phanithans
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Questions about growing Streptococcus Mutans

Postby phanithans » Tue Jan 11, 2022 3:42 pm

Hello! I have a couple of questions I am planning on ordering a live culture from Ward's (Link below - I am ordering the tube with the live bacteria, not the lyophilized one). I want to grow the culture in an MSAT Agar plate for my experiment. My experiment is about testing copper water's effect on the oral microbiome. I have two questions:

1. Would my procedure for inoculating the agar work? I put it at the bottom of the post.
2. I am planning on inoculating the agar once to practice. To do that, I would need to open the culture vial and remove some of it to inoculate. For the actual experiment, can I use the opened vial again? Do I need to store it in low temperatures to use it again? If I cannot use the vial again after opening it, I was planning on removing some of the bacteria from the practice agar plate and subculturing it into another plate for the actual experiment. How would I store the practice agar plate that has already been inoculated so I can remove some of the bacteria and reuse it for the actual experiment?

One more question! Can I post my procedure here with a bunch of questions I have about it and hopefully get some advice on it? I know it's a big ask, but I would really appreciate it if that was possible.

Thanks a bunch in advance!

Link for Streptococcus Mutans:
https://www.wardsci.com/store/catalog/p ... .jsp.order

Procedure for inoculating Streptococcus Mutans:
a. Sterilize half a cup of water in the microwave for 1 minute. Wait for it to cool to room temperature.
b. Light a Bunsen Burner.
c. Put on nitrile gloves for the following steps.
d. Open the test tube of live Streptococcus Mutans and pass the opening through the flame 3 times. Wait for it to cool.
e. Take a sterile pipette and draw up 0.5 ml of the culture from the test tube. Remove the lid of an agar plate and place 0.2 ml into it.
f. Take a sterile cotton swab and dip it into the sterilized water. Press the cotton head against the edge of the container and apply pressure to get rid of excess water. Hold the pipette in another hand while doing this. Do not let it touch any surface.
g. Streak the agar plate in one direction using the cotton swab to spread out the bacterial culture. Rotate the plate 90 degrees and do the same thing again. Repeat the process three more times, then dispose of the swab. Place the lid back onto the agar plate. Let it dry for 5 minutes.
h. Seal the agar plate using Parafilm.
I. Incubate the jar and the agar plate of S. Mutans in a 37 degree Celsius incubator for 48 hours.

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Re: Questions about growing Streptococcus Mutans

Postby brandimiller610 » Mon Jan 17, 2022 11:04 pm

Hi phanithans,

I hope you are having a great day and thank you for your questions!

Your protocol for inoculating S. mutans looks good -- I just have one question. Do you have access to an autoclave? I think it would be best to use autoclaved distilled water to ensure that your bacteria do not become contaminated. Also, make sure that you are using aseptic techniques throughout the entire procedure. As long as you use aseptic techniques, such as the ones you described (i.e. Bunsen burner, sterile pipette tips, wearing gloves, etc.), you can reuse the vial of bacteria for the duration of your experiments. It is best to store your bacteria at 4 degrees Celsius between experiments (anything under 10 deg. C will be sufficient since the bacteria do not grow at 10). However, you did mention subculturing your practice plate and making fresh liquid cultures -- this is also good practice if you wanted to make fresh cultures after your practice inoculation. The decision is entirely up to you! The practice agar plate can also be stored at 4 degrees C after you get colonies. I suggest wrapping the plate in fresh parafilm to prevent contamination and storing it upside down in the refrigerator (4 deg C). You should also incubate your agar plates upside down to prevent condensation from dripping on the plates!

I hope this helps! And yes, please feel free to ask any additional questions you have about your project/procedure! We are happy to help :)

--Brandi

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Re: Questions about growing Streptococcus Mutans

Postby phanithans » Wed Jan 19, 2022 3:38 pm

Thank you for your help! Unfortunately, I do not have access to an autoclave (An autoclave would be really useful, though!)

Also, I have more questions!

1. Is Streptococcus Mutans considered a pathogen?
2. I have a freeze-dried powdered form of Lactobacillus Acidophilus. I want to use equal amounts of both the Streptococcus Mutans Culture and this Lactobacillus Acidophilus to inoculate my plates with. But the Streptococcus Mutans is in a liquid form while the Lactobacillus Acidophilus is a powder. My plan is to dissolve 1 gram/mL of each bacteria in 5 mL of saline, then inoculate each agar dish with 0.2 mL of the solution. Would there be enough bacteria for growth?
3. Should I culture the freeze-dried Lactobacillus powder before inoculating the agar dishes?
4. Lactobacillus Acidophilus grows best in anaerobic conditions, so I was planning on keeping it in a bell jar with a candle inside to remove all oxygen and produce carbon dioxide when I incubate it. But for my experiment, I am testing the effect of copper water on oral bacteria. The mouth isn't always free of oxygen, though. Should I keep the bacteria in the bell jar with the candle, or is an aerobic environment better?
5. How can I sterilize things without an autoclave (My plan for sterilizing the water was using a microwave)?
6. Copper water is water left inside a copper vessel overnight. When I create my copper water, I was planning on transferring it to a beaker to use later. How can I seal the beaker to prevent contamination?
7. Should I view the growth of the bacteria at different time intervals after they have been put into the incubator (Ex. 12 hrs, 24 hrs, 48 hrs)? If I am going to do this, will removing the bacteria from the incubator to take pictures and measurements affect their growth?

I wrote the questions kind of in a hurry, so if anything is unclear/ungrammatical, please let me know so I can clarify. Thank you so much for your help!

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Re: Questions about growing Streptococcus Mutans

Postby phanithans » Wed Jan 19, 2022 3:40 pm

One more question I forgot to mention! How would I keep pipettes, cotton swabs, forceps sterile?

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Re: Questions about growing Streptococcus Mutans

Postby brandimiller610 » Thu Jan 20, 2022 4:39 pm

Hi again,

1. Yes, S. mutans is considered to be a pathogen because it a major bacterium contributing to dental caries and tooth decay. It would be considered an "opportunistic pathogen" because, when present in the right conditions, it becomes pathogenic and may cause harm.

2. You should have plenty of bacteria. Do you know the CFU/gram found in the freeze-dried bacteria or the CFU/mL present in the liquid culture? I would say, based on my experience with such cultures, that you could even do a 1:10 dilution of the powder/pure culture and you would still be inoculating more than enough bacteria. 200 uL is also an appropriate volume to inoculate per plate.

3. Yes, you should suspend the freeze-dried bacteria into liquid media before inoculating the agar plates.

4. Since L. acidophilus is a facultative anaerobe, it should be able to grow well with or without oxygen. My suggestion would be to cultivate it aerobically... this is more practical from a clinical perspective. Because the oral microbiome is always exposed to oxygen, it makes sense to grow it aerobically. I have a lot of experience growing Lactobacillus and I have never had any problems cultivating facultative anaerobes aerobically.

5. Since you don't have access to an autoclave, you can heat the water. If you have access to a hot plate, you could boil the water on there. Otherwise, you can microwave it until it reaches a rapid boil. Once it is done boiling/microwaving, you should loosely cap it to prevent contamination. I am not sure what kind of lab space you have access to, but you should also do all of your microbiology work under a laminar flow hood that has been sterilized with 70% ethanol before and after use. If you do not have access to a hood, be sure to use multiple Bunsen burners and sterilize the lab bench prior to and after each use (with 70% ethanol).

6. You can seal your beaker with 1-2 layers of parafilm or a layer of aluminum foil. Make sure to wrap the parafilm/aluminum foil tightly.

7. I think it would be a good idea to monitor the growth of the bacteria at different time points. I would suggest making a separate plate for each time point so that you don't have to worry about rushing to take pictures/make measurements and returning your plate to the incubator. For clarification, what kind of measurements do you plan to make with the plates at these time points?

8. Pipettes can be kept sterile by simply wiping down with 70% ethanol between uses. Forceps can also be kept sterile in ethanol. For cotton swabs, you should make sure they stay in their packaging until it is time to use them. As I mentioned, it would be best to store everything under the laminar flow hood, if you have access to one. Otherwise, maybe you could get some individually wrapped cotton swabs (single use). I am not sure how accessible these are, but its an idea!

Hope this helps! Good luck on your project!

--Brandi

phanithans
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Re: Questions about growing Streptococcus Mutans

Postby phanithans » Sun Jan 23, 2022 6:20 am

Thank you so much for your quickly reply. I have more questions, though!

1. I contacted the supplier for my Streptococcus Culture (Ward's) and they said this, "Compliments of our specialist, Streptococcus mutans is considered Biosafety Level 1 by the CDC, and Containment Level 1 by the PHAC, so neither agency considers it pathogenic." If Streptococcus mutans is pathogenic, can I still use it in my experiment?

2. I do not the CFU/gram or CFU/mL, but I can find out!

3. Do you know where I can purchase MRS Broth for culturing the freeze-dried Lactobacillus powder or where I can purchase live Lactobacillus Acidophlius culture?

5. Would sterilizing my workspace with Lysol wipes also work, or is 70% ethanol better?

7. I am planning on measuring the zone of inhibition caused by a cotton disk soaked in copper water (Kirby-Bauer Test).

8. Thanks for your idea about individually wrapped pipettes and swabs! That will save me a lot of headache! But how can I keep things like test tubes or the copper vessel that I am using sterile?

9. An extra question! How can I measure the copper concentration of my copper solution created by keeping water in a copper vessel for 16 hours? I know that I can use a spectrophotometer, which I have access to, but what wavelength should I set the spectrophotometer at?

Sorry for bombarding you with so many questions, but I really appreciate the help you give! Thanks so much!

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Re: Questions about growing Streptococcus Mutans

Postby brandimiller610 » Mon Jan 24, 2022 2:34 pm

Hi again,

1. The S. mutans from the manufacturer is probably considered BSL-1 because the strain you are ordering poses relatively no risk to you when contained in the lab and when proper PPE is worn. In a general sense, S. mutans is considered pathogenic because it is well known to be a causative agent for tooth decay/dental caries. However, like with E. coli, some strains are pathogenic (i.e. the strains that cause UTIs), while others are non-pathogenic and even reside in the gut. Please let me know if this makes sense. You can use either strain in your experiments (as long as you have proper protocols in place for BSL-1 and BSL-2), but you likely want to go with the non-pathogenic strain. It's easier to contain, safer, and you can still gather some good, relevant data from your experiments.

2. Great! I am very confident you will have enough bacteria, but it is standard protocol and just a good idea to know how many CFU/gram or CFU/mL you have.

3. Many companies should sell MRS media for bacterial culture. Take a look Sigma Aldrich, Oxoid, or Fisher Scientific (Difco) -- these are some of the most common vendors for reagents. For live bacteria, Carolina (https://www.carolina.com/) is a good place to look. Your instructor/supervisor should also be able to help you with purchasing required items for your project.

4. I think both Lysol and ethanol are sufficient, but I recommend using 70% ethanol instead of Lysol wipes.

5. Using the Kirby-Bauer test sounds good for measuring the zones of inhibition!

6. I am not sure if I already asked you this, but do you have access to a laminar flow hood or biosafety cabinet for your experiments? If so, you could keep materials sterile under there. I know you don't have an autoclave -- maybe you could instead use 15 mL plastic tubes (they come sterile) and only open them under the hood. I am not sure what volume of copper water you are planning to store, but companies also sell sterile plastic bottles. Maybe this is an option? Any pre-packaged plastic is better in this case since you don't have access to an autoclave. Just keep in mind that once you open these plasticwares, they need to be kept as sterile as possible.

7. I found this video on YouTube (https://www.youtube.com/watch?v=xgl56xM2SSM) to answer your last question -- looks like he measured the OD at 800 nm.

Please let me know if you need clarification on anything!

--Brandi

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Re: Questions about growing Streptococcus Mutans

Postby phanithans » Wed Jan 26, 2022 4:29 am

Thank you so much! I have a couple more questions. Why is it necessary to suspend the lyophilized Lactobacillus in MRS broth before inoculation if I am inoculating it in MRS agar?
Also, once I add the lyophlized Lactobacillus to the broth and incubate it, how should I confirm I am using the same amount of Lactobacillus and Streptococcus in my experiment (because once I add it to the MRS broth, it's no longer pure bacteria)?

Once again, thank you!

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Re: Questions about growing Streptococcus Mutans

Postby brandimiller610 » Thu Jan 27, 2022 2:59 pm

1. You will want to rehydrate the lyophilized culture in liquid media before streaking or plating on agar. If you were taking from a frozen glycerol stock, it would be better to streak on agar, obtain pure colonies, and then inoculate liquid media. So, basically, with freeze-dried culture, adding it to broth is important for rehydrating it.

2. There are a couple of ways to measure the relative amount of bacteria in a liquid culture. You can indirectly quantify by using the spectrophotometer and measuring the OD600. The turbidity can be used as an indirect measure of bacterial growth (but keep in mind it does not account for live versus dead cells). If you use this method, be sure to run blanks; the blanks will be the media you are suspending your bacteria in. Second, you can do serial dilutions for your broth cultures and plate 100 uL samples to determine the CFU/mL. Once the plates grow, you can count the colonies and take into account the dilution factor to calculate the number of CFUs per mL of bacterial suspension. This is more direct, but takes more time. I found this YouTube video that explains serial dilution plating for bacterial culture (https://www.youtube.com/watch?v=uIIxNLPUu8g). Hope it helps!

Let me know if you need any clarification or have any more questions!

--Brandi

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Re: Questions about growing Streptococcus Mutans

Postby phanithans » Thu Jan 27, 2022 5:32 pm

Thank you! By the way, what do you think of using a McFarland Standard for making sure I have the same amount of bacteria?

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Re: Questions about growing Streptococcus Mutans

Postby brandimiller610 » Thu Jan 27, 2022 10:37 pm

I think using a McFarland standard is also a good idea! Unfortunately, I do not have experience using McFarland standards, but I believe it might be more accurate than using the OD600. I think a 0.5 McFarland standard (which is equivalent to 1.5e8 CFU/mL) would be suitable for your experiments. You could always do both (using turbidity/McFarland standards and serial dilution plating) just to confirm how much bacteria you have if your time and resources allow.

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Re: Questions about growing Streptococcus Mutans

Postby phanithans » Sun Jan 30, 2022 8:51 pm

Thank you for your advice! I think I will go with the McFarland Standard for time's sake. Here is my procedure for inoculating the bacteria and everything. It's not as detailed as it could be because I wrote it kind of fast so I could post it here, but I will make it more detailed. I will add in steps about cleaning up/disinfecting, and I will be wearing nitrile gloves through the entire process. Could you take a look and see if all of the other steps work, and did I miss anything?

I've been almost constantly inundating you with questions, and I hope you aren't too annoyed. Thanks so much! :D

For Lactobacillus Acidophlius:
1. Open a sachet of Lactobacillus Acidophilus Freeze-Dried Yogurt Starter. Weigh out half a gram of the powder into a test tube and recap it.
2. Open up a test tube of MRS broth and flame the opening for 5 seconds. Wait for it to cool.
3. Pipette 1mL of the broth, recap, dispense into the other test tube. Stir with the pipette. Pipette up the solution and dispense it into the tube of broth. Stir well, recap, and incubate for 48 hours at 37 degrees Celsius.
4. The test tube should be cloudy, indicating bacterial growth. If not, repeat the process. If growth is present, pipette 0.2 ml of the solution into an MRS agar plate.
5. Flame the metal portion of an inoculating loop until red hot. Wait for it to cool.
Use the loop to spread the solution across the plate in a zigzag motion. Turn the plate 90 degrees and repeat the motion. Repeat this process 2 more times.
6. Seal the plate with Parafilm.
7. Incubate the plate upside down at 37 degrees Celsius for 48 hours.
8. Flame the metal portion of an inoculating loop until red hot. Wait for it to cool.
9. Open the agar plate. Remove some bacteria from the plate with the loop.
10. Shake the loop into a test tube with 5 ml of 0.85% saline. Recap the test tube and shake. Sterilize the loop.
11. Shake a 0.5 McFarland Latex Standard to evenly suspend the particles. Compare the turbidity of the bacterial solution to the Standard using a Wickersham card. If the turbidity is less than the standard, add more bacteria into the solution, sterilizing the loop before and after. If the turbidity is greater than the standard, add more saline into the solution. If the turbidity is comparable, then proceed with the next step.
12. Shake the test tube of the Lactobacillus Acidophilus solution. Open it and pass the opening through the flame 3 times. Wait for it to cool.
13. Take a sterile pipette and draw up 0.2 ml of the culture from the test tube. Remove the lid of an MRS agar plate and place 0.2 ml into it.
14. Take a sterile cotton swab and dip it into the sterilized water. Press the cotton head against the edge of the container and apply pressure to get rid of excess water. Hold the pipette in another hand while doing this. Do not let it touch any surface.
15. Streak the agar plate in one direction using the cotton swab to spread out the bacterial culture. Rotate the plate 90 degrees and do the same thing again. Repeat the process three more times, then dispose of the swab. Place the lid back onto the agar plate. Let it dry for 5 minutes.
16. Once the plates have dried, put on another pair of nitrile gloves and sterilize a pair of forceps by holding them in the Bunsen burner flame for 5 seconds.
17. Use the forceps to dip a sterile disk into distilled water. Hold it above the water level to remove excess water. Then place the disk into the plate. Gently press it down with the forceps, then sterilize them with the Bunsen burner flame for 5 seconds. Place the lid back onto the agar plate and seal it with Parafilm.
19. Place a thermometer in the incubator and wait for it to heat to 37 degrees Celsius.
20. Incubate the plates upside down when the temperature reaches 37 degrees Celsius for 48 hours.

For Streptococcus Mutans:

1. Open the tube of Streptococcus Mutans culture. Pass the opening through the flame 3 times. Wait for it to cool.
2. Take a sterile pipette and draw up 0.2 ml of the culture from the test tube. Flame the opening again and recap.
3. Uncap a test tube with 5 ml of 0.85% saline with one hand and place 0.1 ml of the bacterial culture into it. Seal the test tube and shake well.
4. Shake a 0.5 McFarland Latex Standard to evenly suspend the particles. Compare the turbidity of the bacterial solution to the Standard using a Wickersham card. If the turbidity is less than the standard, add more bacteria into the solution, sterilizing the loop before and after. If the turbidity is greater than the standard, add more saline into the solution. If the turbidity is comparable, then proceed with the next step.
5. Shake the test tube of the Streptococcus Mutans solution. Open it and pass the opening through the flame 3 times. Wait for it to cool.
6. Take a sterile pipette and draw up 0.2 ml of the culture from the test tube. Remove the lid of an MSAT agar plate and place 0.2 ml into it.
7. Take a sterile cotton swab and dip it into the sterilized water. Press the cotton head against the edge of the container and apply pressure to get rid of excess water. Hold the pipette in another hand while doing this. Do not let it touch any surface.
8. Streak the agar plate in one direction using the cotton swab to spread out the bacterial culture. Rotate the plate 90 degrees and do the same thing again. Repeat the process three more times, then dispose of the swab. Place the lid back onto the agar plate. Let it dry for 5 minutes.
9. Once the plates have dried, sterilize a pair of forceps by holding them in the Bunsen burner flame for 5 seconds.
10. Use the forceps to dip a sterile disk into distilled water. Hold it above the water level to remove excess water. Then place the disk into the plate. Gently press it down with the forceps, then sterilize them with the Bunsen burner flame for 5 seconds. Place the lid back onto the agar plate and seal it with Parafilm.
11. Place a thermometer in the incubator and wait for it to heat to 37 degrees Celsius.
12. Incubate the plate upside down when the temperature reaches 37 degrees Celsius for 48 hours.

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Re: Questions about growing Streptococcus Mutans

Postby brandimiller610 » Tue Feb 01, 2022 8:32 am

No worries about asking questions! We are here to help :)
I will take a look at your protocols and reply back to you soon!

--Brandi

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Re: Questions about growing Streptococcus Mutans

Postby brandimiller610 » Tue Feb 01, 2022 1:42 pm

Hi again,

I had a chance to look through your protocols. First, just want to comment that they are extremely thorough...so good job!

Instead of pipetting 200 uL onto an agar plate and then spreading using an inoculating loop, you can pick up broth culture with the inoculating loop and spread it straight onto the plate. (https://www.youtube.com/watch?v=bRadiLXkqoU)

In step 5 (first protocol), are you streaking for isolation? Or do you want to make a bacterial lawn? I ask because if you are streaking for isolation (using quadrant method, which is what it sounds like you have described), an inoculating loop is better to use. For making a lawn, I recommend using a cell spreader! It will make your life easier when spreading bacteria all over the plate.

Also, one tiny detail, instead of "shaking" your tubes of bacteria, you should gently invert them or vortex them. But never shake them.

Hope this helps! As always, don't hesitate to let me know if you need anything else!

--Brandi

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Re: Questions about growing Streptococcus Mutans

Postby phanithans » Wed Feb 02, 2022 4:15 pm

Thank you for your help! In step 5, I am trying to create a bacterial lawn, so I will take your advice and try to use a cell spreader!


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