In my opinion the different media you are using interferes in the OD measurements, especially in the relation between the OD change and the real change in cell concentration. It is possible that the cells metabolites expelled to the media make it change in a "different way". I think you will have to construct a new mathematical expresion for each new media.

with different nutrients in the medium growth and metabolism can change and this can influence the turbidity of the medium. So the the same OD will not give the same cell conc. in different media. Therefore each time you change any growing condition you need to have a new curve.

I read and found that by increasing the wavelength which is not absorbed by color of all solution, the effect of solution color changes can be diminished. So the OD readings at that wavelength would be only related to the cell concentration and we can conclude that the different media has not effect on OD measurements. What is your opinion?

Bacteria may have different volumes and shapes depending on the medium, but these may be negligible. Check under a microscope.

Yes exactly eg. Bacteria will flagellate ,capsular etc based on growth condition . The size varies as well if you look under the microscope. So the concentration and OD taken under one condition will not correlate when you do it under a different growth condition.

I think if you want to compare the number of bacteria or their density you should use some other kind of estimation as this might be inaccurate . If the data is only for comparison or because you want to make previous hypotheses this method can work. It all depends on why you want to measure !!

I will agree will most previous answers that it is dependent of the strain, the medium etc. However, for a rough estimation, you could check the cell concentration on a Neubauer chamber under a microscope for your specific culture in several phases of the cultivations and see if there is a constant corellation between OD and cell concentration. Then check if this applies to all similar cultivations (same strain, same temperature, same medium etc.).

HI, in relation with the example that you mentioned, if you change the media or any condition you MUST to change your equation (curve). Extrapolation in mathematic is quite risky, probably in the example proposed here, your equation wont change that much, but if you want to be precise you have to measure again, but if you need just an approximation value, you could use the same equation. Cells (in general) have different behave, even in similar media, "dauther cells" (clonal) from a common "mother cells" can have different response to the media. Read about noise in gene expresion and variability in parameter kinetics. Bb

As has been said before the OD depends, not only of the cell density, but also of the cell shape and in different growth conditions the shape ciuld change. A tipical example is on those bacteria that produce capsule or EPS that depending on the carbon source the amount of EPS would change and the OD consequently change. In my opinion t is better to make a new curve (formula) even with less point that used the previous one.

Not so an expert with yeasts but we never relate on OD measurements for cell concentration until the media doesn't change anymore. We work with different bacteria and media and see differences with media. Not only because of cell shapes but as previously mentioned also with EPS and other excreted substances. So, you need to calibrate with each media you use.

I would also do a new calibration curve combined with viable counting and/or microscopy for different media, even though the effect might be negligible at the end. Another important factor is that your reading is in the linear range to avoid "re-scattering" hence inaccurate results (as a rule of thumb between OD 0,2 and 0,6) i.e. you should dilute the sample accordingly.

Your YE medium may be subjected to bacterial-induced changes in color, which could interfere i the OD reading, depending on the wavelength you're going to use. In this case I would recommend not use a 600 nm WL since it is presumably too close to the basal yellow absorption peaks of yeast extracts. I'd rather use 550 nm, isntead.

Hi you should do a new calibration for that, it can be similar but sometimes it varies. You have to consider that different bugs have different sizes which influences the OD but not the cell count. But if you don't need a really precise cell count it usually works has a good approximation, at least in my experience.

For each medium, temperature, oxygen status, or any differences in growing conditions, you need a calibration curve (OD vs. cell count)

In various culture media, the bacteria may have some morphological changes (changes in the membrane, in the flagella, there may be some compounds secreted to the culture media, or some compounds accumulated into the cytoplasmic that could modufy the OD.

If the microorganism in question produces large quantities of intracellular product (PHA, lipid, etc.) under certain conditions, then OD and CFU can correlate differently, depending on the media.

That is not obvious since the shape of bacteria can change according to the medium and their physiology, then the OD should be considered only as an indication of the bacterial concentration not a definite proof. You need to make a control by plating and counting, at least in a first trial wit ha new medium.

I agree that the shap of bacteria can be different depending on the medium, culture conditions and cell physiology. OD can be used for counting cell concentration of calibration for OD dependence on cell concentration is done by plating and counting. If bacterial cell sizes are large, the correlation between OD and cell concentration will be different and wrong to use for counting.

Physiology of the microorganisms, (e.g. growth rate, production of primary and secondary metabolites, exolpolymers, pigments and ... ) is very dependent to cultivation. including media ingredients, temperature, pH and .... OD is used only as a rough estimate of total cell counts. So, if you to measure biomass concentration with great care, you have to used more accurate methods, including viable cell count (standard plate count).

Sometimes microorganisms uptake acetate under unfavorable conditions. In this case there is no correlation between OD and cell number. The example of this phenomenon can be found in our paper Appl Environ Microbiol 2004, 70: 2959-2965. I would make a new growth curve too!

As many people have already suggested, you should always make growth curves for every media you use.

I agree with all the comments given by the others. The colour of some media can be changed by the secondary metabolites produced by the microbes which will affect the reading of the OD, so be careful with the media used.

OD never gives accurate cell count. and no matter what media you use you must take a control. thats it. :)

more OD indicate more concentration of bacteria: but usefulness of optical density to count bacterial cell numbers cannot be possible: because OD is Qualitative and not Quantitative

OD is only qualitative.It can give approx bact cell nosPlate count is best

As the others already mentioned, not only bacteria may change their shape depending on the conditions, but also the substrates present in the media may affect the OD. Either cell counting under microscope or plating will give proper results. There is no way to reduce the labour :)

Many comments!! All comments are great but we should know that OD measured base on a beam of light passes through an absorbing medium, so if the clarity in both media are same you can use this equation because you are looking for the concentration of bacteria particles in a media , doesn’t matter the affecting of temperature and other biological factors. Meanwhile what does this numbers used for? In bio-assay test like MIC we usually estimate (no detect) the number of bacteria in a media and it should be OK.

I suggest you to stnadardize your OD against the plate count if you are looking for the viable counts in a defined medium grown for a difeined period of time and under known conditions. This if re-checked when you use a different batch of medium, it will be nice. But surely you have to have the curve for different brands of media.types of media under which you grow the bacterium.

Although not very significant at 0.6 nm, it depends upon the contents of the medium when such contents cause cells to alter their morphology. When this occurs (example-filamentation produced by acids), the absorption changes. Suggest looking up morphology/filamentation/ph/etc.

Jacques Thierie. ULB

In some cases, bacteria may form aggregates. So the size may not always reflects a "single" microorganism. Moreover, microorganisms produce very complex extracellular polymeric substances (EPS) that may change OD and may vary with the medium. In my view point, the "universality" of a single relation OD600=f(cell number) is not granted from medium to medium.

Just spin an aliquot of ca. 1 ml in an eppendorf eliminate supernantant and measure protein by any colorimetric method. Cell biomass is about twice the amount of protein measured

I agree with previous responses that the relationship between OD and cfu or DCW can be altered in different media. Theoretically, these relationships (OD versus cfu or DCW) must be performed in the same medium under the same operating conditions. If you change one of these parameters (media or bioprocessing conditions such as temperature or agitation rate), the equation and therefore the estimated growth (cfu or DCW) may be strongly modified. As Francisco Rodríguez-Valera has quoted above, the measure of protein is another approach to estimate the cellular proliferation although the measure of OD is employed more frequently within scientific community.

Your question: I For instance, assume that the cell concentration of S.pasteurii cultivated in an Ammonium YE medium is estimated by the expression 8.59* 10^7(Z^1.363) where Z is the reading at OD600. Now, if I cultivate the S.pasteurii in an Acetate YE medium, is it possible to use the same equation to estimate the number of cells according to the OD readings?

My answer: No, it is not rigt to use the same equation. You have to make a new equation using acetate YE medium if you grow the bacteria in acetate YE medium. This is because different culture medium will give different growth rate and also the bacteria react differently and may change the colour the medium used.

OD-600 is only lineary correlated with CFU/ml in the Lambert-Beer region. Every spectrophotometric method needs a reference. For microbiology the reference is usually the pure medium without bacteria. If you make a correct reference your OD-600 signal will only be linked to bacterial density as your medium signal will be filtered out. So, to me, if you substract your "medium" signal correctly, using the same formula is certainly a very, very good estimateof your true CFU/ml even in different media.One consideration to taken into account is of course the different metabolites which bacteria can make in different media. If these metabolite (e.g. pigments) absorb light of 600 nm, they will interfere with the OD600 measurement and your CFU/ml will be biassed. The best way to verify your OD600 measurement is to calibrate your signal once. To do this I normally measure OD600 and perform in parallel a plate count for different samples of a growing culture. Afterwards you can simply make a graph and a lineair correlation between counted CFU/ml and measured OD600 of the analyzed samples. You will immediately notice wether the theoretical formula is correct or not.

If the uninoculated media have the same OD, YES you do!

No you cannot use the same equation as the growth of the bacteria will vary in different media and also across different growth conditions.

Hamed, it is not possible. Bacterial growth is dependent on several factors including media composition. So, as you change the media,the growth dynamics changes.

Same reading can not be used. There are several factors to be considered, which can change growth and multiplication of bacteria and hence, for different media, same reading can not be used. Also, for different bacteria, OD will be different in different media. It is better to construct a calibration curve for each bacteria for each medium and go for estimation.

Defintely you cannot use the both the methods for experiments. Because of cell cycle stages the growth will be different in terms of number and optical density. This is nothing but a parameter for experimentation. Both methods have their identity for experimentation

Well Hamed, you have got your answer - that you should recalibrate. But no one (I think) has yet pointed out that what everyone is calling Optical Density (quaint old term for Absorbance) is not what you are primarily measuring in assessing cell density. Most of the light entering the suspension is scattered, not absorbed. So the geometry of the spectrophotometer is crucial (how close is the photomultiplier/diode to the cuvette), as is the refractive index of the medium. Scattering can be largely suppressed in high sucrose. Note also that short wavelengths are more scattered than long. I would not myself count cells, but do dry weight or protein assays to calibrate optical methods.

I have yet add an asnwer !!! Where it is ?

I have yet add an asnwer !!! Where is it ? I think that Hugo Moors have make some remarks. I will not answer a second time if you loose my texts !!!

well Hamed, No you cannot use the same equation, different media or bacteria or growth condensation may interfere with OD, so you must be calibrate new media or condition by reference method

It also depend upon the bacterial species. As examples, pH, heat, cold, high ionic strength, etc will cause different responses of Gram-negatives vs Gram positives.

Whereas cocci will continue to replicate but the hydrolysis of the septae does not take place, the replicated cells remain attached to each other forming clusters. These clusters contain geometric progressions of DNA and when transferred to appropriate medium yield the number of cells that represent the number of replications within the cluster. Hence, the OD of a normal population will not reflect the number of cells in the medium that induces this morphological response. The same takes place with Gram-negatives that become increasingly long filaments which when transferred to agar will yield the number CFUs that does not reflect the actual number of genomes present the filament. The questioner is asked to review the articles by Victor Lorian and Leonard Amaral which are available at PubMed.

Many scientists tend to forget that OD (=Optical Density) is completely different from Absorbance! The level of absorbance can be a quantitative measure for the concentration of molecules in a solution. And this goes with a mathematical equation. However, optical density is a measure of light scattering and thus is gives you a relative number of cell density. The amount, size and shape of the bacteria will all contribute to this OD, and so a mathematically equation to calculate cell concentration based on OD is flawed, unless all bacteria are completely equal in size and shape. And that is impossible. Start counting by colony plating, or by Coulter counter!

You must have a standard reference with known cell population and absorbance

Yes , there is very important to measure the OD. with different concentration of media. and you must follow the growth of bacteria under microscope to found the CFUs. thank you

In many of our published studies on determining MICs or MBCs, a bacterial culture in broth adjusted to ~78% transmittance (0.108 OD) at 600 nm almost always contains ~10(8) CFU bacteria /ml. Of course there are some minor differences due to microbial strains and media, but this is a good rule of thumb and an excellent starting point for fine tuning a protocol

Dear Hamed

I think if you measure OD in true way, the equation which you have mentioned can be used to calculate the approximate number of cells according to the OD. Light adsorption for a specific bacteria don't change in different condition. A specific number of bacteria adsorb same light in different culture media.

Yes you can use the same equation in any medium, but you have to make sure that the medium itself doesn't have any turbidity. In fact you may refer McFarland's standards for this purpose.

As I wrote equation can be generalize to all suspension of S.pasteurii bacteria in different media culture but the main point is that "do not forget to calibrate Spectro photometer just before every test." Whenever OD is measured the default OD of pure culture media must be adjusted to zero. After that spectro photometer is calibrated. If optical density (OD) measure in this way, the above mentioned equation can be used for it and you are sure that the turbidity of the culture media is not consider in measured OD of bacterial suspension.

yes there are high correlation between the growth of bacteria,their number and OD because the high number of bacteria high turbidity high OD and this a recommended method in estimating the bacterial growth

you will need to considerer that the maintenance coefficent (PIRT) is going to chage, so the shape and volume of the cells is likely to be different (as the college said), but in order to save time...you migth try if the difference is significant or not, i mean...if you take a sample of the culture on the second culture media with and you know how much biomass do you have there, you can read the OD and try your equation, may be al the effects that everybody says (they are all right) isn't relevant for what you want to do.

and when you want to count bacteria they must be live. if

viable count is what you are looking for then OD may not be accurate

OD measurement is an easy, nonexpensive way to estimate bacterial mass. There are factors influencing the OD value.:

1 strain

2 cultivating conditions (medium, temperature e, inhibitors tc.)

3 growth phase of the given culture

Living cell count is dependent ofthe growth phase and not allways following the samerend as OD change.

Depending on the purpose you want to monitor bact. growth biochemical markers (cell total protein,DNA ) might give more detailed insight.

Yes,there is definitely a relation.

First, one should ask himself what is the objective of the study, because OD will not give you information for the content of living cells in the suspension! This is especially true after the exponential cell growth phase. If you need to know the viable cell mass, than either seeding on agar plates (less accurate) or determination of the intracellular ATP content over time (much more accurate) are the right choices.

Absolutely the answer is yes. Because the growth of bacteria vary from medium to medium according to the nutrients present. Also the colors of media vary which also affects.

I can agree with some of the answers presented here, but definitely not with all of them. Remember that in measuring bacteria in liquid media at 600 nm, you are not measuring light absorption but turbidity. Turbidity is function of particle (bacteria) concentration, size and shape and some surface properties. Anything in the medium that will change the physiological conditions of your bacteria (size?) will have affecton the relationship cell concentration - turbidity. Hence using the same equation in different media is trickky. I would never do it without checking the validity. And if you check it.... you have a new calibration curve anyway.

OD has a relationship with number of bacteria and it differs with different culture media and bacteria. It can used to a moderate reliability with E coli. but its better to plot a standard graph first with other strains and media before using OD to calculate cell numbers.

It would always be better to plate to get accurate numbers.

it depends on what you are using the OD estimation for

A lot will depend on the media characteristics, like viscosity, color, turbidity and also chromophore components. It will be best if you take fresh autoclaved or filtered (method you use to render it microbe free) as blank.

If cells are growing mostly in planctonic form and if cell composition does not change too much (for ex. compounds that have color), it is possible to use OD measurement for cell number calculation. That is quite convenient method. However, it might be good to make "a calibration" of OD in respect of cell number determination (to know deviation and so on).

It is a very good question but I agree with the above answers as the OD will vary at different media because of change of growth patterns. The size & density of microbial cells will change with the change of media which will affect the OD. So different standard curves need to be made for different media.

Usually, OD reading does not differentiate between active cells and dead cells. Accordigly, if the broth contains old cells and dead cells the OD reading will overestimate that obtained by live cell count. On the other hand, if all cells are viable both reading should be in the same range

In addition, the medium content could affect the reading if it contains suspended matter

It is observed that the size & number of cells vary with the change of media which may not match with the standard curve prepared with a given media.

estimate yes but it's a poor estimate. Calibration of OD is depentend on medium and growth conditions. If you vary either you need to make a relevant calibration curve.

I presume we should rather think more about other methods of counting bacteria. OD may not reveal the viable count. any other organic matter can interfere OD readings. Caliberated loop can help in quantifying bacteria

Optical density depends greatly on the composition of the media, so if you change the media in which you are cultivating your microbes , its best to generate a new model for the different system in order for whatever argument you decide to make in future based on your findings to have a solid footing.

Cell counting depend on OD vale is not such strong methods to count cells because OD is not only depend on cell concentration but also for others for example dead cells, secreted molecules by cells etc. However, OD depended cell counting can be considered upto some stage of growth eg, lag phase, log phase but should not be at stationary phase. The stationary phase consist both death cells and alive cells, so OD doesn't reffer the exact alive cell numbers at this stage. Regarding the equation, the same equation should not be fitted for all culture and media.

I agree with the above answers. But my query is why do you want to grow your culture in different media, you experiment is on media or culture? Different media have its own role in bacterial strain, so automatically OD will vary in different media.

Make new curve for every medium and strain. You can use your equation as a rough estimate to start with. Please also remember that OD vs Cell counts holds good upto log phase (before reaching the stationary phase) and they are not in linear relation from the midway of your growth curve. Remaining things are already told by other respected members. Good luck..:-)

I think while choosing a method we should be very selective and minimize as much as possible the drawbacks.

Dear Hamed,

Surely is. Optical density readings are affected by both the number of cells per unit volume and also by the composition (and so the optical properties) of the media.

yes , the relation between OD reading and cell concentration of bacteria different in various culture media is always different because the composition of the media affect the growth .

Yes, there is a co-relation in the absorbance and the cell number for a bacterial culture. It has to be determined empirically by using different optical densities such as 0.2, 0.4, 0.8 and check for the cell number at these respective densities. Once standardised, the OD values can be considered as an internal standard for a particular cell number. However, this is an approximate value and needs a control during each experiment to assert the co-relation.

The magnitude of the microbial growth may be very different, as it depends of the culture medium composition, among other factors. Each culture medium has a specific chemical composition and different concentration. Therefore, the rate and microbial production will not be the same in any case. On the other hand, the microbial measurement by DO is not a precise method in order to determine microbial growth.

The OD is an approximation method that is affected by bacterial type, the stage of growth as well as any effects that media may have on bacterial volume. Given the wide confidence limits that this test would have, you may find that a change in media as long as it has the same purpose i.e. enrichment and is for the same bacterium may not significantly alter the results. If unsure run a bacterial count at different ODs using the two different media.

Dear Hamed

No you cannot use the same equation for both the media. In addition to what Dr. Hipolito has demonstrated, even you cannot use your equation using the same media if you even change the spectrophotometer in which you obtained your equation, as every machine has its inherent error level.

Good day Hamed

One always wishes, that we can decrease our workload, and I do believe that you may find that you can use the same calculation. However, you will have to prove this by running viable plate counts on broths of known OD value. I use the Miles-Misra plate count method as it requires less media (10uL spots on agar - done in triplicate)

Dear Hamed,

You cann't used the same equation , because the different media when you used gave different growth of bacteria and that gave different OD when measured by spectrophotometer at 600 nm.

Shifting gears to simpler matters: Are you blanking the reader with the corresponding culture medium for each readout?

You may even need a new growth curve for each batch. Whilst bacteria will broadly behave in the same way under specific conditions, I'd echo what's been said above regarding parameter change, such as change of media type, but will also vary from subculture to subculture, and between batches of media.

Miles Misra ia a good back up but has many limitations and should always be done in triplicate or more if at all.

Your equation may give you a prediction, but it would highly questionable to use it for anything else.

the turbidimetric determination of cell growth depends on number of particles in i cm light path length. You may get differences between cells/ml vs OD reading in different media -i) if bacterium does not attains full cell shapein all the media ( may some media missing some growth factor or nutrients) or bacterium grows either freely or as aggregated mass ( chain ,bunch etc. )in different media. You may confirm by simple staining

Najafpour G.D , sorry, what you mean "range of Landa" ??

It seems that one important point is missing out of discussion. That is about a correlation between optical density (Absorbance) and cell density (amount of cells per volume). The proportionality between these 2 values exist only for a certain range of values. Sometimes it is considered to be proportional up to the absorbance value of 0.6, sometimes even lower. In every case it is strongly recommended to make a calibration curve to check the proportionality of values. You prepare dense cell suspension in medium (buffer), make dilutions and measure OD, plot measurements and then you might clear see the range of values that are proportional.

The correlation between optical density and cell concentration relies on the Beer-Lambert law A=EcL (ie absorbance = the molar extinction coefficient times the concentration times the path length (1 cm is a convention but not absolute- the same path length in atomic absorption spectrophotometry is 10 or 20 cm). What is critical is that the cells are in exponential growth phase so no debris from dead cells will absorb light and confound the result. It is also a convention to use 600nm (white light). So long as the 'blank' is the same medium as the culture an absorbance of 0.1 at 600nm roughly equates to million cells per ml, but this varies depending on cell size and density etc. Unless the medium is extremely absorbant (eg charcoal yeast extract) then whether you use NB, Mueller-Hinton, or whatever does not matter.

ps I also agree with Alexander - the Beer-Lambert law only holds true at low concentrations. Cells must be diluted to the 0.1 absorbance range for a useful determination, as concentration increases, accuracy and precision decrease.

Basically, the blanking of the spectrophotometer will give better result. Over all calculation results with OD600 is relative, enough for most of the application. For more precise and accurate maybe ordinary manual cell counts under microscope or automated instruments could be used. Regards / Beston

Alexander's answer is good. You simply need to set up calibration curves (lines) for your conditions and plot OD and cell concentration. You will then discover at which density these two factor become dis-proportional. It is also to do with spectrophotometer limitation. Always try and dilute sample to keep to OD around 0.5-0.7.

At least in the well-studied Escherichia coli and Salmonella typhimurium, where cell mass per number of oriC's at the time of replication-initiation is constant and the time taken to replicate the whole chromosome is constant (i.e., at a constant temperature and in the growth rates range of 1 - 3 hr^-1 [doubling times 60 - 20 minutes], average cell size rises exponentially with the growth rate mu and hence you can predict/calculate cell concentrations by having a reference such as you describe provided you determine the mu values under both conditions (the known and the one in question).

Have a thorough look at the article by Schaechter et al., in J. Gen. Microbiol., 1958 (Vol. 19, if my memory does not fail me), then the 1968 articles by Helmstetter, in J. Molec. Biol. and the Cold Spring Harbor Symp Quant. Biol.