Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

The scope of our research is micro viral test for medicine. We're trying to establish a effective SOP for screening and isolation of intestinal bacterial. To date, a total of 18 bacterial strains have been found with glycine function.

The advantage of our protocol is the use of low carbon source medium to induce bacterial activity, and thus increase the success rate of screening. To begin, prepare the solutions A, B, and C separately, using the appropriate reagents. Mix all three solutions in a 1, 000 milliliter erlenmeyer flask, and add distilled water to make up a final volume of 1, 000 milliliters.

Then adjust the pH to 7.2, using a 10%sodium hydroxide solution. Distribute the medium into 250 milliliter erlenmeyer flasks. Next, add one to 2%auger to the general anaerobic medium into one of 250 milliliter erlenmeyer flasks.

Autoclave the medium at 121 degrees Celsius for 30 minutes. After the medium cools down to room temperature, store it at four degrees Celsius. Prepare a low-carbon source medium, similarly, but omit yeast extract, glucose, and soluble starch.

For the reference solutions, weigh five milligrams, each of orientin and luteolin. Dissolve them separately in one milliliter of dimethyl sulphoxide to achieve a concentration of five milligrams per milliliter. To begin, prepare all the required media and reagents for the procedure.

Collect one to two grams of fresh human feces in a disposable sterile stool collection tube, filled with nitrogen gas. Add five milliliters of general anaerobic medium to the tube. Seal the tube thoroughly and incubated at 37 degrees Celsius for 24 hours in a sterile anaerobic incubator.

After 24 hours, transfer 0.5 milliliters of the human intestinal bacterial suspension into a new micro centrifuge tube, containing 4.5 milliliters of fresh GAM. Seal the tube and incubate it at 37 degrees Celsius for 24 hours under anaerobic conditions to obtain active mixed human intestinal flora. Next, mixed 260 microliters of activated intestinal flora with six milliliters of fresh low carbon source medium containing 40 microliters of orienting reference solution.

Incubate the inoculated media at 37 degrees Celsius. Include a control and a blank group in addition to the sample. After 24 hours and 48 hours, pipette one milliliter of the reaction solution into a 1.5 milliliter micro centrifuge tube.

Centrifuge the tube at 13, 400 G for 15 minutes at room temperature to remove bacteria. Add 200 microliters of the supernatant to 600 microliters of methanol. Mix thoroughly, and centrifuge the tube at 13, 400 G for 15 minutes at room temperature to remove proteins.

Now, filter the supernatant, using a 0.22 micrometer microporous membrane. Analyze the filtrate via high performance liquid chromatography, or HPLC. Use acetone nitrol as mobile phase A and 0.1%formic acid in purified water as mobile phase B.Maintain the column of in temperature at 40 degrees Celsius during HPLC analysis and set the flow rate to one milliliter per minute.

To isolate single strains, activate the mixed human intestinal bacterial flora capable of cleaving orientin, as described earlier. Heat the solid GAM to dissolve and pour it into disposable sterile Petri dishes inside an anaerobic incubator. Allow the medium to solidify to obtain solid GAM plates.

Pipette 100 microliters of the activated bacterial solution into a 1.5 milliliter micro centrifuge tube, containing one milliliter of normal saline. Inoculate an appropriate volume of the bacterial solution onto a Petri dish, using a disposable inoculation ring. Seal the dish and incubate it in an anaerobic incubator at 37 degrees Celsius.

After 48 hours of incubation, observe the size, morphology and color of the colonies on the plate. Select distinct single bacterial colonies and culture them in 10 milliliter micro centrifuge tubes, containing 4.5 milliliters of fresh GAM. Incubate under anaerobic conditions at 37 degrees Celsius for 24 hours to obtain single strains.

Verify the activity of single strains as described earlier, and perform activity verification using HPLC. One fecal sample out of 10 demonstrated the ability to deglycosylate orientin during transformation experiments, confirming the feasibility of screening for active samples.