I am interested to understand the rationale behind the method of use (as mentioned in the file attached) as follows:
1. Why we cannot allow the suspension to sit at room temperature for longer than 45-60 minutes?
2. Why is inoculation of the bead into a broth medium not recommended?
I have contacted the manufacturer, but no reply yet.
https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/CryoBeads.htm
1: Following the reference (ATCC Connection newsletter) given in the procedure from your link:
"The cryoprotectant (glycerol) may be toxic to cells if the equilibration time is longer than 60 minutes."
2: Plating onto solid media gives you an easy first stage to checking purity - contaminants can (generally) be easily spotted by eye. Inoculating into broth may allow contaminants to outgrow your desired organism, necessitating additional purification steps.
I fully agree with Joe A. Hawthorne. Having your culture on solid medium is safe and helps you to ensure the purity.
I would like to add some comment concerning you second question. As you may notice, the beads are submerged in a nutrient broth containing particular amount of glycerole. The amount of the crioprotector is important for preventing water crystal development during freezing. Therefore, if you inoculate the beads into the liquid growth medium you will lose you crioprotector. If you add liquid bacterial culture into the tube, you will dilute the crioptotector. Since there is only 15% of glycerol, I would not advise to dilute it. It will definitely cause more damage to your cells and consequently reduce storage time and may affect recovery.
How can I fluorescently label vesicles? and how would I be able to tell if it is successful?
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