, pressure, and treatment time shall be determined according to the following:
    • Test loads of biological waste, which consist of the maximum weight and density of biological waste to be treated, shall be prepared. Separate loads of autoclave bags, sharps containers, boxes, and compacted waste shall be prepared if they are to be treated separately.
    • Prior to treatment, Bacillusstearothermophilus spores are placed at the bottom and top of each treatment container, at the front of each treatment container at a depth of approximately one-half of the distance between the top and bottom of the load, in the approximate center of each treatment container, and in the rear of each treatment container at a depth of approximately one-half of the distance between the top and bottom of the load.
    • If the operating parameters used during the treatment of the test loads demonstrate a minimum Log 4 kill of Bacillus stearothermophilus spores at all locations, the steam treatment unit shall operate under those parameters when placed into service. If the operating parameters fail to provide a minimum Log 4 kill of Bacillusstearothermophilus spores at all locations, treatment time, temperature, or pressure shall be increased and the tests must be repeated until a minimum Log 4 kill of Bacillus stearothermophilus spores is demonstrated at all locations. The steam treatment unit shall be operated under those parameters when placed into service. Tests shall be repeated and new parameters established if the type of biological waste to be treated is changed.
  2. When operating parameters have been established and documented using the criteria outlined above, the steam treatment unit may be placed into service.
  3. The steam treatment unit shall be serviced for preventive maintenance in accordance with the manufacturer's specifications. Records of maintenance shall be onsite and available for review.
  4. Unless a steam treatment unit is equipped to continuously monitor and record temperature and pressure during the entire length of each treatment cycle, each package of biological waste to be treated will have a temperature tape or equivalent test material such as a chemical indicator placed on a non-heat conducting probe at the center of each treatment container in the load that will indicate if the treatment temperature and pressure have been reached. Waste shall not be considered treated if the tape or equivalent indicator fails to show that a temperature of at least 250 degrees F (121 degrees C) was reached during the process.
  5. Each steam treatment unit shall be evaluated for effectiveness with spores of Bacillusstearothermophilus at least once each 40 hours of operation for generators who treat their own biological waste. The spores shall be placed at the center of the waste load. Evaluation results shall be maintained onsite and available for review.
  6. A written log shall be maintained for each steam treatment unit. The following shall be recorded for each usage:
    1. The date, time, and operator name
    2. The type and approximate amount of waste treated
    3. The post-treatment confirmation results by either
    4. recording the temperature, pressure, and length of time the waste was treated, or b. the temperature and pressure monitoring indicator
    5. Dates and results of calibration and maintenance and
    6. The results of sterilization effectiveness testing with B. stearothermophilus or equivalent.
    1. Set the parameters, determined from testing, that provide consistent treatment such as exposure time, temperature, and pressure.
    2. Identify the standard treatment containers and placement of the load in the steam treatment unit.
    3. Provide for and conduct an ongoing program of training for all users.
    4. Provide for a quality assurance program to assure compliance with the biological waste management plan.

    Authorization

    Only individuals trained and authorized by Environmental Health and Safety may sign waste transport manifests.


    How to sterilize agar broth without autoclave or pressure cooker

    Hey! I am a high school student doing a biology experiment at home. I can't seem to find any practical methods on google that allow me to sterilize agar broth before pouring it into sterile agar plates :(( Are there any methods that don't require an autoclave or pressure cooker? If not then what would be the consequences for skipping this step?

    FYI I'm using MRS agar and growing lactic acid bacteria

    A home pressure cooker works perfectly for small quantities and they’re not very expensive, but you really do need one, can you borrow? You need the 15lb weight, (so called because it raises the internal steam pressure to 2x atmospheric pressure, ie about 30psi above zero), thus the water boils at 120C (sorry for mixed units). Make sure you wait to add the pressure weight until steam issues freely from the valve. Follow safety instructions especially after the pressurized heating period, about 15-20 minutes after adding the weight. Cool slowly or you’ll lose all your agar from the loosely capped tubes. Think about pressure/volume relationships or risk bursting bottles if glass, keep screw caps loose until the pressure cooker has cooled. I assume you’ve read up on aseptic inoculation techniques etc or consulted a knowledgeable teacher. Microbiology projects are really not for the totally untrained. They can be fraught with hidden dangers. The problem is any microwave or home oven will boil the agar at only a little over 100C, no matter what temperature you set it at, which won’t kill resistant spores of bacteria or fungi. The pressure cooker stops the boiling even when the higher temperature is reached. The Tyndallisation technique suggested by someone else can work, but it’s fraught with potential problems of contamination every time you uncap and recap the bottles/tubes. (PhD microbiologist (retired) here, AMA if uncertain about anything.)

    alright thanks a lot! ill see if I could get one somewhere

    Microwave is not a fool proof method as far as I'm aware. UV lights strong enough to kill the agar are just plain impractical at home because they're expensive and you need to light them for a while and not be able to go into the room because of the intensity. I would buy a pressure cooker, they're really not that expensive and by far the easiest method to sterilise.

    you could buy pre-poured plates

    Do you have a UV light? Also if you have a sterile work station and plenty of time you can make a plate and see if contamination occurs

    Google gave me this article I personally would never use a microwave but apparently it can be done. and logically it makes sense, but I don't know how it will affect the quality of the broth

    As a microbiologist, I really can't condone this microwave business. You can't heat the media above 100°C in there and, While that is definitely hot enough to kill most bacteria, it's not enough to kill spores no matter how long you microwave it. I've used the microwave to dissolve liquids and to melt previously sterilized (in the autoclave) and solidified media. But I would never use it to do the actual sterilization unless I was trying to grow halophiles. High salt concentrations (above 10%) can inhibit the germination of spore forming bacteria like Bacillus species, which, while it isn't the same as killing the spores, is nearly as good. But, microwaving alone is very unlikely to kill the spores and salt also inhibits the growth of most organisms. The thing is that since she's using a complex, as opposed to defined media where you have to weigh out all of the amino acids and trace minerals individually, and doing it at home, I feel personally like her chances of getting spore contamination is high and her media will likely not turn out well if she uses the microwave to sterilize her media. MRS media has both yeast and beef extract in it, which makes it complex meaning that we don't know exactly what's in the media because it's made from whole cell extracts.

    The only ways to kill spores is to either use a special disinfectant (which would definitely kill other bacteria she's interested in) or to autoclave/use the pressure cooker. She needs to raise the temperature to about 120°C and keep it there for at least 15-20 minutes. She can do this with an at home pressure cooker.

    One thing to think about is home canning. Canning was invented to preserve food and the canning process is specifically designed to kill bacterial spores on food to keep it from spoiling. Pressure cookers are also useful for making rice/super fast curry or whatever. But, it was originally for decontamination of canned food so you could eat vegetables in January before reliable refrigeration or freezers existed.


    Watch the video: Sada Medical High Pressure Steam Vertical Autoclave Operation - (August 2022).