How to do a serial dilution from an agar plate and inoculating loop??

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shireen1212
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Am I suppose to dig the inoculating loop (10 micro litres) into the dish to pick up bacteria and then put that into a test tube with water or do I gently swab it over?, I am very confused. And after putting it into teh water and diluting it, how am I suppose to plate that? do I use the same loop and and swab onto a plate

Pls help and keep it simple as I am very confused
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abi_nicholls
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(Original post by shireen1212)
Am I suppose to dig the inoculating loop (10 micro litres) into the dish to pick up bacteria and then put that into a test tube with water or do I gently swab it over?, I am very confused. And after putting it into teh water and diluting it, how am I suppose to plate that? do I use the same loop and and swab onto a plate

Pls help and keep it simple as I am very confused
Hi,
Don't dig the inoculating loop into the agar, use it to scoop the colony up. Make sure you're using aseptic technique to do this or you will contaminate your samples. Here is a step by step 1) Put your agar plates around the flame 2) sterilise the loop by passing it through the flame 3) wait for it to cool down, you don't want to kill the bacteria 4) use it to scoop up a colony off the plate 5) Add it to the water and mix well 6) pass your loop through the flame again 7) place it into the water (After it has cooled) 8) swipe it over the second clean agar plate using the technique shown here, (https://microbiologyofbrewinglab.wor...ate-streaking/) don't forget to put the lid back on and label. I hope this answers your question? Make sure you don't break the surface of the agar.
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shireen1212
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(Original post by abi_nicholls)
Hi,
Don't dig the inoculating loop into the agar, use it to scoop the colony up. Make sure you're using aseptic technique to do this or you will contaminate your samples. Here is a step by step 1) Put your agar plates around the flame 2) sterilise the loop by passing it through the flame 3) wait for it to cool down, you don't want to kill the bacteria 4) use it to scoop up a colony off the plate 5) Add it to the water and mix well 6) pass your loop through the flame again 7) place it into the water (After it has cooled) 8) swipe it over the second clean agar plate using the technique shown here, (https://microbiologyofbrewinglab.wor...ate-streaking/) don't forget to put the lid back on and label. I hope this answers your question? Make sure you don't break the surface of the agar.
Hi thank you so much for your reply. So when you say scoop up the colony should I just scoop up the pointy bits of the bacteria without the agar underneath? Could you attach a link if you have one so I know how to do this
also the website attached with the streaking once and then sterilising it, wouldnt that just kill the bacteria? what will be the point of that? so if for example , after diluting the bacteria I put my sterilised loop back in, picked up say 40 bacteria, swiped it onto the agar (say 30 were plated and 10 were not), and then sterilise it, wouldnt that kill the rest of the bacteria instead of plating it?
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abi_nicholls
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(Original post by shireen1212)
Hi thank you so much for your reply. So when you say scoop up the colony should I just scoop up the pointy bits of the bacteria without the agar underneath? Could you attach a link if you have one so I know how to do this
also the website attached with the streaking once and then sterilising it, wouldnt that just kill the bacteria? what will be the point of that? so if for example , after diluting the bacteria I put my sterilised loop back in, picked up say 40 bacteria, swiped it onto the agar (say 30 were plated and 10 were not), and then sterilise it, wouldnt that kill the rest of the bacteria instead of plating it?
Yep, just scoop it up. Here is a video I found, have a look on youtube there are lots - https://www.youtube.com/watch?v=u84bTjqrt7k You need to kill the bacteria on the loop to prevent lawn formation (way too much bacteria grows), sterilising it between swipes allows fewer bacteria to be spread across the plate each time, this will form colonies. However, if you're doing a serial dilution you should be using a spreader to coat the entire plate in a small number of resuspended bacteria, here is a video showing that - https://www.youtube.com/watch?v=Ppe_bgnPFHU After you've finished the practical it will show you that a high concentration of bacteria forms a lawn which is unusable.
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shireen1212
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(Original post by abi_nicholls)
Yep, just scoop it up. Here is a video I found, have a look on youtube there are lots - https://www.youtube.com/watch?v=u84bTjqrt7k You need to kill the bacteria on the loop to prevent lawn formation (way too much bacteria grows), sterilising it between swipes allows fewer bacteria to be spread across the plate each time, this will form colonies. However, if you're doing a serial dilution you should be using a spreader to coat the entire plate in a small number of resuspended bacteria, here is a video showing that - https://www.youtube.com/watch?v=Ppe_bgnPFHU After you've finished the practical it will show you that a high concentration of bacteria forms a lawn which is unusable.
oh okay I watched the first video and it said to swab over the single colony whereas you said to pick it up. Just to double check do I pick it up gently without poking into the agar ? could you jsut check over my method pls to make sure everything is ok. So my goal is to figure how many e.coli bacteria there will be on a plate .
1) spray e.coli solution on my surface
2) swab it onto a plate adn wait a few days
3) using an inoculating loop pick up a single colony and dilute it
4) shake the test tube to suspend the organism off the loop, then sterilise the loop
5) pick up 10micro litres and swab onto a plate using the loop (you said Ill need a spreader, is a loop fine to use? and ill spread it using the quadrant method)

Please let me know if its fine, thank you so much for your help
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abi_nicholls
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(Original post by shireen1212)
oh okay I watched the first video and it said to swab over the single colony whereas you said to pick it up. Just to double check do I pick it up gently without poking into the agar ? could you jsut check over my method pls to make sure everything is ok. So my goal is to figure how many e.coli bacteria there will be on a plate .
1) spray e.coli solution on my surface
2) swab it onto a plate adn wait a few days
3) using an inoculating loop pick up a single colony and dilute it
4) shake the test tube to suspend the organism off the loop, then sterilise the loop
5) pick up 10micro litres and swab onto a plate using the loop (you said Ill need a spreader, is a loop fine to use? and ill spread it using the quadrant method)

Please let me know if its fine, thank you so much for your help
Swipe over, pick it up same difference haha. Yes pick it up gently it will come up easily, it is not embedded into the agar it is sitting on top, do not poke into the agar! Your method is good up to step 4 where it gets a bit confusing, here are my tips: 'incubate overnight', 're-suspend the bacteria in water', 'sterilise the loop and then pick up the bacteria!!', are you using a pipette to pick up 10ul? There are different methods don't worry about the spreader if you aren't being provided with one, using the loop is fine.

No worries, what level are you at by the way?
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shireen1212
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(Original post by abi_nicholls)
Swipe over, pick it up same difference haha. Yes pick it up gently it will come up easily, it is not embedded into the agar it is sitting on top, do not poke into the agar! Your method is good up to step 4 where it gets a bit confusing, here are my tips: 'incubate overnight', 're-suspend the bacteria in water', 'sterilise the loop and then pick up the bacteria!!', are you using a pipette to pick up 10ul? There are different methods don't worry about the spreader if you aren't being provided with one, using the loop is fine.

No worries, what level are you at by the way?
oh okay thanks. Am I suppose to incubate after diluting it? as I was going to put the loop with the single colony into the water mix it around. Take it out, sterilise it and then put it back in (to pick solution up) then plate that. And I am using an inoculating loop thats 10ul . Are you asking what level I am because I sound really dumb lol haha
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abi_nicholls
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(Original post by shireen1212)
oh okay thanks. Am I suppose to incubate after diluting it? as I was going to put the loop with the single colony into the water mix it around. Take it out, sterilise it and then put it back in (to pick solution up) then plate that. And I am using an inoculating loop thats 10ul . Are you asking what level I am because I sound really dumb lol haha
Incubate the plates to help the bacteria to grow, ok. No, I'm asking because I didn't know if I had overly complicated my explanations.
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shireen1212
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(Original post by abi_nicholls)
Incubate the plates to help the bacteria to grow, ok. No, I'm asking because I didn't know if I had overly complicated my explanations.
oh okay yes I was going to do that . And I see, I am at university level but we haven't had much practice in microbiology. Thank you so much for your help Youve really eased my terrible terrible anxiety with everything going on right now
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abi_nicholls
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(Original post by shireen1212)
oh okay yes I was going to do that . And I see, I am at university level but we haven't had much practice in microbiology. Thank you so much for your help Youve really eased my terrible terrible anxiety with everything going on right now
Fair enough, I'm also at uni taking biomedicine I'm glad, good luck with your work!!
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