I need a maximum possible volume of initial culture. I wish to use ASTM E 2149 method. It consists of adjusting the OD at 0.28 and then use 50 ml. volume for each textile sample. Now, I usually I start growing colony in 10 ml broth. But considering the larger volume requirements, can I increase the initial broth amount to lets say 25 or 50 ml.? What would be the repercussions?
I am assuming you will have to standardize the overnight culture to a set concentration before making your 1:10 dilutions. This is what I do for other kinds of experiments where I need to make dilutions. Typically, I set the O/N concentration using OD600 readings to a known number (like 10^9 cfu per mL), and then make the dilutions from that. With that method, you can make your overnight cultures any size you need, since you will be standardizing it later (for example, a large culture with 1 loopful of bacteria into 500 mL broth). If you make multiple flasks of the same bacteria, you can always mix them together before standardizing.
You could increase the volume of media. But you would want to have the start up culture in the case of larger volume to be same as in the case of small volume so the growth phase is not altered. Hence I would scale the OD also depending on the volume of medium, ie, if 0.28 OD is used in 50ml, I would use 0.056 (0.28/5) OD in 10ml.
This is a standard method as you know. Firstly, you are required to grow a fresh 18 h shake culture of Escherichia coli in sterile Tryptic Soy Broth at 35 ± 2°C prior to performing the test. Then, the inoculum preparation states - "Dilute the culture with the sterile buffer solution until the solution has an absorbance of 0.28 ± 0.02 at 475 nm, as measured spectrophotometrically. This has a concentration of 1.5-3.0 × 108CFU/mL. Dilute appropriately into sterile buffer solution to obtain a final concentration of 1.5-3.0 × 105CFU/mL. This solution will be the working bacterial dilution", and you will require 50ml of this working bacterial dilution for each specimen. Also, note that it did not state the initial volume of culture to grow. Therefore you are at liberty to any starting volume, so long as you follow the standard procedure stated. Moreover, you should put into consideration the number of samples you are running and considering the larger volume requirements. Hope that helps!
Thanks all for your time and efforts. Thanks especially to Valentine for identifying my problem so well.
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