How to verify the concentration of a biopesticide in a mixture using Spectrophot - (Oct/14/2014 )

Hi, I need advice regarding this matter.

There's a biopesticide called Vectobac WP. It contains Bacillus thuringiensis spore as the active ingredients. From my understanding it forms crystal protein which can be digested to release endotoxin to kill mosquito larvae. It is in powder form and need to be mixed with water at x mg/L before application. The mixture will be dark brown in colour. A client tasked us to check whether their contractor mix the product according to the label rate which is x mg/L. They'll regularly sample their contractor's preparation and send for test. But no one in the lab knows how to do it... 

I have an idea but not sure whether it works.

First is to mix the product according to the label rate and conduct spectro to get the reading. Do it for a few reps.

Then whenever samples are received, we will just do spectro to get the reading and use maybe Chi square test to see whether the reading is significantly different from the standard. Would this work? The mixture is dark brown in colour, will it affect the reading/which wavelength is better?

I was told that I'll need technical grade of the active ingredient to construct a standard curve. However, because I don't need the exact concentration of the sample, I figure that it is not required. Even if standard curve is required, can I use the product with maybe 35% concentration of the ai instead of the technical grade?

I look forward for your kind feedback.

P/S: we dont have a spectrophotometer in the lab  ... that's why I need to need some input before I can ask to buy 1.

My understanding of this (from a quick google search), is that this is a wettable powder - i.e. a suspension, not a solution!  To measure it, a spectrophotometer would probably work well in absorbance mode, but you would need to determine the wavelength to use. If you use an inappropriate wavelength, the measurements you take will be non-linear and not useful. Ideally this wavelength would be one at which other compounds in the solution (wetting agents etc) don't absorb. I suspect that as this is a bacterial species spore, you could also do it by plating the spores on appropriate growth medium and doing colony counts.

You should establish a standard curve, as just doing one measurement and is not ideal, especially when someone is asking (and paying?) you to determine the concentration used.

Another possibility would be spore counts using a cell counting chamber and microscope. But you might need to know the spore number within x milligrams.

Not sure if the same would be possible with plating out the spores on an appropriate medium and count colonies then (there are mediums on which BT is growing), as I don't know if the spores are viable or not. If it's possible you can automat this procedure if the equipment is available and you get the number of viable spores.

bob1 on Wed Oct 15 03:28:15 2014 said:

My understanding of this (from a quick google search), is that this is a wettable powder - i.e. a suspension, not a solution!  To measure it, a spectrophotometer would probably work well in absorbance mode, but you would need to determine the wavelength to use. If you use an inappropriate wavelength, the measurements you take will be non-linear and not useful. Ideally this wavelength would be one at which other compounds in the solution (wetting agents etc) don't absorb. I suspect that as this is a bacterial species spore, you could also do it by plating the spores on appropriate growth medium and doing colony counts.

 

You should establish a standard curve, as just doing one measurement and is not ideal, especially when someone is asking (and paying?) you to determine the concentration used.

Yup it is not a solution. So I'll have to find out which wavelength to use first. But would the colour of the mixture affect the absorbance reading? Since the colour is dark brown, should i start with the wavelength of brown colour first?

hobglobin on Wed Oct 15 09:40:26 2014 said:

Another possibility would be spore counts using a cell counting chamber and microscope. But you might need to know the spore number within x milligrams.

Not sure if the same would be possible with plating out the spores on an appropriate medium and count colonies then (there are mediums on which BT is growing), as I don't know if the spores are viable or not. If it's possible you can automat this procedure if the equipment is available and you get the number of viable spores.

Indeed, I do not know whether the spore is viable. I am still waiting for reply from the supplier regarding the viability but...... well... no answer yet.

Brown isn't a primary colour, it is the result of mixing several colours together, so it isn't much help in determining the right wavelength to use. Your best bet might be to do a dilution series and scan a the full spectrum to see if you can identify a wavelength that gives a line or curve that is easily calculated. Another good starting place would be the literature - see if anyone has come up with methods of measuring concentrations of this (in the purified form if it is the cystals) by spec.

Note that you may need to use a UV spec as well as visible light to get good readings.

I read that at least here (Germany) the formulations against mosquitoes (B.t.i.) only contain inactivated spores as only the toxic protein-crystals are needed. Therefore the spores get a gamma-ray treatment for inactivation. Anyway the spores should be still countable under a microscope.

Check out this: www.who.int/whopes/quality/Bti_eval_spec_October_2012.pdf

There are many protocols in the notes section, perhaps you find something useful.

if you choose to "read" light, and since you are trying to measure a (probably not stable) suspension, you should probably perform static light scattering measurements. this can be performed with a fluorometer or a dedicated light scatter instrument.

if you use a fluorometer then you would use an excitation wavelength with high intensity (ie green, ~500-550nm (this is the wavelength with greatest intensity, at least with older spectrophotometers), unless it is significantly absorbed by the suspension) and read the incident ("emitted" on the fluorometer, but not really) light.

since the suspension is probably not stable, you will either have to make rapid readings or have some mechanism to maintain the suspension (mixing) without interfering with the readings.

you may also be able to use a spectrophotometer. using a wavelength that is not absorbed by the suspended particles, any apparent absorbance would be due to light scatter (deflected from the photoreceptor).