The overall goal of this procedure is to identify plant metabolites that are antimicrobial to anaerobic bacteria. This is accomplished by first separating a plant extract by thin layer chromatography or TLC. Next, the chromatogram is cut into zones comprising one or more compounds.
The zones are then placed on agar inoculated with anaerobic bacteria and incubated. Following the incubation, the zones are removed and the plates are stained to identify compounds that inhibit bacterial growth. The resulting data reveal the presence or absence of antimicrobial plant metabolites.
This method can help answer key questions in the field of ruminant nutrition, such as what are the effects of plant secondary metabolites on nutrient utilization by specific microorganisms. Though this method reveals plant compounds that are inhibitory to rumine bacteria. It can also be applied to revealing compounds inhibitory to other bacteria or fungi.
In this protocol, phenolic compounds from red clover will be used to demonstrate the identification of antimicrobial compounds by contact biography. Start by cleaning the thin layer chromatography or TLC plates working in a fume hood. Prepare enough cleaning solvent to cover the bottom of the TLC developing chamber, as well as the lower edge of A TLC plate.
When set inside the chamber here, ethyl acetate methanol two to one is prepared. Next, wearing gloves and working in a fume hood. Use scissors to cut aluminum or plastic backed flexible silica plates to fit the developing chamber.
Insert the plates into the chamber with the tops leaning against the chamber walls. The plates should not touch each other. Cover the chamber and let the solvent move up the plates by capillary action.
When the solvent has reached the top of the plates, remove them from the chamber and arrange them in a standing position in the fume hood until the solvent has evaporated. Once the plates are dry, check to see if impurities have migrated near the top of the TLC plates. These will appear as a yellow band under visible light or a fluorescent band under ultraviolet light.
If the majority of the plate still has a yellowish tinge, repeat the cleaning process in the same TLC chamber using the same solvent. After removing the TLC plates from the chamber, discard the solvent. Allow residual solvent to evaporate completely before using the chamber for the next part of the protocol.
Next to remove residual moisture that can affect migration of compounds on silica prop the plates upright in a drying oven at 100 degrees Celsius for 10 to 15 minutes for a 20 by 20 centimeter plate, and five minutes for seven by 10 centimeter plates. Once the plates are dry, let them cool to an ambient temperature before loading them with extracts. To prepare the chambers for extract separation, use scissors to cut a piece of filter paper slightly below chamber height and about half the chamber perimeter in width.
This paper will act as a wick to draw solvents up the chamber wall and saturate the chamber with solvent papers, thus improving reproducibility of separations In a fume hood mix the solvents here. The solvents used are ethyl acetate methanol four to one volume per volume. Pour the solvent mixture into the chamber and cover.
Use a pencil to lightly mark the origin at a point on the TLC plate that will be above the surface of the developing solvent in the chamber. If the TLC plate absorbent is soft and easily damaged, make marks at the edges as shown here. Next, dissolve the extracts in enough organic solvent to have a concentrated solution instead of a turbid suspension.
Here the solvent used is methanol. Using capillary micro pipettes or a microliter syringe loads standards and samples as narrow bands while leaving a one centimeter border on the sides of the plate, allow the bands to dry fanning the plate or loading it in a fume hood helps to dry the bands more quickly. Once the bands have dried, load the samples again on the same spot or over spot.
If a greater concentration of sample is needed on the plate, repeat as needed in the TLC chamber. Ensure that the entire wick has become saturated with solvent. Then with forceps or tongs, place the plates inside the TLC chamber.
The plates should not touch the wick. Cover the chamber and allow the plates to develop before the solvent front reaches the top of the plate. Remove the plates from the TLC tank and mark the height of the solvent front with a pencil.
Let the plate dry. In a fume hood, develop any remaining TLC plates in the same TLC chamber, which is generally usable for an entire day if kept closed. If the amount of solvent in the chamber decreases noticeably, make more solvent mixture after the plates are dry.
Visualize the bands under visible or UV light. A viewing chamber with a portable UV lamp is convenient, especially if the lamp can detect compounds at both 254 nanometer shortwave UV and 365 nanometer longwave uv. Use a pencil to delineate the bands using a gel photo documentation system equipped with overhead, UV and or visible lights obtain images of the plates.
Finally, to aid in characterizing the bands, calculate the retention factor or RF values by first measuring the distance traveled by compound, then measuring the distance traveled by the solvent and dividing the former by the ladder. Once the band locations have been marked, the plates may be stored for later. Use in the bioassay.
Wrap the plates in plastic and cover them with foil. Store the plates at minus 20 degrees Celsius. The maximum storage time depends on compound stability because silica is not neutral.
Some compounds may degrade while on the TLC plate. In preparation for the contact biography assay, prepare and inoculate hyper ammonia producing bacteria or HAB medium under anaerobic conditions. The culture used in this case is Clostridium stickland eye strain sr.
Grow the culture to exponential or stationary phase at 39 degrees Celsius. Use sterile anaerobic techniques when working with anaerobic microorganisms. Prepare zero point 75%weight per volume.
Agar in the same medium used to grow the bacteria after the temperature of the agar has decreased to less than 60 degrees Celsius. Inoculate the molten agar with 1%volume per volume of the culture and mix gently immediately place it in an anaerobic chamber and pour into 15 by 100 millimeter plastic Petri dishes with scissors. Cut the TLC plate into zones containing the band or bands of interest.
Using a sharpie marked the back sides of the plates. Place an unused TLC strip onto agar plate as a control. Then place bands face down onto agar plates.
Incubate the plates agar side down in the anaerobic chamber's. Incubator for 24 hours at 39 degrees Celsius with forceps. Remove the TLC bands from the agar plates while they are in the anaerobic chamber.
Add tetra oleum red dropwise onto the surfaces of the agar plates. Allow the color to develop for at least 20 minutes. When the control plate turns completely red, remove the plates from the anaerobic chamber.
Clostridium stick Landi is an anaerobic bacterium, so it will begin to lose viability immediately after removal from the anaerobic chamber, but the color is stable for more than 24 hours. Using a digital camera photograph the plates under visible light on a background that provides sufficient contrast. A bright red color will be observed when living cells are stained.
Dead cells do not stain. Representative silica, TLC separations of red clover extracts containing phenolic compounds are shown here. Separation in ethyl acetate hexane nine to one over 8.5 centimeters resulted in five bands, one in completely resolved from the origin.
The standards loaded were Formin Bio Canon A and Genine. Four bands from this plate were applied to cultures with Clostridium stick Landi, a ruminal hyper ammonia producing bacterium for 24 hours and stained with tetra oleum red as described as shown here. Incubation with band one Bio Canon A and part of band two four Monon nein on bacteria seeded agar resulted in a well-defined zone of inhibition.
However, incubation of the remainder of band two, along with bands three and four did not inhibit bacterial growth. These results demonstrated that bio Canon A was inhibitory to see stickland eye growth, but form aone Nein was not. While attempting this procedure, it's important to remember that the technique is flexible.
Other test microorganisms can be used as long as appropriate media are selected, and controls are included to ensure that the separation chemistry and the detection method are compatible Following this procedure. Other techniques like high performance liquid chromatography and mass spectrometry can be used to help characterize the antimicrobial compounds identified in a bioassay.
