A large variety of media have been designed for the cultivation of cyanobacteria (see Rippka. 1988, for review). For convenience, only three standard media are currently employed for the maintenance of strains in the PCC, with minor modifications for certain strains (Rippka et al., 1979). Medium BG-11 is used for strains of freshwater, soil or thermal origin, and for those isolated from a marine environment, which do not display the ionic requirements characteristic of true marine strains (Waterbury & Stanier, 1981). A modified BG11 lacking nitrate, medium BG-11o, is used for nitrogen-fixing strains after addition of a solution of filter-sterilized NaHC03 (5 mM final concentration). True marine strains, i.e. those having elevated requirements for Na+, CI-, Mg2+ and Ca2+, are generally maintained in medium ASN-III or, more rarely, in medium MN, which has a natural seawater base supplemented with medium BG-11 at half strength. All strains that grow in medium ASN-III can also be maintained in MN. However, if medium MN is recommended, it means ASN-III has proved to result in poor growth. Media ASo-III and MO are the respective media, lacking nitrate, for the cultivation of marine nitrogen-fixing strains.

The purity of the strains may be confirmed by placing an aliquot of cell material provided onto solid growth medium supplemented with glucose (0.2 %) and casamino acids (0.02 %). We test each transfer at the PCC to control the continuous axenicity of the strain. Test plates should be incubated in the dark for 2-3 days at the recommended growth temperature prior to microscopic examination using phase contrast objectives and oil immersion.

The practical way is to first obtain a nice healthy pre-culture, and to then increase the volume at each transfer. Note that not all strains are suitable for massive growth, but some do the same way they bloom in the natural environment. Large-scale cultures should be inoculated with heavily grown pre-cultures at a dilution of approximately 1/20 (v/v). This implies to grow for example a 50 mL culture, to transfer it, once it has reached high density, into a 1L culture. Direct transfer of the small amount of cell material, such as 5 mL provided by us, into culture volumes greater than 50 mL is not recommended.

The most reliable method (though with variable viabilities depending on the strains) of cryoconservation for cyanobacteria we use is storage in liquid nitrogen using 5 % (v/v) of DMSO as cryoprotectant. The DMSO can be bought from Sigma as a sterile solution. The cell suspensions of a healthy grown culture (1.5 mL) are placed into Nunc cryovials (2 mL) and plunged directly into liquid nitrogen. The cryopreserved aliquots are stored in a liquid nitrogen tank or in a -150 °C freezer. Be aware that the planktonic strains, such as Arthrospira, Microcystis and Planktothrix, are more sensitive to cryoconservation. Before freezing them, it is necessary to collapse the gas vesicles by pressure through a syringe for example and of course to keep all that sterile! Recovery is done by quick thawing the cryovials at 37 °C, followed by an immediate transfer of the cell suspension into fresh medium respecting a dilution that results in a carry-over of DMSO of not more than 0.5 % (v/v). For several strains, we keep the freshly inoculated culture into darkness for 48 hours (over the weekend), and progressively give access to light from Monday to Wednesday to give them time to adapt. To recover the nice and healthy culture without any DMSO, two to three successive transfers will be needed. It could be useful to test the recovery of the strain from cryopreservation before stopping the live culture. We always have a batch for testing and we prepare 3 tubes per batch for longer storage. Cyanobacteria are very sensitive to detergents. Sometimes, dry drops of detergent can stay on the Erlenmeyer’s walls after cleaning the glass and can diffuse very efficiently into the medium. Thus, the cryopreserved culture that already does not like the DMSO, once back to life, has to fight also against the detergent...it dies very efficiently: within 12h to 24h the freshly inoculated culture turns from green to yellow (=death). Some strains show good tolerance to cryopreservation and recover within a month; however, some others are really affected, such as the planktonic strains, which recover after four months. Be patient, as we do recover PCC strains, some of which had been cryopreserved in the 70’s. References cited Rippka, R. (1988). Isolation and purification of cyanobacteria. Methods Enzymo 167, 3-27. Rippka, R., Demelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen Microbiol 111, 1-61. Waterbury, J. B. & Stanier, R. Y. (1981). Isolation and growth of cyanobacteria from marine and hypersaline environments. In The Prokaryotes, vol. l, pp. 247-256. M.P. Starr, H. Stolp, H.G. Troper, A. Balows, H.G. Schlegel, Eds. Berlin, Heidelberg, N. Y.: Springer-Verlag. Waterbury, J. B. & Willey, J. M. (1988). Isolation and growth of marine planktonic cyanobacteria. Methods Enzymo 167, 100-105.

The Collection PCC will reply to your request of PCC strains upon reception of the filled “Declaration to order PCC strains” and “Customer account form”. With this information, the Collection PCC will prepare an MTA, named CGU “General Terms and Conditions of Use”. The GCU, with the prices for the strains including shipment fees indicated in Euro, will be sent to you to be signed by the responsible scientist and the institution in which she/he works. The order will be processed only if the CGU is properly signed. In addition, we will also need a corresponding formal Purchase Order from your institution; please ask for it to your administration.

The order limit for these living cyanobacterial strains is generally up to five PCC strains. For five or less strains, it might be feasible to send them all at once. For additional strains, be aware that they might not be ready for dispatching at the same time, and thus will eventually need to be dispatched in separate deliveries.

Once we have the “Declaration to order PCC strains” and “customer account form” filled, we can ask for a quote to the Account Department of the Institut Pasteur.

When we receive the GCU duly completed and signed, and also the payment for the requests from outside the European Union, it takes about one month for living cultures and one to four months for strains that need to be recovered from a cryopreserved stock. We let you know the preservation format of your strains of interest in our first email communication. For dispatch to countries outside the European Union: For shipments out of the European Union, we require payment in advance . As soon as the CGU has been signed, we will send you a pro forma invoice/quote. Make sure to refer to the number of the pro forma invoice on your wire transfer or payment. You must check with your country’s customs office which documents, i.e, import permit, are needed before ordering. Please provide us with such document(s) to be added in the parcel before dispatch. We will send the strain(s) once we receive your payment and the basic documents needed for the dispatch.

We record every request for our quality management; thus, your order is not forgotten, it is under process, notably if you have sent us the requested documents. We ask you before shipment if you will be present for reception of the living strains. When the order is dispatched, we send a warning email to the contact person you have provided, with the tracking number provided by the transport company.

All the PCC strains are shipped alive and pure, either on slants or as liquid cultures (5 ml aliquots in sterile screw-cap plastic tubes), particularly for gas-vacuolated planktonic strains that grow poorly on solid media. The PCC strains are Class 1 organisms and we use regulation-compliant packaging for the transport of this material in compliance with health and environmental safety standards and under applicable laws and regulations.

Upon receipt of a culture from the PCC, an immediate transfer of at least part of the cell material is recommended at a dilution of approximately 1/10 (v/v) to 1/20. Details of culture media and growth conditions are indicated in the online catalogue and also supplied with each strain. PCC cultures that have been delivered cannot be returned.

Details of culture media and growth conditions are indicated in the online catalogue by typing the PCC number and opening the catalogue datasheet and/or culture medium.

Cultures will normally be replaced free of charge if they arrive in an unsatisfactory condition, provided that the PCC is notified within 28 days of dispatch; however, in that case the costs of packaging and transport must be borne by the customer. The PCC declines responsibility for accidental contamination on subculture in the laboratory of the customer. Each strain dispatched from the PCC is presently checked for identity and purity prior to shipment. In the rare event of initial failure to detect the presence of contaminants due to their poor or slow growth, the customer will be advised immediately and will be provided as soon as possible thereafter with an axenic replacement culture completely free of charge.