JIAC26+ : Introducing some new strains to this study
Hello everyone,
So I mentioned in some of my previous lab notes that I'm still working on PCR, which are actually pretty much all done by this time. Many of these reactions, most notably those for the UPA and tufA genes, are repeated reactions for the same strains of algae I originally introduced in previous lab notes, which failed terribly due to bacterial contamination. After I treated them with antibiotics, I am going to see if they will sequence more cleanly now and can produce informative data, or should be ruled out as impractical for any sort of sequenced-based identification protocols whatsoever, due to their incompatibility with wild, contaminating bacterial DNA. (Technically, they've already been pretty problematic, but I'm giving them the benefit of the doubt here - long story which I'll elaborate on as my research continues).
However, I've also mentioned that I've been adding some more strains to my project in a rolling fashion, so that's why my PCR has been dragging out for so long. As I am approaching the end of my budget for this project and I haven't been able to collect water from new sources, my collection has now started to slow its growth, so I figured it would be a good time to introduce a few of these new algae!
And here they are. It's a very large picture, so I highly recommend that you open it up in a new tab and zoom in to get the best detail of each tiled image.
Now that I've been in the business of isolating and describing algae, I know how important it is to record all observations - visual and otherwise - very thoroughly, so for these pictures, I've used a camera specifically designed for microscope use to take these photos. This is now a part of my home setup, so photo quality is much improved and it's possible to make out a lot more detail and in better resolution than before, when I just used a phone to take pictures through an eyepiece. For a dedicated page with all algal-related photos and less raw science, I post on the Instagram @algaenthused, so check that out if you're interested.
Anyhow, to describing the algae at hand. The new cultured strains I've added to my collection for this project are: JIAC26, 27, 28, 13i, 29, 30, 31, 32, 33, 34, 35, and 36. However, of these, 5 strains (JIACs 29, 31, 34, 35, and 36) are all of either the genus Scenedesmus or Desmodesmus, and are likely isotypes (essentially "backup copies") of each other. JIACs 32 and 33 are isotypes as well. This means that they really won't look very special or different, so I boiled the list of pictures down so that each one represents an entirely different species of alga.
JIAC26 is almost certainly a species of algae from the genus Asterococcus, literally meaning "star berry" from its Greek roots. The "star" refers to the pattern of the chloroplast within the cell, and coccus refers to any round-shaped cell, used to describe bacteria as well as fungi and algae. This alga, in the order Volvocales, is closely related to the famous model organism Chlamydomonas reinhardtii and my other strains JIAC4 and JIAC12, the latter two of which form thick and slimy "blobs" along with JIAC26. This is seen microscopically as a slightly-lighter-colored "halo" surrounding colonies of cells, which is composed of a polysaccharide or sugary gel. I believe that this may help cells either attach to surfaces or resist drying out in their natural habitat. JIAC26 was collected from Locke-Hadden Park in Marina, CA, a few minutes north of Monterey, on June 8, 2017, and this photo was taken at 1000x.
JIAC27 was collected at Locke-Hadden Park in the same sample as JIAC26. However, its cells are much smaller, even though its photo is taken at 400x magnification. Because it is a classic "little green ball", referring to these sort of tiny, amorphous green cocci-shaped algae with no identifying characteristics, I cannot say definitively what this is. However, if I had to make an educated guess, I'd say a species of Chlorella. I really hope that it is Chlamydomonas of some kind, but as it has no visible red eyespot or flagellar movement, I doubt that that will be the case.
JIAC28, seen in this picture at 100x, is a filamentous alga, collected from Locke-Hadden Park as fuzzy green colonies on sticks and other submerged material. On agar media, it rarely forms large colonies but instead just clusters of these small "shoots". I really don't know what this is, so my wild guess is Bulbochaete sp., a genus of filamentous branching algae in the order Oedogoniales. I will be very surprised if I get that right, however.
JIAC13i is interesting; it's photographed here at 400x. The "i" designates an isotype, again meaning that it is in simple terms an identical copy of another strain, in this case JIAC13. JIAC14 and JIAC17 are also isotypes of JIAC13 (JIAC17 is now lost). I isolated this strain of algae late this summer from my last remaining plate that was streaked with Chico vernal pool water I collecte in the early spring, during a time when my cultures still suffered from serious cyanobacteria contamination and I was afraid I would lose many more cultures (I lost JIACs 11, 16, 18, and 22 to cyanobacteria). It is thus a strain of Chlorella variabilis, a species commonly present as an endosymbiont in many freshwater protozoa but (as far as I know) not usually cultured on its own in vitro.
JIAC30 is the next strain shown here, in a photo taken at 400x. (Just as a note: all strains on the bottom row of tiled images are shown at 400x magnification.) This one is pretty exciting for me, because I am quite certain it is a species of Selenastrum. The most famous species of this genus has itself undergone several taxonomic revisions, changing names from Selenastrum capricornutum to Pseudokirchneriella subcapitata to Raphidocelis subcapitata, but in any case, its defining features - curved, stubby crescent-shaped cells and a thin polysaccharide matrix surrounding it - are all seen in my culture. This is the first time for me that I have cultured a strain in the family Selenastraceae that is NOT either Ankistrodesmus or Monoraphidium, two genera with very slender and "sharp" crescent- or sickle-shaped cells. JIAC30 comes from Lloyd Lake in Golden Gate Park, San Francisco, when I collected them in late July of this year.
JIAC33 is another new find for me, and again I'm pretty happy I can identify it by eye. I'm confident that it is a species of Dictyosphaerium, an alga in the class Trebouxiophyceae and thus related to Chlorella. It is primarily distinguished visually by these ring-shaped colonies (which can be spherical and extend in 3 dimensions too) which appear to have "stalked" cells. These stalks, which usually branch in powers of two, are actually pieces of old cell wall from old dead cells of the mother colony. JIAC32 is a functionally identical strain. These were both collected and isolated from Lloyd Lake as well.
One of four very similar strains, JIAC35, imaged here, is easily visually identified as Scenedesmus or Desmodesmus. Both genera of algae form four-celled coenobia, or colonies of identical cells, which are instantly recognizable and found in a majority of aquatic ecosystems with slow-moving or still water (most of the coenobia seen here have only two cells because they are old and have started to break apart). The two genera can be distinguished by the presence of small spines on the tips of each cell (not to be confused with the larger "horns", which are used to help the colony float in the water column and best collect sunlight), but I haven't really gone through and examined each strain of Scenedesmus (I call all of them that by default until molecular data proves me wrong) thoroughly. I could, but after reading a variety of articles that details how species of Scenedesmus exhibit morphological plasticity, meaning that they can change their visual appearance depending on environmental conditions, I figured it'd be easier to just let the DNA sequences sort this out for me. JIACs 29, 31, and 34 all appear close enough to JIAC35 to be too boring to show together, and all four of these strains come from Lloyd Lake.
Finally, JIAC36 is another species of Scenedesmus (this I can say with confidence), and if I had to give a guess as to the species, probably S. obliquus or S. dimorphus. Both of these species have been investigated for their biofuel potential, and S. obliquus is one of very few green algae that has also been experimented with for genetic engineering, something I might be interested in trying myself eventually. It can be distinguished from many other species of Scenedesmus and Desmodesmus by its sickle-shaped cells, which resemble those of algae from the family Selenastraceae. In fact, when they are solitary, cells of S. obliquus may be very hard to distinguish from Selenastraceae algae by light microscopy alone; oftentimes, the formation of coenobia is the only way to tell them apart (most of the very skinny Selenastraceae do not form the same 4-celled colonies that Scenedesmus species like to do). I don't actually know where this came from - just an old plate that I suddenly managed to start a culture off of - but I suspect that it is a relic from Chico vernal pool samples I collected in the early spring, like JIAC13i is, although it was not directly isolated from the first few sampled plates but rather resurrected from previously-cultured and abandoned material.
So that's the introduction of the new strains of algae to this project! Again, I am fairly certain that after these have finished their rounds of PCR, I will not be adding any more strains to this project due to financial and logistical constraints. However, that does not mean that I will not add new strains of algae to my collection. I will still be going out regularly to observe, photograph, and isolate algae (again, check my Instagram page @algaenthused for more of this day-to-day and photographic work!), but the project has expanded so much that it's probably for the better I set a limit for myself, in terms of the number of strains whose DNA I can sequence and analyze. In future posts, I'll hopefully return to some of the other topics I've been planning, including my future project ideas and updates on the progress of steps of my research. Stay tuned.
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