Materials and methods for
View Patent ↗The subject invention provides novel and advantageous methods for growing bacteria. The methods of the subject invention are particularly advantageous for growing parasitic bacteria in vitro, without the presence of host tissue. In one embodiment of the subject invention, Pasteuria endospores, such as those that infect the rootknot nematode Meloidogyne arenaria or other host nematodes, are grown in vitro under acidic conditions. The process of the subject invention is highly advantageous because Pasteuria can be grown in the absence of nematode tissue.
1. A method for producing Pasteuria endospores in vitro, wherein said method comprises introducing bacteria Pasteuria into a growth medium that is free of sufficient nematode tissue to support bacterial growth, wherein the pH of said growth medium is less than about 6.0, and growing said bacteria Pasteuria.
2. The method, according to claim 1 , wherein said medium is buffered to maintain a pH of less than about 6.0.
3. The method, according to claim 1 , wherein the pH is maintained using a succinate buffer system.
4. The method, according to claim 1 , wherein the oxidation reduction potential of said medium is reduced compared to the oxidation reduction potential when the medium is saturated with atmospheric oxygen.
5. The method, according to claim 1 , wherein the oxidation reduction potential is between about 50 and about 200 mV.
6. The method, according to claim 1 , wherein the medium further comprises peptone as a carbon and nitrogen source.
7. The method, according to claim 1 , wherein the medium comprises the following components: meat peptone, succinic acid, zinc sulfate and magnesium sulfate.
8. The method, according to claim 1 , wherein the spores are grown to a concentration in excess of 1.0×10 7 spores/ml when the concentration of spores is measured by withdrawing a 20 μL sample from a region of the culture with the highest concentration of sporulated Pasteuria , based on microscopic observation, diluting the sample to 1 mL with sterile deionized water, subjecting the sample to sonication for one minute at 15 watts on a Fisher Sonic Dismembrator Model 60, and then measuring the spore density with a hemacytometer.
9. The method, according to claim 1 , wherein the oxidation reduction potential of the medium is maintained at about 50–150 mV and sporulation is induced by increasing the oxidation reduction potential to about 150–200 mV.
10. The method, according to claim 1 , wherein said growth medium is free of nematode tissue.
11. The method, according to claim 1 , wherein said Pasteuria are grown in vitro for at least 24 days.
12. The method, according to claim 1 , wherein said Pasteuria are grown in vitro for at least 46 days.