We thank the late Dr. Norm Taylor, Dept. Plant and Soil Science at the University of Kentucky, for allowing us to use samples from his red clover plots for this study. This project was funded by the United States Department of Agriculture.

1. Preparation of Plant Extract
<ol>
	<li>See Kagan and Flythe10 for extraction of phenolic compounds from Trifolium pratense cv. Kenland.&#160;</li>
	<li>To extract other compounds in other plants, check the phytochemical analysis literature for plant- or metabolite-specific extraction methods (many are described), or look for protocols such as those of Khurram et al.7,8 which &#160;isolate many compounds with a wide range of polarities.</li>
</ol>
2. Preparation of Thin-layer Plates
<ol>
	<li>Clean TLC plates by developing in one or more polar, neutral solvents, in order to move adsorbed contaminants away from the zone of development.
	<ol>
		<li>In a fume hood, prepare enough cleaning solvent (e.g. 15-100 ml of ethyl acetate-methanol 2:1, v/v) to cover the bottom of the TLC developing chamber, as well as the lower edge of a TLC plate when set inside the chamber.</li>
		<li>Use commercially available glass TLC developing chambers (different sizes available, with lids) or foil-covered Pyrex beakers or preserving jars.</li>
		<li>Use scissors to cut aluminum- or plastic-backed (flexible) silica gel plates, which come in various sizes (20 cm x&#160;20 cm and smaller), to fit the available developing chamber. (Caution:&#160;silica can cause lung damage if inhaled. Work in a fume hood, and handle TLC plates with gloves to avoid getting skin oils onto the silica.)</li>
		<li>Insert the plates into the chamber, with the tops leaning against the chamber walls.&#160;Plates should not touch each other.&#160;Cover the chamber and let the solvent move up the plate by capillary action.</li>
		<li>When solvent has reached the top of the plates, remove plates from chamber and arrange in a standing position within the fume hood until solvent has evaporated.</li>
		<li>Check to see if impurities have migrated near the top of the TLC plate by looking for a yellow band under visible light, or a fluorescent band under ultraviolet (UV) light (see the &#8220;impurity front&#8221; or IF in Figure 2B).&#160;If the majority of the plate still has a yellowish tinge, repeat the cleaning process.</li>
		<li>After removing TLC plates from the chamber, discard the solvent. Allow residual solvent to evaporate completely before using the chamber for Protocol 2.</li>
		<li>To remove residual moisture that can affect migration of compounds on silica16, prop the plates upright in a drying oven at 100 &#176;C&#160; (10-15 min for a 20 cm x&#160;20 cm plate, and 5 min for 7 cm x&#160;10 cm plates).</li>
		<li>If a 100 &#176;C drying oven is not available, heat plates for a longer period of time at lower temperatures (i.e. 40 min at 60 &#176;C).</li>
		<li>After the plates are dry, let them cool to ambient temperature before loading.</li>
	</ol>
	</li>
</ol>
3. Preparation of Developing Chambers for Extract Separation
<ol>
	<li>Use scissors to cut a piece of filter paper slightly below chamber height, and about half the chamber perimeter in width.&#160;This paper acts as a wick to draw solvent up the chamber wall and saturate the chamber with solvent vapors, thus improving reproducibility of separations1.</li>
	<li>In a fume hood, mix solvents (ethyl acetate-methanol 4:1, v/v, for this study). Pour solvent mixture into the chamber&#160;and cover. Wait until the entire wick is wet with solvent, indicating chamber saturation, to put plates into chamber.</li>
</ol>
4. Loading and Development of TLC Plates
<ol>
	<li>Lightly mark the origin with pencil.&#160;If the TLC plate adsorbent is soft and easily damaged, make marks at edges. Compounds should be above the surface of the developing solvent when plates are inserted into the TLC chamber.</li>
	<li>Dissolve extracts in enough organic solvent (in this case, methanol) to have a concentrated solution instead of a turbid suspension.</li>
	<li>Load samples and standards as narrow bands with a microliter syringe or capillary micropipettes, leaving a 1&#160;cm border on the sides of the plate. Allow the bands to dry (fanning the plate or loading it in a fume hood helps).</li>
	<li>If a greater concentration of sample is needed on a plate, &#34;overspot&#34; by loading samples again on the dried bands.</li>
	<li>With forceps or tongs, set plate(s) inside the saturated TLC chamber.&#160;Plates should not touch the wick because it may provide solvent to the plates at points of contact, thus altering the path of compound migration.&#160;Cover chamber and let plates develop.</li>
</ol>
5. Preparation of Plates for Bioassay
<ol>
	<li>Remove plate(s) from TLC tank before the solvent front reaches the top of the plate, and mark the height of the solvent front with a pencil.&#160;Let plate dry in fume hood.
	<ol>
		<li>Develop any remaining TLC plates in the same TLC chamber, which is generally usable for an entire day if kept closed.&#160;Remake solvent mixture for TLC chamber if the amount of solvent in the chamber decreases notably.</li>
	</ol>
	</li>
	<li>After plates are dry, visualize bands under visible or UV light, and delineate bands with a pencil.&#160;A viewing chamber with a portable UV lamp is convenient, especially if the lamp can detect compounds at both 254 nm (short-wave UV) and 365 nm (long-wave UV). 
	Note: At this point or after the next step, plates can be wrapped in plastic wrap, covered with foil, and stored at -20 &#176;C. Maximum storage time depends on compound stability. Because silica is not neutral, some compounds may degrade while on the TLC plate.</li>
	<li>Photograph or draw plates.
	<ol>
		<li>Use a gel photodocumentation system if one equipped with overhead UV and/or visible lights is available.</li>
		<li>If no commercial photodocumentation system is available, photograph plates inside of a box lined with dark paper or cloth, with a camera set on a tripod and a portable UV light clamped to a ring stand17. When photographing plates that have no fluorescent indicator, use a blue light filter over the camera lens to improve band appearance17.</li>
	</ol>
	</li>
	<li>To aid in characterizing bands, calculate retention factor (Rf) values by measuring distances traveled by compound and solvent front, and dividing the former by the latter (Figure 2A).</li>
</ol>
6. Bacterial Culture and Assay
<ol>
	<li>Prepare and inoculate media under anaerobic conditions.&#160;The culture used in this case was Clostridium sticklandii strain SR, which was obtained from the culture collection of James B. Russell, Cornell University, Ithaca, NY.
	<ol>
		<li>See Flythe and Kagan13 and the materials list for a description of HAB media.</li>
	</ol>
	</li>
	<li>Grow the culture to exponential or stationary phase. Use sterile anaerobic techniques when working with anaerobic microorganisms.</li>
	<li>Inoculate the culture (1% v/v) into molten agar after the temperature of the agar (0.75% w/v) has decreased to less than 60 &#176;C.&#160;Mix gently and immediately bring into an anaerobic chamber (95% CO2-5% H2 atmosphere for HAB media) to pour.</li>
	<li>Pour into 15 mm x&#160;100 mm plastic Petri dishes, and allow to solidify.</li>
	<li>With scissors, cut the TLC plate into zones containing band(s) of interest.&#160;Lay bands face down onto agar plates, marking bands on the plate backing to keep track of original orientation on TLC plate.&#160;Lay an unused TLC strip onto an agar plate as a control.</li>
	<li>Incubate agar plates, agar side down, in the anaerobic chamber (24 hr, 39 &#176;C).</li>
</ol>
7. Visualization of Bioassayed Agar Plates
<ol>
	<li>With forceps, remove bands from agar plates while they are in the anaerobic chamber.</li>
	<li>Add 1% (w/v) tetrazolium red, prepared in water, dropwise onto the surfaces of the agar plates.&#160;Allow the color to develop for at least 20 min, or until the control plate turns completely red, before removing from the anaerobic chamber. The anaerobic bacteria will begin to lose viability immediately after removal from the anaerobic chamber, but the color is stable for more than 24 hr.</li>
	<li>Photograph under visible light.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+inhibitors+of+Erwinia+carotovora+and+E.+herbicola+on+thin-layer+chromatograms.">Detection of inhibitors of Erwinia carotovora and E. herbicola on thin-layer chromatograms.</a> J. Chromatogr. 110, 193-196 (1975).</li><li>Betina, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioautography+in+paper+and+thin-layer+chromatography+and+its+scope+in+the+antibiotic+field.">Bioautography in paper and thin-layer chromatography and its scope in the antibiotic field.</a> J. Chromatogr. A. 78, 41-51 (1973).</li><li>Weltzien, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ein+biologischer+Test+f&#252;r+fungizide+Substanzen+auf+dem+Papierchromatogramm.">Ein biologischer Test f&#252;r fungizide Substanzen auf dem Papierchromatogramm.</a> Naturwissenschaften. 45, 288-289 (1958).</li><li>Khurram, M., Khan, A. M., Hameed, A., Abbas, N., Quayum, A., Inayat, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activities+of+Dodonaea+viscosa+using+contact+bioautography+technique.">Antibacterial activities of Dodonaea viscosa using contact bioautography technique.</a> Molecules. 14 (3), 1332-1341 (2009).</li><li>Khurram, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+anticandidal+potential+of+Quercus+baloot+Griff.+using+contact+bioautography+technique.">Evaluation of anticandidal potential of Quercus baloot Griff. using contact bioautography technique.</a> Afr. J. Pharm. 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Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The authors declare no competing financial interest.

This protocol describes a simple method for separating an extract into subsets of compounds and assaying those subsets by contact bioautography.&#160;The method is quite similar to one used by Chomnawang et al.15 to screen for plant metabolites inhibitory to gonorrhea-causing bacteria. The type of bioautography employed to screen for antimicrobial plant compounds depends on many factors, including the test microorganism, the laboratory setup, and the preferences of the person(s) performing the bioassa...

Assaying for antimicrobial compounds in plants requires separating the components of a plant extract, exposing a test microorganism to those components, and determining whether the microorganism&#8217;s growth is inhibited by any of the compounds. Separations by paper or thin-layer chromatography (PC or TLC) are convenient because many compounds can be separated on a planar surface.&#160;Separation is based on polarity, with some compounds binding tightly to the adsorbent (cellulose in the case of PC, and a variety of adsorbents in the case of TLC) and migrating less than others1. Figure 1 provides an example of the relative positions of polar and nonpolar phenolic compounds after separation on a silica TLC plate.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51411/51411fig1highres.jpg" src="/files/ftp_upload/51411/51411fig1.jpg" /> 
Figure 1. Diagram illustrating distributions of compounds of different polarities after separation on a silica thin-layer chromatographic (TLC) plate.&#160;Phenolic compounds of red clover (Trifolium pratense L.) are used as an example.&#160;Polar compounds, such as clovamide, have a strong affinity for a polar adsorbent like silica and remain near the origin (OR), while less polar compounds, such as the three isoflavones near the solvent front (SF), partition more readily into the solvents (which are less polar than silica unless water, acids, or bases are included) and migrate farther up the plate.
After separation of an extract on a TLC plate, test microorganisms can be exposed to all compounds on the plate, thus speeding the identification of the active components of an extract2.&#160;If a fungal or bacterial culture is exposed to the chromatogram, microbial growth will occur everywhere except over areas with growth-inhibitory compounds. Zones of inhibition then can be visualized by observing the contrast between mycelial growth and the growth-free areas if fungi have been applied3&#160;or by spraying with compounds that change color when reduced or hydrolyzed by living cells4.&#160;Although the use of paper or thin-layer chromatograms for antimicrobial assays was first applied to antibiotics5 and fungicides3,6, plant extracts are now frequently screened for antimicrobial compounds with this method, often referred to as bioautography.&#160;The protocols described herein apply to bioautography of thin-layer chromatograms.&#160;TLC is widely used because it is relatively rapid and can be performed on different adsorbents (e.g. silica, starch, alumina), as well as providing good resolution and sensitivity1.
Plant extracts can be prepared for TLC in many ways.&#160;Common methods include extracting plant material in alcohol-water mixtures such as 80% ethanol7,8, possibly with the addition of acid or base9. Following an extraction in such solvents, which contain some water and are possibly acidic or basic, extracts must be concentrated so that they can be applied to TLC plates in a minimal volume.&#160;The concentration of alcohol-water extracts can be achieved by partitioning with water-immiscible organic solvents8&#160;or with a mixture of such solvents, such as ethyl acetate-ethyl ether (1:1, v/v)10,11. Different plant metabolites are extracted into different organic solvents, depending on their polarities.&#160;To ensure that plant organic acids or bases are extracted into organic solvents at this stage, the pH of an alcohol-water extract can be raised or lowered with a water-soluble acid or base to convert dissociated analytes into their nondissociated forms, which are then soluble in neutral organic solvents9. The organic phase can then be evaporated under reduced pressure or under nitrogen&#160;and adjusted to the desired volume for TLC.&#160;The pH of the extract is unlikely to be lethal to bioassay microorganisms due to the partitioning of analytes into neutral solvents, small final volume, and evaporation of the extract on the TLC plate prior to separation.
Both fungi and bacteria are employed as test microorganisms in bioautography of plant extracts2.&#160;Spores of some fungi, such as Cladosporium cucumerinum, germinate on TLC plates (apart from areas with inhibitory compounds) if sprayed onto plates in a nutrient solution and incubated in a moist environment for several days3.&#160;The dark mycelium of C. cucumerinum on noninhibitory zones provides a sharp contrast to zones free of mycelial growth.&#160;Although bacteria have been applied to thin-layer chromatography (TLC) plates in the same manner4,12, bacteria are also poured over TLC plate surfaces in agar overlays13,14.&#160;Yeast, such as Candida albicans, can be applied in agar overlays as well14.&#160;Alternatively, TLC plates can be placed face down onto agar inoculated with bacteria10,15 or yeast8, a method known as contact bioautography2.
We describe a method for contact bioautography to screen for antimicrobial phenolic compounds from red clover (Trifolium pratense cv. Kenland).&#160;The test microorganism is Clostridium sticklandii, a ruminal hyper ammonia-producing bacterium (HAB) and obligate anaerobe.&#160;Although the separations used do not resolve all components of the extract, they facilitate identification of zones of antimicrobial activity, thus narrowing the pool of possible antimicrobial compounds. The protocol utilizes standard procedures for TLC1. The protocol also describes some of the techniques required for culturing obligate anaerobes for such an assay, a usage of contact bioautography15&#160;and a visualization method with a tetrazolium salt, which stains living cells2,4.

<table><tbody><tr><td>Silica F&lt;sub&gt;254&lt;/sub&gt; TLC plates, aluminum-backed, 0.2 mm thickness, 20 cm x 20 cm</td><td>EMD Chemicals</td><td>5554/7</td><td>These plates are coated with silica that contains an indicator fluorescing at 254 nm.&amp;nbsp; Compounds absorbing at that wavelength appear dark on a fluorescent green background.&amp;nbsp; Alternative sources include Analtech, Selecto Scientific, Fluka.&amp;nbsp; Adsorbents other than silica may be needed.&amp;nbsp; Plastic-backed plates may be suitable, depending on the solvents to be used.&amp;nbsp;</td></tr><tr><td>Sharp, heavy-duty scissors&amp;nbsp;</td><td>any sewing supply company</td><td>similar to Fiskars 175800-1002</td><td>For cutting TLC plates.&amp;nbsp; A paper cutter with a sharp blade can be used as well.&amp;nbsp; Do not inhale silica dust.</td></tr><tr><td>Drying oven at 100 &amp;deg;C (mechanical convection)</td><td>Thermo Scientific</td><td>PR305225M</td><td>Quincy Lab, Inc, Chicago, IL (www.quincylab.com); Cascade Technical Sciences, Hillsboro, OR (www.cascadetek.com)</td></tr><tr><td>TLC chamber</td><td>Kimble Chase</td><td>416180-0000</td><td>Alternative sources:&amp;nbsp; Aldrich. Pyrex beakers or preserving jars can be used for small plates (i.e. 5 cm x 10 cm).&amp;nbsp; Cover with aluminum foil (jar lids may contain material extractable by solvent vapors).</td></tr><tr><td>50 &amp;micro;l Syringe with flat needle tip</td><td>Hamilton</td><td>80965</td><td>For loading amounts of standard or sample exceeding 5-10 &amp;micro;l.&amp;nbsp; Alternative sources are equivalent.</td></tr><tr><td>Micropipettes</td><td>Drummond</td><td>2-000-001</td><td>For loading small amounts of standards or samples.&amp;nbsp; Alternative sources:&amp;nbsp; VWR.&amp;nbsp; Also, Pasteur pipets can be stretched to a thinner diameter with a butane torch.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Filter paper (#1 grade)</td><td>Whatman</td><td>1001 917</td><td>Serves as a chamber wick.&amp;nbsp; Other grades of filter paper are OK.&amp;nbsp; This size can be trimmed for the chambers holding 20 cm x 20 cm plates.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Beaker tongs</td><td>Fisher Scientific</td><td>15-186</td><td>For putting plates in and out of a large TLC chamber.&amp;nbsp; Alternate sources: VWR&amp;nbsp;</td></tr><tr><td>Flat-edge forceps</td><td>Fisher Scientific</td><td>10-275</td><td>For putting plates in and out of a small chamber. Alternate sources: VWR &lt;br /&gt;</td></tr><tr><td>Small portable UV lamp with 4 W or 6 W bulbs for short- and long-wave UV light illumination (254 and 365 nm, respectively)</td><td>Ultraviolet Products</td><td>95-0271-01</td><td>Alternate sources: Spectronics Corporation (www.spectroline.net)</td></tr><tr><td>Viewing cabinet for use with hand-held UV lamp</td><td>Ultraviolet Products</td><td>Chromato-Vue C-10E</td><td>UV-active bands are more easily circled if plates can be set in here.&amp;nbsp; Alternate sources: Spectronics Corporation.&amp;nbsp;</td></tr><tr><td>Photodocumentation system with overhead UV lamp and visible lamp</td><td>Kodak</td><td>Gel Logic 200</td><td>&amp;nbsp;Alternate sources: Ultraviolet Products (www.uvp.com).&amp;nbsp; See protocol for homemade alternative.</td></tr><tr><td>Anaerobic Chamber, Type A, Vinyl</td><td>Coy</td><td>7150000</td><td>This chamber is appropriate for anaerobic bacteria, like &lt;em&gt;Clostridium sticklandii&lt;/em&gt;, as described. &amp;nbsp;However, growth conditions must be tailored to organism used in the assay.&amp;nbsp; A biosafety cabinet and other precautions should be taken if pathogenic organisms are used. Alternate sources: Anaerobe Systems, BioRad, Plas Labs, others&amp;nbsp;</td></tr><tr><td>Tetrazolium red</td><td>Sigma-Aldrich</td><td>T8877</td><td>Alternate sources: MP Biomedicals, Santa Cruz Biotechnology, Alfa Aesar</td></tr><tr><td colspan="4">&lt;strong&gt;Ingredients for HAB media&lt;/strong&gt;</td></tr><tr><td>Pyridoxamine &amp;middot; 2HCl</td><td>Sigma-Aldrich</td><td>P9380</td><td>For this and for all the other reagents in this table, alternative sources are equivalent.</td></tr><tr><td>Riboflavin</td><td>Sigma-Aldrich</td><td>R4500</td><td></td></tr><tr><td>Thiamine HCl</td><td>Sigma-Aldrich</td><td>T3902</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N3376</td><td></td></tr><tr><td>Calcium D-Pantothenate</td><td>Sigma-Aldrich</td><td>C8731</td><td></td></tr><tr><td>Lipoic Acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T5625</td><td></td></tr><tr><td>p-Aminobenzoic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9878</td><td></td></tr><tr><td>Folic acid</td><td>Sigma-Aldrich</td><td>F8798</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>B4639</td><td></td></tr><tr><td>Cobalamine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C3607</td><td></td></tr><tr><td>Pyridoxal HCl</td><td>Sigma-Aldrich</td><td>P9130</td><td></td></tr><tr><td>Pyridoxine</td><td>Sigma-Aldrich</td><td>P5669</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>Iron sulfate &amp;middot; 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>F8263</td><td></td></tr><tr><td>Zinc sulfate &amp;middot; 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>Z0251</td><td></td></tr><tr><td>Manganese chloride &amp;middot; 4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M8054</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B6768</td><td></td></tr><tr><td>Cobalt chloride &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C8661</td><td></td></tr><tr><td>Copper chloride &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>459097</td><td></td></tr><tr><td>Nickel chloride &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>203866</td><td></td></tr><tr><td>Sodium molybdate &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>331058</td><td></td></tr><tr><td>Trypticase (Pancreatic digest of casein)</td><td>Thermo Fisher</td><td>B11921</td><td></td></tr><tr><td>Potassium phosphate monobasic anhydrous</td><td>Thermo Fisher</td><td>P284</td><td></td></tr><tr><td>Sodium carbonate &amp;middot; H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Thermo Fisher</td><td>S636</td><td></td></tr><tr><td>Agar</td><td>Thermo Fisher</td><td>50841063</td><td></td></tr><tr><td>Magnesium sulfate &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher</td><td>7791-18-6</td><td></td></tr><tr><td>Calcium chloride &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher</td><td>BP510</td><td></td></tr><tr><td>Cysteine HCl</td><td>Thermo Fisher</td><td>19464780</td><td></td></tr><tr><td>Potassium phosphate dibasic anhydrous</td><td>Thermo Fisher</td><td>P290</td><td></td></tr><tr><td>Sodium chloride</td><td>Thermo Fisher</td><td>BP358</td><td></td></tr></tbody></table>

thin-layer chromatographic (tlc) separations and bioassays of plant extracts to identify antimicrobial compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Representative silica TLC separations of red clover (Trifolium pratense cv. Kenland) extracts, containing phenolic compounds, are shown in Figure 2. Separation of red clover extract in ethyl acetate-hexane (9:1, v/v), over 8.5 cm, resulted in five bands, one incompletely resolved from the origin (Figure 2A). However, Figure 2B demonstrates that about twice as many bands were revealed when a different sample of red clover extract (from the same cultivar, but grow...

Watch this Scientific Journal Video about Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds at JoVE.com

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

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Principles of Chromatography

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67.7K Views.  United States Department of Agriculture. Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper ammonia-producing bacteria (HAB) native to the bovine rumen.

Video: Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Biochemistry

Extraction and Analysis of Taiwanese Green Propolis

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Immunology and Infection

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Biology

Using TLC-Direct Bioautography Assay to Identify Natural Products for Biocontrol

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

Harnessing Gracilaria gracilis: Bioactive Potential for Sustainable Innovation

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we explore the potential of Gracilaria gracilis, an edible red seaweed cultivated worldwide for its commercial interest as a source of agar and other ingredients for cosmetic, pharmacological, food, and feed applications.
G. gracilis growth conditions were optimized through vegetative propagation and sporulation while manipulating the physicochemical conditions to achieve a large biomass stock. Green extraction methodologies with ethanol and water were performed over the seaweed biomass. The bioactive potential of extracts was assessed through a set of in vitro assays concerning their cytotoxicity, antioxidant, and antimicrobial properties. Additionally, dried seaweed biomass was incorporated into pasta formulations to increase food&#39;s nutritional value. Pigments extracted from G. gracilis have also been incorporated into yogurt as a natural colorant, and their stability was evaluated. Both products were submitted to the appreciation of a semi-trained sensorial panel aiming to achieve the best final formulation before reaching the market.
Results support the versatility of G. gracilis whether it is applied as a whole biomass, extracts and/or pigments. Through implementing several optimized protocols, this work allows the development of products with the potential to profit the food, cosmetic, and aquaculture markets, promoting environmental sustainability and a blue circular economy.
Moreover, and in line with a biorefinery approach, the residual seaweed biomass will be used as biostimulant for plant growth or converted to carbon materials to be used in water purification of the in-house aquaculture systems of MARE-Polytechnic of Leiria, Portugal.

Author Spotlight: Exploring Seaweed's Bioactive Compounds for Sustainable Innovations in Industries 

Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts&#39; antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments.

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we ...

Performing 1D Thin Layer Chromatography

Source: Laboratory of Dr. Yuri Bolshan &#8212; University of Ontario Institute of Technology
Thin layer chromatography (TLC) is a chromatographic method used to separate mixtures of non-volatile compounds. A TLC plate consists of a thin layer of adsorbent material (the stationary phase) fixed to an appropriate solid support such as plastic, aluminum, or glass1. The sample(s) and reference compound(s) are dissolved in an appropriate solvent and applied near the bottom edge of the TLC plate in small spots. The TLC plate is developed by immersing the bottom edge in the developing solvent consisting of an appropriate mobile phase. Capillary action allows the mobile phase to move up the adsorbent layer.&#160;As the solvent moves up the TLC plate, it carries with it the components of each spot and separates them based on their physical interactions with the mobile and stationary phases.

Thin Layer Chromatography: Principle, Procedure, Applications

Source: Laboratory of Dr. Yuri Bolshan &#8212; University of Ontario Institute of Technology
Thin layer chromatography (TLC) is a chromatographic method ...