Hello,
I would like to do a science fair project to test the ability of bacteriophages to kill bacteria in a biofilm. I would like to do this using e coli B and the T4r bacteriophage. Is there a different combination of bacteria and phage that might get me better results. Also, I would like to know if I can purchase a biofilm or if I have to grow it myself. If I do have to grow it myself, how would I do so and how long would it take? In terms of the bacteriophage, how should I place the phages on the place to ensure uniform distribution on the plate? If anyone can answer any of these questions I would be extremely grateful. Thank you!
Science Fair Project
Moderators: AmyCowen, kgudger, MadelineB, Moderators
Science Fair Project
How long does it take for an E coli biofilm to grow?
Science Fair Project
Hello,
I am doing a science fair project o to find the minimum dilution of bacteriophage to kill a Pseudomonas aeruginosa biofilm.
I am having trouble finding a phage that will work with this. What phages are best at killing Pseudomonas aeruginosa? I have been looking at multiple phages that are meant for e coli. Would these coliphages also work on P. aeruginosa? My last question is where I would be able to find such phages. I ask this because most websites that sell live organisms, like Carolina Biological and Ward's Science, only supply coliphages and not P. aeruginosa phages. I would be extremely grateful if anyone can answer these questions.
Thank you!
I am doing a science fair project o to find the minimum dilution of bacteriophage to kill a Pseudomonas aeruginosa biofilm.
I am having trouble finding a phage that will work with this. What phages are best at killing Pseudomonas aeruginosa? I have been looking at multiple phages that are meant for e coli. Would these coliphages also work on P. aeruginosa? My last question is where I would be able to find such phages. I ask this because most websites that sell live organisms, like Carolina Biological and Ward's Science, only supply coliphages and not P. aeruginosa phages. I would be extremely grateful if anyone can answer these questions.
Thank you!
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SciB
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Re: Science Fair Project
Well, that depends on the temperature, the availability of nutrients and the material surface on which the bacteria are growing. If you could post again with more specific details we might be able to help you.
Sybee
Sybee
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Re: Science Fair Project
Hello Sybee and Anushatek, you will see that I've merged the separate posts into one thread. Anushatek, it is helpful to keep all of your questions together in one thread so the expert who is helping you can see your posts and see when you have follow-up questions! Thank you!
Moderator
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Re: Science Fair Project
Hello,
Thank you for your response. My original idea was to test the bacteriophages on E coli B. I was planning on using the procedure used in a science buddies project (https://www.sciencebuddies.org/science- ... ia#summary). In this experiment, the goal is to find the minimum dilution of bacteriophages needed to kill all the bacteria on the plate. I have changed the procedure slightly to use an E coli biofilm instead of planktonic bacteria. I will be conducting my experiment at a local community college lab. To grow the biofilm I was planning on using this a procedure similar to this (https://biology.mit.edu/sites/default/f ... ksheet.pdf).
As you can see in the procedure, the biofilms are grown in a petri dish with a basal medium and are grown at room temperature. Could I speed this process up if I put them in an incubator?
One of my posts is about Pseudomonas aeruginosa because, during my research, I found that e coli might not be the best option for growing a biofilm so I tried researching Pseudomonas aeruginosa. This bacteria is good for making biofilms but I could not find any P. aeruginosa phages that I could order online. With this, I went back to the E coli.
In my experiment, I plan on using the T7 coliphage. I am trying to put together a procedure but I am not sure how to place the phages on the biofilm. I have tried researching a procedure like this but I haven't found a way to get the phage on the biofilm. If you have any questions about my project please do not hesitate to ask.
Thank you for your assistance,
Anusha
Thank you for your response. My original idea was to test the bacteriophages on E coli B. I was planning on using the procedure used in a science buddies project (https://www.sciencebuddies.org/science- ... ia#summary). In this experiment, the goal is to find the minimum dilution of bacteriophages needed to kill all the bacteria on the plate. I have changed the procedure slightly to use an E coli biofilm instead of planktonic bacteria. I will be conducting my experiment at a local community college lab. To grow the biofilm I was planning on using this a procedure similar to this (https://biology.mit.edu/sites/default/f ... ksheet.pdf).
As you can see in the procedure, the biofilms are grown in a petri dish with a basal medium and are grown at room temperature. Could I speed this process up if I put them in an incubator?
One of my posts is about Pseudomonas aeruginosa because, during my research, I found that e coli might not be the best option for growing a biofilm so I tried researching Pseudomonas aeruginosa. This bacteria is good for making biofilms but I could not find any P. aeruginosa phages that I could order online. With this, I went back to the E coli.
In my experiment, I plan on using the T7 coliphage. I am trying to put together a procedure but I am not sure how to place the phages on the biofilm. I have tried researching a procedure like this but I haven't found a way to get the phage on the biofilm. If you have any questions about my project please do not hesitate to ask.
Thank you for your assistance,
Anusha
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Re: Science Fair Project
Hi Anusha,
I read the MIT procedure for creating a biofilm of P. fluorescens and I believe it's ok for you to put your Petri dishes of E. coli in a 37C incubator to speed up the biofilm formation. E. coli normally grows at human body temperature, 37C, whereas P. fluorescens grows in soil or water at 20-30C.
One other thing i noticed in the MIT procedure was that they used E. coli as a negative control--meaning bacteria that do not form a biofilm under their conditions. I know E. coli can make biofilms, but I don't know under what conditions. You might want to do a search for this and see if there's a different type of basal medium that you need to use to get the bacteria to produce a biofilm.
As for exposing the E. coli to T7, I would think that all you would do is make a suspension of the phage in broth and dip the slide with the biofilm into it for some length of time. T7 attaches to susceptible bacteria and lyses them fairly quickly at 37C, so experiments are often done at 30C (https://en.wikipedia.org/wiki/T7_phage)
One other thing I read in searching for T7 and biofilms is that if the phage infects the bacteria before they form a biofilm, then they cannot complete it (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136088/). You could try adding T7 to the basal medium with the E. coli and see what effect it has on biofilm formation.
There is a term that phage researchers use that you might want to pay attention to in your experiments and that is 'multiplicity of infection', abbreviated MOI. Put simply, MOI is the number of phage per bacterial cell in your experiment. The measurement is used with liquid [i.e., 'planktonic'] cultures of bacteria and I don't know how you would determine the number of bacterial cells in your biofilm that are exposed to the phage. If you stained the cells with crystal violet and examined the slide at 400X under a microscope you could count the number of cells in a given area and then calculate how many cells there are in the entire area exposed to the T7. I would do some more reading about phage and biofilms and see if published papers give an MOI. If they do not, then you can omit it. I just wanted you to be aware that there is such a measurement and it can be important to interpretation of data.
The next problem is how to measure the killing of bacteria in the biofilm by T7. Do you have a method for this? Maybe all you have to do is count the live bacteria before exposure to T7 then count how many are left after the lytic infection. I don't know if this would work. Have you read any papers that describe how to count bacteria in biofilms before and after some treatment that is supposed to kill them?
Let me know what you think of my suggestions and questions and let's see how we can make your experiments work. Do you have someone at the college lab who can help you with methods and advice?
Sybee
I read the MIT procedure for creating a biofilm of P. fluorescens and I believe it's ok for you to put your Petri dishes of E. coli in a 37C incubator to speed up the biofilm formation. E. coli normally grows at human body temperature, 37C, whereas P. fluorescens grows in soil or water at 20-30C.
One other thing i noticed in the MIT procedure was that they used E. coli as a negative control--meaning bacteria that do not form a biofilm under their conditions. I know E. coli can make biofilms, but I don't know under what conditions. You might want to do a search for this and see if there's a different type of basal medium that you need to use to get the bacteria to produce a biofilm.
As for exposing the E. coli to T7, I would think that all you would do is make a suspension of the phage in broth and dip the slide with the biofilm into it for some length of time. T7 attaches to susceptible bacteria and lyses them fairly quickly at 37C, so experiments are often done at 30C (https://en.wikipedia.org/wiki/T7_phage)
One other thing I read in searching for T7 and biofilms is that if the phage infects the bacteria before they form a biofilm, then they cannot complete it (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136088/). You could try adding T7 to the basal medium with the E. coli and see what effect it has on biofilm formation.
There is a term that phage researchers use that you might want to pay attention to in your experiments and that is 'multiplicity of infection', abbreviated MOI. Put simply, MOI is the number of phage per bacterial cell in your experiment. The measurement is used with liquid [i.e., 'planktonic'] cultures of bacteria and I don't know how you would determine the number of bacterial cells in your biofilm that are exposed to the phage. If you stained the cells with crystal violet and examined the slide at 400X under a microscope you could count the number of cells in a given area and then calculate how many cells there are in the entire area exposed to the T7. I would do some more reading about phage and biofilms and see if published papers give an MOI. If they do not, then you can omit it. I just wanted you to be aware that there is such a measurement and it can be important to interpretation of data.
The next problem is how to measure the killing of bacteria in the biofilm by T7. Do you have a method for this? Maybe all you have to do is count the live bacteria before exposure to T7 then count how many are left after the lytic infection. I don't know if this would work. Have you read any papers that describe how to count bacteria in biofilms before and after some treatment that is supposed to kill them?
Let me know what you think of my suggestions and questions and let's see how we can make your experiments work. Do you have someone at the college lab who can help you with methods and advice?
Sybee
Re: Science Fair Project
Hello Sybee,
Thank you for your suggestions. I have researched more on how to grow biofilms. In my research, I have found a study as well as a video describing how to grow a biofilm. While these procedures aren't specifically for E. coli, I figured that I could change some things to make it more applicable to E. coli. Would you mind looking at these procedures to see if I could use this in my experiment?
Study link: http://journals.plos.org/plosone/articl ... ne.0168615
Video link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/
Also, the study mentioned that they start with ~10^6 cells to grow the biofilm. What do they mean by this? I am planning on using E. coli B for my experiment. Will I have to change the dilution of my bacteria before growing the biofilm?
The procedure needed to quantify the number of cells in the biofilms uses a plate reader. While I do not have access to a plate reader, I figured I could maybe use the procedure you suggested to me. Here is a short procedure that I think may work.
1. grow the biofilms using the procedure mention in (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)
2. stain the biofilms
3. calculate the average amount of biofilm cells in each well by counting the number of cells at 400X
4. grow more biofilms under the same conditions
5. combine the phage and biofilm using this procedure(http://journals.plos.org/plosone/article?
id=10.1371/journal.pone.0168615)
6. After phages have killed the biofilm, stain and calculate the number of cells per well again to find the number of biofilm cells killed by the phages
While my mentor at the community college is a microbiologist, she isn't experienced in growing biofilms. Let me know if you think this could work for my project.
Thank you for your suggestions. I have researched more on how to grow biofilms. In my research, I have found a study as well as a video describing how to grow a biofilm. While these procedures aren't specifically for E. coli, I figured that I could change some things to make it more applicable to E. coli. Would you mind looking at these procedures to see if I could use this in my experiment?
Study link: http://journals.plos.org/plosone/articl ... ne.0168615
Video link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/
Also, the study mentioned that they start with ~10^6 cells to grow the biofilm. What do they mean by this? I am planning on using E. coli B for my experiment. Will I have to change the dilution of my bacteria before growing the biofilm?
The procedure needed to quantify the number of cells in the biofilms uses a plate reader. While I do not have access to a plate reader, I figured I could maybe use the procedure you suggested to me. Here is a short procedure that I think may work.
1. grow the biofilms using the procedure mention in (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)
2. stain the biofilms
3. calculate the average amount of biofilm cells in each well by counting the number of cells at 400X
4. grow more biofilms under the same conditions
5. combine the phage and biofilm using this procedure(http://journals.plos.org/plosone/article?
id=10.1371/journal.pone.0168615)
6. After phages have killed the biofilm, stain and calculate the number of cells per well again to find the number of biofilm cells killed by the phages
While my mentor at the community college is a microbiologist, she isn't experienced in growing biofilms. Let me know if you think this could work for my project.
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SciB
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Re: Science Fair Project
Hi Anusha,
I found a video on making an E. coli biofilm and they used the same medium as in your P. aeruginosa paper--LB broth.
https://www.youtube.com/watch?v=s9YW__bcss4
In the JOVE video, however, the biofilm was produced using a minimal salts medium, M63, which they say gives a more 'robust' [?] biofilm. I would use LB first because it is readily available. It should give you a satisfactory E. coli biofilm. Otherwise you can use an E coli minimal salts medium like M9 to dilute the cells into.
Do you have some polystyrene well plates? These would be very convenient to do this kind of experiment because they are sterile and available with different numbers of wells--a 12-well or 24-well plate would give you lots of space to try different combinations of media and titers of E. coli and T7. You don't need to use a plate reader. You can examine the wells using an inverted microscope (one with the objectives underneath the stage).
The number 10^6 (10 to the 6th power = 1 million) refers to the number of bacterial cells in a certain volume, usually 1 ml. That would refer to the titer of the bacterial culture that you use to make the film. Bacterial titers--the number of bacteria per ml--are determined by making dilutions of the bacterial culture, plating them on nutrient agar and counting the number of colonies after incubating the plates 24-36 hours at 37 C. I am assuming that you have a stationary phase culture of E coli and a culture at that stage in the bacterial growth cycle would have about 10^8 cells/ml. So, in order to get a titer of 10^6 cells/ml you would dilute your culture 1 to 100--pipet 0.1 ml of culture into 9.9 ml of sterile LB.
Here is where you can vary the independent variables to see what happens. If you have enough bacterial culture you can try adding it straight to the well plate, and then you can make a 1:10 dilution, a 1:50 dilution, a 1:100 dilution and a 1:500 dilution and see what effect this has on biofilm formation. It may be easier to count cells if you make biofilms using a lower titer of bacteria.
If you haven't been following everything that I am suggesting for the experiments, then post again with questions and I will explain in more detail.
Your first goal is to make consistent biofilms that you can stain with crystal violet and count bacterial cells on. You need to repeat this several times until you can grow countable biofilms that are all the same. Then you can go on to test the T7 to see how well it can lyse E. coli when they are growing as a biofilm. I don't know what will happen here. You will just have to expose three or more biofilms to T7 and keep three as controls with no phage, then stain them and examine them thru the microscope. I hope your 'scope has a camera attached, but if it doesn't you can take pics thru the eyepiece with a smart phone camera. This is important. You absolutely have to have a visual record of the biofilms with and without T7 exposure. There are instructions online how to take microphotographs with a smart phone camera so just do a search on youtube or google and you will find several.
I'm sure you will have more questions, but I hope my suggestions have been helpful so far. This is a pretty complicated project and you really need someone to work with you on the procedure.
Good luck!
Sybee
I found a video on making an E. coli biofilm and they used the same medium as in your P. aeruginosa paper--LB broth.
https://www.youtube.com/watch?v=s9YW__bcss4
In the JOVE video, however, the biofilm was produced using a minimal salts medium, M63, which they say gives a more 'robust' [?] biofilm. I would use LB first because it is readily available. It should give you a satisfactory E. coli biofilm. Otherwise you can use an E coli minimal salts medium like M9 to dilute the cells into.
Do you have some polystyrene well plates? These would be very convenient to do this kind of experiment because they are sterile and available with different numbers of wells--a 12-well or 24-well plate would give you lots of space to try different combinations of media and titers of E. coli and T7. You don't need to use a plate reader. You can examine the wells using an inverted microscope (one with the objectives underneath the stage).
The number 10^6 (10 to the 6th power = 1 million) refers to the number of bacterial cells in a certain volume, usually 1 ml. That would refer to the titer of the bacterial culture that you use to make the film. Bacterial titers--the number of bacteria per ml--are determined by making dilutions of the bacterial culture, plating them on nutrient agar and counting the number of colonies after incubating the plates 24-36 hours at 37 C. I am assuming that you have a stationary phase culture of E coli and a culture at that stage in the bacterial growth cycle would have about 10^8 cells/ml. So, in order to get a titer of 10^6 cells/ml you would dilute your culture 1 to 100--pipet 0.1 ml of culture into 9.9 ml of sterile LB.
Here is where you can vary the independent variables to see what happens. If you have enough bacterial culture you can try adding it straight to the well plate, and then you can make a 1:10 dilution, a 1:50 dilution, a 1:100 dilution and a 1:500 dilution and see what effect this has on biofilm formation. It may be easier to count cells if you make biofilms using a lower titer of bacteria.
If you haven't been following everything that I am suggesting for the experiments, then post again with questions and I will explain in more detail.
Your first goal is to make consistent biofilms that you can stain with crystal violet and count bacterial cells on. You need to repeat this several times until you can grow countable biofilms that are all the same. Then you can go on to test the T7 to see how well it can lyse E. coli when they are growing as a biofilm. I don't know what will happen here. You will just have to expose three or more biofilms to T7 and keep three as controls with no phage, then stain them and examine them thru the microscope. I hope your 'scope has a camera attached, but if it doesn't you can take pics thru the eyepiece with a smart phone camera. This is important. You absolutely have to have a visual record of the biofilms with and without T7 exposure. There are instructions online how to take microphotographs with a smart phone camera so just do a search on youtube or google and you will find several.
I'm sure you will have more questions, but I hope my suggestions have been helpful so far. This is a pretty complicated project and you really need someone to work with you on the procedure.
Good luck!
Sybee
Re: Science Fair Project
Hello Sybee,
Thank you for your input on the Jove video. I am not sure if there are polystyrene well plates at the lab but I will try to find out as soon as possible.
I have found a procedure that may be good for growing my biofilms. The procedure uses the materials and methods that you mentioned and gives a step by step procedure to follow. I should be able to perform this procedure in the lab at the community college. If you think this procedure is applicable to my project, I could start ordering the materials and try growing the biofilms.
Thank you,
Anusha
Thank you for your input on the Jove video. I am not sure if there are polystyrene well plates at the lab but I will try to find out as soon as possible.
I have found a procedure that may be good for growing my biofilms. The procedure uses the materials and methods that you mentioned and gives a step by step procedure to follow. I should be able to perform this procedure in the lab at the community college. If you think this procedure is applicable to my project, I could start ordering the materials and try growing the biofilms.
Thank you,
Anusha
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SciB
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Re: Science Fair Project
I would get the materials as soon as possible and try your procedure. You may have to make some little changes to get consistently good biofilms before you can do the T7 experiment.
Do you have a microscope with a camera? It is absolutely essential to take good, sharp microphotographs of your stained bacteria so that you can print them to show on your display.
Keep records of everything in your lab notebook with dates and details. Don't trust your memory!
If you have any questions at all, please ask. When you are working with someone in the lab, make sure they understand you and you understand them. One small error can ruin a whole experiment. Also remember to do at least three identical runs of each experiment so you can average the results and do some statistical tests for significance.
Good luck!
Sybee
Do you have a microscope with a camera? It is absolutely essential to take good, sharp microphotographs of your stained bacteria so that you can print them to show on your display.
Keep records of everything in your lab notebook with dates and details. Don't trust your memory!
If you have any questions at all, please ask. When you are working with someone in the lab, make sure they understand you and you understand them. One small error can ruin a whole experiment. Also remember to do at least three identical runs of each experiment so you can average the results and do some statistical tests for significance.
Good luck!
Sybee
Re: Science Fair Project
Hello Sybee,
Sorry, I forgot to link to the procedure I was talking about. This is the link: http://onlinelibrary.wiley.com/doi/10.1 ... 1-bib-0007
Sorry, I forgot to link to the procedure I was talking about. This is the link: http://onlinelibrary.wiley.com/doi/10.1 ... 1-bib-0007
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SciB
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Re: Science Fair Project
Hey Anusha--
That's a great reference you linked to but it is loaded with technical jargon, so when you have questions, make a list of them and post back here and i will try to guide you through it.
Try to get started right away so you can begin to get an idea how this biofilm project is going to work out. You still haven't told me if you have a microscope and camera. I keep saying that this is VERY important and you need it. You can take microphotos with a smart phone if your 'scope does not have a built in camera.
Sybee
That's a great reference you linked to but it is loaded with technical jargon, so when you have questions, make a list of them and post back here and i will try to guide you through it.
Try to get started right away so you can begin to get an idea how this biofilm project is going to work out. You still haven't told me if you have a microscope and camera. I keep saying that this is VERY important and you need it. You can take microphotos with a smart phone if your 'scope does not have a built in camera.
Sybee
Re: Science Fair Project
Hello Sybee,
I am ordering my materials and I am ready to start my procedure. I have a few questions about how I should go about quantifying the cells in the biofilm. You recommended that I examine the biofilm under a microscope with a microscope. My question is how exactly will I calculate the number of cells in each well. If I transfer the biofilm to a slide to look at it under the microscope, what will it look like? Also, how will I know how many cells I am seeing at a certain magnification? I have watched a couple videos about how to measure cells under a microscope but I don't quite understand them. If you could answer these questions, I would be very grateful.
Thank you for your assistance,
Anusha
I am ordering my materials and I am ready to start my procedure. I have a few questions about how I should go about quantifying the cells in the biofilm. You recommended that I examine the biofilm under a microscope with a microscope. My question is how exactly will I calculate the number of cells in each well. If I transfer the biofilm to a slide to look at it under the microscope, what will it look like? Also, how will I know how many cells I am seeing at a certain magnification? I have watched a couple videos about how to measure cells under a microscope but I don't quite understand them. If you could answer these questions, I would be very grateful.
Thank you for your assistance,
Anusha
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SciB
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Re: Science Fair Project
Hi Anusha,
Sounds like you have made good progress. Now you need a way to measure the effect of the phage on the biofilm.
As i think i said in a previous post, there are two tests you could do. One would be to expose the E coli to phage and then see what effect this has on the formation of the biofilm. Preventing formation of a biofilm is desirable. If that can be done, the problem is eliminated.
The other test would be to see if a biofilm can be removed or reduced by phage treatment.
Both of these tests need a way to visualize the bacteria and you have a microscope so you're half way there. Now you need a way to quantitate the effect--meaning measuring changes in numbers of bacteria. You do this by counting the cells in a specific field.
OK. How can you do that? If you use a 10X eyepiece and a 100X objective lens, the resulting magnification is 400 diameters. This is adequate to see and count E coli cells, but as you look through the eyepiece there will be too many cells in the entire field of view to count. The way around this is to insert a round glass disc into the eyepiece that has etched on it a grid of horizontal and vertical lines of known dimensions. Now when you look through the eyepiece you can see the grid superimposed on the field of view. All you have to do is count the number of bacteria in a part of the grid. You use a formula to convert this number into a value for the entire field or grid. Do the same count for each of your slides and you can compare them.
Here are some references that explain this method of cell counting under a microscope:
http://www.microscopenet.com/wf10x18-mi ... -9492.html
https://www.youtube.com/watch?v=8TIyj8OrtWA
Eyepieces and reticules (the grid disc) can be pretty expensive. If you can set up a camera on a tripod or stand so that you can take pictures of the biofilms then you can do a cell count on the photo rather than thru the eyepiece. You would have to make a square hole large enough to include about 200 cells in a piece of paper and tape this onto your computer screen over the area of the image you want to count cells in. As long as your photos were all taken at 400X with the camera at the same position and you opened the image file on the PC at the same size each time then the counts can be directly compared. I haven't tried this, but I think it will work.
Do a search for bacterial counting online and see if you can find more information. In your case, however, you want to count bacteria as they appear in the intact biofilm. Many of the counting procedures require liquid suspensions of bacteria and that would not be practical to do with a biofilm.
Let us know what you decide to do and if you need more help.
Good luck!
Sybee
Sounds like you have made good progress. Now you need a way to measure the effect of the phage on the biofilm.
As i think i said in a previous post, there are two tests you could do. One would be to expose the E coli to phage and then see what effect this has on the formation of the biofilm. Preventing formation of a biofilm is desirable. If that can be done, the problem is eliminated.
The other test would be to see if a biofilm can be removed or reduced by phage treatment.
Both of these tests need a way to visualize the bacteria and you have a microscope so you're half way there. Now you need a way to quantitate the effect--meaning measuring changes in numbers of bacteria. You do this by counting the cells in a specific field.
OK. How can you do that? If you use a 10X eyepiece and a 100X objective lens, the resulting magnification is 400 diameters. This is adequate to see and count E coli cells, but as you look through the eyepiece there will be too many cells in the entire field of view to count. The way around this is to insert a round glass disc into the eyepiece that has etched on it a grid of horizontal and vertical lines of known dimensions. Now when you look through the eyepiece you can see the grid superimposed on the field of view. All you have to do is count the number of bacteria in a part of the grid. You use a formula to convert this number into a value for the entire field or grid. Do the same count for each of your slides and you can compare them.
Here are some references that explain this method of cell counting under a microscope:
http://www.microscopenet.com/wf10x18-mi ... -9492.html
https://www.youtube.com/watch?v=8TIyj8OrtWA
Eyepieces and reticules (the grid disc) can be pretty expensive. If you can set up a camera on a tripod or stand so that you can take pictures of the biofilms then you can do a cell count on the photo rather than thru the eyepiece. You would have to make a square hole large enough to include about 200 cells in a piece of paper and tape this onto your computer screen over the area of the image you want to count cells in. As long as your photos were all taken at 400X with the camera at the same position and you opened the image file on the PC at the same size each time then the counts can be directly compared. I haven't tried this, but I think it will work.
Do a search for bacterial counting online and see if you can find more information. In your case, however, you want to count bacteria as they appear in the intact biofilm. Many of the counting procedures require liquid suspensions of bacteria and that would not be practical to do with a biofilm.
Let us know what you decide to do and if you need more help.
Good luck!
Sybee