It is not accurate to read the OD when above 2 or 3 for a UV specrophotometer

Hi

It is common. Take Candida albicans as an examples. It can easily reach an OD of 30-40 if grown overnight with shaking (200 rpm). What we normally do is to dilute the suspension and time the OD reading to the dilution factor.

Hope this help

repeat the experiment as triplicate, take the average, secondly, take the OD at doubling time, plot a graph and conclude. please do follow standard literature

Thank you everyone. Actually I've got the OD after diluting the sample 10 times and adding that diluting factor . Experiment was performed about 4-5 times and the results showed more or less same. I am very much confused actually.

One more thing, which wavelength will be accurate for determining microbial population , 600 or 660?

Both might work; it depends. The idea is to select a wavelength at which none of the components of your culture media (or excreted metabolites) will absorb light.

And yes, it may be normal. What was your starting OD?

Starting OD was about 0.02-0.05. I was trying to adjust the OD to approx 1.0, but it exceed as above, even after 12 hours incubation the OD shift to 7.0-9.0.

Alejandro Martin, may I know ho to eliminate the absorption by secondary metabolite? during OD scanning from 340 nm to 750 nm, the highest OD showed near lower wavelength and lowest shown in higher region. Should I take the higher wavelength?

Yes, that is the expected behavior, and the reason why people use longish (600-700 nm) wavelengths when using OD as a proxy for turbitdity. It is rare to find a difference between 600 and 660, unless your microorganism is secreting colored compounds.

First of all the term OD is wrong !. Even if it is widely used it is wrong. There are no density units involved. The proper term is Attenuance or Extinction, both terms include absorbance and scattering. Please see:

Braslavsky et al, "Glossary of Terms used in Photochemistry. IUPAC Recommendations." Pure Appl. Chem., Vol. 79, No. 3, pp. 293–465, 2007.

Second, it is obviously only by dilution that you get such numbers, which essentially means that your sample is totally opaque. You will never be able to "eliminate" the absorbance of secondary metabolites, in particular, many cells have coloured compounds. Each culture should be treated differently, i.e., different wavelengths should be used to measure growth, depending of the possible substances produced by the cells.

To add a remark to Silvia, at 600 nm in microbial cultures you're actually determining turbidity. But that's semantics. What the instrument measures (and this is important) is transmittance which is automatically converted to OD or extinction or ... give it a name. This means that at higher ODs the instrument is multiplying very few photons on a photomultiplier tube to a measurable signal and hence creates high signal to noise.

On the other hand, the ODs you're attaining are indeed high and fast (if you are starting at ODs well below 0.5). Are you sure you do not have bacterial contamination in your cultures? They can easily outgrow your yeasts. Plating on a general agar or microscopy will tell you. Faster (but maybe I shouldn't suggest this) is the smell of your cultures.

Freddy Dardenne, no, the stock culture was maintained with amipicillin with 50 micrograms per ml concentration, and now the same concentration was maintained for the experiment.. But since the OD continued to show that high, hence I have started to monitor the cultures for every 3 hours. Lets see, to which extend the readings continue to rise. It is not possible to go for other expensive or time consuming methods right now, so OD reading is the only way left for me.