I am currently incubating liquid medium (seawater) with bacteria and some clay/calcite seeds. I am interested to quantify how much EPS produced by the bacteria. The problem is I included yeast extract in my medium to promote bacterial growth. Therefore, measurement of protein and polysaccharide as a proxy for EPS wouldn't work in my case because yeast extract in the medium comprises these soluble stuff (protein + polysaccharide etc). So, is there any literature about the quantification of exactly how much EPS produced by the bacteria (not something from the yeast extract)? Your thoughts and suggestion are very much welcome. Thank you very much!
Hello Yik,
EPS composition of planktonic cells determined by using Conventional spectrophotometric methods, Scanning Electron Microscopy (SEM), Fourier transform infrared (FTIR) and confocal laser scanning microscopy (CLSM) which observes the main components of the biofilm extracted from the cultures.
FTIR used to determine the variation in the chemical groups in the EPS due to different growth conditions. SEM apply to check the surface structure of EPS. It's easy to quantified by measuring the total cell volume from a 3-D image of the biofilm using CLSM after staining cells with a fluorescent dye.
M.A.Redmile-Gordon., P.C.Brookes., R.P.Evershed., K.W.T.Goulding., P.R.Hirsc. Measuring the soil-microbial interface: Extraction of extracellular polymeric substances (EPS) from soil biofilms.
Soil Biology and Biochemistry. 2014, 72, 163-171
Article Quantification of extracellular polymeric substances in biof...
Hello,
You can try a method wherein you can first assess the soluble stuff (protein + polysaccharide etc) as you have mentioned; in yeast extract in the amount and volume which you are adding.
You can then subject your EPS for the same and deduct the value from that obtained in yeast extract.
The following articles may be of some use to you
Article Is biofilm removal properly assessed? Comparison of differen...
Article Characterization of extracellular polymeric substances (EPS)...
regards
Varsha
Hi Mr.LIN, At first, you should separate supernatant from your culture by centrifugation and then precipitate EPS from the supernatant with adding of two or three volumes of cold 96%(v/v) ethanol and incubate them for 24h at refrigerator. After incubation, discard the upper phase and transfer precipitated EPS for centrifugation. After centrifugation, discard supernatant and freeze EPS pellets at -80c freezer for overnight. At the end, freeze-dry the froozen PS with a freeze-drier apparatus untill onstant weight was obtained and weigh your freeze-dried EPS. You can determine sugar purity of your EPS with phenol - sulphoric acid method that demonstrated by dubois ( It's a colorimetric assay).
If you need extra purification of EPS from smaller molecules, you can utilize dialysis tubing with low cut off (eg. 3.5 K.Da)
Dear Chin Yik Lin, did you solve your problem? I have very similar problem in my culture. I'm thinking of the following solution.
In my case, more than 70% the yeast extract has the MW 500 Da (I'm cheating, I know), I'll simply quantify the portion of bacterial EPS that larger than 500 Da by using a dialysis membrane with MWCO = 500 Da.
The bacteria is put inside the dialysis membrane. The medium inside the dialysis membrane doesn't include Yeast Extract, only the medium outside the dialysis membrane including Yeast Extract. Yeast Extract still provide the growth promoting effect to your bacteria because 70% of them can freely transfer through the dialysis membrane. However, bacterial EPS (only the portion that >500 Da) will be keep inside the membrane dialysis and you can analysis this portion. However, we must be sure that the cultivation time is long enough so that bacteria consume all the Yeast extract that is
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts