cell suspencion and medium - (Apr/18/2011 )

if we are going to prepare cell suspension of microbes (e coli, yeast, bacil ....), how we need to prepare it in the standard way. in how much medium, how much of cells are required for best results or performance in later propagating.
if we are using the colonies, how many colonies needed to dilute in how much volume of medium ?
and if you say some certain amount, please explain why you choose that particular ones.

how long the cultured plate with the isolated cultures and cell suspension can be kept and still ideal for doing experiments?

and how long can we keep the medium? if the medium was prepared long ago, would it be losing its nutrition and not fresh enough for microbes to consume and so, it would give the impact on their growth. ?

all these always confuse me.

thank you very much for your answers in advance.

Your questions are too general.

Preparing bacterial colonies for what?
You need to specify more and about the plates: it also depends, are the plates with antibiotics? if so, wich antibiotics.... etc...

.....

Are these homework questions ?

pito on Mon Apr 18 21:36:22 2011 said:

thanks
let's say, for ecoli. after i had plated out on lb agar, 1 colony is collected from the plate and dissolved into the LB broth 10ml. Is this suitable with the number of colonies and broth volume ? no antibiotics is concerned for that, its just for general.
yes. i am doing the experiment in college and just wondering about it.

and i think the medium prepared should not be used after one week that left in outside instead of in fridge due to unfreshness. does it make sense ?

Thank you

MicroGeek on Tue Apr 19 15:06:02 2011 said:

if you start with one colony, you are sure to have the same "genes" (same bacteria) and yes, you can then add that into 10ml for example to grow untill it reaches OD=0,6 to transform the cells.. or..

medium prepared can be stored in fridge for a very very long time.
And if you dont keep the medium in the fridge.. you can still use it, it doesnt really matter, but you would get contamination faster... and/or break down some of the products in the medium.. I dont know what you have in your medium, but its possible there are some products in it that would react with sunlight or with the heat... But for example: if its just water + sugar... nothing much will happnen with it if you store it outside a fridge (but I am oversimplifying things here).

Its like how you keep your food: you store certain foods in the fridge, others in normal room temperature... its the same for the "food" you give to the bacteria.


And to be honest: I never heard of people storing prepared medium in a room (outside a fridge).

MicroGeek on Tue Apr 19 15:06:02 2011 said:

Ever notice how the vast majority of food says "Refrigerate after opening." It's mostly the stuff that comes straight from animals that needs refridgeration - milk, eggs, cheese, meat, etc. Just like like the salsa you buy at the store, the LB you make is sterilized. Keeping it at room temperature shouldn't have any effect on it. It's after you open it (salsa) or add something that you know will decompose (ampicillin) that you'd want to refrigerate it to extend it's shelf life.

We prepare many flasks of media and just leave them setting out on the shelf or in a cabinet. It's better that way because you'll be alerted to contamination pretty quickly when stuff starts to grow for no good reason. Same thing with LB only plates. It's a decent idea to keep them at RT rather than 4C to ensure they aren't contaminated.

Usually I left my prepared media (except those with antibiotics)in Room temp/inside incubator... so that if there is contamination, I can see it after few days...