I used leaf explant and inoculated onto solid MS media after 4 weeks of incubation i transferred it into liquid media, after 3 days of incubation the media turned turbid and i could see round white mass of fungi.
Dear Mithun,
do you mean bacteria or fungi?
The reason is that you have some (1-2) fungi spores in your tissue (explant surface), which start to growth in the liquid. There are almost no way to remove fungi, except restarelization of addion of some preservatives (look on companies web-site). There are no antibiotics against fungi...
Dear Mithun,
In my opinion, the explants has endophytic bacterias.
Thanks for your opinion sir. Is there any method to remove the contamination like antibiotic wash or removing the infected parts.
Dear Mithun,
do you mean bacteria or fungi?
The reason is that you have some (1-2) fungi spores in your tissue (explant surface), which start to growth in the liquid. There are almost no way to remove fungi, except restarelization of addion of some preservatives (look on companies web-site). There are no antibiotics against fungi...
Dear Mithun,
I agreed with Mr. Pedro, this contamination coming from explants source, and during this stage its very difficult to remove it. I faced many problems like this situation. and you can reduce it by:
1- Rinse explant by fungicide for 12-24 hours before inoculation onto solid media.
2- add antibiotic such as tetracycline to your media
3- select young and healthy leaf explants.
Dears,
Since the growth of the microorganism is so quick (2-3 days), I think that the source of contamination is an endophytic bacteria.
Another possibility is to use an isothiazolone derivate (Luna et al. 2013. In Vitro Cell.Dev.Biol.—Plant 49:326–332.
In my opinion,
the explant leaf may not contain the fungal spore which germinated into the mycelium.
This is because the explant was in culture for 4 months free of fungal growth. Fungi or bacteria develop quickly in in vitro ocnditions with in 2/3 days.
Your 2nd medium-liquid medium must had the fungal spore which resulted in the mycelial growth, in which case the medium was not properly autoclaved. Or during the subculture, the fungal spore must have got into the medium.
Fungal growth can be controlled by the fungicide Bavistin. Some times, when we know the explant is collected from a humid environment, we add Bavistin
( eg,0.1mg/ml) to the medium before the medium is autoclaved.
But Bavistin will also have affect on explant/callus/regenerants.
Thank you for your question. In my opinion the culture having some fungal spore and it will grow very faster in liquid medium. In some cases, some endophytic fungus or bacteria will create problem in liquid medium. Before you state experiment, you may filtrate your liquid medium through 0.45 uM sterile microfilter. But if you are culturing in either liquid medium or semisolid medium you may use carbenicillin. (50 mg/l) to check the fungal infection.
I do agree with Mr Rout. More possibilities are handling errors during transfer
The culture may be having some fungal spore. It will grow very faster in liquid medium. In some cases, some endophytic fungus or bacteria will create problem in liquid medium. Before you state experiment, you may filtrate your liquid medium through 0.45 uM sterile microfilter. But if you are culturing in either liquid medium or semisolid medium you may use carbenicillin. (50 mg/l) to check the fungal infection.
Hi Mithun,
It might be possible that some fungal spores might be present on your explant which is creating problem of contamination. To avoid such contamination you should modify the protocol of surface sterilization of explant by using multiple wash of fungicides.
It is also possible that there might be fungal contamination in the liquid medium due to improper sterilization. There are some articles regarding use of fungicides in plant tissue culture media. So you may add some fungicides with antibiotics in the medium to prevent contamination. However it may reduce the growth of the explant which depends on the fungicide you use and plant species of which the explant is.
hi mithun
this may be an endophyte. if this is the case then you have to find out another explant source. sometimes endophytes will make their appearance on transferring to the liquid medium.
For surface sterilization i have used 3% Sodium hypochlorite and 0.1% Mercuric chloride for 5 minutes and 2 minutes respectively.
Rinse the explants with 20% glorix for 10 minutes before transferring to liquid media.
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