I am trying to determine a bacterial cell growth curve. I wonder which method will be preferable for me to be able to obtain appropriate/more-reliable data or results from either of the following; dry-weight measurement or viable plate count. I'm also a bit puzzled about the specific or most correct absorbance to be used in reading the OD. Most journals I read used 760nm, 660nm or 600nm.
If I understand your question correctly you need a method to establish a reliable bacterial growth curve for the organism of choice. Numerous methods are available to you but each measures a different parameter. Plate counts are very reliable and give you information about live cell number over time. These measurements correlate with dry weight, total Nitrogen content etc from stationary through late log phase however as the bacterium enters stationary and in particular the log heath phase (as is the case with some bacteria) bacterial number and mass are lost i.e. log death phase. The spectrophotometric assay you inquire about above is referred to as the McFarland method for determining bacterial concentrations on a McFarland Scale. Please see http://www.microbiol.org/resources/monographswhite-papers/measurement-of-cell-concentration-in-suspension-by-optical-density/ It is easy to perform and standardise and when calibrated against the organism in question (selection of absorbance between 420 and 660 which gives highest absorbance reading for your organism correlate cell viability with cells harvested mid log phase it is very reliable. Your data should include two methods with one being viable cell count by plate method over time and an alternate measurement. I use the spectrophotometric method of McFarland described herein.
Hi,
I would suggest you to use 600 nm for measuring the optical density of bacterial growth but to determine its biomass it will be difficult from the growth curve as it will also have dead cells therefore i would suggest you to correlate your ate with the organic matter content of bacterial cells.
If I understand your question correctly you need a method to establish a reliable bacterial growth curve for the organism of choice. Numerous methods are available to you but each measures a different parameter. Plate counts are very reliable and give you information about live cell number over time. These measurements correlate with dry weight, total Nitrogen content etc from stationary through late log phase however as the bacterium enters stationary and in particular the log heath phase (as is the case with some bacteria) bacterial number and mass are lost i.e. log death phase. The spectrophotometric assay you inquire about above is referred to as the McFarland method for determining bacterial concentrations on a McFarland Scale. Please see http://www.microbiol.org/resources/monographswhite-papers/measurement-of-cell-concentration-in-suspension-by-optical-density/ It is easy to perform and standardise and when calibrated against the organism in question (selection of absorbance between 420 and 660 which gives highest absorbance reading for your organism correlate cell viability with cells harvested mid log phase it is very reliable. Your data should include two methods with one being viable cell count by plate method over time and an alternate measurement. I use the spectrophotometric method of McFarland described herein.
I dislike autocorrect on spell check. Line 7 should read:
the bacterium enters late stationary and in particular the log death phase etc
Dear Omoregie,
Similar type of question has been asked in RG. you can check the answers there...here is the link
https://www.researchgate.net/post/How_can_I_convert_optical_density_OD600_value_of_rhizobium_to_biomass_mg_L?exp_tc=tprc
Prof. John Kilbane have given the nice explanation.
Hope this help you...Thanks
you can do that using a set from McFarland standards and measure the O.D. for these set to blot the standard curve. with each point about 2 hours interval you must check the viable count and then you can get the best results
Thank you all for your contributions. The information given were very helpful.
http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=142693
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