Streaking Technique: Reville?

[mesq]

Reville: In streaking a dish to isolate a culture in most texts i read one must flame the loop before moving on to the next quadrant of the dish..however I bought plastic inoculation loops and now Im wondering If I should use the same loop for each qaudrant of a plate or use a new one??

Also should I make a spore slurry to dip the loops in or should I just scrape it off the print?

Cheers.

[shroomy]

you could try wiping your loop with medi swabs i bought a packet the other day containing two hundred for $10. they are saturated with 70% isopropyl alcohol. seems to take care of contaminents for me.

[occidentalis]

wouldn't it 'take care' of mushroom spores too?

[squiresk]

Yeah, plastic loops are generally disposable.

But as Shroomy said, you could just resterlise them in 70% EtOH.

k.

[mesq]

I thought alcohol kills mushroom spores...

I mean flaming the loop in between streaking a new sector of the same petri dish... aaanway Im going to get a box of wire ones..

[occidentalis]

I have been using good old electrical fuse wire.

[shroomy]

Mesqualero:

I thought alcohol kills mushroom spores...

I mean flaming the loop in between streaking a new sector of the same petri dish...

yeah hot flame shouldnt hurt the spores aaaaannnyway ,But if im missing somthing here mesqualero could you let me know. I thought the idea of flaming the loop was to sterilize it therefore preventing the spread of contaminents thats why i sugested wiping your plastic ones with alcohol. but if there is another reason for flaming(sterilising) please point me to some info on it.

[ 14. April 2003, 14:08: Message edited by: shroomy ]

[mesq]

Ok... basically the technique calls for flaming the inoculation loop and cooling in the agar.. then scraping some spores off a print and streaking a corner of a petri dish. Then Flame the loop, cool in the agar and make one line through the first streak to catch some spores and streak a corner and repeat...

What this does is it gradually dilutes the amount of spores so that single colonies can emerge and be isolated...The reason the loop is flamed is to kill any spores still hanging onto the loop so that the only spores streaked in the next quadrant are the ones being picked up from the previous streak. So if I were to use alcohol then I would kill more spores rather than pick them up for another streak..

this article shows what i am trying to do...

http://www.umsl.edu/~microbes/streakplates.pdf

Compared to say inoculating with a syringe it is better... I have about 5 dishes I inoculated with a syringe but they are turning out to be a messy entanglement of contams and mycelia.. I will definitely follow the streaking methods through next time...

Hmm all this talk of streaking....

"Oh my god .... it's Max!! It looks like a baby's arm holding an apple!"

hehe

[ 14. April 2003, 17:41: Message edited by: Mesqualero ]

[shroomy]

Ok hand off your apple for a start. the flaming is meant to sterilise the loop and avoid spreading to much culture so alchohol will do the same and also evaporate(so you shouldnt kill any spores on your next streak) quicker than it takes the loop to cool im guessing. but your right if there was alcholol left on the loop you would be defeating your purpose, but i was thinking with the small amount of alcohol on a medi swab that it would vap off your loop very quickly leaving it clean and sterile for your next streak. nice pdf file by the way thanks .

[mesq]

Yeah you are right I guess :D

[squiresk]

Actually, what you can also do is:

A bit difficult to describe, but useful if you're in a hurry or using a limited amount of disposable loops.

With one loop, make a well of spores/bacteria. i.e a just spread bunch around a bit, keeping the loop at the same angle.

Then with the loop at the same angle, smeer, say 5 lines across the agar, then,

turn the loop over(so you've got a clean side, smeer 3-4 lines across the first 5 lines. Then wiggle a line using the edge of the loop, (i.e the smallest surface are of the loop), acroos the 3 latest lines.

Hope that makes sense,

Kai.

So less farting about with disinfecting etc..

Practice, and you'll get better, and develop your own technique.

[mesq]

Thanks for the advice squiresk. A thing I have found difficult is getting dry spores off a print with a loop... what should I do.. I havent had much success with using a syringe...

[squiresk]

Use the agar. Dip the loop into the agar so it breaks the surface, and it'll get kinda wet/sticky. Then start your well away from the hole in the agar. (The loop will get stuck and tear the agar further and it'll get all messy)

I'm gonna have to read the rest of this post, to figure out what you are actually trying accomplish.

"""I have about 5 dishes I inoculated with a syringe but they are turning out to be a messy entanglement of contams and mycelia"""

Thats the whole point. Pick a bit of mycelia, and start that on a new clean plate.

Um, one thing that you may have to look out for. The strain may get 'used to' your agar/ingredients and find it difficult to jump off onto another substrate. If you put a small bit of the final target substrate into you purity plates, that may help. Also If you keep on subbing onto new plates, your original culture may not be as vigourous as you hope. (Senescence (sp) ) I think this is called. i.e getting old.

What is it your actually trying to do? What have you got and what do you want?

Kai.

There is another methods of diluting stuff to obtain pure cultures, ah, nevermind...

[spiders]

flame your scalpel, then dry it with an alcohol swab. Wait for this to evaporate, then swipe it over your print in a sterile environment (glove box/flowhood if your rich) and then just swipe it across.

as soon as the germinate, transfer the best growth to h202 agar.

Then test them buy rhizomorphes and invitro pinning.

[Rev]

3 ways to go about it it seems

the never mind technique- forget the whole dipping between method and just wipe through each

the dipped in alcohol technique (plastic)

and the make your own wire loop technique - in which case it can be flamed

i prefer options 3,1 and 2 in that priority

scalpels are suitable too but i prefer to have a committed tool

the fewer spores the better, but do more plates- the problem youll likely have is that youll have so many isolates youll never get round to testing them all to see which one is best

this can be a problem , not so much for the alsready domsesticated spore races but very much when trying to bring wild strains into cultivation - there is a lot more diversity and environment specific genes active in these than in indoor types

if working with wild types i recommend cloning followed by taking sterile prints followed by multispore innoculations into cakes - then cloning off the successful fruitbodies - that come from the most well adapted isolates from the multiisolate cake

and so on and so forth until you have a domesticated race

[Rev]

or as BM says there are invitro clues to strain quality

[mesq]

Squiresk:

I have a print from a wild panaeolus (inactive) that I wish to culture on agar... suprisingly I think the dish that I did the panaeolus hasmore mycelium than the others...I did Tasmanian and thai as well..

I am having difficulties telling the difference between cobweb mold and mycelia and Im sure there is mycelia mixed in with cobweb...

I have noticed some areas where there is white fluffy growth that looks like mycelia has turned the agar a dark amber colour... its all an interesting learning experience for me@!

[mesq]

wow ... when you do a google search, and then find some dumb questions on SAB you asked from many many years ago...

I don't see why I thought just alcohol swabbing wouldn't be good enough with the plastic loops. Ah well... the benefit of hindsight

So much patience and helpful responses though!

Edited June 26, 2015 by mesq

[Zen Peddler]

Haha