Culturing Protocols

Chytrid and blastoclad fungi in culture are primarily saprobic heterotrophs, with notable pathogenic exceptions on algae, plants, insects, other fungi, and, in the case of Batrachochytrium, amphibians. Consequently, many types of culture media are necessary to maintain this diversity. The recommended medium for each culture is recorded in the database. Here we discuss maintenance by sterile transfer and how to avoid contamination, list culture media recommended by the CZEUM for culturing zoosporic eufungi, and describe our cryopreservation protocol.

A. Sterility and Transfer

Unless otherwise requested, we provide CZEUM cultures on agar medium plates without antibiotics. Though zoosporic eufungi are by nature aquatic organisms, we recommend that cultures on agar media be kept inverted and void of free-flowing water for routine maintenance. Without excessive drying, the surface of the agar is an acceptable environment for limited zoospore dispersal. Excessive moisture can precipitate and permeate into seams between the plate and lid, potentially making contact with the outside environment and bringing in contaminants. To maintain sterility, always transfer zoosporic eufungi in a laminar flow cabinet. To inoculate a new agar medium plate, remove an inoculation plug from the culture plate using a sterile scalpel, place the plug inverted on a new plate such that the thalli of the zoosporic fungi are in contact with the fresh agar surface, and push the agar plug around the surface of the plate to remove some of the thalli from the plug. Pushing the agar plug along the surface of the new plate may also physically displace any contaminants that were also transferred and may also increase the area on the new plate that is covered by the fungal culture. Monitor growth of the fungus of the closed agar plate with either a stereo dissecting microscope or invert the plate and observe the culture through the agar with a compound microscope at low magnification.
In order to stimulate zoospore production 1-2 ml of sterile distilled water can be added to a plate once the first zoospores are observed. After 30 minutes of saturation, cultures often will release copious zoospores into the water which can be harvested and used.

B. Removing Contamination

Unless otherwise requested, we provide CZEUM cultures on agar medium plates without antibiotics. If cultures arrive with minor contamination, observe the plate with a microscope to determine if the contamination is restricted to a certain area. If this is the case, transfer a plug of the non-contaminated agar with zoosporic eufungi to a fresh agar medium plate with antibiotics; we recommend 200mg/L penicillin G and 300mg/L streptomycin mixed into the medium post- autoclaving. Place the inoculation plug such that the thalli of the zoosporic fungi are in contact with the fresh agar surface. Pushing the agar plug along the surface of the new plate may also physically displace any contaminants that were also transferred and may also increase the area on the new plate that is covered by the fungal culture. Monitor growth of the fungus, repeating the above process if necessary, until contamination is no longer observed. At this time, it should be safe, but not required, to transfer the fungus to media without antibiotics.