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Cleaning out Agar from Autoclave Reservoir

Cleaning out Agar from Autoclave Reservoir


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I have a Tuttnauer EZ10 autoclave where the previous user poured m-Endo Agar into the reservoir. Using vinegar, we've been able to remove most of it, but there is a thin film of purple residue coating the interior walls. To get the autoclave safely working again, would you recommend I 1) pour water in and do a steam cycle, or 2) buy the Tuttnauer Chamber Brite and use it?

P.S. I can't make an agar, autoclave, reservoir, maintenance, or cleaning tag, so would someone please make those too?

Thanks in Advance!


Autoclaving Guidelines

Autoclaving is the most effective and reliable means of sterilizing laboratory materials. Autoclaving sterilizes material using saturated steam under pressure (“moist heat”). Due to the use of pressure, steam and high temperatures, there is significant risk for injury, so it's important for individuals to be properly trained on operational procedures.

Autoclaves may be used to sterilize equipment/products prior to use in an experiment or to render items non-infectious prior to disposal. The University of Iowa’s Biohazard Waste Guidelines states that cultures, plates, and vials containing pathogenic organisms must be autoclaved prior to disposal. The reason for autoclaving infectious waste is that it must be handled several times during transport proper containment and treatment at the source reduces the potential for an accidental exposure. The necessary treatment to achieve sterility will vary in relation to the volume of material treated, its contamination level, moisture content and other factors.

Contact information and areas of expertise can be found on the Contact Us page.


Completely Sterile Agar Plates

  1. Measure out your agar and broth and place in a clean Erlenmeyer flask that measures twice the volume of the agar you are making. So, if you are making 200 mL, use a 400 mL flask.
  2. Add your desired volume of water to the flask using a graduate cylinder.
  3. If you have it, place a magnetic stir bar in the flask.
  4. Place foil over the top of the flask.
  5. Place autoclave tape on the foil.
  6. Autoclave for 25 minutes.
  7. Remove the flask using autoclave gloves. If you are using a stir bar, place the flask on a stir plate and stir gently.
  8. Prepare flow hood and plates using sterile technique.
  9. Once the flask is at room temperature, or close to it, add any desired antibiotics and move to the flow hood.
  10. Using sterile technique, lift the lid of the top plate at an angle and gently pour your desired volume. Cap the plate, lift it, and gently place in the back left of the flow hood, creating rows of capped plates as you go.
  11. Let the plates dry in the laminar flow hood, then either individually close each one with parafilm or place in sterile bags. Store at 4°C.

8 Tips for Pouring Perfect Agar Plates Every Time

1. Use a Recipe

Make up the medium according to the recipe, then add the desired amount of agar (normally about 1% w/v) and stir. If you autoclave without stirring, with the agarose still floating on top of the liquid, you get an agarose cake in the medium. Interesting, but useless.

When making up the agar, only use 3/4 of the volume of the bottle. This allows space for bubbles to rise while the agar is melting in the microwave (and saves you cleaning up overflowing agar from the microwave!).

2. Autoclave

Autoclave your medium for 25 minutes. After autoclaving, you can of course store the medium-agar mix in a toughened glass bottle then melt it in a microwave or water bath when needed. Make sure you use toughened glass bottles, or disaster (see #2) can strike.

3. Cool It!

Cool the medium-agar mix to 55°C. For routinely consistent results, do the cooling for a couple of hours in a 55°C water bath. Agar starts to solidify at about 50°C. Using the water bath means you can consistently cool the mixture to just above the solidification temperature.

Before I used a water bath, I used to just cool it in the air, but would inevitably forget about it and come back to find solidification had already started – lumpy plates are no good for spreading!

4. Supplement It

You can now add any antibiotics or supplements, and be confident that the agar is at a suitable temperature because you have cooled it in the water bath.

5. Pour the Plates

Use about 30 mL of the agar-medium mix for each plate when using a 100 mm diameter plate. The less agar-medium mix in each plate, the more easily they will dry out. 30 mL is a good amount for long-term storage, 10–20 mL is fine if you are going to use the plates relatively soon.

For consistency, I’d recommend using a serological pipette. Suck up 2–3 mL more than you need to minimize blowing bubbles into the plate.

6. Let It Set

If there are any bubbles in the plates, briefly pass the flame over to pop them. Classic error: trying to move the plates before they’ve set is just asking for trouble. Just leave them alone (and maybe admire your perfect agar plates while you wait)!

7. Get Dry

Dry the plates in the laminar flow hood with the lid slightly off for 30 minutes (or in a 37°C incubator for 2–3 hours, or room temperature for 2–3 days). Drying the plate is very important for storing the plates and growing colonies on them.

If you don’t dry the plates, the moisture will evaporate and condense on the lid during storage or incubation and give you horrible wet plates. At worst the moisture can affect the plating of your cells. Use a timer to remind you when the 30 minutes are up as – in my experience – it is very easy to forget about your plates and come back to find your plates have turned into agar crisps/chips. Tasty.

8. Use It or Store It

Once you’ve poured your perfect agar plates, you can use them immediately or seal them for later use. You can use Parafilm, or pop them in the bag that the plates came in for easy storage. Store the plates at 4°C. Guidelines suggest using agar plates within approximately 2 to 4 weeks.

Depending on the additives you have included, the shelf life of the prepared plates might be shorter – make sure you check this before you start so you don’t end up wasting your time (and resources) making too many plates.

A quick way to label your plates is to have a color code for each antibiotic and medium type you tend to use (e.g. red for ampicillin, black for kanamycin, green for LB, blue for M9). Stack the plates and use the appropriately colored lab marker to draw a line down the whole stack. Make sure you keep the color code to hand though.

Now you should have perfect agar plates every time. If you’ve got any further ideas or additions to this protocol, please leave a comment.

Originally published July 5, 2011. Reviewed and updated February 2021.


Working Principle

Essentially, sterilization refers to the process through which microorganisms (bacteria, viruses, etc) on the surface of a given substance or material etc are destroyed thus making the substance sterile. Here, the agent used to sterilize is known as the sterilant.

Generally, in many homes and even in some laboratories, given material can be placed in boiling water for about 10 minutes as a means of sterilizing them. However, this method has several drawbacks.

Boiling at 100 degrees C is not enough to kill all microorganisms. While some microorganisms will be destroyed by boiling for several minutes (particularly the non-sporing organisms), it does not completely eliminate all the microbes.

One of the other issues with boiling is the fact that material/substances have to be immersed in boiling water for sterilization. For this reason, some of the material/substances (e.g. waste material) cannot be sterilized using this method. As compared to boiling water, steam as a sterilant has several advantages.

One of the biggest advantages of using steam is that unlike boiling water, steam creates high pressure with increasing temperature. As mentioned, water is added to a certain level so that the material in the basket sits just above the water level. Once the water is added, the lid is tightly closed. As the water is heated, it turns to steam (the gaseous form of water) which occupies more space as compared to water.

As the temperature increases, pressure builds up in the inner chamber. The increasing temperature causes the molecules to vibrate faster. This not only causes the gaseous molecules to take up more space but results in increased pressure.

* Compared to steam molecules, water molecules are tightly packed together which allows water to occupy less space compared to steam.

In the inner chamber, very high pressure is exerted on the microorganisms on the surface of material/substances being sterilized. This is why it's important to ensure that the material are not overcrowded in the inner chamber. Overcrowding prevents steam from penetrating certain areas. Apart from the high pressure created in the chamber, the heat within the chamber also kills the microorganisms present.

When the hot steam comes into contact with the surfaces of the material being sterilized, the steam condenses into a small volume of water. Given that these surfaces are cooler than the steam, latent heat from steam molecules is released to the cooler surface as they condense to water.

As a result, the condensed water molecules become cooler which in turn results in suction of more hot steam to the site. This continues until the site becomes as hot as the steam. In the process, microorganisms located on the surface of the materials/substances are destroyed.

* In order to ensure that all the microorganisms present on the material being sterilized are killed, it's important to sterilize them for a given period of time. generally, this takes about 15 minutes.


Agar plates, otherwise known as Petri dishes, serve as an integral and irreplaceable part of microbiological research, particularly in their functional and utilitarian role in culturing bacteria, fungi, and other microorganisms.

Though relatively easy to prepare and requiring a minimal amount of equipment to culture, clean-up and disposal can be a messy affair, requiring the user to undertake procedures to sterilize and minimize the risk of potentially harmful microorganisms contaminating both inside and outside the laboratory environment.

Standard protocol requires the use of autoclaves to sterilize agar plates, as only high heat and pressure can effectively kill the full range of microorganisms, which can persist even under unusually harsh conditions. Alternative agar plate sterilization methods not requiring the use of autoclave sterilizers, such as microwave ovens or caustic chemicals have been recommended in place of using autoclaves. However, these methods have been proven ineffective due to the resistance of certain microorganisms.

The method in which agar plates are placed in biowaste/biohazardous waste bags and sterilized in autoclaves has become the standard protocol at many laboratories. However, this method comes with downsides, as liquified agar mixed with biomaterial can easily leak and cause a large mess, within the laboratory or on its way to waste disposal areas and beyond.
When paired with the correct autoclaving accessories, the autoclave sterilization method for plastic agar plates/Petri dishes can be both effective in deactivating harmful microorganisms and preventing a large mess.

Please find below the following plastic petri-dish agar plate sterilization and disposal method using an autoclave sterilizer, broken down into a few easy steps.

Collect used petri dishes / culture plates with agar. Cultured agar plates / petri dishes should be left as is with agar and covered to prevent the spread of microorganisms and reduce the risk of contamination.

2. Load Agar Plates / Petri Dishes

Load used culture plates in the plastic petri dish / agar plate sterilization basket, removing covers and placing them aside before loading the agar-filled portion in the specialized perforated basket. Place dish / plate covers on top of the agar-filled portions at the end.

3. Set Up Bucket and Stand

Set up the stand inside the specialized solid bucket and fill with water to prevent melted media from solidifying inside the bucket. The stand will be used to place the specialized perforated basket on top of the water to allow the melted media to fall through. Fill water to about half of the height of the stand.

Place the water filled bucket inside of the autoclave. Next, place the perforated basket with disassembled plastic petri dishes / agar plates, resting the basket on the stand.

Close autoclave and run a normal sterilization cycle for at least 20 minutes. Please note that the sterilization time differs with the amount of plastic petri dishes / agar plates.

After sterilization cycle is complete and the autoclave temperature has cooled down to a safe level, remove perforated basket. Even though the temperature is registered at a safe level, please be mindful of hot steam, liquids, and media.

Note that the plastic petri dishes / culture plates and lids have melted and re-forged into a large chunk. Some of the agar material has been trapped / fused into the plastic, and though it should be sterile, avoid touching parts where agar is exposed to prevent a mess. Most of the agar has melted and mixed with the water inside the bucket.

Dispose the plastic chunk and pour water into a biohazardous liquid receptacle or a drain where it can be safely disposed and eventually processed. Safe, easy and no mess!

​As you can see, petri dish sterilization can be done safely and easily with little mess, drastically reducing the risk of contamination.

If you are interested in the aforementioned agar sterilization accessories or the compatible TOMY SX-Series autoclaves, please fill out a contact form and one of our sales representatives will get back to you shortly.


Need advice on cleaning charred agar in flask

I melted agar gel by heating it on a hot plate. The agar overboiled and now I have charred agar stuck at the bottom of the flask. Traditional soap + scrubbing is not an option because it is in a narrow neck flask. Soaking in 37% HCl for two days didn't help much. The color of the HCl solution darkened but the charred agar is still there. Any advice?

Step 1 - throw flask in lab broken glassware container.
Step 2 - buy/order new flask.

You've created what's generally referred to as "indelible black tar," a generic term for residue that can't easily be removed from glassware. Iɽ recommend filling it halfway with water or water + a little NaOH and then seeing if autoclaving it will loosen it. That, or maybe GooGone and a bottle brush. If you don't have a bottle brush, get one (or a dozen). They come in really handy for glassware cleaning, because you can scrub the inside of Erlenmeyer flasks and the like with ease.

If you are close to a chemistry lab ask them for some help using piranha solution (a mixture of H2SO4 and H2O2), it will oxidize C-C bonds into C02. So you will have clean glassware. Now, the note of common sense. This thing it is crazy dangerous on contact, do not even try to do it by yourself if you do not have experience working with hazardous /corrosive chemicals.
Now, this thing will save your glassware. But normal flask are cheap, cheaper than the treatment of a chemical burn.


Handling and Storing

To prevent breakage when rinsing or washing pipets, cylinders, or burets, be careful not to let tips hit the sink or the water tap.

Dry test tubes, culture tubes, flasks, and other labware by hanging them on wooden pegs, placing them in baskets with their mouths downward and allowing them to dry in the air, or placing them in baskets to dry in an oven. Drying temperatures should not exceed 140 °C. Line the drying basket with a clean cloth to keep the vessel mouths clean.

Dry burets, pipets, and cylinders by standing them on a folded towel. Protect clean glassware from dust. This is done best by plugging with cotton, corking, taping a heavy piece of paper over the mouth, or placing the glassware in a dust-free cabinet.

Store glassware in specially designed racks. Avoid breakage by keeping pieces separated.

Do not store alkaline liquids in volumetric flasks or burets. Stoppers or stopcocks may stick.

Proper care and handling of Pyrex ® and PyrexPlus ® labware will greatly increase its life and increase the safety of your work place.

Cleaning Specific Types of Glass Labware PyrexPlus ® Labware Autoclaving:

PyrexPlus ® labware can be successfully sterilized using liquids or dry cycle sterilization which involves no vacuum or low vacuum (<5 inches Hg).

Recommended cycles for automated autoclaves are:

CAUTION: Always autoclave vessels with loose caps or closures.

Steam sterilization time should not exceed 15 minutes at 121 °C (250 °F). Drying time should not exceed 15 minutes at 110 °C (230 °F). The actual cavity temperature of the autoclave should be checked to be sure the autoclave temperature does not exceed the recommended sterilization and drying temperature.


How it works

The MEDIACLAVE product range allows the rapid and gentle preparation of 1 – 30 L culture medium. Precise controlling and monitoring of temperature, time and pressure during the sterilization process guarantee constant high quality.

Safety

MEDIACLAVE is equipped with several independent monitoring systems for pressure and temperature, guaranteeing highest safety standards for the user and the working environment. The vessel lid is equipped with an autonomous overpressure safety valve and a burst disc in case all other electronic monitoring systems fail.

1) Adding Port
2) Overpressure Safety Valve
3) Dispensing Port
4) Temperature Probe Pt1000

Fast Heating and Rapid Cooling

Powerful heating elements permit fast media processing. This minimises thermal stress and ensures high fertility of your culture medium. Pressure and temperature controlled deaeration guarantees saturated steam in the vessel.

Rapid cooling is enabled by an efficient plate heat exchanger. The separation of the cooling water system and the sterilization chamber makes it unlikely that culture media gets contaminated by cooling water.

1) Standard mode: Heating up, sterilization and cooling down to dispensing temperature.
2) Chocolate agar mode: After the first sterilization phase, blood is added and the medium heated up again.

Reliable Sterilisation

The large and strong magnetic stirrer with adjustable speed and reversing rotation direction ensures homogenous media preparation over a wide viscosity range. A Pt-1000 temperature probe and microprocessor control of process parameters allow reproducible and complete sterilization of culture media.

Dispensing

MEDIACLAVE can be quickly and conveniently connected to the dispensing tubing of the automated Petri dish filler MEDIAJET. Alternatively, the peristaltic pump DOSE IT can be used to fill containers of more unusual volumes or shapes, e.g. quadrangular Petri dishes, bottles or flasks.

Large containers such as bottles can be filled by pressure dispensing. Medium is dispensed by automatically building up pressure inside the sterilization chamber.

Process documentation and validation

The MEDIACLAVE sterilization process is monitored and can be stored on a PC. A digital signature according to FDA (21 CFR Part 11) / EU (GMP Annex 11) is automatically added to the electronic process file. All logfiles can also be stored using the MEDIACLAVE USB port.

If equipped with an optionally available dot matrix printer, high quality printouts insusceptible to fading can be archived.


Incubation(7)

Place each Petri dish inside a zip lock bag to prevent drying out and to control odors. Turn the plates upside down and put them in a warm place. For many microorganisms, the ideal temperature for incubation is 32°C or 90°F. Bacterial growth should start to become visible in 2-3 days.

For those growing bacteria at home (for example, investigating bacteria growth at various places around the house), you may use a homemade "light bulb incubator" in place of a laboratory incubator. This page describes how to construct a "light bulb incubator:" http://www.umsl.edu/


Sterilize using one of the methods described on the Sterilizing Liquids page.

One advantage of high-salt media is that typical contaminating microbes won&apost grow on it, so media with a salt concentration of at least 10% can be sterilized by boiling.

Make sure the agar dissolves completely. In media with 15% or more salt, the agar may be slow to dissolve. The media may look cloudy, or you may see small, translucent lens-like objects floating in it. Continue boiling until the media is completely clear this may take longer than 15 minutes. Incompletely dissolved agar will leave your media squishy or fragile.


Watch the video: Autoclave Cleaning Kit - Solving spillage of agar and solid substance (October 2022).