Autoclaving is a sterilization technique in which equipment is sterilized at 121° C 2 atm for 20 minutes in a high-pressure steam sterilizer (Autoclave Machine) where bacteria are killed by steam at elevated temperature and pressure. This is the most efficient sterilizing approach if you wish to eradicate the pipet or dispenser tip infection.
By heating and sterilizing at 100 °C, it is also helpful in eliminating germs, spores, or pathogens that are difficult to eradicate using standard decontamination procedures.
When different microbes (bacteria, fungi, viruses, and so on) infect a microbe sample, the results can be influenced. Pipette and its tips, various research apparatus, and culture medium to be used in tests should be autoclave sterilized to avoid infection and cross-contamination.
Remember that autoclave sterilization is ineffective in removing contamination from particular bacteria with extreme temperature tolerance and enzyme proteins such as RNase, which are hard to neutralize at extreme temps.
Ensure the pipette tips you buy will be autoclaved before you buy them. Not every tip can be autoclaved. If you autoclave tips that shouldn't be autoclaved, you'll end up with a massive problem and potentially ruin your autoclave.
The time required for an autoclave session refers to the overall steam penetrating period: the time needed for the complete autoclave load to achieve the prescribed degree and the time necessary to maintain that temperature. Ensure you choose the best sterilization cycle time; the tips will not be sterilized when you abide by the complete cycle duration.
Whenever the autoclave tape achieves a temperature of 80 degrees Celsius or more, it will signal. It will not state that the right time was reached at this temperature. Biological or chemical indications, in addition to autoclave tape, can be used to check that the autoclave is working. These additional signs track temperature as well as time spent at that temperature.
If the pressure in the autoclave isn't zero, don't open it. Because the pipette tips will be heated, use thermal gloves while collecting them.
To sterilize new pipette tips, fill old pipette tip containers. This not only simplifies autoclave loading but also allows you to utilize the tips straight from the box after sterilizing. Otherwise, you'll have to put the tips in a glass beaker and wrap them in tinfoil.
The operator may easily remove and reassemble the pipette's lowest half (Tip section). When required, dismantle and rinse. Treat the trash fluid and garbage created in line with the facility's government rules and standards.
When working with low-boiling point (very volatile) solvents, vapor can readily leak into the tip even if you don't accidentally ingest it into the body by accident. Regular cleaning of the tip and the exposed internal components are suggested. When cleaning, employ a mild cleanser and wash entirely with purified water. After washing, thoroughly dry before using.
If you accidentally intake acid or alkali into the tip, properly clean the nozzle and all exposed internal components with pure water, after washing, thoroughly dry before assembling.
If the central section area has been polluted, clean it thoroughly with filtered water, 70% ethanol, or a mild cleanser.
If you've inhaled within the tip, properly clean it and any exposed internal parts using filtered water, 70% ethanol, or a mild cleanser. Finally, make sure there is no radiation and that it is at a healthy level. Lastly, check to see if the radioactivity has been reduced to a safe level.
When RNase is generated from bacteria or an enzyme reagent is polluted at significant levels, the whole cleanup is challenging. Intrinsic RNase is the most common cause of specimen RNA deterioration. The approach presented here is for washing and preventing contamination of pipettes used in RNA research regularly (Note, 2018). To begin, remove the nozzles and soak the interior and exterior of the nozzles, as well as the insides, in a 0.1 percent dilute solution of diethylpyrocarbonate (DEPC) up overnight. After that, sterilize each component. Un - reacted diethylpyrocarbonate is rendered inactive. After autoclaving, allow drying before assembling. It would be best to clean with 70% ethanol produced with DEPC sanitized water to hasten the drying process.
False positives are caused by infection by nucleic acids obtained from PCR samples in specific. It concerns regular sterilizing, wiping the tip and interior components with 3 percent sodium hypochlorite and 70 percent ethanol for DNA elimination afterward. Because RNA breaks quickly, it is scarcely noticed. Because RNA breaks rapidly, there is usually almost nothing to notice.
UV light with a wavelength of roughly 254 nm is recognized as efficient in neutralizing all bacteria (including viruses, mycoplasma, and molds). Pipette tips marked "UV Resistant" in the catalog and elsewhere can tolerate Uv light. The radiation exposure period varies depending on the strength of the illumination and the proximity to the object. Still, up to 30 minutes in a sterile workbench with a sterilizing light is usually enough.
Nevertheless, because UV light does not penetrate the pipet, remove the tip section and treat individual components to UV light while cleaning interior parts with this approach. (UV-resistant O-rings and seal rings should be cleaned using different procedures.)
There are other gas sterilization techniques such as (ethylene oxide gas) and sterilization ways such as gamma radiation or electron beam. However, as a common contamination elimination technique, labor, expense, or equipment can be fair. The eradication and control of everyday contamination of pipettes may be accomplished by appropriately choosing from all the previously described ways. Finally, Pipettes tips should be reserved solely for RNA or RI experiments and must not be mixed with cultured cells or bacteria cultures.
Furthermore, with PCR, you must usually produce a negative control. The most efficient technique to detect and avoid cross-contamination of samples is to be diligent.
The above methods can help you make proper sterilize pipette tips.
Le Rouzic, E. (2006). Contamination-pipetting: Relative efficiency of filter tips compared to Microman® positive displacement pipette. Nature Methods, 3(6). https://doi.org/10.1038/nmeth887
Note, A. (2018). How to avoid pipeting contamination.
Senger, C. C. D., Kozloski, G. V., Bonnecarrère Sanchez, L. M., Mesquita, F. R., Alves, T. P., & Castagnino, D. S. (2008). Evaluation of autoclave procedures for fibre analysis in forage and concentrate feedstuffs. Animal Feed Science and Technology, 146(1–2), 169–174. https://doi.org/10.1016/J.ANIFEEDSCI.2007.12.008
Soltani, E., Rezaee, M. A., & Gholizadeh, P. (2019). How to Reuse Pipette Tips and Tubes in PCR and Electrophoresis Procedures? A New In-house Method Development. Journal of Research in Medical and Dental Science, 7(2), 210–213.