My field, biological control, is served by several culture collections of fungi, bacteria, viruses and nematodes (collated at WDCC). In these collections it is possible to find a natural enemy of just about any insect pest. Collections also support conservation of earth’s biodiversity in the face of globalization, habitat loss, and climate change–pressures that threaten microbial species as much as whales and tigers.
Culture collections are expensive to support, as they require special equipment and continuous attention in order to maintain fungal cultures without losing their pathogenicity or virulence. Two examples of these collections are the USDA-ARS Collection of Entomopathogenic Fungal Cultures, with more than 5500 cultures of over 350 species of fungi from 900 hosts. The International Entomopathogenic Bacillus Centre in the Institute Pasteur has nearly 3500 strains of Bacillus thuringiensis, the most important bacterium used in biocontrol. Cultures are typically either freeze-dried in a process called lyophilization, or stored in liquid nitrogen at ultra-low temperatures. Both techniques require intense labor and expensive equipment.
At the International Center of Tropical Agriculture (CIAT, in Colombia), expenses were reduced with a novel, reliable and cheap technique. The dry filter paper technique was developed by Rosalba Tobon and Ximena Aricapa in the early 1980s and can be used for preservation of cultures of insect pathogenic and plant pathogenic fungi as well as many molds.
The first step is to isolate the fungus into pure culture. A tiny pinch of the fungus is taken directly from the insect or plant host and added to a Petri dish containing culture media. If the identity of the fungus is known, selective culture media might be available; if not, general media such as PDA (Potato Dextrose Agar) can be used for the initial isolation. Lactic Acid or Chloramphenicol can be added to any standard medium in order to reduce contamination by bacteria. After a pure culture has been obtained, the fungus is grown for 5 to 10 days. Now the storage process can be initiated.
In the second step, pieces of filter paper of about one square centimeter are cut and sterilized in an autoclave. They are then placed on the agar surface of the same selective or general medium used to isolate the fungus.
Fresh spores or a little piece of the pure culture is cut from a fresh colony, and placed on top of each piece of filter paper (be sure to work in a sterile environment). The Petri dishes are sealed and placed in an incubator–the fungus typically grows more slowly on filter paper, needing approximately 10 to 15 days to fully colonize it.
Once the fungus begins to sporulate on its filter paper, the individual pieces of paper bearing fungus are separated from each other and the underlying medium, then placed in new Petri dishes without any culture medium. After that the Petri dishes are put again into the incubator until the paper and fungus are completely dried, approximately 20 to 30 days.
The drying process is most crucial because if it is too fast, the fungus can lose pathogenicity and virulence or be killed; and if it is too slow it can become contaminated by other fungi or bacteria.
As soon as the fungus is dried, 10 to 12 pieces of paper filter are put in a sterile glassine envelope. Each envelope is labeled, bagged in a plastic bag and it stored in a plastic container at 4Â°C or -20Â°C depending on the facilities that are available. When a fresh culture is needed, one small piece of filter paper is removed from the envelope and placed on fresh medium.
The new technique is not only reliable, it is very inexpensive and easy to use in any laboratory with few resources. CIAT uses this method to store about 500 cultures of insect pathogenic fungi and 1000 cultures of plant pathogenic fungi and bacteria. Evaluations of purity, pathogenicity and virulence were performed on fungi stored between 5 to 10 years. With a few exceptions the fungus was recovered easily and with the same characteristics of pathogenicity and virulence it had when first stored. This technique has been successfully implemented in other institutions with great results. Research studies at CIAT are adapting this methodology to work with bacteria and viruses.