The close of the covers of the test tubes or the vessels may be un good and uncluttered , and the climate changes help in the growth of certain types of fungi and bacteria, and the interruption of electricity during the sterilization may also affect the sterilization process

Check your autoclave maybe the problem with it, or you don't seal the container well.

I agree with  Arwa Tawfiq. You need to have a certified company to come check on your autoclave. Some worn rings or parts can change the temperature or steam pressure.

Thank you very much to all of you for your comments and suggestion.

All these answers are very important  and have to be followed.

Nevertheless last year my tubes got contaminated. And finally with the help of a lupa the mites could be seen. Thus the whole growthroom had to be sprayed.

Try to find out this aspect too.

Where did you place the tubes? does the storage place has a standard procedure as storage place after being autoclaved?

Did you check the cover after autoclaved? 

My experience when I did tissue culture, that I had to spray the outside environment around tubes with ethanol at least once in a day to prevent some contamination after autoclave. And the storage place was 24 hours under the lights and about 16 degree celcius (or something, I didnt know much about the temperature setting, but it's little bit colder from the outside room). I don't know if it is a proper way or not, but it did work well.

Are you using sanitation tape ? Is it turning color ?  Check the water level and the Wrap your media container w/ parafilm to make sure the container remains sealed. If it  shows contamination then the Autoclave does not maintain temp / psi / time balance and should be examined.  

Dr Saiful Islam, follow the following steps to reduce the contamination levels in media storage room as well as test tubes.

1.  Cheek the  autoclave pressure and temperature scale onace. 

2.  If not possible increase temperature 125 oC  at 20 psi pressure for 20 minutes.

3. Store the media for 3-4 days then after check the contamination levels.

4. Finally contamination is not controlled, you can use Plantomycine (Aries company) 100 mg/ litter media.

         If you follow these steps you can definitely we can control the Contamination in test tubes. 

The pH of the medium was adjusted to 5.8 and autoclaved at 121 oC for 15 lbs./cm 2 for 15 min. The cultures were incubated in a sterile growth room at 25 ± 1 oC with cool fluorescent light (irradiance 80 mol m−2 s−1) at 16 h photoperiod with 50–60% humidity.

My suggestion to avoid some sources contamination in tissue culture media. To avoid such un-noticed sources contamination. 

1. Thoroughly close (even while it is hot) the leads of the bottles after autocalving and seal them with parafilm or saran-rap 

2. Transport the bottles covered with clean plastic sheet or closed container. During summer, there are more dust particles that could be sources of contamination than clean air in the winter 

3. Check your laminar hood by placing open LB plate for 30 min and incubating them for 3 days at 37 degree. The device during inspection may not detect some sources of contaminates in the hood itself 

I agree with the suggestion of Prof Abebi. Whatever bacterial and fungal contamination that are seen in the cultures after autoclave is from laminar airflow hood or come along with dusts that settle during transportation. To avoid this, cultures are need to be kept in dust free cool room adjuscent to inoculation room. Media are to be utilized within 2-3 days of autoclave and proper surface sterilization of explants ensures no or  minimum contamination in cultures.

Regards 

I agree with above suggestions. What kind of culture vessels are you using? Autoclaving time depends upon the culture vessels size and medium volume. You can increase the time as per your medium volume.

Best, 

Hello

I agree with Dr. Abebie's opinion. I had the same experience and I realized that there is a problem with laminar filters.

SOmetimes people are able to publish papers on that too. No doubt changing HEPA filters should solve the problem, or might be the lab environment is itself not that clean air. I hope you don't have autoclave within the lab itself.. Try a big pressure cooker rather the autoclave to check if its the autoclave with the problem.

I agree with all above suggestions,,,, I hope you follow them,,,with all these just try to fumigate your laboratory ipossible.

Have you tried using Plant Preservative Mixture (PPM)? PPM is a is a heat stable preservative/biocide that, based on the dose, effectively prevents or reduces microbial contamination in plant tissue culture.

Works well with prevention and elimination of contamination.

https://www.plantcelltechnology.com/

I think that you should clean the growth room