How to Culture Bacteria from Capsule

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ryanum
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How to Culture Bacteria from Capsule

Post by ryanum »

Hi guys, in my science experiment I am using a probiotic supplement of Lactobacillus Acidophilus to tests it against an antibacterial agent. Since the supplement will be in the form of a pill I want to culture it in a nutrient broth so that I can then streak the bacteria on nutrient agar plates. Basically, I would like the bacteria to be in a test tube where I can easily use a sterile swab to streak it on my media plate. For example like this: http://www.hometrainingtools.com/escher ... i-bacteria

My question is: what procedure should I follow to make a culture of this bacteria that I can streak onto my nutrient-agar media plates?

Thanks for the great help! :D
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Re: How to Culture Bacteria from Capsule

Post by SciB »

Hi,

It sounds like you have it pretty well figured out already. Just open a capsule of lactobacillus and pour some of it into 5 mL of nutrient broth in a sterile culture tube. Shake the tube gently to dissolve the material then put the culture tube in a warm (24-30 C, 75-86 F) dark place and check it after 24 hours. You should see a cloudiness in the broth that indicates bacterial growth. Then just pipet a drop of the culture onto a Petri dish and spread it evenly over the surface to make a bacterial 'lawn'. I would use a spreader rather than a swab because the spreader makes a more even, uniform lawn. The method for making a spreader from a paper clip is here: https://www.sciencebuddies.org/science- ... #procedure

Does that answer your questions? If not post again.

Sybee
ryanum
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Re: How to Culture Bacteria from Capsule

Post by ryanum »

Hi, Sybee!

Thank you for your response. I am having difficulty finding a procedure for the cultivation of bacteria in a capsule form. Should I pound the capsule/pill into powder first? And what should the ratio of broth (5mL) to the powder of bacteria be? Again, thanks for the help.
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Re: How to Culture Bacteria from Capsule

Post by SciB »

Hy Ryan,

Are you testing a capsule or a tablet? If it's a capsule the stuff inside is already in powder form so you can just sprinkle it into the broth. If you have a tablet, just put it into a small glass dish and crush it with something hard and smooth like the bowl of a metal spoon. If the tablet is very hard put it on a cutting board and carefully split it into smaller pieces with a knife.

Well, you just asked the big question that i don't have a good answer for--how much powder to add to the broth. I said 5 mL because that is what I use for starting cultures [called an 'inoculum']. Into this i would put some bacteria scraped from a colony on a Petri dish and incubate it overnight at 37 C. The next day I would have a nicely grown broth culture to use to inoculate a larger amount of broth or to streak on a plate.

OK. That still doesn't answer your question! What I am trying to say is that I can't tell you how much to add because I don't know how much bacteria is in the capsule or tablet and I don't know how quickly it recovers and grows from being dried out and processed. The tablet contains fillers and binders as well as bacteria so you can't tell how much actual bacteria is there.

You could set up 3 or 4 tubes with 5 ml of broth in each and add different amounts of lactobacillus powder to each. Leave the tubes in a warm, dark place and check them in 24 hours. If you hold the tube up to a light and swirl it gently you should be able to see some bacterial growth if the culture is viable. You don't need a really heavy culture. It is best to use the cells when they are in what is called logarithmic growth--doubling every 0.5-1 hour, but I can't tell you how to know this. Just try it. That's what scientists do. Test your lactobacillus before you do the actual experiment to make sure you can get a good lawn of bacteria. We often have to do several mini-experiments before we can do the real experiment.

Good luck and keep posting!

Sybee
ryanum
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Re: How to Culture Bacteria from Capsule

Post by ryanum »

Hi again!

Thank you for your response, I find it very helpful. I am just wondering what is the case if I do not have an incubator to culture the bacteria? Would I be able to make the broth culture at room temperature? If so, how long should that take?
Thanks!

Ryan :)
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Re: How to Culture Bacteria from Capsule

Post by SciB »

Hi Ryan,

I'm glad you found my answers helpful! We like feedback so we can keep improving.

You don't have to have an incubator to grow lactobacillus but it is easy to improvise one if you have a lamp with a 20 W incandescent bulb. Put the lamp inside a cardboard box with a thermometer and measure the temperature after an hour or so. You want it to stay at 24-30 C (75-86 F). Once you have a stable temperature you can wrap the tubes in aluminum foil to keep them dark and put them inside the box.

If you have a room that stays warm then you don't need to bother with the box, but your bacteria will grow a lot faster if you keep them warm.

Good luck and do keep us posted on your progress.

Sybee
ryanum
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Re: How to Culture Bacteria from Capsule

Post by ryanum »

Hi Sybee,

Thanks your reply! I might try homemade incubator. :) I do actually have another question regarding the best alternative to an inoculating loop. If I were to use a metal paper clip, can this be sterilized with a gas flame from a bunsen burner? And are you aware of any other ideal methods to streak the bacteria? Thanks again!

Ryan
ryanum
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Re: How to Culture Bacteria from Capsule

Post by ryanum »

Hi!

I was also wondering... The E. coli liquid culture I will buy will have 5 mL of the bacterial strain. On the website http://www.hometrainingtools.com/escher ... i-bacteria it says, it will provide sufficient bacteria for 10-15 petri dishes, is that accurate? If so, I should buy two tubes of the specimen and make double the amount of Lactobacillus A. liquid culture? Thanks!

Ryan
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Re: How to Culture Bacteria from Capsule

Post by SUS »

Hello,

A paperclip can be tricky because you could scrape up the nutrient-agar plate as you are streaking out the bacteria. A wooden stick may work better. Below is a link of the type I am referring to:

http://www.amazon.com/Wood-Craft-Dowels ... oden+dowel

I am not entirely sure about your second question. Usually it should be enough, since 5mls of a bacterial strain is a lot of cells. Hopefully Sybee can be of more help for this question. Let us know if you have any more!

Regards,
Surya
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Re: How to Culture Bacteria from Capsule

Post by SciB »

Hi Ryan,

Yes, you can sterilize the paperclip by holding it briefly in a flame. Be sure to use a large paper clip so the ‘handle’ is well away from the flame and doesn't get hot and burn your fingers. You only need to hold it in the flame a few seconds. Then wait a few seconds for it to cool off before doing the spreading. Hold it gently and loosely at an angle to the agar surface and spread carefully to avoid digging into the agar as Surya said. You can practice spreading a drop of sterile water on a couple of plates just to get the feel of it.

The size of the drop that you spread should be 0.1 to 0.3 mL, so 5 mL of bacterial culture will give you enough for 25 plates. Do you have a pipet to measure the volume of the drop or are you using a dropper? One drop from a dropper should be about 0.2 mL. Spread the drop evenly over the surface of the agar until the liquid is all absorbed.

The E coli culture you buy from Carolina Bio should have plenty of live bacteria to make a good lawn using a drop of about 0.2 mL, so you only need one 5 mL tube of broth culture unless you are planning to spread more than 25 plates.

For the lactobacillus I don't know how much to use because it depends on the brand and type of supplement. You may have to spread a test plate just to make sure you get a nice lawn of growing bacteria for your experiment.

I can estimate the amount of bacteria in a liquid culture by looking at the turbidity (cloudiness) of the liquid. There are actually optical instruments that do this by shining light though a tube and measuring how much is scattered by the bacterial cells. The lactobacillus supplements, however, contain binders and fillers that could cause cloudiness; so what you are looking at in the liquid after mixing in the powder is not all bacteria.

I hope this information is helpful. Keep us up to date on your progress and post again if you have any questions.

Good luck!

Sybee
ryanum
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Re: How to Culture Bacteria from Capsule

Post by ryanum »

Hi!

I am just about to order the materials for my project and I am decided on most of my materials. The only thing I need to know before ordering everything is how I can take Lactobacillus Acidophilus and Bifidus capsule powder and be able to grow colonies on a petri dish with nutrient agar? This is instead of using a liquid nutrient broth to make a liquid culture. Basically, I want to grow the bacteria on the petri dish and form colonies, which can then be transferred to many other petri dishes. I can imagine the powder can not be swabbed directly onto the nutrient agar plate. But anyways, is my idea possible? Or do I have to use some sort of liquid broth? Thanks!

Ryan
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Re: How to Culture Bacteria from Capsule

Post by SciB »

Hi Ryan,

Trying to spread powder on moist agar is not going to work very well. You need liquid.

Since you are not culturing lactobacillus, you don't need nutrient broth. I would just get some distilled water at the grocery store and use that to make a normal saline solution to suspend the bugs in. Normal saline is 0.9% sodium chloride and you can make it easily by dissolving exactly 1/2 teaspoon of table salt in one cup of distilled water.

As I said before, I don't know how much lactobacillus powder to use and I would recommend testing a measured amount of powder in a measured amount of saline on a Petri dish to make sure you get a lawn of bacteria. If it turns out to be too little you can add more, or if too much add less, but try it first so you will have an idea how well it grows. Set up your incubator as we talked about before so you can keep the plate warm and the bacteria will grow much faster.

Sybee
ryanum
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Re: How to Culture Bacteria from Capsule

Post by ryanum »

Hi! Thanks your help. Its been awhile now and I'm really close to beginning my project. I do have an off topic question, however. I was wondering if using regular tape for sealing my petri dishes are okay? I heard stories where the dishes do not get sealed properly, which results in contamination. Any ideas on how to seal the dishes properly? Thanks!

Ryan
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Re: How to Culture Bacteria from Capsule

Post by SciB »

Hi Ryan,

The purpose of the tape is to prevent the lids from falling off accidentally. Any tape will work. All you need are two small strips one on each side of the lid taped to the bottom. I would not be concerned about contamination because you are adding enough lactobacilli to coat the entire surface of the agar.

What antibacterial agents are you testing and how are you doing it--by the zone-of-inhibition method? If you are not sure about something, now is the time to ask before you start the experiment. It is always a good idea to run your plans by another person because they may think of something that you should include or spot something you are doing incorrectly.

Keep us posted on your progress.

Good luck!

Sybee
ryanum
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Re: How to Culture Bacteria from Capsule

Post by ryanum »

SciB wrote:Hi Ryan,

Trying to spread powder on moist agar is not going to work very well. You need liquid.

Since you are not culturing lactobacillus, you don't need nutrient broth. I would just get some distilled water at the grocery store and use that to make a normal saline solution to suspend the bugs in. Normal saline is 0.9% sodium chloride and you can make it easily by dissolving exactly 1/2 teaspoon of table salt in one cup of distilled water.

As I said before, I don't know how much lactobacillus powder to use and I would recommend testing a measured amount of powder in a measured amount of saline on a Petri dish to make sure you get a lawn of bacteria. If it turns out to be too little you can add more, or if too much add less, but try it first so you will have an idea how well it grows. Set up your incubator as we talked about before so you can keep the plate warm and the bacteria will grow much faster.

Sybee
I did not have time to test different amounts of powder and their lawns unfortunately. However, I made very concentrated powder solutions and very diluted solutions. A range of 1-4 capsules/50 mL of saline solution. I can still alter my range, as they are newly growing, any thoughts? Also, which would you recommend using the more concentrated or less, is there a risk of having too much as you said? I want to ensure the bacteria will grow, so I am thinking of using the higher concentration over the lower concentrated bacterial solution. Thanks Sybee!

Ryan
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