Streaking vs Spreading Plates - (Feb/11/2015 )
Hi all. I have 2 different questions here actually.
After I've done transformation of plasmid into e.coli, the protocol I referred to suggest to spread the recovered bacteria suspension on the antibiotic plate instead of streaking plate.
1. I wonder what's the difference? When do we streak, when do we spread, how to know?
One more thing. When I prepared a 100mg/ml erythromycin stock, dissolved in absolute alcohol. I noticed precipitation after it's kept in cold at -20c. I have to shake it and slightly warm it up with my hands to make sure it dissolve nicely before I can use it.
2. Is it normal for erythromycin to precipitate in cold? I once used those prepared by others before and did not see such problem.
Sorry I just started my master work. Microbiology is totally new to me. Would really appreciate if people with experiences can share their opinions with me.TQ 
I have no experience specifically with erythromycin, but in general cooling solutions will sometimes precipitate material. You can make your stock more dilute, or simply warm the stock as you are doing. For spreading after transformation, I'd recommend using 5 mm glass beads. They can be autoclaved and kept sterile, then added by pouring 10-30 carefully onto the plate surface. After pipetting 20-200 ul of recovered transformation onto the plate and covering, they can be stacked and shaken for a minute (it takes a while) to cover the plate uniformly. You can either incubate the plates with the beads or (as I do) tap the beads out after shaking. Streaking is used when you have a high density culture (liquid or frozen glycerol) that you want to prepare single colonies from. Spreading such a sample will produce a lawn of bacteria. You want very dilute samples. You could do this with serial dilutions then spreading (which can give you quantitative counts of cfu/ml) but usually you want to avoid that and simply want single colonies. Streaking does that for you. You can get cheap 5mm glass beads from
https://www.fdglass.com/products/details/12
phage434 on Thu Feb 12 16:29:39 2015 said:
I have no experience specifically with erythromycin, but in general cooling solutions will sometimes precipitate material. You can make your stock more dilute, or simply warm the stock as you are doing. For spreading after transformation, I'd recommend using 5 mm glass beads. They can be autoclaved and kept sterile, then added by pouring 10-30 carefully onto the plate surface. After pipetting 20-200 ul of recovered transformation onto the plate and covering, they can be stacked and shaken for a minute (it takes a while) to cover the plate uniformly. You can either incubate the plates with the beads or (as I do) tap the beads out after shaking. Streaking is used when you have a high density culture (liquid or frozen glycerol) that you want to prepare single colonies from. Spreading such a sample will produce a lawn of bacteria. You want very dilute samples. You could do this with serial dilutions then spreading (which can give you quantitative counts of cfu/ml) but usually you want to avoid that and simply want single colonies. Streaking does that for you. You can get cheap 5mm glass beads from
Thanks for the explanation. Correct me if i am wrong, so the reason why we do spreading instead of streaking after transformation is because the recovered transformed bacteria is not very concentrated?
That's right. Challenging ligations and transformations can produce only a small number of colonies, but you need only one correct one.
phage434 on Thu Feb 12 16:29:39 2015 said:
I have no experience specifically with erythromycin, but in general cooling solutions will sometimes precipitate material. You can make your stock more dilute, or simply warm the stock as you are doing. For spreading after transformation, I'd recommend using 5 mm glass beads. They can be autoclaved and kept sterile, then added by pouring 10-30 carefully onto the plate surface. After pipetting 20-200 ul of recovered transformation onto the plate and covering, they can be stacked and shaken for a minute (it takes a while) to cover the plate uniformly. You can either incubate the plates with the beads or (as I do) tap the beads out after shaking. Streaking is used when you have a high density culture (liquid or frozen glycerol) that you want to prepare single colonies from. Spreading such a sample will produce a lawn of bacteria. You want very dilute samples. You could do this with serial dilutions then spreading (which can give you quantitative counts of cfu/ml) but usually you want to avoid that and simply want single colonies. Streaking does that for you. You can get cheap 5mm glass beads from
you really add 10-30 beads per plate?
It seems so much, I only add 3 beads per plate.
I aim for around 10, but sometimes more come out. I'm sure 3 would work, but would require more shaking. This is obviously not critical.