Agar Plate Experiments.

[Lolita]

Righto, hi there!

 

I'm conducting an experiment to identify microbes in water. So i've been give two agar plates--- the control's in the classroom. I've chosen water as my subject, and i have to grow microbes/bacteria on them. I was thinking about pond water (found in a disgusting hole (?) at school) and normal, bottled water, most likely Mount Franklin Bottled water.

 

So i know what to do... just applying it onto the agar plates is the bit.. D:

I've read numerous methods, and they all involve THE LOOP. D: I do not possess such an item at home, and i doubt the school will lend me one... >.<

So i thought, after researching some more-- why not swabs?

 

After that breakthrough of the century, i discovered another problem: I have no idea how to actually put it on.

 

So, here's my question: Do i just dip the swab into the water, and quickly slide it over the agar, then incubate? or do i have to some complex burning and sterilization and put it in a bottle, burn mouth and etc and etc?

 

Please, point me in the right direction.

[Fuzzwood]

You could make one out of a safety pin, or even a paper clip. Then heat it over a fire till it glows red (loop only) and dip it into the water so you have a fleece inside the loop, not unlike that of a bubble blower.

 

 

Step one, place the fleece onto the agar plate so you have a nice drop in the top right 1/3th of the plate, and strike it out all over that top 1/3th. Do NOT apply any pressure on the loop, or you will cut into the plate. Just let it rest on top and angle it towards the direction you are sliding.

 

Step 2, sterilize the loop by holding it into a flame till red glowing, and let it cool.

 

Step 3, Strike out 5 lines from the bottom half of the top 1/3th part towards the bottom left

 

Step 4, sterilize the loop by holding it into a flame till red glowing, and let it cool.

 

Step 5, Strike out 5 lines upwards to the left from the bottom half of the first 5 lines you made

 

Step 6, sterilize the loop by holding it into a flame till red glowing, and let it cool.

 

Step 7, Strike out 5 lines from the top half of the second 5 lines you made to the upper right, and wiggle the bottom line towards the center of the plate. Be careful NOT to touch the parts you already hit with the loop.

 

Step 8, sterilize the loop by holding it into a flame till red glowing, and let it cool.

 

And you are done. This ensures you will end up with free colonies instead of having a big blob on your plate.

Edited November 9, 2009 by Fuzzwood

[CharonY]

You could also use sterile swabs or pipette tips in lieu of an inoculation loop. Or toothpicks, if you blunt and sterilize them. The key is to make sequentially diluting strikes as pointed out above. If high titers are expected I would do it slightly differently, but the principle is the same.

[Mr Skeptic]

If you have a sterile swab, you can use that. The swab cannot be flamed to sterilize it; you just discard it. A swab can give you what they call a "confluent lawn" ie cover the whole plate with bacteria. The loop, on the other hand, is reusable and allows you to do as Fuzzwood said, getting areas of increasing dilution of bacteria.

[Lolita]

Thanks for the help!

 

So i've obtain the sterile swabs (from my sister)... but it seems like getting those nice consistent strikes on the agar is the key to this experiment... will Q-tips work the same? i can get those from my local GP.

 

@Fuzzwood: Can you explain or show me a diagram of how this 'fleece' should look like? D: Your information is fantastic! <3

 

@CharonY: If i use toothpicks... to sterilise them, don't i have to burn them?! D: Or do i dump it in a bucket of cleaning detergent/bleach to sterilse? Is that even sterilising?! D:

 

@Mr Skeptic: I like the sound of that confluent lawn... XD I have about.... 8 or 9 of these sterile swabs.. but i think i'll use the loop, once i make one... D:

 

Thanks for the help and keep it coming!

[CharonY]

It depends on what you have available for sterilization. Ideally use an autoklave or a heat sterilizer.

Though your question implies that you do not have access to any of them. But if you get access to anything sterile it is probably easier to use that instead. Sterile Q-tips could also work. The key is really not consistent strikes but rather the ability not to make too broad ones. But starting with a low enough dilution even that is not an issue. Just make broad strokes on around 1/3 of the plate (just to distribute your sample) then draw out a single line from the end of that area. Then continue to strike it out into a free area of the plate. Then start from the end of that strikes and continue.

[Maria Angelica]

I've been given the same experiment to conduct and i'm not sure what food i should test for identifying microbes. Help!?!!

Are there any specific conditions if i use yoghurt?

 

 

Thanks guys.

[CharonY]

Bacteria living in milk are usually acidophilic. You should take care to use a suitable medium.

[Maria Angelica]

oh okay. we've been given nutrient agar plates. uhm i've chosen to do milk and tomato. both are off date. would these produce microbes in a nutrient agar plate?? and how would i incubate them?

[Mr Skeptic]

You should use fresh milk and tomato; they will have bacteria anyhow and it would raise more eyebrows.

[CharonY]

You will need to find more info on the agar plates. What medium does it consist of? All agar plates contain nutrients, but the question is what precisely do they contain. One guess is that at least one is LB (containing tryptone, yeast extract and NaCl). It is a very rich medium and quite a number of bacteria, including E. coli and Salmonella. Especially with the latter I'd be careful, though.

The thing is, everything will have bacteria on it. The question is only whether it will grow on the medium presented. Also the question is whether the teacher wants you to know what to expect on any given foodstuff (quite tricky) or what will grow on a given nutrient plate (a bit easier though only with a limited view).

[Lolita]

I've been given the same experiment to conduct and i'm not sure what food i should test for identifying microbes. Help!?!!

Are there any specific conditions if i use yoghurt?

 

 

Thanks guys.

 

Tell me you're not from the same school as i am.

 

D:

 

*cough* anyway, thanks to you guys, i am now growing a beautiful microbe from chicken. I ended up using the sterile Q-tips.

 

Alright, my microbe, which apparently, is a fungus--- is growing only one plate, but that's alright. Anyway, here are the characteristics of my "fungus"

 

Form: Circular

Elevation: Raised

Margin: Entire, but it maybe curled...

Texture: Soft, fluffy-like

Colour: white

Size: 3cm in diameter, but i can' be sure, since it's sticking near the edge of the plate.

 

I also enquired on YahooAnswers, and some poster told me it may have/be hyphae...?

 

@Charon: The thing is, that our teacher wants us to identify the microbe, and only told us that we're not growing pathogenic microbes. But i searched up on microbes that may grow on chicken, and i keep getting Salmonella. >.>

 

Anyway, anyone out there that has a good website on identifying food microbes?

 

All help is loved <3

 

@Maria Angelica: Does your school have an incubator? You should use that. But for us, we had to find our own way of making it warm. Luckily for us, it's been a hell of a hot week in Australia (New South Wales area) so i just placed it in a Maccers cardboard drink holder, covered it with newspaper and put it on the window still---although this can't be as good as an incubator, and may hinder the investigation, you can still talk about this in your discussion and gain extra points/marks for identifying that problem.

Remember, as my teacher says: This is science, and sometimes, it just doesn't work.

[CharonY]

First thing is to determine what kind of microorganisms are supposed to grow on the plates you used. This will limit the overall possibilities. The problem with fungi is that they tend to be a tad unspecific if grown on bacterial media. The diameter is itself not diagnostic as they continue to grow, of course.

 

To put in other words, if the medium is highly selective and if what you have was a bacterium then the colony morphology itself could provide good hints on what possibly could be there, but if it is a fungus and the medium is just a full medium I am not sure how the teacher can expect proper identification without further analyzes.

 

And btw. fungi tend to be a bit more unspecific in their habitats (or so it appeared to me). It is quite possible that the fungi were growing due to some contamination (i.e. they were not actively growing on chicken). And if the medium is highly selective it is possible that you won't get anything grown from chicken (I assume it is not a live one).

 

Tell me you're not from the same school as i am.

And only now do I realize that actually two people have asked the same question.

Edited November 23, 2009 by CharonY