Recently, we’ve started testing the ability of previously isolated bacteria to fix nitrogen. For that reason, we have prepared a solid N-free medium (namely NFb) (http://collection.ibppm.ru/nfb-medium.html). We adjusted BTB to be 3x as proposed by modern literature. After 7 days of incubation a Bacillus spp. strain seems to exhibit growth, yet the medium doesn’t turn blue (or even blueish).
We have already made sure that the protocol of the medium preparation was followed to the letter and there were no nitrogen contaminations. Moreover we have tested the ability of the medium to change colour by using a strong KOH solution. We have also already ruled out the possibility of AcdS activity as proposed by Professor Ann M. Hirsch in a previous answer of hers (https://www.researchgate.net/post/How-to-know-nitrogen-fixing-ability-of-bacteria-on-Nitrogen-free-media), since we have the sequence of the full chromosome of the isolate, and such a gene or genes encoding for said enzyme are nowhere to be found.
Could anyone please propose a different interpretation of this result? Your help would be greatly appreciated!
Jakub Grzesiak Originally, it has a very light green tint that doesn't change at all after 7 days in the incubator
I suspect that the strain does not fix nitrogen with malate as a C source and/or under aerobic conditions. Here is an interesting paper: https://www.idosi.org/wjas/wjas3(5)/9.pdf
and a matching recipe:
Azotobacter paspali Medium
Composition per liter:
Agar ............................................................................................ 20.0g
Sucrose........................................................................................ 20.0g
CaCO3 ........................................................................................... 1.0g
MgSO4·7H2O................................................................................ 0.2g
KH2PO4....................................................................................... 0.15g
K2HPO4....................................................................................... 0.05g
CaCl2........................................................................................... 0.02g
Na2MoO4·2H20 .......................................................................... 2.0mg
Bromthymol Blue solution ......................................................10.0mL
FeCl3 (10% solution) .................................................................0.1mL
pH 7.0 ± 0.2 at 25°C
Bromthymol Blue Solution:
Composition per 10.0mL:
Bromthymol Blue ......................................................................... 0.5g
Ethanol.....................................................................................10.0mL
Preparation of Bromthymol Blue Solution: Add Bromthymol
Blue to 10.0mL of ethanol. Mix thoroughly.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15
psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.
Good luck 👍
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