We would like to determine the absortion spectrum of a bacterium. I have, however, never done something like that. It's to see if there are any absorption peaks present and if the bacterium would thus for example produce Bchl a.
Online I have found that many people test this in different solvents, like methanol. Is it also possible to do this with whole cells and avoid making extracts? Thus, is there a fast and easy way to determine the spectrum? There is little biomass available and the strain grows rather slow. So if there would be a simple and fast protocol that does not need a lot of material, that would be great!
Thanks!
Dear Guillaume Tahon,
I think Fluorescence is suitable for your case, As well have a lot of benefits of use this approach, In details: this method have a wide Concentration Range, which can be used without modification on the cell. Furthermore, Fluorescence is simple and sensitive due to the small sample size. In fact, it is a cheap method due to Low cost of equipment comparing with HPLC. In addition, Fluorescence can be work on broad wavelength range 200 to 1000 nm for some hybrid approaches.
Best regards
Musab Tr
If you want to avoid the extraction you can try to do fluorescence measurements of whole cells.
I have a question for the questioner. What wavelength range of light are you considering?
PLEASE define your bacterium !! When you write :
We would like to determine the absortion spectrum of a bacterium. WHAT A KIND???..
I have, however, never done something like that. It's to see if there are any absorption peaks present and if the bacterium would thus for example produce Bchl a.
What is the easiest way / Please define your possibilities and spectrometer (single or double beam or diodearrays) / to determine the absorption spectrum of a bacterium?
Very important:
Please launch Google Scholar and NOT Google and insert the part of your question:
You 'll earn a lot of hits. Among them :
http://www.sciencedirect.com/science/article/pii/0003986160901934
Concerning the question posted from Douglas :
The author of the cited paper (link) worked between:
Absorbancy changes (390–600 mμ) in the purple sulfur bacterium Chromatium have been studied by means of a double-beam spectrophotometer.
Please answer Douglas and me !
For the answer of Jaroslaw: read the link
http://www.pnas.org/content/83/14/5121.short
Please read the another hits and repost your question with more infos.
JRG
You can try to do photoluminescence measurements of whole cells.
Dear Prof. Pease,
Considering your question, this would be between 300 and 1000 nm. For measurements, we have two spectrophotometers in the lab. These function with cuvettes, 96/384-well plates or even a single droplet.
As for the bacterium, all I can say is that it is a Gram negative bacterium. Other information I cannot give due to the novelty of the bacterium.
Kind regards,
Guillaume Tahon
Dear Guillaume Tahon,
I think Fluorescence is suitable for your case, As well have a lot of benefits of use this approach, In details: this method have a wide Concentration Range, which can be used without modification on the cell. Furthermore, Fluorescence is simple and sensitive due to the small sample size. In fact, it is a cheap method due to Low cost of equipment comparing with HPLC. In addition, Fluorescence can be work on broad wavelength range 200 to 1000 nm for some hybrid approaches.
Best regards
Musab Tr
I think there is no use to make an UV-VIS spectrum of a specific bacterium. If it produces special extracellular or intracellular compounds it may make sense, however, likely purification would be useful.
The easiest (and only way that I am aware of) to measure the spectrum of a bacteria without purification is using a dual beam integrating cavity absorption spectrophotometer to eliminate the effects of light scattering from the particulate bacterial suspension. The only commercial instrument available is an OLIS CLARiTY spectrophotometer as used in this Frontiers in Microbiology pub from 12/2016: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5143472/
To see comparisons of conventional spectrophotometers vs. an integrating cavity spectrophotometer with bacteria, whole cells, phytoplankton and liposomes, see the spectra presented at: http://www.olisclarity.com/comparisons
In further detail, from the Results section of the attached publication:
The principal features of the ICAM (Integrating Cavity Absorption Meter) employed herein to conduct absorbance measurements on intact cells in turbid suspensions were described earlier (Blake and Griff, 2012; Li et al., 2015). This novel design for a spectrophotometer imposes two consequences. First, the multiple transversals around the interior of the observation cavity means that the incident exciting light experiences a much longer effective path length than it would in an equivalent linear spectrophotometer where the transmitted light experiences only one pass through a filled cuvette. Consequently, absorbance measurements in this ICAM result in a much greater sensitivity than would equivalent measurements in a standard linear spectrophotometer. Second, to the extent that the exciting incident light can be made to be totally diffuse, there are no longer deleterious consequences to absorbance measurements from turbid suspensions that scatter light. If the exciting light is already randomly scattered to a maximum extent, then additional light scattering by the turbid sample will have no immediate consequences on the integrity of the absorbance measurement. Thus it is possible to conduct absorbance measurements in situ in whole cells under physiological solution conditions.
Article In situ Spectroscopy Reveals that Microorganisms in Differen...
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