INDOOR ALGAE STOCK CULTURES
The indoor algae room is separated from the rest of hatchery in order to maintain a sterile environment and minimize the opportunity of contamination by rotifers, bacteria, protozoa and other algae. It is air-conditioned at 20-25oC to maintain cool temperatures. Twenty –four hour illumination (2500-5000 lux) supplied by fluorescent lamps throughout the room provides the light needed for growing monocultures of N.oculata Aeration is supplied by PVC air lines with enough adjustable air valves for each culture, indoor algae cultures consist of five- phases: stock culture in test tubes, 250 Ml Erlenmeyer, 2 L Erlenmeyer flasks, 20 L carboys, and 100 L fiberglass cylinders
ASEPTIC TECHNIQUES
Ideally, the algae room technician should complete all indoor algal duties prior to starting any other hatchery work. This ensures that contaminating organisms are not introduced into the algae room from outside sources. If other work must be done before starting the indoor algae duties, the technician should shower and change into fresh clothing before entering the algae room. Other personnel are generally prohibited from entering unless absolutely necessary. Supplies and equipment are cleaned before being brought into the algae room.
In starting and maintaining algae culture, all supplies are soaked overnight in detergent, washed and scrubbed well, then rinsed thoroughly to remove any soapy residue. Glassware is acid washed with 10% HCl, rinsed thoroughly and allowed to air dry overnight before use
Culture vessels and seawater medium must be sterilized. Seawater is filtered through 5and 1-micron cartridge filters. Sterilization of culture vessels occurs either by use of an autoclave or by chlorination, glassware and its algal medium is sterilized in an autoclave
The algae room is sterilized weekly by flooding the floor of the room with fresh water and sprinkling it with powdered chlorine. The water is allowed to evaporate over night and/or empty through floor drains.
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CULTURE METHODSIt starts by transferring pure colonies from the Petri dish to the test tubes. After the growth period (From 7 - 10 days) transfer algae from test tubes to the other different stages
** Test tube stock cultureStock cultures are started in 15 ml test tubes equipped with scrow top caps. Cultures are allowed to grow for one month before transferring. A clean test tube culture with no contamination is selected to laterally transfer into four new test tubes.
Clean test tubes are first filled with 10 ml of sea water and f/2 media. They are then sterilled by autoclaving. Caps are loosely scrowed on prior to autoclaving to allow steam to escape during the process. After autoclaving, the test tubes are cooled and caps are tightened.
Inoculation of new tubes is accomplished with sterile, microbiogical techniques.Transfar are carried out a fume hood equipped with a Bunsen burner and an ultraviolet (UV) light which kills most micro-organisms. The UV light should be turned on 15 minutes prior to use and turned off while the technician performs the inoculations under the hood. The surface of the work area should also be wiped clean with alcohol before use.
A harvestable tube and a new tube are uncapped and the lip of each test tube is waved back and forth over the flame of the Bunsen burner several times. Two milliliters of algae from the harvestable test tube are removed with a sterile pipette and transferred into the new tube. The lips of the test tube are flamed again, and the test tubes are loosely recapped. This is repeated to produce as many new test tubes as possible from the original test tube. A new, sterile pipette should be used for each transfer. Inoculated test tubes are placed on the flask shelves. These test tubes are inverted daily to resuspend algae cells which allowed cells to receive more light, thereby increasing growth.
TWO-Liter Flask Cultures
Flasks are assembled with glass rods, a rubber stopper and air line tubing. The (air outlet tube ) which allows air to be released from the flask, is a short piece of glass rod bent at a45-degree angle which extends over the flask lip to minimize contamination.
Before autoclaving, flasks are assembled and filled with 700 ml seawater and media. Aluminum foil is used to loosely cover the top of an assembled flask and is taped at the nick. The flask is autoclaved and then cooled overnight before use. The contents of the autoclave flasks will appear cloudy as a result of precipitation of the nutrients
To start a new flask culture, a single, clean, uncontaminated test tube culture is selected to inoculate
Tow flasks .these first two are allowed to grow for two weeks to a density of 100 million cell / ml .F each flask is then transferred into two other 2 L flask following inoculation, the initial densities are 10 to 20 million cells / ml which increase to 100 to 150 million cell / ml after 7 days. Once the cultures attain these harvest densities, 400 ml from each of the four flask is laterally transferred to two flasks while the remainder of the culture is used to inoculate a 10 L carboy. Once the flask cultures have been established, lateral transfers of flasks should accrue daily with the oldest cleanest culture, still in the log phase, being chosen each day to transfer. It is highly recommended that at least 14 flasks be cultured at the same time. This number is more than necessary, but provides insurance against algae culture crashes.
To laterally transfer one flask into another flask, the foil from a clean autoclaved flask is removed and air line a fixed to the long glass tube. The flask is aerated vigorously for several minutes so its contents are well mixed before inoculating. With the rubber stopper in place, the exterior of a harvestable flask is rinsed with tab water to remove sedimentation from the upper portion of the flask. Tap water is flushed through the air line of the harvestable flask to replace the amount of water evaporated during the culture period. Approximately 400ml of algae from this flask is then used to inoculate the new flask and the remainder is used to inoculate carboy .aeration should be sufficient so that all algae cells are exposed to light
It is very important that laterally transferred flask flask culture be slowly replaced be new stock every three month to reduced the chances of contamination .new batches of flasks should be inoculate with tube cultures. Old batches of flasks are slowly “phased out" and should be used to inoculate carboys, they should not be used for lateral transfers.
**20 Liter carboy culturesThe carboys are filled with 16 L of filtered seawater and chlorinated with liquid bleach for 24 hours, then neutralized with sodium thiosulphate and gentle aeration for at least 48 hours. The aeration is then turned until they are ready to be inoculated. Nutrients are added just prior to adding the inoculums.
The carboys are inoculated at 7 to 10 million cells /ml and reach harvest densities 40 to 70 million cells/ml after seven days.
** 100-L fiberglass CylinderThe Cylinders filled with filtered seawater and chlorinated with liquid bleach for 24 hours, then neutralized with sodium thiosulphate and gentle aeration for at least 24 hours. The aeration is then turned until they are ready to be inoculated. Nutrients are added just prior to adding the inoculum.
The carboys are inoculated at 4 to 6 million cells /ml and reach harvest densities 30 to 40 million cells/ml after seven days. A harvestable Cylinders is then used to feed the larvae and rotifer
** NUTRINT MEDIA
The medium used is the F/2 formulation of GUILLARD 1962
STOCK SOLUTION
1- SODIUM NITRATE (NaNO3)75 g dissolved in 1000 ml D.W
2- SODIUM DIHYDRGEN ORTHOPHOSPHATE (NaH2PO4.H2O)5 g dissolved in 1000 ml D.W
3- SODIUM SILICATE30 g dissolved in 1000 ml D.W
4- EDTA – TRACE METAL5 g Fe-EDTA +
1 ml CoCl2.6H2O
1ml MnCl2.4H2
1ml NaMoO4.2H2O
1 ml ZnSO4.7H2O
1 ml CuSO4.5H2O
5-VITAMINS1 ml THIAMINE HCl
1 ml BIOTINE
1 ml B12
THE MEDIA IS USED WITH ADATION 1 ML FROM EACH (NITRATE, PHOSPHATE, SILICATE, EDTA – TRACE METAL)
NOTES:SODIUM SILICATE is used for ISOCHRYSIS and DITOMES
** HOW TO PRERAR STOCK SOLUTION OF TRACMETALS
TRACE METAL STOCK SOLUTIONCuSO4.5H2O 4.9 g dissolved in 100 ml D.W
ZnSO4.7H2O 2.2 g dissolved in 100 ml D.W
CoCl.6H2O 1.0 g dissolved in 100 ml D.W
MnCl2.4H2O 18.0 g dissolved in 100ml D.W
Na2MoO4.2H2O 0.63 g dissolved in 100 ml D.W
** HOW TO PREPARE THE VITAMINS
VITAMIN STOCK SOLUATION
THIAMINE HCL 0.1 g dissolved in 1000 D.W
BIOTINE 1.0 g dissolved in 1000 ml D.W
B 12 1.0 g dissolved 1000 ml D.W
