The media composition: Glucose (10g), yeast extract (5g), Peptone (5g), K2HPO4 (1g), MgSO4 (0.2g), Na2CO3 (10g), dw 1L and agar 2%. The pH of the medium was automatically adjusted to 10pH. The media was yellow initially but after autoclave it turns to black. Is the Horikoshi media itself black or anything wrong I did during media preparation?
Glucose can't be autoclave, you must add it to media after autoclave by using syringe filter.
Thanks Dr Reza Khakvar and Dr Pete Holt. It means Glucose is sole responsible for blackening of the Horikoshi medium?
Many substances such as glucose, vitamins, antibiotics and etc are heat-sensitive and in the higher temperatures their molecular structure can be change. 121C (autoclave temp.) can burn glucose and turn it color to brown
Don't need to be worry if glucose turn brown during sterilization process.
Hope you are not autoclaving sodium carbonate with the medium... it will decompose and will be lost as CO2... the pH will rise too. Check the attached file from the ATCC on the Horikoshi medium.
Also, autoclaving for too long usually causes "darkening" of complex organic media
Additional recommendations: autoclave not long (10-15 minutes). Adjust pH after autoclaving (as mentioned in ATCC medium). Glucose - filter sterilization is highly recommended (1( and keep glucose solution at 4C (2). Glucose is reducing sugar and in the solution under air is slowly oxidized (some functional groups are oxidized) . 10 g/L is big amount of glucose and it "masked" the losses of glucose during autoclaving and storage at room temperature.
Actually it is not the glucose turning black. Glucose do not char at autoclave temperature and pressure in aq. solution. It is the peptone which got hydrolyzed at pH 10 and then the peptides polymerized to give a dark compound bythe well known method called as Maillard Reaction.
It was also proposed that sugars are also involved in Maillard Reactions.
It's not the glucose that is responsible for turning media to black but autoclaving the media containing sodium carbonate. Try autoclaving the media with sodium carbonate (to prevent changing color) and glucose (to support better growth of microbe) separately.
Abhas Kumar Maharana
To prevent Maillard Reaction due to high temperature in alkaline conditions, 100 ml of a 10% Na2CO3 sterile solution should be aseptically added to the medium after autoclaving, and check the final pH to be about 10.0
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