How to inoculate bacteria? - (Nov/11/2009 )

I'm having trouble because we use the 150ml? flasks to inoculate ecoli which for me are harder to inoculate than the 15ml tubes where I can twirl the loop to get bacteria off the end. But I'm not sure whether to use a pipet tip or use the loop to inoculate. When I use the tip, it doesn't always work and also when I used the loop it sort of works. The colonies I'm scraping from are very small which could be the problem. Anyhow I can't get the colony to come off the tip or the loop and into the liquid. Any suggestions? Also, what is the correct way of doing this because I know certain people have different preferences.

Normally you can simply scrape the loop or tip on the side of the flask, then swirl the flask to cover the region you have scraped with liquid. Alternatively, you can put a small amount of liquid in an eppendorf, put the loop contents into the eppendorf, then add the eppendorf contents to the larger flask. Often a 10 ml overnight culture is made from single colonies, which is often a better way to start a large culture.

phage434 on Nov 11 2009, 07:42 PM said:

Oh I usually use 10-15ml but for some reason the people in my lab use flasks since it aerates in the shaker (which is why we don't used capped 15ml tubes). I don't know...

So anyway, using the pipet tip is pretty unconventional?

For me, I do use autoclaved pipette tips and pick the colonies, after which I just use the pipette to 'eject' the tip by force into the media and leave the tip inside the media.

jiajia1987 on Nov 11 2009, 08:07 PM said:

You leave the tip in the media overnight?

jiajia1987 on Nov 11 2009, 08:07 PM said:

I'm surprised that doesn't contaminate your culture -- your pipette barrel must be way cleaner than mine...

If I was having trouble inoculating a culture due to the size of the flask, I would do either of phage434's two suggestions -- resuspend the initial colony in a small amount to media and pipette that in, or subculture from a smaller overnight culture.

We routinely use sterile cotton swabs to inoculate cultures -- pick the colony up, submerge the swab in the media, and swirl.

Thanks for all the input! Is inoculating sometimes difficult simply because of how small the selected colony is? It's as if it disappears when you are getting it onto the loop and you really can't see what's happening.

HomeBrew on Nov 12 2009, 11:12 AM said:

I often use a DNAzap to clean my pipette barrels. Plus, I do not put my pipette barrels into the opening of the flasks. I simply put the tip at the opening and by ejecting using the pipette ejector, it will be 'forced' into the media.

jiajia1987 on Nov 12 2009, 07:27 AM said:

we do something very similar but just use the sterile pippette tips without the pippette! works very well and you can always work around the tip in biger cultures.

jiajia1987 on Nov 12 2009, 02:27 AM said:

I understand that this must work for you or you wouldn't be doing it, but I'm still confused -- unless the outer surface of your pipette barrel is sterile, then the inside surface of your pipette tip could become contaminated where it contacts the barrel, and carry that contamination into your culture when you eject it into the media. How do you protect against this?