How to Culture Bacteria from Capsule

[SciB]

OK. Just go with the more concentrated suspension so you will be sure to have enough bacteria to make a good lawn. Be sure to spread at least three plates for each set of tests so you can take the average of the zones of inhibition. Keep the plates in the dark at a temperature of 24-30 C (75-86 F) and check them in 24 hours. I don't know how long it will take for the Lactobacilli to grow. Take photos of the plates after you have spread them and again at 24 hours and at the final time when you do the measurements.

You haven't said what antibacterial agents you are testing or how you are testing them so I am assuming that you are going to put the agents on filter paper disks that are placed on the surface of the agar after you spread the bacteria. Is that right? If the agents kill the bacteria or stop them from growing then there should be a clear area around the filter disk that you measure as the zone of inhibition (ZOI). Is that how you will do the experiment?

Hope everything goes well!

Sybee

[ryanum]

That's right. I am using the Kirby Bauer Diffusion Disk Susceptibility Test method.

I am a little tight on time, do you think the bacteria will be able to grow within 48 hours under room temperature conditions? Also, I might not have access to the bacteria during the growing period, are there any specific observations and/or data I should collect that would advise me to visit the experiment? Thanks!

Ryan

[SciB]

Well, it depends on how cold the room is! E. coli will grow within 24 hours at 37 C but if the room temp is lower than 22 C they may not make it by 48 hours. I've never tried to grow Lactobacilli from capsules so I really don't know if 48 hrs is long enough. Make it warmer if you possibly can just to be safe.

It isn't necessary to check the growth at 24 hours but it is a good idea. Can't you incubate them at home where you have access to them? The plates are sealed so there's no risk to anyone. Just dispose of them in 10% Clorox after the experiment is over.

Sybee

[ryanum]

My bacteria from the saline solution are not growing on the agar plate. Any idea what could possibly be wrong? I can not tell if there is growth in the saline solution as the colour of the solution has remained the same since the first day I poured it. This is quite disappointing. Thanks for the help.

I carefully made the saline solution and measured the right amount of salt to distilled water. I made different amounts of bacteria powder calculations: 1 capsule/50 mL saline, 2 capsules/50 mL saline, 3 capsules/50 mL saline, and 4 capsules/50 mL saline. I streaked and tested each solution on the nutrient agar plates and they were no colonies on any of them. I speculate that the bacteria I bought, Weber Naturals Lactobacillus supplement, was dead when purchased or the agar does not work. The agar was prepared from dehydrated nutrient agar powder and was autoclaved. I am now testing bacteria from the floor on a different petri dish with the same agar. And I will streak my solutions of bacteria again on a petri dish with different agar, which came prepared instead.

What else should I do? I have invested a week of precious time waited for growth and found nothing.

Ryan

[SciB]

Well, I think that you are right that the bacteria in these capsules or tablets are dead. The saline is just a vehicle for suspending the bacteria. It does not support growth. For that you would need a nutrient solution. You can try suspending them in just water but 0.9% saline is better because it maintains the correct osmolarity of the solution.

The agar is most likely not the problem. Commercial nutrient agar such as LB will support good growth of many types of bacteria but not all. I suggest that you get some plain yogurt from the store that says on the label--LIVE cultures of Lactobacillus. You could even get a couple different brands for comparison. Make sure the plates are in a warm place.

Did you SPREAD a couple drops of the bacterial suspension? In your post you said you STREAKED it. That is a different way of applying bacteria to a plate and is NOT correct for this experiment. In order to get an even and uniform growth of bacteria over the whole agar surface you must spread 0.2-0.3 ml of bacterial suspension [shake it before you spread!] on the agar using the paper clip spreader I told you about earlier.

Also, you did not say what temperature you had the plates at. If it isn't warm the bacteria won't grow very fast.

Post again with more information and I'll try to help.

Sybee

[ryanum]

Hi Sybee!

Thanks for the advice. I was able to get growth on my dishes from the saline solution. It just took time. I have another question, not exactly related to the topic, but it's how would I be able to grow bacteria from a raw meat, for example, ground beef or poultry? I have already tried swabbing the meat and spreading the swab onto a petri dish. Anything else I could try to receive nicely growing bacterial colonies on my dish? Thanks!

Ryan

Update: I'm trying a saline solution with pieces of raw meat placed inside.
If anybody has input I'd greatly appreciate it!

[SciB]

Hi Ryan,

Glad your bacteria are finally growing! I hope you get some measurable zones of inhibition.

If you want a lawn from the meat bacteria on an agar plate then I would suggest cutting the meat very fine, putting it in saline as you said, and then filtering out the bits with a coffee filter. The bacteria are so small that they will go right through the fibers in the filter.

You could leave the meat in saline at room temp for an hour or so to dislodge the bacteria then filter the 'soup' and use the paper clip spreader to spread a couple drops over the agar. Add your antibacterial disks, SEAL the lids to the bottoms of the Petri dishes and put them away in a warm, dark place. After a couple days you should have a really good lawn.

Be especially careful with bacteria that originate from meat because these can contain serious human pathogens like Salmonella and Clostridium. When you are working with bacteria, always wear disposable nitrile gloves, safety glasses and a lab apron. Do NOT remove the lids from the dishes. Take photos of the results through the lid then make your ZOI measurements on a printed copy of the photo.

After you get all the data from the plates, put them into a pail containing a 1 gal of 10% Clorox bleach [12 oz of Clorox plus 116 oz of water] and leave them over night to kill the microbes. Then you can dispose of them in the regular trash.

Please let us know how your results come out and if you have any feed-back for someone else doing this project.

Good luck!

Sybee

[adityach1993]

Hey,
I need to know how to activate a culture from a Lactobacil plus capsule. Given the contents:
L. acidophilus + L. rhamniosus=1.5bn
B.longum + B.bifidum=1.25bn
S.boulardii = 0.1bn
FOS=100mg
What should be the ideal concentration of serial dilution for this to serve as a fermentative inoculum?