Please find enclosed some documents in this direction... 

Hi Puskar, I don't know the layout of your lab, but you can do simple steps to at least hopefully limit contaminations.

1) Do experiments in tissue culture hoods that is sprayed with 70% isopropanol before use and after use.

2) Make everybody wear a facemask, gloves, labcoats and a hairnet. Choose ~3-4 people that you trust to do the experiments. Nobody else should be allowed to enter the lab.

3) Have only one entry and exit into the lab. All instruments that go in (pipetters, specs, etc) should stay there and never go out.

4) Autoclave everything that can be autoclaved.

My cultural lab size is 2987 cubic fit and growth chamber is of same size. I went through the paper 38859PDF.pdf, calculation says that 1972gm of KMNO4 and  3584 cc of formalin for each room. Isn't this very huge amount? 

Hi Puscar, personally I don't know why permanganate is recommended as a disinfectant for a TC lab.  It is very good to clean glassware and should be kept in fume hood.

But KMNO4 is a salt and it is corrosive to everything in the lab, except glass.

And this salt has to be washed away in order to get things growing again.  And when you use water to get rid of the permanganate, you can re-introduce contaminants.

I would recommend that you first try 70% isopropanol on everything. It kills everything and it evaporates. No need to wash things that have been treated with 70% isopropanol that has evaporated.

I gather you have HEPA filtered hoods. Filters do not do well with permanganate or formaldehyde..

Try killing flies with a fly swatter before using flame throwers..   

I am not so sure the contamination is really from the environment or the experimental materials or the equipment was not up to bar. As people already suggested, the equipment need to be checked periodically. In our Institute, the HEPA filters are changed after a period of time, and the flow rate and other factors in the hood are also checked by a certified company. The institute schedules this kind of check for every lab every 6-12 months. The parts of an autoclave can be worn out after repeat use as well. This also need to be checked. I have not seen any 'lab fumigation' in our institute for as long as 10 years (in the US).

But, I am not sure whether the climate is a major player in this contamination--for here it is always drier than some other geographical regions.

@Yuan!

How do that certified company checked flow rate and other factor? What are included in other factor?

I found contaminated culture after three days of fumigation? Does fumigation does not sterilized culture chamber? Or fumigant gives negative impact on explant over fumigation duration? 

I think fumigation is not the option for cultural chamber sterilization. What should be the other option?

Hi Puskar Thapa,

1. I am sorry that I don't know the details of the machine they used. Once they mentioned testing 'flow rate', that is why I remember it. I regret now that I didn't ask them the details, because when they came, we just let them did their job and tried not to bother them. They have a machine with a 'probe'. They stick the probe into the hood when the air was blowing. Then some numbers will show up on the machine. They would stick a label on the hoods if the hoods are good. I have done plant tissue culture myself for more than 10 years and watch other people doing the same things for many years too. But, I have not encountered that the labs have to be fumigated.

2. You said, " found contaminated culture after three days of fumigation?" This can be (1) something wrong with the plant tissue culture sterilization technique or equipment problem (as mentioned earlier), rather than the 'environment' you are in. (2). The culture could have already been contaminated before fumigation. It just took 3 days to show the contamination symptoms.

Hi Puskar Thapa,

I just went online to check Laminar Flow Cabinets "Testing/Maintenance Service". Here is one example. http://www.escoglobal.com/resources/certification-matrix/69/

You can see what they are testing or maintenance for Laminar Flow Cabinets. Here lists some services from their Certification Matrix for different types of laminar flow.

Airflow Balancing

ULPA / HEPA Filter Integrity

ULPA / HEPA Filter Replacement

Inflow / Face Velocity

Laminar Flow Velocity

Particle Count

Airflow Visualization

Airflow Alarm Calibration

Blower Replacement

Electrical Troubleshooting

Electrical Safety

GFCI Performance

Work Chamber Pressure

Enclosure Integrity

Product Ingress / Egress

Recovery Time

Glove Breach

Decontamination

Hi Pushkar, please calm down and review what could be wrong. From what you describe, this is not an accidental problem, but a chronic problem.

Therefore, you have to approach this as a systemic or structural failure of your lab design and management.

1) Is your lab under negative pressure of positive pressure? Negative pressure will force outside air into the lab, while positive pressure will force inside air out. All TC labs should have positive air pressure.

2) TC hoods can only give a limited protection. If you have many people walking in and out of the lab, they not only disrupts the positive air pressure but also disrupts the flow that the TC hoods use to separate outside air from inside air.

3) limit the number of people that can go into the lab. No schoolchildren or dignitaries allowed. The people working in the lab should have labgowns, hairnets, facemasks and gloves. And regularly spray every thing (exept cultures) with 70% isopropanol.

I have worked at TC facilities in academia, small biotech startups and big pharma. These simple procedures work.

 If this problem still persists, then there is something seriously wrong with your facility.

You should do Fumigation by formaldehyde with potassium permanganate...