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1. Subculture methods

1.1. Microalgae, protozoa, and freshwater red algae

You will receive the culture strains in a screw-cap test tube. Slightly loosen the screw cap and keep the test tube in an appropriate place, as indicated in individual strain data. If you want to maintain the culture strain, please transfer the culture into fresh medium according to the following methods.

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1.2. Charales

You will receive several pieces of thallus. As soon as you receive them, transplant them into fresh culture media according to the following methods.

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2. How to prepare stock solutions and media

2.1. Stock solutions

Media are generally composed of three types of components; macronutrients, trace metals, and vitamins. For convenience we recommend to prepare stock solutions of these components in dark glass bottles. Stock solutions of trace metals and vitamins are prepared at extremely low concentrations, and therefore required dilution steps. The following methods are currently used at the NIES-Collection.

2.1.1 Macronutrients
Prepare stock solutions of individual macronutrients separately at a concentration of 10 mg/mL, and store them in a refrigerator (5℃).

2.1.2. Trace metals
These elements are prepared as either separate stock solutions or mixed stock solutions.

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2.1.3. Vitamins
Vitamins requirement is in majority fulfilled with three vitamins; vitamin B12, biotin, and thiamine HCl. Therefore, most of the media contain only these three vitamins. However, several media contain additional vitamins.

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2.2. Media preparation

Two categories of media are usually used; synthetic and enriched. The former is used for maintenance of all freshwater algal cultures and some marine ones and the latter for most marine ones. Most of the media are dispensed to test tubes and autoclaved before use, whereas some media must be filter sterilized.

2.2.1. Synthetic medium for freshwater algae

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2.2.2. Synthetic medium for marine algae

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2.2.3. Enriched seawater medium

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2.2.4. Filter sterilization
MNK medium must be filter sterilized by using a filter apparatus with a filter (Millipore 0.22μm), which is previously autoclaved (121℃, 20min). Then, the medium is dispensed into previously sterilized test tubes by using a sterilized syringe or dispenser under aseptic conditions.

Filter sterilization

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2.2.5. Agar slants
Agar is usually added at a concentration of 1.5% after liquid medium has been prepared, and before autoclaving.

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2.2.6. Medium for protozoa
These media contain organic matter to encourage multiplication of bacteria as a food source for protozoa. For media containing wheat or rice grains, these cereals should be sterilized by dry heat (150℃, 30min) in advance, and kept in a cool place. For use, one grain of cereal is added to 10mL of medium.

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2.2.7. Medium for Charales

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3. Media list

Please click here to go to Media list.

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4.4. Methods of cryopreservation

A two-step freezing protocol is used in the NIES Collection: algal culture is cooled to -40℃ by a programmable freezer and then cooled rapidly to 196℃ in liquid nitrogen. Most cyanobacterial strains, some strains of green and red microalgae, and some strains of freshwater red algae are cryopreserved by the methods described in 4.1 and 4.2. Detailed methods for microalgae are also explained in Mori et al. (2002) and Mori (2007).

REFERENCES
Mori, F., Erata, M. & Watanabe, M. M. 2002 Cryopreservation of cyanobacteria and green algae in the NIES-Collection. Microbiol. Cult. Coll. 18:45-55.
Mori, F. 2007 Cryopreservation methods of microalgae. Microbiol. Cult. Coll. 23:89-93. (In Japanese)

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4.1. Cryopreservation of microalgae

4.1.1. Materials and instruments
i) Culture: late log or early stationary phase cultures.
ii) Medium: appropriate sterile medium for each strain.
iii) Cryoprotectant: 6% dimethyl sulfoxide (DMSO) for cyanobacterial strains, and 10% DMSO for green and red algal strains dissolved in the appropriate media. These concentrations are double the final concentration. DMSO is previously sterilized by filtering through an alcohol-stable filter (Millex-LG).
iv) Laminar-flow cabinet and materials for aseptic treatment.
v) Cryovials: 2-mL presterilized polypropylene cryovials, pre-labeled with the strain number and date.
vi) Programmable freezer (e.g. Planer Kryo 320-1.7 is used in the NIES-Collection).
vii) Liquid nitrogen Dewar vessel: 10-L wide-neck Dewar vessel (Shattle Drum JIK-S10).
viii) Long forceps (19cm), cryogloves, a cryoapron, and goggles.
ix) Nunc polycarbonate storage boxes, 8-decker stainless-steel racks, a liquid nitrogen tank (Taiyo Nippon Sanso DR-245LM; vapor phase).
x) Water bath (e.g. As-One-Corp. Thermal Robo TR-1).

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4.1.2. Freezing
i) The processes ii)-iv) should be done under aseptic conditions.
ii) Dilute the cryoprotectant with appropriate medium to obtain double the final concentration, and cool it on ice.
iii) Dispense 0.5 mL of cell suspension (late log or early stationary phase culture) into each labeled 2-mL-cryovial.
iv) Add 0.5mL of the cryoprotectant (diluted and cooled) to each cryovial and mix well.
v) Leave the cryovials at room temperature for 15 min.
vi) Place the cryovials in a programmable freezer (Photo 9), and start cooling at -1℃/min to -40℃.

vii) Hold the cryovials in the programmable freezer at -40℃ for 15 min.
viii) Transfer the cryovials rapidly to the Dewar vessel containing liquid nitrogen (Photo 10). ix) After 1 h, transfer the cryovials in the Dewar vessel to a storage box and place the box on a stainless-steel rack set in the vapor phase of liquid nitrogen in a liquid nitrogen tank (Photo 11).

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4.1.3. Thawing
i) Preheat a water bath to 40℃.
ii) Shake the cryovials well in the water bath until the last ice crystal in the cryovials has melted (Photo 12).

iii) Under aseptic conditions transfer the contents of the cryovials into test tubes each containing fresh liquid medium. Incubate under dim light for a few days (depending on the strain), and transfer to ordinary culture conditions as suggested in the strain data.

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4.2. Cryopreservation of freshwater red algae

4.2.1. Materials and instruments
i) Culture: several thalli cultured for at least 2 weeks after the last transplantation. If a thallus is large, cut it into small pieces with scissors or tweezers, and culture for more than 2 weeks (for recovery), before use.
ii) Medium: sterile Bold 3N medium.
iii) Cryoprotectant: 40% dimethyl sulfoxide (DMSO) for cryopreservation of Thorea okadae, T. hispida, and Nemalionopsis tortuosa; and 30% methanol for N. tortuosa. These concentrations are double of the final ones. DMSO and methanol are previously sterilized by filtration through an alcohol-stable filter (Millex-LG), and dissolved in sterile Bold 3N medium.
iv) Instruments: same as the instruments for microalgae.

4.2.2. Freezing
i) Dilute the cryoprotectant (DMSO or methanol) with medium to obtain double the final concentrations (40% or 30%, respectively), and cool it on ice.
ii) Dispense a 0.8 mL aliquot of culture into each of the cryovials.
iii) Add 0.8 mL of 40% DMSO or 30% methanol to ii), and mix well. In the case of DMSO, leave the cryovials at room temperature for 15 min.
iv) Then same as 4.1.2 vi) to ix).

4.2.3. Thawing
i) Preheat a water bath to 40℃, and cool appropriate amount of medium in ice water.
ii) Shake the cryovials well in the water bath, and transfer the cryovials into ice water just before the last ice crystals have begun melting.
iii) Transfer the contents of the cryovials quickly into 50-mL centrifuge tubes, add 40 mL of cold medium, and leave the tubes until the thalli have settled to the bottom.
iv) Remove the supernatant with a pipette.
v) Add 40 mL of cold medium again, and again remove the supernatant with a pipette after the thalli have settled.
vi) Transfer the thalli into 60 mL of fresh media in 100-mL conical flasks, and incubate under the culture conditions suggested in the strain data.
vii) All manipulations from iii) to vi) should be done under aseptic conditions.

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