I have often done plating and inoculation and all that, but right now I am facing a problem of the contamination in my media even when it is growing in unstreaked plates. I have changed the plates and bought new ones, cleaned the autoclave machine, autoclave media properly, changed the deionized distilled water too, sterilized the incubator with ethanol, sterilized laminar hood every time at the time of inoculation (with UV sterilization for 30 minutes and ethanol sterilization).
Could anybody suggest where my mistake is.
When it comes to sterility, the key rule is “NO SHORTCUTS & STRINGENT PROTOCOLS”.
Ensure the followings are periodically checked (once in a few months):
1) Particle count in laminar flow. Use a BSC II if you have it.
2) Check sterility of the autoclaved items.
Prior to working:
1) Ensure all items: media, pipette tips are autoclaved.
2) The pipettes you use must be sterile too. If possible do not share the pipettes. Or at least, keep one set of pipettes for use in the laminar flow/ BSC II only.
3) UV the laminar flow / BSC II. Spray the surface with VIRCON disinfectant or Clidox-s (ensure you leave the disinfectant for appropriate working time before you wipe them off). Spray 70% alcohol and wipe it clean. Remember to disinfect and clean the walls too.
4) Ensure all sterile packaging are in proper seal (petri dish, pipettes). Wipe them with 70% alcohol prior to placing them into the working cabinet. Remember that once your cabinet is sterile, you must maintain the sterility by ensuring there are no cross contamination. Your gloves need to be disinfected with 70% alcohol too prior to placing your hand inside the sterile space.
While working:
1) Avoid repeatedly taking things in and out. Keep your media capped when not in use.
2) Never obstruct the air flow. Place your instruments in the centre, with at least 2inches away from any wall of the cabinet.
Please check how old is your HEPA filter in Laminar Hood...Get it cleaned or repalced..
I would also clean and check all pipettes and pipettors (check filters inside), maybe the contamination comes from there.
Surely you autoclave your tips, but if the contamination comes from inside the pipette/pipettor due to improper handling- I don't know if you have your personal set of pipettes- even autoclaved tips don't avoid contamination.
I absolutely agree with Junaid and Elisabeth.
Plus their comments, I heard the mold does not die with EtOH.
So, my lab cleaned up clean bench and incubator with diluted cresol soap.
Take other people's opinions into consideration, too.
When it comes to sterility, the key rule is “NO SHORTCUTS & STRINGENT PROTOCOLS”.
Ensure the followings are periodically checked (once in a few months):
1) Particle count in laminar flow. Use a BSC II if you have it.
2) Check sterility of the autoclaved items.
Prior to working:
1) Ensure all items: media, pipette tips are autoclaved.
2) The pipettes you use must be sterile too. If possible do not share the pipettes. Or at least, keep one set of pipettes for use in the laminar flow/ BSC II only.
3) UV the laminar flow / BSC II. Spray the surface with VIRCON disinfectant or Clidox-s (ensure you leave the disinfectant for appropriate working time before you wipe them off). Spray 70% alcohol and wipe it clean. Remember to disinfect and clean the walls too.
4) Ensure all sterile packaging are in proper seal (petri dish, pipettes). Wipe them with 70% alcohol prior to placing them into the working cabinet. Remember that once your cabinet is sterile, you must maintain the sterility by ensuring there are no cross contamination. Your gloves need to be disinfected with 70% alcohol too prior to placing your hand inside the sterile space.
While working:
1) Avoid repeatedly taking things in and out. Keep your media capped when not in use.
2) Never obstruct the air flow. Place your instruments in the centre, with at least 2inches away from any wall of the cabinet.
Well thanks to all for sharing such kind of meaningful information to me.
Check the performance of your autoclave. It seems far out, but we had this problem when sterilization in one of our autoclaves was actually incomplete. To check, we are using Sterikon plus ampoules (Merck) containing Geobacillus stearothermophilus spores. When you incubate the ampoules after autoclaving, and the color changes from blue to yellow, the spores are growing. In this case, prolong your autoclaving cycle until there is no more growth. Also, give the instrument a service checkup.
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