HBSS Components – trial 2
In these discoloration tests, I looked at the components of HBSS. HBSS consists of NaCl, KCl, 0.048 mg/mL Na2HPO4, 0.06 mg/mL KH2PO4, glucose and 0.35 mg/mL NaHCO3. In a previous post, I showed that NaCl, KCl and glucose didn’t cause discoloration (previous post). So, I made HBSS from scratch and I combined the various phosphate buffers with NaHCO3. For homemade HBSS, I autoclaved one batch (called “homemade HBSS”) and sterile filtered the other batch (called “homemade and filtered HBSS”). I also looked at homemade HBSS without NaHCO3. I compared all of these to HBSS that we bought from Invitrogen (Gibco). All of the concentrations matched the HBSS formulation.
The only solution that caused discoloration was Gibco HBSS, even though I made HBSS to exactly match the formulation sheet from Gibco. This suggests that there is some proprietary chemical in Gibco HBSS that speeds up discoloration. I’m going to email Invitrogen to see what they say.
I also followed the pH of these solutions.
So, the starting pH of these solutions varied quite a bit, but after a day in the incubator, all of the pH’s came down to ~ 7. The exception was HBSS without the NaHCO3 buffer.
It’s odd that the effect is so striking. Can we have a sample analysed in some way? I wonder if it has something to do with how it is sterilized? Do you think they autoclave it or expose it to something before the bottle is sealed?
If we have a solution of NaHCO3 at the same concentration as DMEM, will it dissolve the membranes? I know DMEM is complex, but if we mix a solution of say the top 5 components, I wonder if it will replicate the dissolution characteristics of DMEM. I wonder if there is a mystery component there as well…
OK. So throw out Gibco HBSS and the confounding result it provided. This simplifies things again.
Doesn’t all our data fit the following?
I wondered about sterilization, too. That’s why I looked at autoclaved homemade HBSS and sterile-filtered HBSS. The sterilization technique, in my hands, didn’t affect discoloration.
A solution of NaHCO3 alone at DMEM concentration (3.7 mg/mL) will discolor membranes – I have a post on Sep 10. You might argue that it discolors slower, but barely.
Jim – I think your statement rings true.