5. General Culture Procedures
5.1 Stock Culture of Rotifers
Culturing large volumes of rotifers on algae, baker's yeast or artificial diets always involves some risks for sudden mortality of the population. Technical or human failures but also contamination with pathogens or competitive filter feeders are the main causes for lower reproduction which can eventually result in a complete crash of the population. Relying only on mass cultures of rotifers for reinoculating new tanks is a too risky approach. In order to minimize this risk, small stock cultures are generally kept in closed vials in an isolated room to prevent contamination with bacteria and/or ciliates. These stock cultures which need to generate as fast as possible large populations of rotifers are generally maintained on algae.
The rotifers for stock cultures can be obtained from the wild, or from research institutes or commercial hatcheries. Before being used in the production cycle the inoculum needs to be disinfected. The most drastic disinfection consists of killing the free-swimming rotifers but not the eggs with a cocktail of antibiotics (e.g. erythromycin 10 ppm, chloramphenicol 10 ppm, sodium oxolinate 10 ppm, penicillin 100 ppm, streptomycin 20 ppm) or a disinfectant. The eggs are separated from the dead bodies on a 50 mm sieve and incubated for hatching. The offspring are used for starting the stock cultures.
If, however, the rotifers do not contain a lot of eggs (can be the case after a long shipment) the risk for loosing the complete initial stock is too big. In this case the rotifers will be disinfected at sublethal doses: the water of the rotifers is completely renewed and the rotifers are treated, either with antibiotics or disinfectants. The treatment is repeated after 24 h in order to be sure that pathogens which might have survived the passage of the intestinal tract of the rotifers are killed as well. The concentration of the disinfection products differs according to their toxicity and the initial condition of the rotifers. Orientating concentrations for this type of disinfection are 7.5 ppm furazolidone, 10 ppm oxytetracycline, 30 ppm sarafloxacin, or 30 ppm linco-spectin.
Figure 3.4. Stock cultures of rotifers kept in 50 ml centrifuge tubes. The tubes are fixed on a rotor. At each rotation the medium is mixed with the enclosed air.
(click to see the video)..(click to see Real Player movie)At the Laboratory of Aquaculture & Artemia Reference Center the stock cultures for rotifers are kept in a thermo-climatised room (28°C ± 1°C). The vials (50 ml conical centrifuge tubes) are previously autoclaved and disposed on a rotating axe (4 rpm). At each rotation the water is mixed with the enclosed air (± 8 ml), providing enough oxygen for the rotifers (Fig. 3.4.). The vials on the rotor are exposed to the light of two fluorescent light tubes at a distance of 20 cm (light intensity of 3000 lux on the tubes).
The culture water (seawater diluted with tap water to a salinity of 25 ppt) is aerated, prefiltrated over a 1 mm filter bag and disinfected overnight with 5 ppm NaOCl. The next day the excess of NaOCl is neutralized with Na2S2O3 and the water is filtered over a 0.45 mm filter.
Inoculation of the tubes is done with an initial density of 2 rotifers.ml-1. The food consists of marine Chlorella centrifuged and concentrated to 1-2.108 cells.ml-1. The algal concentrate is stored at 4°C in a refrigerator for a maximum period of 7 days, coinciding with one rotifer rearing cycle. Every day the algal concentrate is homogenized by shaking and 200 ml is given to each of the tubes. If fresh algae are given instead of the algal concentrate 4 ml of a good culture are added daily.
After one week the rotifer density has increased from 2 to 200 individuals.ml-1 (Fig. 3.5.). The rotifers are rinsed, a small part is used for maintenance of the stock, and the remaining rotifers can be used for upscaling.
Also after some months of regular culture the stock cultures will be disinfected as described earlier in order to keep healthy and clean stock material.
1. Introduction
2. Morphology
3. Biology and Life History
4. Strain Differences
5. General Culture Procedures
