What may sound like a very simple question is driving crazy 2 experienced lab technicians. We run growth inhibition tests with green algae using 5 ml incubation volume and low algal density. At the end of the incubation, the incubation media has to be removed from the samples and algal cells are analyzed for various biochemical markers. Intuitively, centrifugation seems to be the method of choice, however, algae vary a lot between the controls and toxic treatment in their ability to sediment at 16 g, no matter how long we spin them. This is clearly depending on the biochemical composition of the algae (amount of sugars produced?), but this is what makes it absolutely crucial to get a similar sedimentation efficiency in treatments and controls.
Filtration on GFF is not an option, because at the end the pellet has to be resuspended in a very low volume of liquid, which will be impossible with the filter.
We have been struggling with this forever...
Dear Elena
the literatures below may help you ::))
Barnabé, G. 1990. Harvesting micro-algae. In: Aquaculture, Volume I. Barnabé, G. (Ed.). Ellis Horwood, New York, pp 207-212.
Fox, J.M. 1983. Intensive algal culture techniques. In: CRC Handbook of mariculture. Volume 1. Crustacean Aquaculture. McVey J P (Ed.). CRC Press, Inc., Boca Raton, Florida, USA, pp 43-69.
Thanks, I will take a look if I find those...
the problem is that we are dealing with small volumes and densities, whereas all these intensive culture techniques, like flocculation with various additives or disc-centrifuges, are designed for mass production...
Is it possible under your experimental conditions to use flocculation? Chitosan would be a good choice.
Elena, unfortunately i cannot help you. We have been having the same problem with our Chlorella cultures. They seem to create an oily substance and adhere to the walls of whatever container we use. I'll be following the discussion. hopefully you get some good input that we both can use!
Dear Elena: we also work with very small volumes, both TEM and SEM preparations and to extract DNA. Sometimes we have had interference with the mucilage secreted by algae of various groups and this is "the law" if it is cyanobacteria. The mucilage enables them to "float" instead of centrifuged to form a pellet. Perhaps in your research could also wash the material to dissolve the mucilage, if this were the case.
For this there is a range of useful products, including enzymes. The wall and the internal structure are not altered, but I do not know what happens to the chemical content, only that the DNA is not altered. I hope this information serves you and if you're interested, you can extend it with this article that explains in detail and can be downloaded directly here at RG: "Tavera & Calderon. Use of CTAB as a cost-effective solution to an old problem: the interference of the mucilage of desmids for scanning electron microscopy "
Best regards,
Rosaluz
I think this may be concerned with the size and density gravity of algae. If you want to seperate the algae from the water, you can try some flocculants, such as PACl, Polyaluminium Chloride, Polyaluminium Sulfate, under the low temperature. These flocculan agents do not destroy the cell morpholoy and DNA consist. And they can sediment algae quickly within several hours.
Dear Elena, have you considered using small reusable syringe filters? You can choose the required pore size. After removal of the filter you can resuspend your cells. This may not be 100% efficient in terms of cell recovery.
Dear Elena,
I was going to suggest the same than Louis. If you cannot use GFF filters you can also try to filter in polycarbonate ones and then resuspend your cells in a small volume. We do that all the time in our lab.
Hi mam i worked with pseudokirchineriella i followed the centrifugation method in lowest rpm to get pellete of cells and to resuspend it again inthe medium,filtering the cells through the filters also i followed but it will causes the lose of some amount of cells because algal cell will adhere to the material of filter.
If centrifugation method you are going to be followed means be aware to use plastic thing because cells will adhere to the walls.
Thank you all!! I am really impressed with the breadth and quality of replies. I should have probably explained that we are measuring enzyme acticies, proteins and lipids in these algae using fluorescence based methods. Therefore, any addition of enzymes or polymers (natural or synthetic) may affect either the quantity/activity of the measured compounds or detection sensitivity (polymers would have a high background fluorescence, for example). Therefore, adding organics is generally not advisable. Flocculation FeCl3 is the direction we are testing right now, and are also thinking of try ingmagnetic nanoparticles, I will tell how it goes...
Louis & Maria: polycarbonate filter might be an option, do you know what is a humanly possible lower limit for the recovery volume?
Many thanks again!
to Rentikota: yes, it is Pseudokirchneriella we are fighting. It was a good point to try slow speed centrifugation, thank you.
and how exactly do you recover algae from the filter? Is this a syringe filter with a reusable plastic cover, or the one that is usually used to collect the filtrate?
The cell disruption is also a challenge:) the best results we get with FastPrep and ceramic beads, but Pseudokirchneriella is notoriously difficult to homogenize compared to other algae. It works reasonably well...
I am currently working with a similar algae (Keratococcus) what I have done is: filter the culture with GFF 0,7 um (25 mm) filters (with a previous step in the muffle furnace to eliminate organic matter from the filter), after that I put the filter in an eppendorf tube with 1 mL of my extraction buffer and ultrasonicated it, 3 pulses of 20 s each in ice bath. That protocole worked for me and I am studying the effect of different treatments on protein, carbohydrate, chlorophyll and some ROS indicators concentration.
to Juan: thanks, it certainly makes sence, if you have sufficient cell densities or large volume... we have low algal densities (
Elena,
Sorry for my ignorance. What is wrong with simple evaporation method
The ability of a component to sediment depends from its density as compared to that of the suspending medium. Why not trying to float the algae instead to sediment them by using gradient density centrifugation or just increasing substantially of density of the medium by addition of Percoll which allow to maintain the osmolarity of the preparation? Best wishes.
Michel Spina
to Richard: evaporation under low pressure at room temperature takes too long (hours), and using of high temperature is impossible because the enzymes will be destroyed.
Elena, just for clarity, in your original post, your are writing 16g. Is this correct? What is the density of the medium? Is diluting with saline, tap water, PBS or whatever an option to achieve a medium that is substantially less dense than the algae?
I am sorry for the typo, it is 1600 rpm. The medium is not saline (Z8 - medium for fresh water algae). Stressed algae produce more lipids and less carbs which makes them more buoyant. We are kind of hesitant to play with adding more chemicals than absolutely necessary, because this may have effects on the substances measured or the detection limits.
Elena, do you have any chance to spin the algae at higher speeds like 16.000 rpm in an eppi-style tabletop centrifuge (15...20.000g or so)? When you chill them on e.g. ice before adding cold water (to slow down or even stop their biochemistry), might that help?
Don't know if that might work for you, as the density of your cells seems to very similar to that of the medium:
For recovering pancreatic beta cells after a labelling experiment, we centrifuged them through a layer of a dodecane/n-bromododecane mixture (not miscible with water, the ratio defines the density), to have the cells in the pellet and the aqueous layer on top. However, we did not need to recover viable cells, we even froze the eppis to get rid of the aqueous layer.
to Elena: Percoll is not composed of PVP. According to the Amersham booklet it : possesses physiological ionic strength and pH, has low viscosity, is non-toxic, will not penetrate biological membranes, is supplied sterile and is resterilizable, is compatible with biological materials, is easily removed from purified materials, and so on.
to Michel: According to GE, it does... [Percoll is composed of colloidal silica coated with polyvinylpyrrolidone (PVP),.. http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/sv/GELifeSciences-SE/products/AlternativeProductStructure_16963/17089101 ]
Whereas PVP is generally non-toxic to animal cells, in autorophophs it may contribute to sublethal toxicities (depending on co-occurring compounds in the media, particularly colloids, NPs, etc.). We have all the weird stuff in our incubations and having Si-PVP there would require extra controls...
to Elena: In fact you are right. It's silica but coated with PVP. Good luck.
to Elena: We used Polyaluminium Chloride or Polyaluminium Sulfate in flocculation of harmful algae. When we detected the in vivo fluorescence of algae, we found it showed no influences on the detected sensitivity. I think the main effects of the high background fluorescence caused by polymers is the excitation wavelength and the fluorescence-detected wavelength. You can try this inorganic compound.
Elena, I work with 20 mL cultures and a final cell density of 10^4 or 10^5 depending on the effect of the treatment on the cells. Initial inoculum is always around 10^4. Have you tryed F2 (non saline) medium for growing?
to Juan: We are rather happy with our Z8, why do you think F2 can help in solving the problem? Low cell densities (they are 10^4, not 10^3 as I wrote earlier) and volumes (5 ml) are used because these are optimal (=provide highest growth rates) in our incubation conditions (light, temperature, tubes). Our lab techs have tested different media and volumes...
- Badges
- Science topic