Culture Maintenance Guides

General Conditions

Although achieving the growth of large quantities of algae in a short time is desired by many who receive UTEX cultures, such goals are not consistent with the needs of the Collection. On the contrary, it is more efficient for us to grow small cultures slowly, so as to minimize the use of space, materials and staff work, all of which contribute to cost. With this thought in mind, the following account of how UTEX cultures are maintained is not to be interpreted as our recommendations for the best growth conditions and media to achieve optimal growth rates and large quantities of algae. Such information must be acquired in other literature or through careful experimentation. Those who receive cultures are encouraged to duplicate within reason the conditions used in the Collection, when handling newly acquired cultures, to reduce the chance of losing the culture. After a stock culture is established, subcultures may be used for testing other conditions.

The Environment in the Room Temperature: 20 °C Light Source: mix of cool-white/warm-white fluorescent lamps Intensity: 3200 lux (maximum) Periodicity: 12/12 hour L/D (diurnal)

Set up: with the exception of Volvox, all cultures are maintained in a single room having approximately 500 square feet of floor space. The temperature of the room is kept at 20 °C using a chilled water cooling system and air circulator. The relative humidity is kept at 50 ± 1%. Racks of metal shelves having no lights are placed on the north wall near three windows, and cultures placed on these shelves may benefit from this natural light. The remaining cultures are maintained in five incubators located on the perimeter of the remaining walls. The temperature in four of the incubators is kept at 20 ± 1 °C. The fifth incubator is kept at 25 ± 1 °C and maintains Dasycladales and some tropical chlorophytes. Four workstations, one each parallel to the east and west walls, and two parallel to the north wall, are equipped with a pair of 4 foot cool-white fluorescent light tubes mounted approximately 10 inches above each of three shelves. During the life of the cultures, none are aerated or agitated.

Of note are cultures which are never placed under bright lights, e.g., the colorless strains of the euglenoids (Astasia, Menoidium, Rhabdomonas and some Euglena strains), the greens (Polytoma, Polytomella and Prototheca); heterotrophs (Ochromonas, Poterioochromonas and Chlorella cultures 1663-1671); and other light-sensitive algae, which are adversely affected by high light intensity. All reds except Porphyridium, the browns, and the macrophytic marine greens grow well in the dimly lighted areas of the culture room or in front of the windows. However, blue-green algae are especially troublesome, because they may bleach out under high light intensity or otherwise achieve little growth in dimly lighted areas. It has been found that most do well on the lighted shelf when the light intensity is diminished by placing a neutral density filter such as a sheet of white bond paper between the cultures and the lights.

Culture Production

The serial transfer of all agar cultures into new medium is performed in a laminar flow hood. Transfers are made with metal bacteriological loops appropriately sterilized with the flame of a Bunsen burner or with autoclavable wooden sticks. Culture tubes are also flamed before and after they are entered with the loop or stick. The serial transfer of all axenic liquid cultures is made in the laminar hood also. An aliquot of the culture suspension is inoculated into fresh medium using a Pasteur pipet and 1 mL bulb. Flaming is not necessary. All xenic cultures are transferred in open work areas of the culture rooms.

Newly transferred cultures are usually placed on a lighted shelf and allowed to grow or reproduce asexually until the desired size or population density is achieved. Then the culture is placed on one of the shelves away from the lights until it is needed. This initial growth period usually takes one to two weeks, but more or less time may be required. The frequency at which cultures are transferred varies from two weeks to nine months and is determined through experience and close observation of the cultures.

Cryopreservation

Most soil algae and many freshwater algae that are maintained in the Collection are now permanently cryopreserved. We have not extensively studied cryopreservation protocols for marine algae. Handling liquid nitrogen need not be a serious risk for a prepared user, but because it is so cold and because it displaces air from the atmosphere, those who utilize cryopreservation procedures must be aware of the hazards and use proper precautions. A broad variety of methods employed at the CCAP, at UTEX, and elsewhere, can be found in Day, J.G. and Brand, J.J. Cryopreservation Methods for Maintaining Microalgal Cultures, in Algal Culturing Techniques, ed. R. A. Andersen,Elsevier, Amsterdam, 2006. 

A relatively simple method that uses a minimum of specialized equipment, yet allows a large variety of microalgae to be cryopreserved with high viability, is described in the Cryopreservation of Microalgae page. Newly transferred cultures are usually placed on a lighted shelf and allowed to grow or reproduce asexually until the desired size or population density is achieved. Then the culture is placed on one of the shelves away from the lights until it is needed. This initial growth period usually takes one to two weeks, but more or less time may be required. The frequency at which cultures are transferred varies from two weeks to nine months and is determined through experience and close observation of the cultures.