LB + amp agar plate is used in bio labs often. Preparing agar plates is a good skill to have. Below are the steps or protocol to do it but I also an online protocol as reference. The LB in powder form should have proportion information there. The one I have say dissolve 40g of mix in 1 liter of water. The 40g is a mixture of NaCL 10g, Trypton 10g, Yeast Extract 5g, and Agar 15g. The plate I use is 100mm x 15mm. Below is the scale for reference:
I have 10 of these 100mm x 15mm plates and each will take 10mL to 15mL of agar. If I select to do 10mL per plate so we need 10mL x 10 = 100mL of Agar. If I select to do 15mL per plate then I would need 15mL x 10 = 150mL. I feel 15mL is better to give me extra margin or error.
I can take a 500mL bottle and will it up to 200mL of water + agar powder. So, 40g/1.0 liter where 1.0 liter equals to 1000mL. I can do the following algebra:
40g/1000mL = Xg/200mL then X = 8g. To check this math we can do 40g/1000mL = 8g/200mL or 0.04 = 0.04. So, the proportion is worked out and checked. Incidentally, gram to milliliter conversion formula is as follow:
milliliters = grams ÷ ingredient density
For example, here's how to convert 5 grams to milliliters for an ingredient with a density of 0.7 g/mL.
5 g = (5 ÷ 0.7) = 7.142857 ml
In our case, we don't need to make gram to mL conversion because the agar container already say the proportion we need. We just need to measure up 8g of agar powder and put it in 200mL of water.
Now, we need to figure out the amount of antibiotic we need and where and when to mix that in. The antibiotic in the lab is 1000x stock solution. Since 1 milliliter equals to 1000 microliter and we need 200mL of agar, this means we need to put 200microliters into the agar. Use a 200mL or 1000mL Pipette should work. The 200mL has a little extra room beyond 200mL so it should still work. Let's formulate the 1000x stock solution of antibiotic: 1000x means 1000 times more dense than we need. Then how much antibiotic per mL do we need. If I take 1mL of antibiotic concentrate and put it into 1mL of agar, that means I have 1000 times more than I need. To make the proportion right, I would need to divide the antibiotic concentrate 1000 times. 1mL / 1000 = 0.001mL = 1microL. Since I have 200mL of agar, 200mL * 1mL = 200mL:
1mL / (density multiple) * (number of mL agar)
(1mL / 1000) * 200 = 200 microliter
When weighing 8g of agar powder, take the plastic plate and put on the scale first to calibrate the scale with the plate sitting on it to let it level to zero. There is a re-zero button on the scale. Once you put the plate on the scale, just press the re-zero button once. It should read 0.00g.
Take the 8g of agar powder and put it into the 500mL flask glass bottle. Pure still water into the flask bottle until it reaches 200mL. Stew until mixed. Put an autoclave take on it and put a foil or cover leaving it loosely covered and put it in an autoclave. Set the autoclave to 250F for 15 minutes. The autoclave will take 40 minutes to compress and decompress. After it is done, the autoclave tape should show strips which indicate autoclave is successful. The bottle should be just warm to the touch. If taken out too early, it might be hot to the touch.
The 1000x ampicillin antibiotic we have at the lab is usually stored in -30F freezer. It needs to be thaw at room temperature and mixed 200microliter of it into the 200mL agar while the agar is warm. Pour 15mL of the mixture onto each plate while inside a bio safety cabinet. Wait until the liquid agar solidify inside the bio safety cabinet. Put the cover back onto each plate and shield them with Parafilm tap. Sit them upside down where the agar is hanging on top. This allows water condensation to fall down to the cover and key the agar out of excessive moisture. Then you can put the plate in a frizzer or out in room temperature depends on how long you want to keep them.
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