WHAT are new rules on culturing micro-organisms

[snoopy]

Went to a CSIRO excursion with biology students, and they plated samples of E.coli, which are now in my incubator. The facilitator used parafilm around the edge of the plates to seal them, one peice stretched the whole way round. Had never thought of this, we just use sticky tape. Does anyone else use parafilm?

Cheers
Snoopy

[RosalieM]

Yep. I won't let them use sticky tape. There was discussion on here some time ago about the advantages of parafilm. The one I remember was that it allows for transfer of air from inside to out which prevents a nasty anaerobic environment developing in the dish (whether it is true or not I don't know but it sounded good! And makes sense).

[Ian]

If the plates are taped around the edges after exposure and never untaped and then autoclaved, is the dreadful stuff grown from door handles etc ok? Will be trying peppercorns for biol but Yr8 microbes will probably still be looking at what grows from what and where!!!!

I think that it is not so much what might happen if everything is done correctly, that we need to worry about. Rather what might happen id something goes wrong. The "Wild Cultures" are OK if kept in sealed Petri Dishes until autoclaved, but what if somebody drops one and accidentally steps on it? or if some kid who knows no better opens it up? Something like Bacillus Subtilis is actually known to be BENEFICIAL to humans rather than harmful, and it can be effectively killed with household disinfectant. No need to autoclave!

Cheers
Ian

[RosalieM]

PEPPERCORN INFUSION

* Soak approximately 20g of peppercorns in 200ml of water in a conical flask. Stopper with cotton wool.
* Stand at room temperature for several days or place in incubator overnight

At what temperature do you put the peppercorn water in the incubator? Also, what temperature do you then incubate the plates?

I am hoping to give this a go with some plates I have left over from a class a week or 2 ago if they aren't too dried out. On that note, is there a way to rehydrate plates that have been in the fridge too long? Can you just pull out the agar and add some extra water, reheat, sterilise and re-plate it? I have never tried.

I will try the antibiotic rings we have in the fridge on a couple of them too, and see if they work, and also the disinfectants we have that we test (and usually they don't kill anything the kids manage to grow! If only it was so easy to keep their pot plants alive!)

[Ian]

Hi Rosalie,
Our incubator is pretty old and not very accurate, so it does not keep temperature very well, but the general rule is NOT to let the temp get to close to blood temperature. Mine is generally somewhere between 25deg and 32deg. Any warmer, ands I start to get nervous about growing human specific pathogens, and any cooler, just takes a longer time. The Peppercorn broth can be left at room temperature, but it takes several days to grow, compared to over night in the incubator.

The person who told me this recipe also suggested making simulated anti biotic disks by soaking a tiny bit of filter paper in HomeBrand disinfectant and placing it on the plate.

cheers
Ian

[RosalieM]

Thanks Ian, we have a brand spankin' new incubator with digital temperature control!! I love it When the kids did their plates a few weeks back I set it on 30. Someone came in and changed it to 37!!! So I changed it back... and it has since stayed there. I will try at 30 overnight for the broth and see how it goes. Thank you so much for this idea. Let's hope the teachers are happy! Too late for this year, but next year I might be able to promote it (If I start promoting now I might have them convinced by then!).

[RosalieM]

OK so I did a trial. It didn't look nearly as good as the pictures on here! What did I do wrong??? Yr 12 Bio is going to be trialling it tomorrow (Tuesday) so I want them to be able to get it right. I brewed the peppercorns overnight in the incubator (maybe not long enough?) but this time I have put them in a flask on Friday, covered with Parafilm and have left it sitting on the bench. How did you plate the peppercorn broth? I used an innoculating loop. Now that I think about it I dipped it in metho first to sterilise (as they are plastic, so can't go in the bunsen flame) so perhaps the metho killed the peppercorn? We have some metal ones (which were in hiding when I did my trial but have since returned...) so is that the way to go? Or do you use a pipette and drop plenty on there and just swirl it around? But how to do you sterilise a pipette? Anyway, thanks in advance!

[nickyw]

We just use sterile cotton buds to innoculate our plates. Dip in bacteria and wipe onto the plates.

[RosalieM]

I have not had much success with sterilising cotton buds. I tend to just burn them! I wrap them in a paper bag and put them in the pressure cooker but I guess I leave them too long. Might just try for the metal innoculation loops.

[DavidPeterson]

I've got a batch of peppercorn broth 'brewing'. It sounds like a great idea - I've never heard of it before. Are there any other natural products that yield pure starins of bacteria?

[bindibadgi]

Instead of spreading with a loop or cotton bud, I inoculate the sterilised agar with the bacterial broth before I pour the plates. That way the bacteria is actually in the agar, all the way through, & grows a nice thick lawn.

Once I've sterilised the agar, I leave the bottles out on the bench to cool, & when I can just comfortably hold a bottle with a bare hand, I add a couple of ml of bacterial broth, swirl gently to mix, then pour. The agar should be about 45-50 degrees & still pour easily.
You have to be careful to check the temp of the bottles frequently so the agar doesn't cool too much & go lumpy & impossible to pour, but I like this method because you end up with consistent growth across an whole batch of plates, & nice clear inhibition zones.

[RosalieM]

That's a good idea Bindi Might try that with some of the junior classes at some stage. I think for the seniors the physical inoculation of the plates is part of the prac. Plates are poured so hopefully we will get some good ones out of it.

[bindibadgi]

Our senior haven't swabbed/streaked their own plates for years!
Not part of the assessment here, so the teachers just get me to provide the already inoculated plates.
I doubt the kids would finish the prac if they had to streak their own. They use 4 or 5 plates each, with 3 discs per plate....so in a single 50 minute lesson it's a push....if we had double lessons I'd love to have them actually learn the technique, but alas it's not the case

[DavidPeterson]

Most of our classes are 'doubles' (we have 2x double plus 1xsingle per week) but I still inoculate plates for the students - it eliminates one source of error. What I do couldn't be referred to as 'streaking' (and I reluctantly studied Microbiology as part of my degree although I got out of there at the first opportunity) - I use a cotton ball (or one of those make-up removing pads) with forceps, take a swab from the plate containing the pure bacteria. Then on the students plates, I start from the middle, and twist & swirl out to the edge of the plate. This (usually) ensures a complete lawn over the entire surface of the plate.

[RosalieM]

Interesting... Perhaps I will suggest this to the teachers. Maybe (like in many things) they only do it because "that's the way we've always done it". Kids are in the process of this prac now. The teacher decided to just put some drops of the peppercorn broth on it and swirl it around. Hopefully they will grow. I can use this other method (peppercorn mixed before pouring) if their's don't grow.

[Ocean Breeze]

Sunray18

You can still do the wild cultures. Remember that you will still need a Risk management /assessemt - for health and safety.

Wild cultures are generally safe, as long as plates are not opened once grown, disposed of properly etc.

Can someone please tell me about the peppercorn plates? Does the peppercorn inhibit microbial growth in the same way that antibiotic disks do? Or are the peppercorns the SOURCE of the bacteria?

Thanks in advance

[DavidPeterson]

From my understanding (which is limited as I only Googled it yesterday) peppercorn infusion is a source of Bacillus subtilis a gram +ve bacterium. I believe that Anton van Leeuwenhoek (1632 –1723) described micro-organisms he found in peppercorn infusion.

[Ocean Breeze]

Does the liquid that the pepper corn is boiled in, then go on to produce a baciilus selective medium? Is that whats happening ?

Anyone have a recipe? that would help me asking random questions

[RosalieM]

Hi Ocean Breeze

It is on page one of this thread. All I know about it I got from here! Otherwise I would attempt to provide more info

[Ocean Breeze]

OOPS! Thanks Rosalie