EE - Project Eradicating Bacteria/Biofilm

[TGH904]

Hello all,

I have a question about the electrical engineering necessary to design an experiment in which one would test various electrical parameters, such as high voltages, low voltages, low currents, varying signals (direct current, square waves, etc) on biofilms, and bacteria.

I have asked a similar question on the Arduino forums asking how I can implement an Arduino in a power supply design to control the electrical parameters, but now I am wondering more about the bacteria as part of the circuit.

Essentially the bacteria will be grown, and stored on a petri dish, or a biofilm grown on a 'coupon', and with some power supply system, providing measurable, and controllable voltage, current, and signals will be directly applied to these substances.

My questions are:

1) Can someone point me in the right direction of a power supply that will provide for such control, or the schematic for building my own? (There may be more ways to tackle this question I am open to suggestions)
2) What kind of engineering equations in the electrical world will I need to understand, and know for evaluating my experiment? (In the electrical engineering world, not the microbiology evaluation techniques)
3) What impact will the bacteria have as a circuit element?*

*For the last question I am not sure if it is best to ask a microbiologist as they would know more about the fundamental properties of bacteria/biofilms, yet they may lack the basic, or advanced understanding of electrical engineering fundamentals and how to apply those concepts.

Does this make sense? I will provide links to various articles where other researchers have conducted their own experiments using electrical stimulation on bacteria/biofilms, so that anyone interested in this project/topic may review them. As well as a link to the Arduino forum where I have an on going discussion there as well.

I appreciate your time, and help. Thank you very much and I look forward to hearing back from you.

https://forum.arduino.cc/index.php?topic=488615.0

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.548.8043&rep=rep1&type=pdf

http://www.rehab.research.va.gov/jour/04/41/2/pdf/Merriman.pdf

http://aac.asm.org/content/52/10/3517.full

 

[berkeman]

Welcome to the PF;; nice first post!

What standard lab equipment do you have available? Do you have any of the equipment shown in the papers you linked to?

For example, some signal generators will be able to give you medium voltage outputs, like this:

http://www.transcat.com/rigol-dg102...MIuIqKlZuJ1QIVxZd-Ch3-tQD2EAQYAyABEgI6XvD_BwE

 

[TGH904]

Welcome to the PF;; nice first post!

What standard lab equipment do you have available? Do you have any of the equipment shown in the papers you linked to?

For example, some signal generators will be able to give you medium voltage outputs, like this:

http://www.transcat.com/rigol-dg102...MIuIqKlZuJ1QIVxZd-Ch3-tQD2EAQYAyABEgI6XvD_BwE

View attachment 207159

Hey thank you for your reply!

I am a penniless researcher essentially, so my equipment is not the best. I am current looking into power supplies, but I am interested in the ones around 50 - 100 dollar range. Which I have yet to find a 'good' power supply capable of producing various signals, so I have decided to settle for a power supply with voltage/current control where I would then use maybe a 555 timer, so produce a square wave of my liking, and I understand there is some necessary circuity design to go along with this, but everything is all still in prototype/paper design phase.

In short I do not have any of the equipment these researchers have, and I have found they really don't know much about the equipment other than some electrical engineers built and designed it for them, told them how to use it, and they conducted their experiments with that knowledge.

I don't think they really were involved with the design of the electrical system.The equipment I have/used would be:

WON Smart DS7102V oscilloscope: Used to look at my signals
Craftsman Multimeter: Used for measuring current/votages
Arduino Unos: Used as an attempted power supply
TENS Unit 7000: Used as power supply/signal control

and lots of little components (resistors, capacitors, transistors, 555 timers, etc.)

 

[berkeman]

TENS Unit 7000: Used as power supply/signal control

How controllable is that TENS unit? Can you use it as an arbitrary waveform generator?

 

[TGH904]

How controllable is that TENS unit? Can you use it as an arbitrary waveform generator?

It is controllable. I used the scope to measure the output voltage, and this is controlled by a little knob on the top, but it ranges between 0 - 150 Volts maybe? It is analog control so I can't exactly program any specific output voltage which makes repeating an experiment difficult and is not ideal. The other settings are controlled digitally using some press buttons for controlling the type of mode like whether its pulsating(BURST), or gradually getting stronger (MODULATION) etc, and the last three settings are 'time' (duration of the device to be on), 'width of pulse' in micro S, and 'Rate' measured in Hz.

Here is a link to the exact device I have:
https://www.tenspros.com/tens-7000-...-JaC6p_uHAlntFdW_2VJZmARjzM_UyHTmDBoC-DXw_wcB

I had purchased two of these. One for deconstruction to see how the design of the circuit, because I originally thought I may need to replicate this product, but implement my own controllable features, or rather somehow make the voltage be controlled digitally so I can reproduce exact voltage levels, but I have been unsuccessful in doing that. The other one I have used for just a 'I want to see if this will even kill bacteria on the highest setting experiment', so it is mostly for mock experiments until I can actually design a working system like these other guys.

I found that using the TENs on the highest setting doesn't kill bacteria with electrical stimulation alone, but the gel electrode pads I had used actually had more antimicrobial effects, and killed the bugs on the plate (petri dish) instead. So I then decided to run the experiment again, without the gel electrodes, and just use 'wires' (basically the two leads without the gel pads attached), and I was still unsuccessfully at killing anything at all!

Which is why I am wondering if it is a connection issue, and that I am not actually completing my circuit, and my current isn't running through my bacteria.

 

[Windadct]

I do know the US Dep of Ag (USDA) had developed a RF sterilizer for things like Apple Juice a few years ago.

https://www.researchgate.net/publication/222674784_Radio_frequency_electric_fields_processing_of_orange_juice

Another Example

This is focusing on the NON-Thermal processes.

https://ucanr.edu/repositoryfiles/ca6004p192-69377.pdf

The energy levels were pretty high, but the benefits ar NOT heating and affecting the quality of the food.

 

[TGH904]

I do know the US Dep of Ag (USDA) had developed a RF sterilizer for things like Apple Juice a few years ago.

https://www.researchgate.net/publication/222674784_Radio_frequency_electric_fields_processing_of_orange_juice

Another Example

This is focusing on the NON-Thermal processes.

https://ucanr.edu/repositoryfiles/ca6004p192-69377.pdf

The energy levels were pretty high, but the benefits ar NOT heating and affecting the quality of the food.

Hey, thanks for those interesting articles. I have a couple of questions as I have also been interested in stimulating bacteria with RFEF, expect I am not sure even how to construct a circuit like this...

How would you design such a circuit to simulate an object with radio frequency electric fields?

I understand if I have a coil of wire with current running through it and I move a conductive rod through the coil I generate an electric field. My understanding of electric fields is very basic, could you elaborate or point me in the right direction to learn this topic in more detail.

Also, are these E.F. strengths of 15 and 20 kV/cm safe?

How do you have frequency control of these signals?
Thanks again.

 

[Windadct]

In the project I came across - they just used an H bridge and fed the field with a Square Wave (From an RF standpoint I would call this sloppy due to the high frequency spread, however this could by why it was effective..). But it was 7 - 8 years ago. ( OH - it was 700V and ~200 A - not trivial )

Try pulling some key words out of the articles and see if you can Google up more info.

 

[TGH904]

In the project I came across - they just used an H bridge and fed the field with a Square Wave (From an RF standpoint I would call this sloppy due to the high frequency spread, however this could by why it was effective..). But it was 7 - 8 years ago. ( OH - it was 700V and ~200 A - not trivial )

Try pulling some key words out of the articles and see if you can Google up more info.

So you're saying they were effective because they used highly lethal amounts of current, and voltage levels?

Thanks again.

 

[Windadct]

Well they were for commercial application, not a lab, so it required a good mount of power, and the system would not apply to a Petri...

So what is the background on your research - is this an college thing, or commercal? --- I may be able to dig up a contact.

 

[TGH904]

Well they were for commercial application, not a lab, so it required a good mount of power, and the system would not apply to a Petri...

So what is the background on your research - is this an college thing, or commercal? --- I may be able to dig up a contact.

I am researching on my own. I have connections with a lab at school that can produce some bacteria for me. I mostly am researching the 'modes' of the biocidal effects of electrical current on bacteria. I want to optimize test methods, and control test parameters, so I can understand the effects of electrolysis in biofilm cells.

 

[Windadct]

Current will be difficult ( IMO ). Biological systems (structures) are chaotic, and that will lead to chaotic current pathways - so the outcomes will be inconsistent. If a colony "bridges" the electrodes vs one that does not, will have very different current levels, and thus outcomes. If you homogenize the samples it may be different.

 

[TGH904]

Current will be difficult ( IMO ). Biological systems (structures) are chaotic, and that will lead to chaotic current pathways - so the outcomes will be inconsistent. If a colony "bridges" the electrodes vs one that does not, will have very different current levels, and thus outcomes. If you homogenize the samples it may be different.

How about this test experiment idea.

I want to measure the amount of current that will pass through my medium, and the resistance is also contains. The experiment would be to use a medium delivered to stimulate the bacteria to form a biofilm growth.

I would then use a simple series circuit to measure current, voltage, and calculate resistance of my medium.

Vin --- resistor ---- LED ---- medium ---- Gnd

The resistor/led will allow me to confirm with just the medium I am completing my circuit.

Then I would use a multimeter to measure across my medium to measure voltage, and put my multimeter in series with my medium and Gnd to measure the current leaving the medium.

If I am successful in doing so, then introducing bacteria to the equation could allow me to obtain information in whole about specific densities of bacteria population.

I understand that the medium, and bacteria will have two different resistance levels, there the electrons moving through the circuit will treat the two 'circuit elements' as a parallel resistor circuit. If I know what current I have across my medium with varying voltage amounts, and noting down the various current levels I could then assume this would be the amount of current following through my bacteria in a parallel circuit, correct?

I am not sure if this is plausible, or will work. I will run some experiments on just medium broth today.

Let me know what you think, or if you have any way you would revise this to obtain more accurate results.

 

[Windadct]

Very quickly becomes - pretty complex, and many variables. As you have seen the gel on the electrodes had an effect on the samples. So - electrodes is the first item, what is the most benign electrode - how do you validate or quantify the effect of the electrodes?.

My concern is the effect of current over time, low current may stimulate growth, high current may cause a fuse effect, where a colony creates a "fuse" and is killed by a spike in current that is not observed - and then not understood. ( needing an analog trigger to take a reading - possibly)

Well worth the study, or learning effort, just not trivial.

 

[TGH904]

Very quickly becomes - pretty complex, and many variables. As you have seen the gel on the electrodes had an effect on the samples. So - electrodes is the first item, what is the most benign electrode - how do you validate or quantify the effect of the electrodes?.

My concern is the effect of current over time, low current may stimulate growth, high current may cause a fuse effect, where a colony creates a "fuse" and is killed by a spike in current that is not observed - and then not understood. ( needing an analog trigger to take a reading - possibly)

Well worth the study, or learning effort, just not trivial.

So, I did an experiment where I had a TENs device with some adhesive electrodes. The idea was just to see if I could kill bacteria using a TENs 7000 Unit and a med range setting. I measured out with an oscilloscope the average voltage wave (pulsating square wave) at 50 Volts. The time duration of experiment was 60 minutes, the rate of frequency was 5.0 Hz, and a pulse width of 100 micro S. The outcome with this experiment was that with, or without the device on I killed bacteria. I wondered why, and I realized that these adhesive pads which are about 1" by 1" where naturally antimicrobial. The next experiment I used the same settings on the TENs device, and eliminated the electrode pads by using the metal conductors without attaching the probes. It will inevitably have a greater current density at a more localized point where it made contact with the bacteria. Therefore, I thought I would see an excess amount of kill around the electrode probes, and maybe an arced line of kill connecting the probes, but in the end I didn't have any kill, and the results were inconclusive.

This is why I want to confirm I am actually making a connection through the medium/bacteria so I am hitting them with the current. All of the papers I have read have listed a wide range of parameters for killing bacteria in their experiments, but I think I have settled on just trying the lowest direct current I can achieve a successful kill with.

I have a new experimental idea involving electrodes at a fixed distance inserted into a flowing medium where the surfaced attached bacteria are located directly in between the two probes, but not touching the electrodes. I will attach a copy of sketch I made for this experiment, please tell me what you think, or how I should revise it.

Also the picture may be upside down.

 

[Windadct]

Still - I am more suspicious of the metallic ions you put into the sample than the current itself.

I am checking with an expert on this...literally.

 

[TGH904]

Still - I am more suspicious of the metallic ions you put into the sample than the current itself.

I am checking with an expert on this...literally.

OK. Let me know what you and your expert think on this one. I am currently in the process of mapping out current levels in relationship with distance of some Tripic Soy Broth. I have measured out a petri dish with .5 cm marking across the diameter of the dish which will indicate where I play my probe.

 

[Windadct]

OK - you asked. It helps to have a microbiologist brother...

"
Hi ,

I am not sure from your colleague's post if there intention is to kill the bacteria, or the push juice through them for another reason.

There are a couple of things to consider. Most bacterial growth medium is salty (quite conductive, 100-200 mS/cm), so assessing if the current is flowing through the medium or through them (or a combination) may be hard. They will get hot though.

We use a technique to get DNA into bacteria called electroporation; it applies a ~10-20 kV/cm between two electrodes for a few milliseconds (is an exponential decay from capacitors, so we monitor the time constants). Those cells are carefully prepared in clean water and made at a very high concentration (forcing juice through them). An old reference for the technique can be found here:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC336852/pdf/nar00156-0416.pdf
When that technique is performed, you blast away ~90-99 % (or more) of the cells; the survivors are then recovered for evaluation. It is believed that a subset get transient holes ripped into them, but survive.
Another side to the story is the bugs themselves. A lab standard like E. coli maintains a 100-200 mV potential across its inner membrane and this potential is necessary to stay alive. There are staining methods used to monitor maintenance of this potential and to assess whether the cells are alive or not. So, a strong current will certainly mess that up. Curiously, it was recently discovered that E. coli "blinks" this potential for unknown reasons (maybe to communicate, but probably to help remove waste); see:
https://www.ncbi.nlm.nih.gov/pubmed/21764748
(sorry, don't have Science subscription on this computer, but the movies of them blinking is really cool)
Aside from this potential, if a bacterium is respiring (dumping electrons onto an exogenous molecule), there will be an electron transport chain of conductive molecules in the inner membrane (this transport is one of the ways they set up that outside-inside potential mentioned above; there is also a proton gradient formed). Depending on the input and output chemicals' redox potentials, this chain may hold a voltage potential of ~0.1-0.4 volts (insulated from the potential across the membrane). So, holding a voltage potential against this chemical potential will alter the ability of the microbe to run its metabolism.
Finally, many bacteria are constantly searching for a place to dump their electrons from metabolism, and some have evolved very cool ways to search for the right redox potentials on the surfaces around them. If you take a mixed microbial population, say from mud, and culture them in a medium with electrodes holding different potentials (in the + 0.1 - 0.5 V range), some will migrate to the surface that has the best potential for their needs. Different bugs go to different places. This stepping of metabolic redox potentials is one the ways a community of microbes can be more efficient that a single form. Rather than having one cell synthesize all possible outputs for electrons from all possible inputs, each microbe can specialize to adapt to a few food/drain sources; but collectively, a much larger redox environment can be maintained and some very hard chemistries can be accomplished. Too much juice through one bug's membrane would fry it (or leak electrons too much), but collectively they can take on the world.
I hope that helps.
Cheers,
S"

 

[TGH904]

OK - you asked. It helps to have a microbiologist brother...

"
Hi ,

I am not sure from your colleague's post if there intention is to kill the bacteria, or the push juice through them for another reason.

There are a couple of things to consider. Most bacterial growth medium is salty (quite conductive, 100-200 mS/cm), so assessing if the current is flowing through the medium or through them (or a combination) may be hard. They will get hot though.

We use a technique to get DNA into bacteria called electroporation; it applies a ~10-20 kV/cm between two electrodes for a few milliseconds (is an exponential decay from capacitors, so we monitor the time constants). Those cells are carefully prepared in clean water and made at a very high concentration (forcing juice through them). An old reference for the technique can be found here:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC336852/pdf/nar00156-0416.pdf
When that technique is performed, you blast away ~90-99 % (or more) of the cells; the survivors are then recovered for evaluation. It is believed that a subset get transient holes ripped into them, but survive.
Another side to the story is the bugs themselves. A lab standard like E. coli maintains a 100-200 mV potential across its inner membrane and this potential is necessary to stay alive. There are staining methods used to monitor maintenance of this potential and to assess whether the cells are alive or not. So, a strong current will certainly mess that up. Curiously, it was recently discovered that E. coli "blinks" this potential for unknown reasons (maybe to communicate, but probably to help remove waste); see:
https://www.ncbi.nlm.nih.gov/pubmed/21764748
(sorry, don't have Science subscription on this computer, but the movies of them blinking is really cool)
Aside from this potential, if a bacterium is respiring (dumping electrons onto an exogenous molecule), there will be an electron transport chain of conductive molecules in the inner membrane (this transport is one of the ways they set up that outside-inside potential mentioned above; there is also a proton gradient formed). Depending on the input and output chemicals' redox potentials, this chain may hold a voltage potential of ~0.1-0.4 volts (insulated from the potential across the membrane). So, holding a voltage potential against this chemical potential will alter the ability of the microbe to run its metabolism.
Finally, many bacteria are constantly searching for a place to dump their electrons from metabolism, and some have evolved very cool ways to search for the right redox potentials on the surfaces around them. If you take a mixed microbial population, say from mud, and culture them in a medium with electrodes holding different potentials (in the + 0.1 - 0.5 V range), some will migrate to the surface that has the best potential for their needs. Different bugs go to different places. This stepping of metabolic redox potentials is one the ways a community of microbes can be more efficient that a single form. Rather than having one cell synthesize all possible outputs for electrons from all possible inputs, each microbe can specialize to adapt to a few food/drain sources; but collectively, a much larger redox environment can be maintained and some very hard chemistries can be accomplished. Too much juice through one bug's membrane would fry it (or leak electrons too much), but collectively they can take on the world.
I hope that helps.
Cheers,
S"

Well, this is beyond helpful, and thank you and your brother for all of the insight on this project. I want to inform you of my experiment yesterday and my results.

As I had mentioned I was going to run an experiment with a simple programmed Arduino to output between 0 - 5 V using a keypad. I only used an output of 5 V for this experiment,

Vin (5V) ---- 1k Ohm resistor ----- 1.3 V drop Blue LED ------- Tryptic Soy Broth Medium ----- Multimeter to measure current ---- GND

I measured out a small amount of TSB into a petri dish, and measured the diameter of the petri dish (8.7 cm), and marked .5 cm across the length of the petri dish where I would move my probe to measure current at various marked distances .5 cm's away from one another. I made a make shift Aluminum foil probe measuring 1.2 cm across which was stationary at the 0 cm mark, and the other probe was a stainless steel probe from the multimeter. I essentially used a method called 'spot sampling' across the length of petri dish and noted down the current at 5 V at different distances 0 - 8.7 cm.

I then removed the resistor and LED and ran straight 5 V into my TSB, and did the same exact experiment. What I found though was that galvanic corrosion had been taken place on the Aluminum foil and in relatively short time I had eroded the stationary probe away. Looking under a microscope I could see this happening.

Today I will experiment with different probe materials. The first one will be a carbon fiber thread. I will take apart the original adhesive electrode I used in previous experiments and strip it down to the carbon thread material lining the inside of the square pad, and see if I experience any galvanic corrosion with this in such short time.

I do think I have been successful in achieving a steady draw of current in this experiment. When I get my hands on some more bacteria I will be able to kill bacteria using only 5 V, as the most current I had drawn across the medium was only 33 mA when the probes were .2 cm away from each other. Other researchers had observed bacteria kill at 500 micro A. I can then limit current even further by adding more resistors in series with this experiment.