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Hi everyone. I want to do an experiment on the growth of a certain kind of bacteria in acidic and basic environments. I have 2 questions:

 

1. How do I obtain a pure culture of just one kind of bacteria, without any contamination (preferably a non-pathogenic bacteria)? Do I need a sterilized lab for this?

 

2. How do I go about setting up the acidic and basic environments? I have pre-prepared agar plates at school that I will use. Do I just dump acid or base all over it? Or do I use some other way (like using the little discs of filter paper)?

 

Thanks and excuse my ignorance. I know NOTHING about this.

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Hey, Oak Ridge National Labs, I live near there.

 

Have you tried a supply company to get your bacteria?

 

https://www2.carolina.com/webapp/wcs/stores/servlet/CategoryDisplay?catalogId=10101&storeId=10151&categoryId=1696&langId=-1&parent_category_rn=1285|83&catLevel=2&bottom=N&top=N&pageNum=1&count=24

 

They probably have some kits on there that would help you get an idea of how to do your experiment, too.

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First a word of warning: fooling around with random bacteria can be dangerous. There's always a chance that you'll find a pathogenic type of bacteria, even in samples that seem harmless, like soil samples. Through isolation and culturing, you are allowing the bacteria to multiply freely and fast, and so you should take precautions to make sure they don't get released in the enivonment. So don't do this at home; and ask your biology teacher how to handle media, samples and bacteria.

 

Hi everyone. I want to do an experiment on the growth of a certain kind of bacteria in acidic and basic environments. I have 2 questions:

 

1. How do I obtain a pure culture of just one kind of bacteria, without any contamination (preferably a non-pathogenic bacteria)? Do I need a sterilized lab for this?

You don't need a sterilized lab. The agar plates that you use need to be sterile, though, and also the tools that you use. You also need proper techniques to make sure that nothing unwanted gets into your petri dishes, and that nothing gets out. Ask your teacher about this! Also, when you're done with with the experiments, make sure the used petri dishes get sterilized again, to kill off all the bacteria that you grew.

The trick to get a pure culture, is to dilute your sample a lot, so that only a few bacteria are present in each milliliter. A milliliter of the diluted sample is then spread out on agar plates. If everything goes right, after incubation, a few bacterial colonies will have grown on each plate; each colony is the progeny of a single bacterium. If you transfer a little of a single colony to a new agar plate, you will obtain a pure culture.

Whatever sample you use, there is always a chance that you obtain a pathogenic kind of bacteria.

 

2. How do I go about setting up the acidic and basic environments? I have pre-prepared agar plates at school that I will use. Do I just dump acid or base all over it? Or do I use some other way (like using the little discs of filter paper)?

Normally, biologists use specially prepared media for this kind of experiment. Those media already have the acidity they're aiming for, and also contain a buffer. Growing bacteria can influence the acidity of their environment substantially, through the nutrients that they take up and the waste products that they excrete. But if a buffer is added to the medium, the acidity still stays more or less the same.

The pre-prepared agar plates that you want to use probably already are buffered for a certain pH, so it's not going to be easy to change their acidity. I think the best thing you can do is to ask your teacher if it is possible to order different media, that already have the (buffered) pH that you are aiming for.

 

Airmid.

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Hi everyone. I want to do an experiment on the growth of a certain kind of bacteria in acidic and basic environments. I have 2 questions:

 

1. How do I obtain a pure culture of just one kind of bacteria, without any contamination (preferably a non-pathogenic bacteria)? Do I need a sterilized lab for this?

 

In addition to sterile techniques (in the easiest case you can work in the vicinity of on open flame to reduce contamination) you need a means to sterilize your medium. Moreover, you should try a medium that reduces the risk of getting pathogens. Avoid too rich media as well as media that selects for pathogens (e.g. brain heart infusion, blood agar). As mentioned above you should make agar dilutions (streak dilutions, get single colony, streak again, repeat), for anaerobes you can also do deep agar shakes.

However, as mentioned above, with unknown bacteria you can easily enrich pathogens.

 

 

2. How do I go about setting up the acidic and basic environments? I have pre-prepared agar plates at school that I will use. Do I just dump acid or base all over it? Or do I use some other way (like using the little discs of filter paper)?

 

Thanks and excuse my ignorance. I know NOTHING about this.

 

To test for acid/base sensitivity you can test with the filters, but then you already need an isolated bacterium for Ph you have to check what buffer is in the medium. LB is an often used medium that is essentially only buffered by the tryptone. Problem with just dumping acid or base is that the reached ph might be very unstable. For this, you should add a buffer with a fitting pka. You might e.g use bis-tris for slight acidic and tris for basic media. Hoiwever, many buffers are toxic to microorganisms in too high concentrations. Especially for soil bacteria carbonate buffers are more physiological.

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