Could radio waves be used in a similar to light in Spectrophotometry

[large_ghostman]

Hi,
This might seem a bit daft but I thought I would ask anyway.
As you know light is normally in spectrophotomtry to give a rough estimate of cell count/density, it works well enough especially using a curvette and machine designed to do the test. Sometimes it would be handy to be able to do something similar using the actual reaction vessel instead of taking a sample out.
There are a couple of seemingly simple experiments I have on the go using bacteria/yeast/'other' Methanogens, the problem is taking a sample can cause havoc and is generally destructive to the sample in the conditions I have inside the vessel. I dont have anyway to take sample and return the cells to the vessel. The following are some of the problems I have............
The reaction vessel is anoxic conditions, I can maintain this using a septum and syringe to take the sample. The problem is however I often have to do serial dilutions in order to get a clear enough sample for spectrophotometer to work properly, by the time the sample is dilute enough and a reading is taken many of the cells are dead and I cant return them.

I need to return the cells as part of the experiment is dependent on not altering cell density artificially. I do take samples for viability study etc, but this is once every three weeks. I need a quick easy way to do a general density count every hour preferably (if I can automate it) or 3 time a day at absolute minimum.
I did make a vessel photometer that wasnt great accuracy wise but did fit on the side of the reactor, the problem is now I have to use a light free reactor!
So using something that shines light through the sides isnt going to work.
The original vessel I used was a very dark glass, however this proved to be not dark enough! I then tried stainless steel reactor but this isnt going to work for light or radio waves! So I now have a HDPE reactor thats working well but I can only measure density and cell count every three weeks.

So to get to the point.
Can anyone think of a way to use a low radio signal/wave that I could send through the vessel and measure? Actually the big question is would cells be enough to scatter radio waves like they can light waves and could this be measured?
I would be interested in knowing first of all could cell density scatter/affect radio waves? If I can get an answer to that then I will at least know if its even worth looking at. I have been given £700 to do these experiments and I have around £145 left, I need around £15 of this however to keep the experiment going (reagents blah blah blah).

 

[jpanhalt]

Hi,
This might seem a bit daft but I thought I would ask anyway.
As you know light is normally in spectrophotomtry to give a rough estimate of cell count/density, it works well enough especially using a curvette and machine designed to do the test. Sometimes it would be handy to be able to do something similar using the actual reaction vessel instead of taking a sample out.

Why do you have to remove a sample? Reaction vessels with built in cuvettes have been known for a very long time.

There are a couple of seemingly simple experiments I have on the go using bacteria/yeast/'other' Methanogens, the problem is taking a sample can cause havoc and is generally destructive to the sample in the conditions I have inside the vessel. I dont have anyway to take sample and return the cells to the vessel. The following are some of the problems I have............

Not quite sure what all the dots mean to say, but in general, performing any experiment on a biological specimen disqualifies it for re-incorporation into the untouched population.

The reaction vessel is anoxic conditions, I can maintain this using a septum and syringe to take the sample. The problem is however I often have to do serial dilutions in order to get a clear enough sample for spectrophotometer to work properly, by the time the sample is dilute enough and a reading is taken many of the cells are dead and I cant return them.

See above. Are you simply trying to count them?

I need to return the cells as part of the experiment is dependent on not altering cell density artificially. I do take samples for viability study etc, but this is once every three weeks. I need a quick easy way to do a general density count every hour preferably (if I can automate it) or 3 time a day at absolute minimum.

How does removing a sample affect the "density?"

I did make a vessel photometer that wasnt great accuracy wise but did fit on the side of the reactor, the problem is now I have to use a light free reactor!
So using something that shines light through the sides isnt going to work.
The original vessel I used was a very dark glass, however this proved to be not dark enough! I then tried stainless steel reactor but this isnt going to work for light or radio waves! So I now have a HDPE reactor thats working well but I can only measure density and cell count every three weeks.

Why does it need to be light free? Will IR work? If they are sensitive to any EM radiation (BTW, an experiment I did in high school on antimicrobal sensitivity), how will using radio help?

So to get to the point.
Can anyone think of a way to use a low radio signal/wave that I could send through the vessel and measure? Actually the big question is would cells be enough to scatter radio waves like they can light waves and could this be measured?
I would be interested in knowing first of all could cell density scatter/affect radio waves? If I can get an answer to that then I will at least know if its even worth looking at. I have been given £700 to do these experiments and I have around £145 left, I need around £15 of this however to keep the experiment going (reagents blah blah blah).

It would be extremely helpful, if you would define two thing ins one sentence each:
1) What is the size of the cells you need to test; and
2) What do you need to measure?

John

 

[large_ghostman]

Why do you have to remove a sample? Reaction vessels with built in cuvettes have been known for a very long time.

Yes I agree, however I dont have one and the one I am using is non standard, it was put together for the experiment. The reaction vessel itself has a heating jacket and various input/output ports for nutrients etc, having a reactor custom built with a curvette inside is outside the budget by a long way.

Not quite sure what all the dots mean to say, but in general, performing any experiment on a biological specimen disqualifies it for re-incorporation into the untouched population.

Again normally I would agree, this is one reason I am trying to avoid removing organism's/cells in the first place. The other reason being on occasion various cells/organisms have shown changes and I would like these cells to remain in culture.

See above. Are you simply trying to count them?

I cant count them as such especially using a spectrophotometer, it gives a rough idea of the cell number but when I need to 'count' I use a hameocytometer as its more effective to count viable cells rather than all cells.

How does removing a sample affect the "density?"

Density is a function of cell number, remove a number of cells and the culture density (number of cells not SG) is lowered. However the main reason for not removing is to leave the many mutated (I cant think of the correct word, but its close enough) in the culture, in part its the mutations I am after as part of the end goal.

Why does it need to be light free? Will IR work? If they are sensitive to any EM radiation (BTW, an experiment I did in high school on antimicrobal sensitivity), how will using radio help?

Some of the organisms have proven to be extremely light sensitive, also the new reaction vessel is black high density plastic and wont allow light through.
If radio waves scatter or loose intensity in a culture where the number of organisms/cells increases or decreases then hopefully I can pass a radio wave through the vessel and get an idea how the culture is changing regarding overall density/numbers, admittedly it still wont tell me if the culture is viable or if the change is due to many dead cells

It would be extremely helpful, if you would define two thing ins one sentence each:
1) What is the size of the cells you need to test; and
2) What do you need to measure?

Various size as more than one organism is present but from small Archea (8um rougly) upto around a little bigger than yeast.

I need a rough idea how many cells/organisms are present and the rate of increase/decrease, like a yeast culture this culture can go from almost clear to opaque and fairly dark.

 

[jpanhalt]

Your interpretations are wrong on several counts. For example:

I cant count them as such especially using a spectrophotometer, it gives a rough idea of the cell number but when I need to 'count' I use a hameocytometer as its more effective to count viable cells rather than all cells.

How does a hemocytometer count "viable" cells? Answer, it doesn't. The usual way to determine viability is subculture. I don't know any other way. Do you?

Density is a function of cell number, remove a number of cells and the culture density (number of cells not SG) is lowered. However the main reason for not removing is to leave the many mutated (I cant think of the correct word, but its close enough) in the culture, in part its the mutations I am after as part of the end goal.

When you remove cells, you also remove an equivalent volume of medium. Thus the cell count/medium volume doesn't change. If your culture is not evenly distributed, then it really doesn't matter either, but how can you then be sure the sample you take and/or measure is representative?

If you want a size distribution, withdraw an aliquot and use flow cytometry. Your presumed need to determine size distribution is a new criterion you have just added.

You have not answered my questions.

John

 

[large_ghostman]

How does a hemocytometer count "viable" cells? Answer, it doesn't. The usual way to determine viability is subculture. I don't know any other way. Do you?

Yes its called selective staining, you can do it two ways. First way live cells take up the stain and dead cells dont, so you count the live cells. The other way is the opposite with the stain only taken up with dead cells as the live cells metabolize the stain, this is often done with yeast cultures. In my culture the stain's are different to what you use with yeast but same principle. This explains how to use a haemocytometer https://www.abcam.com/protocols/counting-cells-using-a-haemocytometer

When you remove cells, you also remove and equivalent volume of medium. Thus the cell count/medium volume doesn't change. If your culture is not evenly distributed, then it really doesn't matter either, but how can you be sure the sample you take and/or measure is representative?

As explained I have more than one reason I would like to avoid removing cells, the culture is both stirred and several times a day pumped around the vessel.

This is a similar vessel except most of it is plastic and glass.

So any idea if this can be done with radio waves? I have the biology side very well covered, and while yes flow cytometry is accurate it isnt applicable in this instance, as stated cell size isnt uniform and I dont need the accuracy, also I cant fit one in the vessel let alone afford one

 

[jpanhalt]

Vital staining has been around for years. Do you have the data to support your claim that it completely differentiates viable cells from non-viable cells in your system? As you know, there is a difference between viable and simply having preserved some processes of life.

Your equipment looks impressive, but equipment alone does not a valid experiment make.

I don't see why you cannot remove a small aliquot from a stirred culture. I have done it many times.

Anyway, you still have not answered my two questions. It seems you want to pursue radio waves to do something, as yet not well defined, without any firm theoretical basis or previous studies to suggest it will work.

Please let us know how your experiments turns out. Good luck.

John

 

[large_ghostman]

Vital staining has been around for years. Do you have the data to support your claim that it completely differentiates viable cells from non-viable cells in your system? As you know, there is a difference between viable and simply having preserved some processes of life.

Yes. I would rather supply for yeast as I dont want to give enough information to identify the exact strain and species I am using.
https://link.springer.com/article/10.1007/BF00132172 ,
BittonG,Ivanoff DB,Koopman B(t991)INT reduction as a basis for determining baker's yeast viability.J Food Sci Technol 26:307-311.

there are however MANY papers dealing with it on google scholar. Viability in this case only has to show the cell is still metabolizing, this is accomplished in my case by using stains that are metabolized (or not, as I use both methods to check). I am not trying to be difficult in not discussing the exact method or combination of methods used, it wouldnt take a genius to work out each of the nine organisms/strains I am using if I supplied the methods and/or dyes being used. I can say this isnt a half a@@ed experiment, i have done a great deal of research and had help both from my dad (a great Biologist and scientist) and from people who work in a university lab.

(1) Various size as more than one organism is present but from small Archea (8um rougly) upto around a little bigger than yeast.

(2) I need a rough idea how many cells/organisms are present and the rate of increase/decrease, like a yeast culture this culture can go from almost clear to opaque and fairly dark.

I thought the above had answered both questions, I have added numbers in case that makes it clearer. If you need further information i can try and make it clearer but I am not always great at getting my questions across.
This may help but is more than 2 sentences sorry.

Lets use yeast as an example, although its not a great comparison. Yeast cultures can start off fairly clear and passing light (visible or IR) through a known amount of the solution will scatter depending on the density of the culture. As a yeast culture grows the amount of light it scatters and lets through to the detector decreases. This can be measured and give a reasonable idea of how the culture is behaving, if done correctly you can determine if the culture is in a heavy growth phase or on a decline.
Again as stated I have more than one reason to try and remove as few cells as I can, also removing cells takes time. A method where it could be done hourly would be of great benefit, it isnt possiable for me to access the culture once an hour and even if I did the small aliquot soon adds up.
I do already take samples, as I said earlier once roughly every three weeks I obtain samples. Taking samples is a problem and the equipment allows more than one method.

The equipment I am using is a mixture of professional grade from my dads lab at both home and university, and some I have made or had made. The picture is of one of the vessel's I was using in an earlier experiment, the new reactors are a little different but the stirring and heating mechanism is the same.
The experiment came about from something I observed in a unrelated experiment, I discussed this with a contact of my dads who gave me some money to help me follow it up. It may well lead to a dead end but if it dosnt then it will have been worth all the effort and time I have invested in it.

Its late so I might be off until tomorrow

 

[large_ghostman]

Thanks anyway but never mind I got an answer, and a pretty good solution

 

[large_ghostman]

Ok turned out not to be a good solution after all, light is therefore OUT! If anyone understands the question do you think its viable to measure cell quantity/density using some form of radio wave?

Where have all the radio brains gone?

 

[Les Jones]

I do not believe that radio waves by themselves will work. The nearest way I think it could work is using nuclear magnetic resonance. The sample has to be in a very strong magnetic field and a radio frequency field at the same time. The frequency at which molecules resonate is a function of the magnetic field strength. I do not think it would be possible to use this method for your experiments as the sample needs to be in very controlled conditions. In my first job after leaving school (At Liverpool university.) I used one of these machines. (A Varian A60) The sample was placed in a thin glass tube (About 4mm dia.) and this was placed in the magnet assembly and rotated at high speed using a compressed air jet. The output was a graph of absorbsion against frequency or magnetic field strength. (I can't remember which was varied for the scan.)

Les.

 

[large_ghostman]

I do not believe that radio waves by themselves will work. The nearest way I think it could work is using nuclear magnetic resonance. The sample has to be in a very strong magnetic field and a radio frequency field at the same time. The frequency at which molecules resonate is a function of the magnetic field strength. I do not think it would be possible to use this method for your experiments as the sample needs to be in very controlled conditions. In my first job after leaving school (At Liverpool university.) I used one of these machines. (A Varian A60) The sample was placed in a thin glass tube (About 4mm dia.) and this was placed in the magnet assembly and rotated at high speed using a compressed air jet. The output was a graph of absorbsion against frequency or magnetic field strength. (I can't remember which was varied for the scan.)

Les.

Thanks for that, I feared it wouldnt be as easy as it seemed. I tried with something alot like a sensitive TDS device, the problem with that being the various nutrients when added mess up the readings.
Time to get really creative, I might take a look at gas make up with the Gas Chromatograph when I do my 3 weekly samples. No idea yet but maybe there is a clue in the gas make up that relates to cell density or rather numbers of active cells, I can sample the gas easy enough although not hourly. Some of the organisms respire different gasses as well as other metabolites, taking actual culture samples causes real problems. But I have to vent the gas and analyze it often anyway, the downside is it makes the experiment even more complex and I was trying to keep it as simple as I could.
The cost soon starts to ramp when you start tracking more parameters, but it might be worth it in the end and its certainly interesting and educational.