I am currently studying growth kinetics of a staphylococcus spp., as per the standard growth curve, the phases are lag phase, log phase, stationary phase and decline/ death phase. In the stationary phase the O.D. remains nearly constant, i.e. number of cells formed equals the number of cells that die.The nubber of dead cells out passes the the no. of cells alive in the death phase.
My doubt: If the O.D. is affected by the state of the cell (live or dead), does that mean the bacteria undergoes morphological changes after death ?
or do they float on the surface, thus escaping the spectrometer analysis?
Yes, dead Gram-positive cells lyse (autolysis). So, concentration of cells really decreases. In the case of Gram-negatives, before lysis dead cells swell (refractive index decreases). This process also leads to decrease of OD.
OD600 nm light is much less phototoxic and at this wavelength organic materials like live cells readily absorb light. As the dead cells are disintegrated and precipitated that is may be we are getting low optical density.
I have monitored growth curves by mean of OD600 using microplates in Bioscreen up to 48 hours and I have never observed a significant decrease in the OD600 after the stationary phase. As far as I know you can not observe death phase by mean of OD, you need to do UFC or use some technique that allow you to see viability of cells.
Have you done a growth curve by mean of OD? Because in real life you do not see a decrease in OD after stationary phase...
Dear
Most of the time OD of bacteria decrease due to autolysis. But be careful the OD value can be constant when the medium is less toxic and autolysis very low. Sometime it we observe gluconeogenesis phenomenon which rise again suprising OD value. To be sure we recommand you Cell of Thomas count by using colormetric process.
Hoping to be helpful.
Please you can reinforce your understanding by checking the following links.
http://demonstrations.wolfram.com/DiauxicGrowthOfBacteriaOnTwoSubstrates/
http://www.biologydiscussion.com/bacteria/growth-of-bacteria/growth-in-bacteria-4-main-phases/55128
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268280/
https://www.ncbi.nlm.nih.gov/pubmed/8650219
In death phase, number of dead cells are more than number of live cells, These dead cell undergo cell lysis and then cells contents precipitate and do not continue suspended as cell suspension and OD measure basically absorbance of suspended cell, this in turn decrease OD
Death of cells does not mean the instant disappearance of those cells. This is one disadvantage of the OD method of counting bacteria. It will not differentiate between live and dead cells giving false longer stationary phase
OD is not a good method to determine with precision the death cells. There are many factors involving in the turbidity of a media growth. It is true that many bacteria suffer auto-lysis when they died but it is true too that the bacterial surface charge modified with the state of the bacteria and aggregation varies. But surface forces in a non-colloidal system can avoid the precipitation of the bacteria and give wrong measurements. So, the best way to verify if the cells are really death is still the plate count.
During decline phase (viable count declines) OD can stay level for a while, but may decrease if dead cells start to autolyse, releasing nutrients that survivors can use to maintain viablility.
It would be better to approach your study with the ATP assay as described by Traci above since optical density will not be accurate enough to measure changes caused in the population as a result of death or autolysis. The spec will continue to measure based on the standard wavelenght and absorbance. So try out plate counts for further confirmations
Upasana, yes, bacteria undergo morphological changes but NOT after death: they change their shape and the status of cell in response to environmental conditions. The conversion factor from OD to cell number or cell mass concentration drops after death because cell membrane is damaged and cytoplasm leaks out, as a result refractive index decreases and therefore OD drops because of smaller scattering of light.
I do not agree that ATP is the best method to asses viable population: conversion factor from ATP to cell number/mass is highly variable parameter. And it is technically longer and more expensive than OD or plating. Tool box is much wider. If you have $$ purchase Live/Dead kit based on fluorescence microscopy: dead cell will look red, viable - green. If $$ pool is low, use microcultivation technique and count microcolonies derived from viable cells versus single cells (do not grow and therefore are either dead or Viable-but-not-culturable (VBNC).
Optical density measurements can be used to determine the biomass concentration in a suspension and so frequently used for monitoring growth of a culture of microorganisms. In this way, as autolysis of dead bacteria is most likely and their product w'd not be available in the suspension thereafter as most of it will settle down under force of gravity, O. D. reading must go down in the death phase ordinarily.
Dear Upasana Mangrolia
It is well known that the spectrphotemetric assay for follow up of bacterial growth is measured both of dead and live cells (OD is not affected by the state of cell either dead or live as mentioned above in your question). To measure viable cells only, you can used total viable plate count method which measures viable bacterial cells only.
Kind and best regard
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms
- Bacteria