Autoclaving is a sterilization method that sterilizes equipment at 121° C · 2 atm · 20 min in a high pressure steam sterilizer (Autoclave Machine) and kills microorganisms by steam at high temperature and high- pressure. It is the most effective sterilization method when you want to eliminate contamination of the micropipette or dispenser nozzle entirely.
It is also effective in destroying microbes, spores, or viruses that are hard to eliminate using conventional disinfection methods by boiling and disinfecting at 100 °C.
In experiments of microorganisms (bacteria, fungi, viruses, etc.), if other microorganisms contaminate the experiment, they may affect experimental results. Therefore, autoclave sterilization of Micropipettes, other experiment equipment, and culture media to be handled in experiments is important in preventing contamination/cross- contamination.
Nichiryo’s autoclave-compatible Micropipettes are made with materials resistant to autoclave sterilization, they are sterilized without disassembling, the main body in one piece is repeatedly autoclavable. When autoclave (121°C, 20 minutes) sterilization is performed, please follow the procedure of the instruction manual attached to each model.
*Large volume Micropipettes (1000, 5000, 10000 μL) are equipped with filters at the nozzle tip, the filter must be removed before autoclave sterilization.
Please note that the effectiveness of autoclave sterilization is low in eliminating contamination of special microorganisms with high temperature-resistance and enzyme proteins like RNase that are difficult to inactivate even at high temperatures.
100 μL of G. stearothermophilus spore medium was aspirated directly from nozzle tips of two Nichiro’s autoclave-compatible micropipette models (Nichipet Premium LT and Nichipet EXⅡ) to artificially contaminate the interior. After sealing the tip of the nozzles and wrapping the entire Micropipettes with aluminum, they were placed in an autoclave (121°C, 15 minutes) for sterilization, then 3 mL of separate medium was similarly suctioned into the interior and the discharged recovery solution was applied to agar medium to observe the presence or absence of G. stearothermophilus growth.
The results are shown in the table below. The test confirmed that the G. stearothermophilus spores inside the Nichiryo’s micropipette were eliminated by autoclave sterilization.
This study was performed by the NPO corporation Biomedical Science Study Group.*
|Amount of spore
from Positive control
+：Growth was confirmed. -：Growth was not confirmed. NT：No test.
The same spore medium was used on the 1-3, 4-5, 6-8, 9-10 experiment, respectively.
*Test Report: "Bactericidal test of contaminating bacteria in Micropipettes." BNR 28-19 (Issued on January 24, 2017.)
The lower part (nozzle section) of the Nichiryo pipette is easy to disassemble and reassemble by the user. Please disassemble and wash when necessary. Properly process the waste liquid and waste generated, in accordance to government laws and regulations of the facility.
Even if you do not aspirate into the main body by mistake, vapor can still easily flow into the nozzle when handling low-boiling point (highly volatile) solvents. It is recommended to clean the nozzle and the accessible inside parts on a regular basis. Use neutral detergent for washing and thoroughly rinse with distilled water. After rinsing, dry well before assembly.
If you aspirate acid or alkali into the nozzle, rinse the nozzle and the accessible inside parts thoroughly with distilled water. After rinsing, dry well before assembly.
If the main body surface is contaminated, please wipe off the surface well with distilled water, 70% ethanol, or neutral detergent.
If you have aspirated inside the nozzle, wash the nozzle and the accessible inside parts thoroughly with distilled water, 70% ethanol, or neutral detergent. Finally, check whether radioactivity is present and is at a safe level.
Finally, check whether radioactivity is attenuated to an acceptable range.
Complete removal is difficult when RNase derived from microorganisms or enzymatic solution is contaminated at high levels. In most cases, degradation of sample RNA is due to endogenous RNase. The method described here is for routine cleaning and contamination prevention of Nichiryo pipettes assigned for RNA experiments. First of all, disassemble the nozzle part and immerse the inside and outside of the nozzle and the internal parts overnight in 0.1% aqueous solution of DEPC (diethylpyrocarbonate). Next, autoclave each part. This inactivates unreacted DEPC. Dry after autoclaving and assemble. To speed up drying, it is advisable to wipe with 70% ethanol prepared with DEPC treated water or Nuclease-free water.
Contamination by nucleic acids derived from PCR templates, in particular, is a factor of false positives. In addition to periodic autoclaving, it is advisable to wipe the nozzle and internal parts with about 3% sodium hypochlorite, followed by 70% ethanol for DNA removal. Since RNA degrades rapidly, it is rarely to be noted. As RNA degrades quickly, there is generally little to be noted.
It is known that UV at around 254 nm is effective for inactivating all microorganisms (including viruses, mycoplasma, and molds). Nichiryo pipettes with notation of "UV Resistant" in our catalog and etc., can withstand UV irradiation. The irradiation time depends on the intensity of the light source and the distance to the target, but 30 minutes to 60 minutes in the clean bench with the sterilizing lamp is sufficient.
However, since UV light does not reach inside the micropipette, when disinfecting internal parts by this method, please disassemble the nozzle part and expose individual parts to UV light. (O-rings and seal rings are not UV resistant, so please clean them by other methods.)
There are also gas sterilization methods using EOG (ethylene oxide gas) etc., and sterilization methods by using gamma ray or electron beam, but as a contamination removal method done on a daily basis by ourselves, it cannot be said that it is reasonable in terms of labor, cost and equipment . By properly selecting from all the previously mentioned methods is enough for the removal and prevention of daily contamination of the Nichiryo pipettes. Finally, Micropipettes should be allocated exclusively for RNA experiment or RI experiment use and should not be shared with cell culture and bacterial culture use.
In addition, you should always prepare negative control in PCR.
To be thorough is the most effective way to check and prevent cross- contamination to samples.
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