In many of my culture plates of likely Rhizobia I get that the colonies take color or become central black spotted after 3-4 days. Is it contamination?
Dear Syed,
During rhizobia growth in both starter and broth culture it is very important to look for
contaminants and control the rhizobial density. It seems that the black spots seen are a result of contamination.
The following text describes a method in which contamination can be avoided:
For production of broth culture, vessels or fermenters in different sizes are often used. It is important that all equipment are sterilizable and that inlet air is also sterile. In most ASEAN countries simple autoclavable vessels of glass or stainless steel with capacity of 2-20 L are used in broth culture production. For sterilizing the inlet air, a compressor or a small aquarium pump with sterile filters are used.
Vessels are filled with media to one-third to two-third and sterilized in an autoclave at 121 o C. After the medium is cooled to room temperature, liquid starter culture is inoculated to the vessel with the ration of 1-3 % (v/v) of media. Time required for growing rhizobia range from 3 to 7 days, depending on the rate of growth of rhizobial strains. Starter cultures are best prepared in Büchner flasks. Two 1 L Büchner flasks are connected at the arms with latex tube, one filled with YMA medium and another with distilled water. After sterilization, the agar surface is inoculated with the desired rhizobial strain aseptically and then incubated at 28 o
C until the growth of rhizobium is observed. Pour water from the second flask to the first flask via the connecting latex tube. Ensure suspending maximum rhizobium cell from agar surface to the water by shaking. This technique has an advantage over a broth culture, because an experienced operator can see evidence of contamination, if any, on the agar surface and discard contaminated preparations. During rhizobia growth in both starter and broth culture it is very important to look for contaminants and control the rhizobial density
For more details, please see attached file.
Hoping this will be helpful,
Rafik
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