SB5k - Antibiotics

Hey,

Could anyone please help me understand the SB5k Antibiotics practical.

Previously this has been 'faked' but I really want to master this whilst I have got the time.

Teacher is wanting to demo it w/c 29th and I am hoping can get it organised by then.

I need to purchase some items.
The bacteria - which I have asked Blades for the lead time.

Sterile pipettes, wrapped
Sterile spreader, wrapped
Any advise where to get these from?

I have petri dishes - can I use these or do you need to buy sterile ones?

Once I have made my bacteria slope for stock - how often do I change this?

When I sterilise my nutrient agar - do I just put it in a beaker in the pressure cooker or is there a particular container I should put it in?

I have attached a 'go to' document I have put together for myself - if anyone could check it looks ok it would be massively appriciated.

Thanks in advance.

Emily :)
 

Attachments

It looks like you have a lot to learn -
your 'go to' doc first:

No need to make a stock bacterial slope as you buy the culture already in that form - You can reuse it as long as it doesn't get contaminated (I buy a new one every term)
You do need to get your bacteria growing in broth this is called sub culturing. Your 'go to' is okay but you use a metal inoculation loop (not a spreader) to take some of the bacteria off the slope and into the broth - this must be done without introducing unwanted micro organisms from the air - so called 'aseptic technique'.

I suggest you google sub culturing, pouring agar plates, making a bacterial lawn and aseptic techniques. If you are a member of CLEAPSS then visit their help guides
here are some youtube videos:

I make a lawn by spreading some broth culture over a set agar plate aseptically the next video is an alternative method

With pouring plates use a half full nutrient agar 250ml flask sealed with cotton wool and foil over the top (to stop the steam from the pressure cooker wetting the cotton wool). That should be enough to pour 10 plates. If they are pouring their own then use McCartney bottles half full with agar one for each plate.
When you take the agar out of the pressure cooker you need to keep it molten so put it in a water bath @60 degrees until ready to pour

Sterile pipettes/spreaders - you can do these yourself if they are glass - just wrap them in aluminium foil and autoclave.
My petri dishes are plastic and come already sterile in a plastic bag - remove them carefully without letting the lids come off.

One tip make the lawns just before you run the practical as antibiotic discs stop bacterial division so if the lawn is fully grown then the discs won't work.
 
Last edited:
It looks like you have a lot to learn -
your 'go to' doc first:

No need to make a stock bacterial slope as you buy the culture already in that form - You can reuse it as long as it doesn't get contaminated (I buy a new one every term)
You do need to get your bacteria growing in broth this is called sub culturing. Your 'go to' is okay but you use a metal inoculation loop (not a spreader) to take some of the bacteria off the slope and into the broth - this must be done without introducing unwanted micro organisms from the air - so called 'aseptic technique'.

I suggest you google sub culturing, pouring agar plates, making a bacterial lawn and aseptic techniques. If you are a member of CLEAPSS then visit their help guides
here are some youtube videos:

I make a lawn by spreading some broth culture over a set agar plate aseptically the next video is an alternative method

With pouring plates use a half full nutrient agar 250ml flask sealed with cotton wool and foil over the top (to stop the steam from the pressure cooker wetting the cotton wool). That should be enough to pour 10 plates. If they are pouring their own then use McCartney bottles half full with agar one for each plate.
When you take the agar out of the pressure cooker you need to keep it molten so put it in a water bath @60 degrees until ready to pour

Sterile pipettes/spreaders - you can do these yourself if they are glass - just wrap them in aluminium foil and autoclave.
My petri dishes are plastic and come already sterile in a plastic bag - remove them carefully without letting the lids come off.

One tip make the lawns just before you run the practical as antibiotic discs stop bacterial division so if the lawn is fully grown then the discs won't work.
There is no denying I’ve got a lot to learn :p.

Thank you for your reply.

I was aware they came on a slope but I thought could transfer onto new slope periodically to save repeat purchases. But sounds much easier to just purchase as and when I need. which in my case is only once a year.

I will look at purchasing pipettes and spreaders I can sterilise- must more environmentally friendly.

I don’t know why I didn’t think of YouTube videos. Thank you for that suggestion - it will be really helpful.

I have been reading up on Cleapss - imagine how clueless I’d be without it ;).

Thank you again for the help and advise - it’s much appreciated ☺
 
On a practical note: to save yourself embarrassment and time, when you are buying equipment that will need sterilising, make sure it will fit in your autoclave in at least reasonable quantities, if not full class sets. Sero pipettes are particularly vexacious in this regard.
 
On a practical note: to save yourself embarrassment and time, when you are buying equipment that will need sterilising, make sure it will fit in your autoclave in at least reasonable quantities, if not full class sets. Sero pipettes are particularly vexacious in this regard.
good point
 
Sterile pipettes/spreaders - you can do these yourself if they are glass - just wrap them in aluminium foil and autoclave.
I didn't realise these could be steralised this way. When on the CLEAPSS course they said about the oven wrapped in paper method but not this. Do they need to be wrapped loosely to allow the steam to penetrate and then tightened up as they are removed? Can one of each be wrapped in the same package to come as a set? Any idea how long they will remain sterile?

Sorry for all the questions, these are the items I have the most trouble providing sterile.

Thanks.
 
I didn't realise these could be steralised this way. When on the CLEAPSS course they said about the oven wrapped in paper method but not this. Do they need to be wrapped loosely to allow the steam to penetrate and then tightened up as they are removed? Can one of each be wrapped in the same package to come as a set? Any idea how long they will remain sterile?

Sorry for all the questions, these are the items I have the most trouble providing sterile.

Thanks.
If you are autoclaving them them greaseproof paper is better as it lets the steam through. but dry glassware should really go in an oven at 180C for a couple of hours for Dry heating sterilisation.

don't forget to plug the end with cotton wool.
 
I didn't realise these could be steralised this way. When on the CLEAPSS course they said about the oven wrapped in paper method but not this. Do they need to be wrapped loosely to allow the steam to penetrate and then tightened up as they are removed? Can one of each be wrapped in the same package to come as a set? Any idea how long they will remain sterile?

Sorry for all the questions, these are the items I have the most trouble providing sterile.

Thanks.
You can wrap them individually or as a set. I learnt this method at long time ago and it works well. They will remain sterile until you open up the foil. I put a strip of foil down on the bench, place the items in the middle and then fold the foil in and twist the ends - like a sweet wrapper. Always oversize the foil to make sure there are no gaps. Even double wrap if you want.
 
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