We have isolated two bacterial isolates producing urease enzyme on Urea Broth/Agar growth medium with phenol red as indicator. During sub-culturing the colony from one broth to freahly prepared medium (same composition) some times similar culture shows urease production (by changing the color of the medium) and some times the culture grows but not change the color of the medium (*orange to pink color). Incubation temperature is 30 degree C with shaking @ 120 rpm.
What is the reason for that?
Also, when we isolate urease producing bacteria on Urea Agar from soil, we got few colonies and the color of the agar changes from orange to pink (that shows urease production by some isolate). Morphologically different colonies were selected and reinoclulate in urea broth containing phenol red. Few isolates changes the color of the broth but others not. After sub-culturing of the positive isolates (that changes the color of the medium) the isolates grew but instead of changing the medium color to pink .... they change it to yellow. I am surprised to see this?
Please tell me what are the possibilities for this transition? Should I change the medium or I have some error in my procedure?
Thanks.
I think, Please try with other growth media, but not left any experimental error as it is. Scientific job is to solve the problems. Wil it may take a week on experiment and can find excellent results.
Wishing you all the best
A mon avis il faut suivre le pH de votre culture bactérienne. Il faut aussi s'assurer de la pureté de votre isolat bactérien. Les conditions de culture doivent être réaliser en respectant l'aseptie pour éviter les contaminations et l'échange de matériel génétique entre les souches bactériennes.
In my opinion it is necessary to follow the pH of your bacterial culture. It must also ensure the purity of your bacterial isolate. The culture conditions must be carried out in accordance asepsis to avoid contamination and exchange of genetic material between bacterial strains.
Hi Kunal,
I am also working urease producing bacteria. i will suggest u consider the following;
1. Use Christensen’s Urea Agar to test for production of urease enzyme.
Christensen’s Urea agar base is a solid media used as a qualitative urease assay and to rapidly screen for urease producing bacteria. Urea agar base is composed of (g/L); Peptone-1.0, Glucose-1.0, Sodium chloride-5.0, Disodium phosphate-1.2, Potassium dihydrogen phosphate-0.8, Phenol red-0.012, Agar-15.0. if your media changes from orange to pink its considered urease -positive and if it changes from orange to yellow its considered urease negative.. (if your medium changes to yellow in presence of urea it indicates that your bacteria can degrade urea substrate which is a source of nitrogen for the microbes however, they are unable to produce urease enzyme.
2. I will suggest you change the incubation factors. try 32C or 37C for the detection of urease production and also i will suggest you increase the agitation rate to 150 or 170rpm.
3. the urea substrate concentration you add in your growth media is is sufficient enough ? what mM of the urea do you use.
4. in order to confirm your qualitative measures of urease production i will suggest you use an analytical approach by checking for urease activity using a conductivity meter.
All the best with your research
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