Hello,
I am trying to determine hydrogen peroxide production of my lactic acid bacteria isolates. The method I used is that: ''10% skim milk powder medium was prepared and distributed into 30 ml dark bottles and sterilized by autoclaving at 110°C for 15 minutes. Lactic acid bacteria were activated in MRS liquid medium at 30°C for 24 hours and then inoculated into 2% skim milk powder medium and incubated at 30°C for 48 hours. At the end of incubation, samples were completed to 30 ml with distilled water and centrifuged at 5000 rpm for 15 minutes. After centrifugation, the supernatant liquid was taken and filtered in a dark environment using Whatman 42 filter paper. 8 ml of the filtered part was taken, 1 ml of sulfuric acid, 1 ml of ammonium molybdenum and 1 ml of potassium iodide solutions were added to them, mixed and measured in a spectrophotometer at a wavelength of 350 nm. The measured sample values were compared with the standard hydrogen peroxide curve to determine the hydrogen peroxide production by the bacteria.'' Here, do I need to inoculate bacterial isolates into 30 ml skim milk medium? If so, how I will complete to 30 ml with distilled water, because it is already 30 ml? Also, how can I prepare blank sample, the same procedure without inoculating the bacteria?
If you can help me, I would be grateful. Thank you.
Dear Dr. Zeynep Seyda,
For your blank you may add LAB or skip it altogether.
If you prefer to add LAB then immediately sterilize it by autoclaving the same. The sterilized sample won't produce any H2O2 as there is no active bacterial culture present in it. Alternatively try to adjust the pH to extreme alkaline/acidic range so as to prevent LAB activity.
If you wish to skip LAB in your blank then in that case add equivalent quanity of distilled water instead of bacterial culture.
Hope these suggestions would be helpful to you.
Thanks and regards,
Arun Padhye Dear Dr. Padhye,
Thank you very much for your answer. I will try your suggestions. But I am also confused about how much ml skim milk powder do I need to use to inoculate bacteria? 30 ml or less? Is it okay if I inoculate in 10 ml and after incubation completing it to 30 ml? Thanks for your help.
Dear Zeynep Seyda,
I suppose you first should find out the optimum quantity of skim milk powder by carrying out varied quantity of it such 10, 20, 30 ml etc. At any given point of time during LAB fermentation it shouldn't be substrate limiting reaction or else the assay will be erroneous. Also find out the reported method from standard assay protocol.
Hope you would find this bit helpful.
N.B.
If you find out that 10 ml itself is not substrate limiting for maximum production of H2O2 then further dilution upto 30 ml with water is alright.
Regards,
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