Disease prac

[ellice]

Hi all,
We are doing Disease with Yr 12 Bio at the moment, and doing lots of pracs with agar plates. One of our pracs involves E Coli and Staph Albus, and the antibiotic mastrings, which we have done every year with mixed success. I use the standard agar (peptone, salt, sugar, vegie, agar), and was wondering should I be using some other media.
Any help would be appreciated.
Ellice.

[Jazz]

hi
I mix in some nutriant agar, give them more food

[Ross]

Hi Ellice,

Can you please clarify what you mean by "mixed success". It might be a couple of different reasons depending on what unexpected results you got. Unfortunately some of these pracs can be a little susceptible to slight variations in procedure.

Ross

[ellice]

Hi Ross,
In the past, we have had clear rings around the antibiotic areas, but the last few years have been a bit of a non event. Don't know whether it's the kid's technique or what. I'm going to try the suggestion of adding nutrient agar. Thanks for that tip.
Ellice.

[JudyM]

Ellice,
It sounds as though your mastrings might be the problem.
Have you had them very long and do you refrigerate them? We also do this prac every year and I always do a demo first so there's more chance the students will get good results.

My nutrient agar recipe is
Boil 500ml distilled water.
Weigh 10g agar, 2.5g peptone & 1.5g beef extract into an 800ml beaker. Add enough cold distilled water to form a slurry.
Pour in the boiled water & stir.(preferably using a magnetic stirrer/hotplate)
Pour nutrient agar soln into boiling tubes to ~4cm from the top. Cover with foil then autoclave. One tube will be enough for 2 plates. & the labelled tubes can be kept in the fridge for a couple of years.

Hope this helps
Judy

[Ross]

Ellice,

I agree with Judy, sounds like the mastrings. If the bacteria lawn grows, then this is most likely the culprit. The only other thing that I can think of is the mastrings have not stayed incontact with the agar.

Cheers
Ross

[kerryw]

Hi. We are about to do this prac next week. In the past I have always made up the plates by using nutrient agar which is available from any of the science suppliers. We have had success with this prac each time. I feel very lucky this year because the Science Co ordinator said instead of making the plates I could buy them already made and sterilized. 90 of them!

Kerry

[lygray]

Hi Judy,
Would you be able to give me more info about this whole experiment. Our biology teacher has given me vague instuctions about antibiotic rings. I see supplies are available from Southern Biological.
I am new to this job, but loving it so far.
Any help much appreciated
Lyn

[JudyM]

Hi Lyn,
You can also buy the bacterial broth (we use E coli & B subtilis) from the same place or you can try making a peppercorn infusion to obtain B subtilis. You can do a search on chemtalk for this. I purchased the broths ~ a year ago & storing them in the fridge I've found they are still active.
1.Prepare nutrient agar plates.
2.Swab work surface with 70% ethanol soln.
3.Working next to a bunsen apply 1 drop of the bacterial broth to the centre of the agar plate using a sterile pasteur pipette (you can buy plastic ones wrapped individually). Don't take the lid off the broth & put it on the bench, you'll need to hold it and replace it as soon as you've taken your drop out. You should also only lift the lid on the petrie dish as much as you need to so as to avoid introducing any bacteria from the air. Replace the pipette into the labelled pkt if other students need to use it.
4.Sterilize a glass spreader (you can make this from a glass rod by heating over a bunsen & bending into an angular ? shape or buy plastic ones) by dipping into another petrie dish containing iso- propyl alcohol & then hold over the flame for a few seconds(not too long as the glass will break)
5.Lift the lid slightly & use the spreader to cover the entire surface of the agar.You may need to rotate the plate a couple of times. Pass glass spreader through the flame briefly.
6. Sterilize a pair of forceps in the same way & use these to place a mastring in the centre of the plate.
Press down slightly in a few places on the white ring to ensure the antibiotics are in contact with the agar. Pass through flame.
7. Plates need to be labelled around the edge of the plate ie not top or base, & sticky taped. Place in incubator upside down ~ 30 deg. Check in ~ 48hrs.

Good luck with it.
Judy

[lygray]

Hi Judy,
Thank you so much for all this info, will be happy to have a go at it now. i know the basic sterile procedures and we have a pressure cooker to disinfect before disposing.
Thanks again
Lyn

[Labbie]

Also a good thread for Peppercorn & Agar plates