Hi Aisha,

Has your laminar flow hood been checked periodically by a certified specialist? Has the filter in your laminar flow been changed periodically? Ours are checked and filters are changed periodically. You can place a dish with solid medium in the hood (open lid) as a control (without your plants) to find out, with airflow on. If your medium grows mold, then probably the 'autoclave' machine or the 'laminar flow hood' has some kinds of problems.

In general, contamination occurs from glassware, medium, explants, handling and this appears quickly after culture, while internal infection appears after a period of incubation, so you have to emphasize from the plant, is it endophytic infection or others. In special cases, it occurs from the cover of glassware due to water evaporation from the medium because of temperature rise and gets down with spores.

You can do one more thing firstly give UV for 40 minutes after washing the whole laminar, after completing UV again clean it with 70% ethanol. Then put every materials that is necessary for your work into the LAF by spraying them with 70%. Then again give UV for 1 hour. After that you can start your work. Please remember that you should not enter anything into the LAF after starting your work. May be by this way you will be able to solv your problem.

I also had similar problems. Sucrose in the media makes it more harder. Try PPM, it helps to get rid of fungi as well as bacterial contamination. https://www.plantcelltechnology.com/about-ppm/

Hi Aisha,

Has your laminar flow hood been checked periodically by a certified specialist? Has the filter in your laminar flow been changed periodically? Ours are checked and filters are changed periodically. You can place a dish with solid medium in the hood (open lid) as a control (without your plants) to find out, with airflow on. If your medium grows mold, then probably the 'autoclave' machine or the 'laminar flow hood' has some kinds of problems.

Dear Aisha Khan it's important to determine the origin of contamination. As Yuan-Yeu Yau said, an open plate without plant material would help to know if contamination occurred because of laminar hood by itself or by operations performed in it. However, I'm going further, you might also left sealed plates without plant materials either in and out of growth chamber. Remember that the flame that came from burning alcohol is not so esterilant as it seems, thus check the cleaness of forceps and blades. If you like you can make an extra plate touching agar with instrumental after flame them and before to manipulate plant materials. If all these tests are negative (or positive, in some cases), probably you could face the posibility that mites could be the origin, which's a big problem to sove.

What plant species and tissue are you using for tissue-cultruing? I don't know how fast your plant can grow in the medium. If they can grow quick enough in the medium, you can sterilize their seeds (using ethanol and/or bleach) and grow them into a plantlets in the medium, and use their tissues for your next step (such as callus induction, instead of inducing callus from greenhouse-grown plants), while you are tackling the contamination issues. I am not sure how fast you can identify the contamination source. You need to have some back-up plans, as time is tight for graduate students or students.

contamination could be coming from plant material itself, you can sterile the seeds/leaves using sodium hypochlorite or bleach before growing in the medium.

contamination can be also caused if your laminar flow is not working properly. check if the laminar flow is working, if not, it can be the source of contamination.

you can also take extra precautions by turning on UV light for 30 minutes before starting your experiment.

Would it be possible to have these informations.

1: timing, that is the delay between observable contamination and last manipulation (In days).

2: a picture representing the typical contamination: 1 contamination or more/Petri dish, with a scale + info on the number of days between picture and inoculation + localisation of the contamination (near the side of the dish or in the middle).

3. Which species?

Biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from the laboratory environment. Laboratory contamination is caused by plant‐associated, environmental, and human‐associated bacteria; yeasts; and microarthropods.

Chapter Micropropagation

Chapter Contamination Detection and Elimination in Plant Cell Culture

Technical Report Plant tissue culture contaminants identification and its res...

Chapter Contamination Detection and Elimination in Plant Cell Culture

https://www.actahort.org/books/560/560_66.htm

some times the source of contamination is endophytes ( a bacterium or fungus, that lives within a plant). I recommend to add PPM in your media, use a small petri dish and put just one explant in each dish. For sterilise the explant add 3-4 drops of tween 20 or 80 with sodium hypochlorite.

Hi Aisha Khan ,

I also experienced the same problem when I was working with Carissa opaca and I had to re-look and ensure the following things by making a check list and checking it with each step:

I hope these basic points may help you and good luck!

Biological contamination in plant tissue cultures originates from two sources:

1- from Explants (seeds or tissue) used to initiate the culture.

2- from the laboratory environment.

you can sterile the Explant (seeds/leaves) using sodium hypochlorite or bleach by increasing time.

Hi..first of all mention the plant species on which you are working as thn it will be easier for many to guide you for sterilization treatment or which type of antibiotics you can use. Second as suggested by @ Yuan-Yeu Yau sir, you can develop seedlings from which explants can be taken as it reduces the contamination. Lastly I would also sugges to give your plant sample to microbiology expert to check whether it has endophytes or not as I have seen that it hinders the work. All the best

Did you sterilize your explant with. 10%Sodium hypochlorite or 1% mercuric chloride for desired time followed by washing with ddw.??? Secondly, wheter HEPA filters were working or not..u can chk it???..

You have to eradicate biological contamination also, sterilize your explant too..

To eradicate the contamination you need to optimize the surface sterilization process...alternatively, you may apply meristem culture which is effective in eliminating endogenous infection (fungal, bacterial or viral)...

For meristem culture, you may refer to this channel:

https://www.youtube.com/channel/UC61bPc54xu8-nmb0_5N1xhw?view_as=subscriber

please look for meristem culture from my channel, Plantastic

You may subscribe my channel if you found it useful and I will share more information about plant science

Biological contamination in plant cell culture comes from the tissue used to initiate the culture, and Laboratory contamination is caused by plant‐associated, environmental,,bacteria; yeasts; and micro arthropods.

If the contamination is of endophytic origin, there will be problem of infection during the culture even if u surface sterilized the explant well. One way of controlling it is to do subculturing at regular intervals and use antifungal agent in sterilizing solution if there is fungal contamination.