**5.1 Water**

Daphnia can be maintained in reservoirs of natural water; however it is suggested an environment of synthetic water and should be standard quality reconstituted in order to generate predictive results and allow an adequate reproduction and growth of the culture. Hardness water recommended is 160-180 mg CaCO3/L, dissolved in deionized water and aerated for few hours. pH closed to 8.0. Normally stays between 7.0 and 8.6, no monitoring or adjustment necessary. Requirements to prepare the hard water: 192 mg/L of NaHCO3, 120 mg/L of CaSO-4, 120 mg/L mgSO4, and 8.0 mg/L of KCl (American Public Health Association, 2002).

#### **5.2 Food and fed**

Daphnias usually are fed *in vitro* with different type of algae, generally *Ankistrodesmus falcatus, Selenastrun capricornutum,* and *Clorella.* A mixture is prepared in sterilized water, adding two drops of solution per each Daphnia adult culture. Another diet to use is adding 6.3 g of trout pellet, 6.2 g of yeast and 0.5 g of alfalfa. Mix for 5 minutes with sterilized water, gauge to 500mL, decant for an hour and get rid of the surplus. Freeze 50 to 100 mL portions and do not save it for more than eight days. Use It to feed only 0.5 mL per each 1000 mL of culture medium, with a 3 times a week frequency; in an aerated environment and replaced every week (American Public Health Association, 2002). The yeast (*Saccharomyces cereviseae*) is a probiotic. FAO (2001) declare that probiotics are "living microorganisms, when managed in adequate amounts, present to the host health benefits" (Austin & Brunt, 2009, as cited in Montet & Ray, 2009).

#### **5.3 Temperature**

Temperature changes can induce death or induce ephippium production or sexual eggs. The ideal temperature is between 20°C and 25°C.

#### **5.4 Light**

Light intensity variations between 538 and 1076 lux and the prevalence of the light during the day/night cycles; do not affect the reproduction and growth of Daphnia cultures significantly. Try to provide a minimum of 12 hours of light / 12 hours of darkness.

#### **5.5 Containers**

Glass or plastic containers should be used, in order to observe easily the cultures, they should have a capacity of 3 L, with 2.75 L of medium, and 30 Daphnias, will be able to produce approximately 300 neonates and young Daphnias per week. It is necessary to clean weekly, removing the food left and the death Daphnias. Wash the containers monthly with detergent and replace the medium completely.
