Its better to wash the ex-plant repeatedly with DW after bavistin treatment. Now u can take ur ex-plant in to LAF where u can again wash the material in 70% ethanol for 30 seconds and repeated washing with DW. The washedex-plants can be surface sterilized with 0.1% HgCl2 for 3-5 min.

Ascorbic acid and citric acid in combination can also resolve the problem encountered by fungal contamination. U can also try it.

Bavistin treatment  followed by mercuric chloride ( 0.1%-1.0% for 2- 8 min ) treatment in addition to frequent subculture can reduce the fungal contamination.

Wash  your explants with tween 20 (detergent). Treat your explants with Bavistin (0.1-0.5%) for 30 min - 2 hour. Wash with double distilled water in combination with tween-20 again for 15 minutes followed by washing for around 5-6 times with double distilled water. Then in LAF treat your explant with 0.1%HgCl2 followed by 70% ethanol( 30 sec - 3 minutes) and finally with autoclaved doubled distilled water for 3-4 times. 

If endophytic contamination is troubling your media, you can also use PPM (plant preservative mixture)(0.1-0.5%) as it is a heat stable preservative/biocide reducing microbial contamination in PTC medium.

Good luck

Shiwani Kaushal

All mentioned is good, if contaminant is not endophytic microbe. Try to use elevated temperatures for incubation of your plants at 50C for several days and do use meristematic tip culture.

Of course, with use of antifungal agent (s).

After bavistin u can use 0.1% Hgcl2 

Bavistin treatment can be given for 10 minutes followed by several washing with sterile distilled water. After washing, you can use o.1%  HgCl2, which is to be washed again several times with sterile distilled water.

Contamination of cultures depend on plant species and season. Generally during winter season infections are low compared to rainy and summer. Select ex-plants from vigorous mother plants growing in controlled environments (Green houses). Try to collect ex-plants in the cool time of the day and the same time ex-plants should not contain due & dust. After collecting ex-plants start pre-treatment process by placing them under running tap water for 20-30 min. Then immerse in 0.2 % Bavistin + 0.2 % Ridomil+ 1-2 drops of tween-20 + 200 ppm 8-HQ for 30 min to 2 hr depend on the plant (Use shaker for effective treatment). Rinse with distilled water 3-4 times  in LAF . Treat with 0.1 % HgCl2 for 2-5 min. depend on your plant type. Again wash the ex-plants with sterilized distilled water 3-4 times. This procedure can work for most of the ornamental and horticulture crops.

ALL THE BEST

Ravindra Kumar.K

Hi Please use tetracycline and streptomycene mix or two antibiotics one controlling gram negative and one gram positive .  Dissolve one capsule each in the medium 

A various mixture of antibiotics can be tried 

with regards

Yous sincerely

Ashwani Kumar 

There are many ways to control fungal growth. The most important step is to eliminate them during surface disinfection. As suggested by researchers here, you may use fungicide, mercury chloride, clorox, ethanol, detergent with a few drops of tween-20, etc. Starting material is also vital. 

If you facing contamination problem during subculture / maintenance, you may include antifungal agent in your medium (beware of the high concentration that may affect plant growth). You may disinfect the contaminated explants but usually it will not help much.

All the best!

this article could help you find a suitable solution for your problem,

Contaminants of plant-tissue and cell cultures, By C. Leifert, J. Y. Ritchie, W. M. Waites, World Journal of Microbiology and Biotechnology, July 1991, Volume 7, Issue 4, pp 452-469

http://link.springer.com/article/10.1007/BF00303371

you might want to look up work with Cyclosporin but it is usally hazardous to the plantlets and so its use is questionable

Hi, you can use Plant Preservative Mixture (PPM) both in substrate and to sterilize plant material

Yes sometimes you can have problems in rigeneration, but you can solve it transferring your explant in a new medium, without PPM, after about 20 days.

The active ingredient of Bavistin is carbendazime. The product can be autoclaved together with the media without any loss of activity. However, this chemical might affect the growth and development of your plant.

Article Carbendazim as an alternative plant growth regulator in tiss...

You can use Metronidazole as antifungal with your media

In addition to antifungal agents use suppressive temperatures (many fangi are suppressed above 30C or more) to suppress possible contaminants, proper sterilization of media, air, desinfection of rooms walls, flor and tables, filter change in laminar flow cab and have proper caps with filters on your jars and tubes. Than, possibly, you may isolate not infected plant cells from shoot tip apical meristem.

Fungal contamination may be seasonal, observe the seasonal changes of contamination frequency

The following steps can be useful to get rid from fungal contamination

1. Select freshly emerged plant material as starting material

2. Thoroughly rinsed with sterilized distilled water for 30 min, use vortex if possible

3. Treat the explants with 70% ethanol for less than 60 s, which will remove the external fungi

4. Treat the explants in systemic fungicide solution, which can remove internal fungi

3. Surface sterilize the explants with 0.1% (v/v) HgCl2 for 5 min under aseptic conditions.

Optimize the treatment duration according to observation.

I must first say that the most important issue is to identify the source of the contamination. Contamination can always have several causes, but it is usually either due to the mistake of the culture operator or due to environmental contamination (laminar hood, autoclave, blade, plate, glass or petri dish, and growth chamber) or explants (failure of sterilization or endogenous fungal contaminants).

Overall, there are two types of contaminants (Exogenous and endogenous fungal contaminants). Exogenous fungal pathogens are easy to eliminate from the stock plants using many fungicides such as systemic fungicides (e.g. Benomyl) or other disinfectants (sodium hypochlorite). Endogenous or endophytic fungi become pathogenic to the host plants when the plants are stressed, for example, when the cell walls are weakened or under other unfavorable in vitro conditions. Mature stock plants are highly loaded with fungal pathogens and spores, but used due to their maturity, especially in fruit trees.

For further guidance, some of these chemicals include: 1. Nystatin (toxic to fungi ); 2. Copper oxychloride (fungicide ); 3. Sodium & calcium hypochlorite; 4. Formaldehyde (aldehyde); 5. Benomyl and Captain; 6. Teepol (detergent). 7. Silver nanoparticles; 8. Titanium dioxide (TiO2); 9. Ampicillin 10. Alcohol (70%) and so on.

Also, you can use meristem tip culture technique with several sub-culturing.

I hope the content is effective and useful.

With luck

Reza Ghahremani

you may try PPM (plant Preservative Mixture)