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Strain Information, Culturing & Media FAQ

1. What is the salinity of your seawater?
The Gulf of Maine Seawater we offer is filtered seawater for the base of our prepared marine media L1, f/2, K and Prov50 is approximately 31-34 ppt (parts per thousand) salinity. Sargasso Seawater salinity, which we offer unfiltered or in Pro99 media, is approximately 35 ppt.

2. How is your seawater filtered?
We have a state-of-the-art, continuously flowing seawater system, extracting seawater from the adjacent bay. It is pumped through three bag filters into a 3,000-gallon holding tank at a shore facility; it is sand filtered then a variable frequency drive (VFD) pump moves the water up to the seawater laboratory. There it is filtered through a three-stage cartridge filter system down to 1-micron prior to use.

3. Can I modify the recipe of the culture media when using one of your media kits?
Yes. Feel free to use our kits as the base for your media. You may eliminate components you feel unnecessary i.e. silicate if not growing diatoms. You may vary the concentrations for experimental purposes or you may add additional components. You may also wish to make reduced salinity media as we do at NCMA for some strains. We do, however, recommend following the recipe if you are unsure. Our strain information will list the optimum media as best we know for strains in our collection.

4. May I re-sterilize the components in the kit after I use them?
In most cases, yes. However, if you autoclave them, you will lose a slight amount of liquid thereby increasing the concentration of the component proportionally. Autoclaving ammonia will drive some off thereby decreasing the ammonia concentration. Vitamins should be refrigerated. Autoclaving the vitamins repeatedly will degrade them. See culturing methods page.

5. Should I add the components in my media kit aseptically or may I add them to water and then autoclave the media?
You may do either as long as the final product is sterile. We recommend handling all nutrients aseptically as they can become contaminated. See culturing methods page. At the NCMA, nearly all our media are prepared by autoclaving the nutrients components in their water base. All of the prepared marine media that we offer for sale is made by adding sterile nutrients aseptically to autoclaved seawater. See culturing methods page.

6. I don’t have access to an autoclave. How can I keep everything sterile when I make my culture media?
You may purchase ready to use sterile media from the NCMA. You would then just need sterile culture vessels, pipettes, and other items that will come into direct contact with your algal cultures. These sterile items are available from scientific supply houses such as VWR. You can also sterilize equipment and media with good success in a microwave. The reference at the end of our FAQs describes the microwave sterilization protocol. Using the best sterile technique is always the best practice. See culturing methods page.

7. What does L1-Si (or f/2 –Si) mean?
This refers to our L1 and f/2 media that are made without silicate. Silicate can cause precipitates in media, which can inhibit the growth of some strains. Silicate is only necessary to culture diatoms or other organisms that have a silica requirement. See culturing methods page.

8. Can I use f/2 instead of L1?
In most cases these two are interchangeable. The difference between f/2 and L1 is the trace metal component; f/2 has fewer components in its trace metals than L1. Please see our media recipes for details.

9. Can I use artificial seawater?
See culturing methods page. At the NCMA, we maintain our marine strains using enriched seawater media only, and therefore are not able to advise about the use of artificial seawater media. We have been told that some species do not grow well in artificial seawater media, but this must be discovered empirically.

We strongly recommend, using enriched seawater media when you first receive your cultures. If you do not have access to natural seawater, you can purchase natural seawater or prepared media from the NCMA or any number of sources. Once you have your strain established, you can try acclimating it to the artificial SW medium you wish to use.

10. What is the content of your medium kits?
Media kits include enough sterile nutrients components to make the quantity designated on the kit. That is, a 50L L1 medium kit will have enough components to make 50L of that L1. The medium recipes on our website are included in our kits. Those recipes describe not only the chemical makeups of the kit components, but also advise as to preparation methods. See culturing methods page.

11. What type of growing vessel should I use?
See culturing methods page. Please note that we primarily use borosilicate glass tubes. Occasionally we use tissue culture "T" flasks.  If you wish specific information as to which strain grows in what vessel, please contact us.

12. Can I keep my cultures in the refrigerator?
No, photosynthetic organisms need light and the correct culture temperature.

13. Can I put my cultures in continuous light?
Phytoplankton are photosynthetic organisms and adapted to the natural rhythm of day and night. Some organisms will tolerate continuous light, but we maintain ALL our strains on 13:11 light dark cycle. We do not recommend 24 hour continuous light exposure upon receipt of strains from the NCMA, and you will have to discover empirically if your strain of interest will tolerate continuous light.

14. What lighting does the NCMA use?
We use soft white fluorescent bulbs. Our light levels range from 10µEinsteins (for just a few strains that require low light levels) to 230 µEinsteins, with the majority of our strains being grown at approximately 80 µEinsteins. Every single species, and even different strains within a species, has different optimal light requirements. We use a 13:11 light dark cycle. We do not grow any algal strains in conntinuous light. Please see FAQ number 13 for more details.  

15. Should I, or can I aerate or agitate my culture?
We do not agitate or aerate our stock cultures at all here. We aerate large volumes such as carboy size (5L or more) with sterile filtered air.

Do not aerate small volumes with CO2 because it will lower the pH and may kill the culture.

Some algal species such as dinoflagellates and large diatoms will not tolerate agitation. Great care is advised should you decide to grow such species in larger volumes.

16. Can you tell me how to grow my algae?
The process of culturing algae involves not only good sterile technique, but also knowledge of the growth conditions (light, temperature and media), growth habit, and the rate of growth that your strain of interest demonstrates. To succeed, you must have a working knowledge of these parameters.

We offer a consultancy service, and can assist you with basic algal maintenance, etc. For more information please contact us.

17. Can strain be grown under conditions other than those at the NCMA?  (i.e. different temperature, medium, etc.)?
 Yes, but determining alternative conditions is an empirical task. Information about the water sample your strain came from (collection site, water temperature, time of year, salinity, etc) can furnish clues about tolerated growing conditions.

18. Can I grow algae from harvested cells?

No.

19. What is a toxic strain?
NCMA defines toxic strains as strains that belong to species that have been known to produce toxins. We do not routinely test for toxins in cultures, therefore, we consider all strains that are members of a toxin producing species as potential toxin producers. We consider best practices to handle the transfer of all strains in the same way:

  • We consider them biolevel 1
  • We use good asceptic technique, including a clean bench, when handling them
  • We use proper laboratory protocol when handling them, such as no pipetting
  • We dispose of our cultures in a way that does not contaminate the water supply
We follow these steps when working with 18ml cultures.  We cannot answer for working with larger volumes of culture, where greater volumes of waste are created and the possible creation of aerosols.

The Bigelow Analytical Services group (BAS) has the ability to test for a range of algal toxins. If you are interested in confirming (or not) the toxicity of the strain you've purchased, please reach out to the BAS directly. 

20. What does “axenic” mean?
Not contaminated by or associated with any other living organisms, essentially a pure culture. HOWEVER, that definition is only as good as the testing protocol (see 21). NCMA defines axenic as a pure culture based on lack of bacterial/fungal growth on our test medium (see 21). It should be noted that this is not a foolproof method, only sequence analysis of (e.g.) ribosomal markers will give a definitive determination of a culture’s axenic state.

21. How do you test for bacteria at the NCMA?
We routinely test our axenic marine algal strains by subculturing strain to be tested TM and f/2PM liquid medium simultaneously. View recipe page.We use these media to enrich for possible bacterial and fungal contaminants. We then incubate these subcultures in the dark for at least three weeks to allow any contaminants to grow. We check them for such growth at least twice a week. We do not do DNA analysis on our strains; thereby our definition of axenic is limited to what we can culture in our test media. In the same way we test our marine strains, we test our axenic freshwater strains, but using a freshwater test medium. View recipe page.

22. Will my culture grow on agar?
We maintain few strains on agar. Our generic agar recipe can be found on our medium recipe page. View recipe page. Most green algae, coccoid algae, and pennate diatoms will grow on agar but that has to be determined empirically.

23. Why is my culture not growing?
Your culture may be growing but biomass may not be visible to the naked eye. Examination with a dissecting microscope or a light microscope can determine if there is actual growth.

If there is living material, but not growing at the rate that you expect, your culture may still be recovering from its journey. Or the problem can be any of the following: a. your medium b. your seawater c. your temperature d. your light e. the state of your glassware (was detergent used to clean it?)

24. I was looking at one of your strains on your website just the other day and now it’s gone, where did it go?
If it is no longer on our website, then it is no longer available for sale. This can be due to a number of reasons such as poor growth, contamination with a eukaryote, etc. When the issue is resolved, it will be visible on the website again.

25. What is a cold strain?
A cold strain is one that is grown at 4 ° C, and is likely from a water sample collected in a polar region.

26. What is an aquaculture express strain?
There are 14 strains that we maintain for next day shipment. These strains are robust, good travelers and are used often by the aquaculture industry.

27. What’s an easy strain to grow?
Click here to see our “Robust” strains.

28. I would like to cryopreserve my strain; can you provide me with the protocol?
On our website, associated with each strain record, you will find a broad outline of how the strain was cryopreserved if we were able to cryopreserve it.

We have been cryopreserving algae at the CCMP/NCMA since 1996. We use a controlled rate freezer and store all of our cryopreserved material in large dewers in liquid Nitrogen vapor. We use DMSO as our primary cryoprotective agent and, second most common, methanol.

We cryopreserve most diatoms in 12% DMSO. Our success with small centric diatoms is about 60%. We cryopreserve haptophytes using DMSO (6 and 9%) with about a 70% success rate. Our success with marine Plantae is about 98%, using DMSO as well as MeOH. We have had more limited success with large organisms, macrophytes and other algal groups. All the above are generalizations. Cryopreservation of algae is an empirical process with every strain of every species potentially requiring different protocols.

Click here for general description of cryopreservation, materials, and equipment requirements.

29. Why can’t it be stored at -80°C?
Temperatures lower than -137°C (the glass transition temperature)  is essential for archival storage of cryopreserved material. There is no crystal growth at that temperature or lower, and therefore no damage to cells stored at those temperatures.  Appropriate storage temperatures can be achieved by storing in liquid nitrogen (LN2) vapor, in LN2 (not recommended) or in mechanical freezers. 

30. Why is the strain species name sometimes different than when I ordered it before?

From time to time, you may see different scientific names (or species identifications) for the strains that you may routinely order. This can be because the strain was originally mis-identified. More likely, new scientific names are a result of recent genetic research using, among other things, DNA analysis. Though the species identifications may change, the strains themselves and the original CCMP strain number have not.

For example, CCMP1332 and CCMP2092 formerly, Skeletonema costatum, have been assigned to the Skeletonema species marinoi (CCMP1332 in publication in  Protist 159.2 (2008): 177-193) and CCMP2092 identified by Sarno, private communication, 2011).  CCMP1324 and CCMP463 have been assigned to a new genus and species, Tisochrysis lutea (publication in J. Appl Phycol 2013).

 

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