For performing bacterial growth, OD must be taken at 600nm, and the result should be within 1. In case, if it exceeds, you can diluute the bacterial culture and perform again. As it is concentrated, it exceeds above 1

The best method is Spectrophotometric method

Albino Wins Thank you, what is the ideal value or range to be expected

0.5 to 1.0 will be the ideal range

At a wavelength of 600 nanometer. However, when it's greater than 1, there is high bacterial density, active growth phase or potential overgrowth

The absorbance should be in general in the range 0.1 - 1, beceause outside this range, it's less than 10% of light that is absorbed or passes through. However, some spectrophotometers may work up to 1.5 or 2.

If your asborbance reaches 1, you can easily dilute it and measure. However, in the past, I observed weird phenomenon, when I measured OD of Pichia, that after dilution, the OD was lower than it should have been. So it's better to keep the dilution the same from the start.

An OD600 of around 0.1 to 0.5 typically indicates an exponential growth phase, while higher values suggest a denser culture.

At 600nm and the value may varies between 0.1 to 1 or 2. However if two values show similarities after 2 consecutive incubation time then it would be considered as stationary phase

Higher OD probably means the culture has high cell density or it may be contaiminated.

I usually measure from OF650 from 0.05 to 1. Above 1, I dilute the bacterial culture 1 in 10 dilution before taking the reading

Different labs use different wavelengths to measure, usually from 590-650. They all work fine so it doesn't really matter so long as you consistently use the same. Traditionally 600nm has been used but not by everyone, I think because it matches the old color filters of Klett meters that used to be used.

When you get to optical densities above 1, the amount of light being scattered vastly exceeds the amount passing through, so the accuracy of the measurement goes way down. As the other responders said, the solution is to dilute your culture and take your reading so that it is below 1.

Usually, we measure the OD values at 600nm in a bacterial culture. If the value exceed 1, it means your culture is too dense. Such a situation we cannot obtain a linear graph between OD value and the cell concentration. Linear graph is important to estimate the cell count. Therefore, you have to dilute your cultures and check the OD values again. It will solve your problem.

Usually, the O.D. we measure is at 600 n.m., however, if you measure it between 590-650 it won't make much difference. Secondly, if your O.D. exceeds 1 that means your culture is dense and 90% or more light is blocked. If you are analyzing the growth curve then start from 0 hour reading which may be 0.1-0.3 O.D. and follow untill the decline in O.D. starts, in this process your O.D. might exceed from 1 and may also reach 2.5-3 in a couple of days.

At every one hour interval time starting from zero time. For growth curve, You must be started with 0.3. OD= 1 , or less or more during growth curve is normal values resulting form bacterial growth.

Usually bacterial growth is measured in 600nm. Usually if the growth of the culture attains stationary phase, it will cross 1 OD

Usually recommended bacterial density is between (OD) 0.4-0.6 at 600nm. If the OD is greater than 1, you dilute by adding more sterile diluent until it lowers to the acceptable range

Usually bacterial density is between (OD) 0.4 - 0.5 at 600nm and sterile nutrient solution is used to reduce the concentration

Hi Azzeddin,

OD600nm is correct for Bacteria growing tracking. You should reach Log phase at OD600nm 0.4-0.6. If your OD is>1, just dilute your samples 1/2 to measure it.

Did I catch well your concern?

Standardly, the OD is between the range 590-650nm. If it exceeds 1, then dilution can be made to reduce the density/ conc.

Bacteria culture OD measurements range from 590nm to 650nm. As you read in the articles, Most Labs measure the OD at 600nm. If the value exceeds 1, you need to dilute it to obtain consistent results.

OD (Optical Density) is used as an approximation of bacterial growth, as bacterial cells scatter light, thus the more bacterial cells or other substances you have in a culture, the more light particles will get scattered and the higher the OD will be. While growing in a liquid shaking culture bacteria go through four growth phases: lag phase, log phase, stationary phase and death phase. In the lag phase bacteria are growing slowly because it takes time for them to sense that they are in a nutrient rich, low bacterial concentration environment and it takes time for the bacterial cells to sense this change (if they are coming from overnight cultures, high bacterial cell concentration, low nutrient environment, high waste build-up) and to adapt their internal machinery for rapid growth and proliferation. Once they are able to grow quickly in their planktonic state, they enter the log phase, also called the exponential phase. In this phase they rapidly multiply and if you plot the measured OD values along time points and draw a line through these points you will see that this phase is basically a straight line at a diagonal. At some point the nutrition in the liquid medium becomes depleted, the concentration of bacterial cells becomes high and waste build-up from the bacterial cells becomes high. The bacterial cells switch into stationary phase, where they turn off their machinery for rapid division and instead focus on maintenance and survival. This is characterized by a plateau in the OD measurements as plotted along time points. At some point when all of the nutrients are depleted and waste build-up is maximized the cells begin to die off and when more cells die then are dividing this is known as the death phase. Since the spectrophotometer cannot differentiate between live and dead cells, one cannot detect the death phase. The log phase corresponds to OD 600nm up to 0.8 and above that is considered stationary phase.

The optimal measuring range is dependent on your specific spectrophotometer and that specific information should be provided in the user manual. But as others have answered before it is typically between 0.1-1.0 at around 600nm. To get accurate measurement of your OD you first have to blank the device. This means that you measure the growth medium without your bacteria, since the media will on its own have a light scattering effect and this value is then subtracted from your bacterial measurement. In this way your OD measurement will only reflect the light scattering effect of the bacterial cells and nothing else. Most spectrophotometers have a blank function, so you just have to place 1 mL of your medium in a cuvette, put that in the machine (making sure the arrows match, the cuvette is in the correct direction) and press the ‘blank’ button. The machine will then automatically subtract the medium value for you in future bacterial measurements. Now you can measure your bacteria with 1 mL volumes. If your value is too high (above 1, depending on your spectrophotometer) you can then dilute the measurement 1:2 or 1:10. For a 1:2 dilution you add 500uL bacterial culture to 500uL of growth medium, you then measure and multiply your OD value by 2 because it is a 1:2 dilution, to get the actual OD value.

As I mentioned at the top, the OD can be used to estimate the bacterial cell population and this is dependent for the bacterial strain that you are using. For example: 1.0 OD600 = 2.66 x 10^9 cells/mL of E. coli.

Swapnil Srivastava Absorbance values between 0.1 and 1 are commonly used for measuring bacterial growth in cultures. Absorbance value greater than 1 may lead to inaccurate measurements due to light scattering and non-linear behavior, so cultures should be diluted if OD600 exceeds this range to get reliable readings.

I have measured optical density of bacterial cultures at 650nm. Above, 0.8 I perform a 1 in 10 or 1 in 5 dilution of my culture to get accurate results.

Keep in mind if your culture is overgrown your reading will be measuring both live and dead cells (intact dead cells show up as optical density).