Name: the effect of the application of thyme essential oil on microbial load during meat drying
Uploaded: 2018-03-14T13:30:00
Description: Watch this Scientific Journal Video about The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying at JoVE.com

This work was supported by the Internal Grant Agency of the Faculty of Tropical AgriSciences, (project number: 20175013) and the CIGA 20182023&#160;both grants, from the Czech University of Life Sciences.

1. Meat Preparation
<ol>
	<li>Obtain a short loin of beef (fresh beef from biceps femoris) from a local butchery and transfer it to the lab. 
	NOTE: It is recommended to transport the loin of beef at ambient temperature (20 - 25 &#176;C), for a period not longer than 20 min in a hermetic sealed bag.</li>
	<li>To sterilize the outer surface of the beef muscle, in a laminar safety cabinet, wash the muscle by spraying with 70% (v/v) ethanol for 10 s using a squeeze bottle of 500 mL. Apply 0.025 g of ethanol ...

<ol>
	<li>Eklund, M. W., Peterson, M. E., Poysky, F. T., Paranjpye, R. N., Pelroy, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+bacterial+pathogens+during+processing+of+cold-smoked+and+dried+salmon+strips.">Control of bacterial pathogens during processing of cold-smoked and dried salmon strips.</a> J. Food Prot. 67 (2), 347-351 (2004).</li><li>Mahmoud, B. S. 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=Preservative+effect+of+combined+treatment+with+electrolyzed+NaCl+solutions+and+essential+oil+compounds+on+carp+fillets+during+convectional+air-drying.">Preservative effect of combined treatment with electrolyzed NaCl solutions and essential oil compounds on carp fillets during convectional air-drying.</a> Int. J. Food Microbiol. 106 (3), 331-337 (2006).</li><li>Rahman, M. S., Guizani, N., Al-Ruzeiki, M. H., Al Khalasi, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microflora+Changes+in+Tuna+Mince+During+Convection+Air+Drying.">Microflora Changes in Tuna Mince During Convection Air Drying.</a> Dry. Technol. 18 (10), 2369-2379 (2000).</li><li>Faith, N. G., 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=Viability+of+Escherichia+coli+O157:+H7+in+ground+and+formed+beef+jerky+prepared+at+levels+of+5+and+20%+fat+and+dried+at+52,+57,+63,+or+68+C+in+a+home-style+dehydrator.">Viability of Escherichia coli O157: H7 in ground and formed beef jerky prepared at levels of 5 and 20% fat and dried at 52, 57, 63, or 68 C in a home-style dehydrator.</a> Int. J. Food Microbiol. 41 (3), 213-221 (1998).</li><li>Hierro, E., De La Hoz, L., Ord&#243;&#241;ez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Headspace+volatile+compounds+from+salted+and+occasionally+smoked+dried+meats+(cecinas)+as+affected+by+animal+species.">Headspace volatile compounds from salted and occasionally smoked dried meats (cecinas) as affected by animal species.</a> Food Chem. 85 (4), 649-657 (2004).</li><li>Nummer, B. A., 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=Effects+of+Preparation+Methods+on+the+Microbiological+Safety+of+Home-Dried+Meat+Jerky.">Effects of Preparation Methods on the Microbiological Safety of Home-Dried Meat Jerky.</a> J. Food Prot. 67 (10), 2337-2341 (2004).</li><li>Greig, J. D., Ravel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+foodborne+outbreak+data+reported+internationally+for+source+attribution.">Analysis of foodborne outbreak data reported internationally for source attribution.</a> Int. J. Food Microbiol. 130 (2), 77-87 (2009).</li><li>Eidson, M., Sewell, C. M., Graves, G., Olson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beef+jerky+gastroenteritis+outbreaks.">Beef jerky gastroenteritis outbreaks.</a> J. Environ. Health. 62 (6), 9-13 (2000).</li><li>Allen, K., Cornforth, D., Whittier, D., Vasavada, M., Nummer, B. <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+high+humidity+and+wet+marinade+methods+for+pasteurization+of+jerky.">Evaluation of high humidity and wet marinade methods for pasteurization of jerky.</a> J. Food Sci. 72 (7), (2007).</li><li>Levine, P., Rose, B., Green, S., Ransom, G., Hill, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+testing+of+ready-to-eat+meat+and+poultry+products+collected+at+federally+inspected+establishments+in+the+United+States,+1990+to+1999.">Pathogen testing of ready-to-eat meat and poultry products collected at federally inspected establishments in the United States, 1990 to 1999.</a> J. Food Prot. 64 (8), 1188-1193 (1990).</li><li>Keene, W. E., 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=An+outbreak+of+Escherichia+coli+O157:H7+infections+traced+to+jerky+made+from+deer+meat.">An outbreak of Escherichia coli O157:H7 infections traced to jerky made from deer meat.</a> JAMA. 277 (15), 1229-1231 (1997).</li><li>Oliver, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viable+but+nonculturable+state+in+bacteria.">The viable but nonculturable state in bacteria.</a> J. Microbiol. 43, 93-100 (2005).</li><li>Oliver, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+findings+on+the+viable+but+nonculturable+state+in+pathogenic+bacteria.">Recent findings on the viable but nonculturable state in pathogenic bacteria.</a> FEMS Microbiol. Rev. 34 (4), 415-425 (2010).</li><li>Khamisse, E., Firmesse, O., Christieans, S., Chassaing, D., Carpentier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+cleaning+and+disinfection+on+the+non-culturable+and+culturable+bacterial+loads+of+food-contact+surfaces+at+a+beef+processing+plant.">Impact of cleaning and disinfection on the non-culturable and culturable bacterial loads of food-contact surfaces at a beef processing plant.</a> Int. J. Food Microbiol. 158 (2), 163-168 (2012).</li><li>Li, L., Mendis, N., Trigui, H., Oliver, J. D., Faucher, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+viable+but+non-culturable+state+in+human+bacterial+pathogens.">The importance of the viable but non-culturable state in human bacterial pathogens.</a> Front. Microbiol. 5, 258 (2014).</li><li>Hern&#225;ndez, H., Claramount, D., Ku&#269;erov&#225;, I., Banout, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+modified+blanching+and+oregano+essential+oil+on+drying+kinetics+and+sensory+attributes+of+dried+meat.">The effects of modified blanching and oregano essential oil on drying kinetics and sensory attributes of dried meat.</a> J. Food Process. Preserv. , (2016).</li><li>Garc&#237;a-D&#237;ez, J., 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=The+Impact+of+Essential+Oils+on+Consumer+Acceptance+of+Chouri&#231;o+de+vinho+-+A+Dry-Cured+Sausage+Made+from+Wine-Marinated+Meat+-+Assessed+by+the+Hedonic+Scale,+JAR+Intensity+Scale+and+Consumers'+&amp;#34;Will+to+Consume+and+Purchase.&amp;#34.">The Impact of Essential Oils on Consumer Acceptance of Chouri&#231;o de vinho - A Dry-Cured Sausage Made from Wine-Marinated Meat - Assessed by the Hedonic Scale, JAR Intensity Scale and Consumers' &amp;#34;Will to Consume and Purchase.&amp;#34.</a> J. Food Process. Preserv. 41 (4), (2017).</li><li>Govaris, A., Solomakos, N., Pexara, A., Chatzopoulou, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+effect+of+oregano+essential+oil,+nisin+and+their+combination+against+Salmonella+Enteritidis+in+minced+sheep+meat+during+refrigerated+storage.">The antimicrobial effect of oregano essential oil, nisin and their combination against Salmonella Enteritidis in minced sheep meat during refrigerated storage.</a> Int. J. Food Microbiol. 137 (2-3), 175-180 (2010).</li><li>Holley, R. A., Patel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+in+shelf-life+and+safety+of+perishable+foods+by+plant+essential+oils+and+smoke+antimicrobials.">Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials.</a> Food Microbiol. 22 (4), 273-292 (2005).</li><li>Petrou, S., Tsiraki, M., Giatrakou, V., Savvaidis, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+dipping+or+oregano+oil+treatments,+singly+or+combined+on+modified+atmosphere+packaged+chicken+breast+meat.">Chitosan dipping or oregano oil treatments, singly or combined on modified atmosphere packaged chicken breast meat.</a> Int. J. Food Microbiol. 156 (3), 264-271 (2012).</li><li>Ballester-costa, C., Sendra, E., Viuda-martos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Antioxidant+and+Antibacterial+Properties+on+Meat+Homogenates+of+Essential+Oils+Obtained+from+Four+Thymus+Species+Achieved+from+Organic+Growth.">Assessment of Antioxidant and Antibacterial Properties on Meat Homogenates of Essential Oils Obtained from Four Thymus Species Achieved from Organic Growth.</a> Foods. 6 (8), 59 (2017).</li><li>Hern&#225;ndez, H., 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=The+effect+of+oregano+essential+oil+on+microbial+load+and+sensory+attributes+of+dried+meat.">The effect of oregano essential oil on microbial load and sensory attributes of dried meat.</a> J. Sci. Food Agric. 97 (1), 82-87 (2017).</li><li>Garc&#237;a-D&#237;ez, J., Alheiro, J., Falco, V., Fraqueza, M. J., Patarata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+characterization+and+antimicrobial+properties+of+herbs+and+spices+essential+oils+against+pathogens+and+spoilage+bacteria+associated+to+dry-cured+meat+products.">Chemical characterization and antimicrobial properties of herbs and spices essential oils against pathogens and spoilage bacteria associated to dry-cured meat products.</a> J. Essent. Oil Res. 29 (2), 117-125 (2017).</li><li>Cavanagh, H. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+Activity+of+the+Volatile+Phase+of+Essential+Oils:+A+Brief+Review.">Antifungal Activity of the Volatile Phase of Essential Oils: A Brief Review.</a> Nat. Prod. Commun. 2 (12), 1297-1302 (2007).</li><li>Tajkarimi, M. M., Ibrahim, S. A., Cliver, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+herb+and+spice+compounds+in+food.">Antimicrobial herb and spice compounds in food.</a> Food Control. 21 (9), 1199-1218 (2010).</li><li>Nedorostova, L., Kloucek, P., Kokoska, L., Stolcova, M., Pulkrabek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+properties+of+selected+essential+oils+in+vapour+phase+against+foodborne+bacteria.">Antimicrobial properties of selected essential oils in vapour phase against foodborne bacteria.</a> Food Control. 20 (2), 157-160 (2009).</li><li>Burt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+oils:+Their+antibacterial+properties+and+potential+applications+in+foods+-+A+review.">Essential oils: Their antibacterial properties and potential applications in foods - A review.</a> Int. J. Food Microbiol. 94 (3), 223-253 (2004).</li><li>Ramanathan, L., Das, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+control+of+lipid+oxidation+in+ground+fish+by+some+polyphenolic+natural+products.">Studies on the control of lipid oxidation in ground fish by some polyphenolic natural products.</a> J. Agric. Food Chem. 40 (1), 17-21 (1992).</li><li>Yamazaki, K., Yamamoto, T., Kawai, Y., Inoue, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+antilisterial+activity+of+essential+oil+constituents+by+nisin+and+diglycerol+fatty+acid+ester.">Enhancement of antilisterial activity of essential oil constituents by nisin and diglycerol fatty acid ester.</a> Food Microbiol. 21 (3), 283-289 (2004).</li><li>Garc&#237;a-D&#237;ez, J., Alheiro, J., Falco, V., Fraqueza, M. J., Patarata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+activity+of+essential+oils+from+herbs+and+spices+used+on+meat+products+against+food+borne+pathogens.">Synergistic activity of essential oils from herbs and spices used on meat products against food borne pathogens.</a> Nat. Prod. Commun. 12 (2), 281-286 (2017).</li><li>Hussein Hamdy Roby, M., Atef Sarhan, M., Abdel-Hamed Selim, K., Ibrahim Khalel, K. <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+antioxidant+activity,+total+phenols+and+phenolic+compounds+in+thyme+(Thymus+vulgaris+L.),+sage+(Salvia+officinalis+L.),+and+marjoram+(Origanum+majorana+L.)+extracts.">Evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (Thymus vulgaris L.), sage (Salvia officinalis L.), and marjoram (Origanum majorana L.) extracts.</a> Ind. Crops Prod. 43, 827-831 (2013).</li><li>Gouveia, A. R., 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=The+Antimicrobial+Effect+of+Essential+Oils+Against+Listeria+monocytogenes+in+Sous+vide+Cook-Chill+Beef+during+Storage.">The Antimicrobial Effect of Essential Oils Against Listeria monocytogenes in Sous vide Cook-Chill Beef during Storage.</a> J. Food Process. Preserv. 41 (4), (2017).</li><li>Chen, C., Nace, G., Irwin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+6+x+6+drop+plate+method+for+simultaneous+colony+counting+and+MPN+enumeration+of+Campylobacter+jejuni,+Listeria+monocytogenes,+and+Escherichia+coli.">A 6 x 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli.</a> J. Microbiol. Methods. 55 (2), 475-479 (2003).</li><li>Herigstad, B., Hamilton, M., Heersink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+optimize+the+drop+plate+method+for+enumerating+bacteria.">How to optimize the drop plate method for enumerating bacteria.</a> J. Microbiol. Methods. 44 (2), 121-129 (2001).</li><li>. Practical food microbiology Available from: <a target="_blank" href="https://drive.google.com/file/d/0BzyVOLllJ0B1YmlEemZ5M1RZekU/view?ts=590d8019">https://drive.google.com/file/d/0BzyVOLllJ0B1YmlEemZ5M1RZekU/view?ts=590d8019</a> (2003)</li><li>Smith-Palmer, A., Stewart, J., Fyfe, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+properties+of+plant+essential+oils+and+essences+against+five+important+food-borne+pathogens.">Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens.</a> Lett. Appl. Microbiol. 26 (2), 118-122 (1998).</li><li>Burt, S. a., Reinders, R. 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Previous research has shown that microorganisms causing foodborne diseases survive drying10. It is therefore necessary to apply preservatives before drying to assure food safety. In this study, we focus on using TEO. The reason is twofold: First, there is a high demand from consumers to use natural products as alternative additives to improve food quality16; Second, a previous study demonstrated positive results after using OEO during the meat drying process<sup class="xref...

Drying as a traditional method to preserve foods has been used since ancient times. Nowadays, there is a growing interest in drying as an effective method for food preservation1,2,3. It is used to make a variety of specially processed meats. One of the most well-known is jerky.
Jerky, one of the oldest methods for meat preservation, is based on curing and drying to lower water activity and therefore to extend its shelf life4. Nowadays, jerky as a preserved cured meat is still very popular, where food safety, flavor, and texture are essential. Jerky preparation can be used for almost any type of meat, including beef, pork, poultry, or game5, and it requires chopping the meat in lean strips and drying it. Usually, marinating the meat in a curing solution or smoking are used along with drying to give the jerky its characteristic flavor6.
Despite the vast interest of drying to truly preserve food, the risk of foodborne outbreaks by E. coli from poorly dried meat is critical and needs to be controlled. There are some studies reporting foodborne gastroenteritis outbreaks particularly with E. coli O157:H7, attributed to inadequate heat processing during home-drying. Similar cases have occurred even in commercially prepared jerky7,8,9. Levine et al.10 proposed that foodborne microorganisms can survive moderate drying conditions (approximately 60 &#176;C) used by commercial jerky producers. E. coli O157:H7 outbreaks of foodborne diseases in the middle of the 1990s were attributed to ground dried meat products6,11. Interestingly, in all the previous cases, the main risk is caused by bacterial pathogens recognized as viable but non-culturable (VBNC). Under various stresses such as temperature changes or starvation, the E. coli cells could enter a particular state known as the VBNC state12,13. The VBNC cells may then be resuscitated back to culturable cells by exposure to suitable conditions and then present a threat to human health due to foodborne contamination14,15. This means that if the meat is consumed immediately after drying the product it is safe. However, in the case of inadequate storage, such as increased humidity, there is a high risk of reactivation of pathogens and microbial growth.
Besides drying and marinade methods, there is a high demand from consumers to use natural products as an alternative to additives to improve food quality16,17. There has been a particular interest in the application of natural food additives for meat instead of classical synthetic preservatives18,19,20,21. Even though there is a lack of sufficient experimental evidence in the use of essential oils when drying the meat, early research in this field already demonstrates positive results22,23.
Since the Middle Ages, people have recognized Essential Oil Compounds (EOCs) for their antimicrobial, insecticidal, and antiparasitic chracteristics24,25,26. Today, EOCs are part of one of the most important group of bioactive natural compounds. Among the different EOCs, thymol is one of the most well-known. It is composed of more than 85% of TEO23. This phenol prevents microbial and chemical deterioration when added to food. Additionally, its antibacterial properties might be improved in combination with other natural preservatives2,27,28,29,30. Nowadays, thyme (Thymus vulgaris), a herb that belongs to Lamiaceae family, has been recognized as a flavoring agent as well as a very effective meat preservative31. A study by Garc&#237;a-D&#237;ez et al.30 on meat products found that TEO displayed a wider inhibition pattern against foodborne pathogens when compared to other essential oils. Therefore, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process.
In this protocol, we present a novel method of applying TEO during meat drying, specifically using it in vapor form directly in a drying chamber. For evaluation, we use the MIC to determine the absence of pathogenic bacteria in treated samples compared with raw ones. The preliminary results show that this method is a highly effective alternative to synthetic preservatives and that it significantly reduces microbial load in dried meat.

<table>
	<tbody>
		<tr>
			<td>Meat cutter</td>
			<td>Kalorik</td>
			<td>KP 3530</td>
			<td>from Miami Gardens, FL, USA</td>
		</tr>
		<tr>
			<td>Laminar safety cabinet</td>
			<td>Faster s.r.l</td>
			<td></td>
			<td>from Italy</td>
		</tr>
		<tr>
			<td>Squeeze bottle of 500 mL</td>
			<td>Merci</td>
			<td>632 524 325 025</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Standard laboratory drier UFE 400</td>
			<td>Memmert</td>
			<td>DE 66812464</td>
			<td>from Germany</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>BT 120</td>
			<td>N/A</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Refrigerator and Freezer</td>
			<td>Bosch</td>
			<td>KGN34VW20G</td>
			<td>from DE</td>
		</tr>
		<tr>
			<td>Densitometer</td>
			<td>Biosan</td>
			<td>220 000 050 122</td>
			<td>Latvia; supplier Merci, CZ</td>
		</tr>
		<tr>
			<td>Escherichia coli ATCC 25922</td>
			<td>Oxoid</td>
			<td>CL7050</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Chromservis</td>
			<td>22008013</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Sterilized plastic tubes 15 mL</td>
			<td>Gama</td>
			<td>331 000 020 115</td>
			<td>from CZ, supplier Merci</td>
		</tr>
		<tr>
			<td>20 mL injection vial</td>
			<td>Healthy vial</td>
			<td>hvft169</td>
			<td>from China</td>
		</tr>
		<tr>
			<td>20 mm sterile butyl rubber stopper</td>
			<td>Merci</td>
			<td>22008013</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>20 mm aluminum cap</td>
			<td>Healthy vial</td>
			<td>N/A</td>
			<td>from China</td>
		</tr>
		<tr>
			<td>Thyme essential oil</td>
			<td>Sigma Aldrich</td>
			<td>W306509</td>
			<td>from St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>Mueller Hinton Broth</td>
			<td>Oxoid</td>
			<td>CM0337</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Penta</td>
			<td>16610-31000</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Oxoid</td>
			<td>LP0034</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Polysorbate 80 (Tween 80)</td>
			<td>Roth</td>
			<td>T 13502</td>
			<td>from DE, supplier P-lab</td>
		</tr>
		<tr>
			<td>Shaker SHO-1D</td>
			<td>Verkon</td>
			<td>DH.WSR04020</td>
			<td>from CZ,&#160; 10 - 300 rpm. 350 x 350 mm with a platform for flasks</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Bioferm</td>
			<td>N/A</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>MacConkey Agar</td>
			<td>Oxoid</td>
			<td>CM007</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Plate Count Agar</td>
			<td>Oxoid</td>
			<td>CM0325</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Merci</td>
			<td>480 622 080 040</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 250 mL</td>
			<td>Simax</td>
			<td>610 002 122 636</td>
			<td>from CZ; supplier Merci CZ</td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>Socorex</td>
			<td>S852820</td>
			<td>from Switzerland; supplier P lab, CZ</td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td>Gamma</td>
			<td>V400916</td>
			<td>CZ</td>
		</tr>
		<tr>
			<td>Microlitre pipette 100-1000 &#956;L</td>
			<td>Eppendorf</td>
			<td>333 120 000 062</td>
			<td>from Germany; supplier Merci, CZ</td>
		</tr>
	</tbody>
</table>

the effect of the application of thyme essential oil on microbial load during meat drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

We had first previously developed this method by using oregano essential oil (OEO) to enhance food safety and increase the value of dried meat. In general, the preceding experiments showed that E. coli goes into the VBNC state during drying as a survival strategy. This is demonstrated by the fact that there were no culturable bacteria after the drying finished22. Therefore, the pre-enrichment process for 6 h was necessary to allow the counting of the strai...

Watch this Scientific Journal Video about The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying at JoVE.com

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

The overall goal of this experiment is to evaluate if thyme essential oil, applied in vapor phase, can serve as effective preservative during meat-drying process. This method can help answer questions in the field of food processing and safety, such as how to effectively apply natural preservatives instead of synthetic ones. The main advantages of this technique is that enables fast and equal distribution of thyme essential oil while the meat dries and that all vapor can be used in lower volumes than in case of liquid oil application.The use of this method can provide insight into improving the safety and quality of dried meat and also can be applied for treatment of other food products against other unfavorable microorganisms. Generally, individuals new to this method will struggle because the drop plate methods used to count the number of the bacteria requires properly prepared plates and practice. Obtain a short loin of beef from a local butchery, and transfer it to the lab in a sealed bag at ambient temperatures within 20 minutes of purchase.In a Laminar safety cabinet, sterilize the outer surface of the beef muscle using a 10-second application of 70%ethanol, sprayed on from a squeeze bottle. Then, using sterile technique, cut out the outer surface of the muscle so that no ethanol remains on the preparation. Then pack the muscle into a sealed bag, and freeze it for one day at negative 18 degrees Celsius.The next day, thaw the frozen muscle at four degrees Celsius over six hours. Then, in a Laminar safety cabinet, use a meat cutter to make 0.5 centimeter thick slices. And further reduce the size of the slices into 5x2.5 square centimeter rectangular samples.For storage, pack the samples into plastic bags and keep them in the freezer. In a Laminar safety cabinet, prepare a standardized inoculum of E.coli ATCC 25922 to inoculate the meat samples. For the inoculation, arrange at least four samples on an aluminum foil sheet.Then load just as many samples on a second foil sheet to serve as uninoculated controls. Next, inoculate each sample by applying 400 microliters of inoculum onto one side and gently spreading it over the surface using a sterile cell-spreader. Then, let the samples dry for 10 minutes.Next, flip the samples over with tweezers, and repeat the procedure for a total of 800 microliters of inoculum per sample. After this, check the inoculation efficiency of two of the inoculated raw samples and two uninoculated controls. And the rest of the samples, wrap them in foil.Wash the raw samples in peptone water for 10 minutes at room temperature with 140 rpm of shaking. After the wash, on a Microtiter plate, make five 10-fold serial dilutions of the bathwater which now contains bacteria. Next, apply five microliter drops of each dilution by using a multichannel pipette onto plates with MacConkey agar and plate count agar using the 6x6 drop method.The drops should absorb quickly into the agar. For the cultivation of the dilutions by the 6x6 drop method, ensure that the plate count agar and MacConkey agar plates are appropriately dried so that the drops will absorb quickly into the agar. Then, culture the inoculated plates at 37 degrees Celsius for 24 hours.Prepare a meat drying oven for the application of thyme essential oil by vapor. Soak a 12x20 centimeter filter paper with the desired volume of TEO containing 79%thymol. Now take the samples left in the foil and transfer them together with the filter paper to the prepared drying oven with TEO to dry for six hours.In parallel, treat a control set of samples in a meat drying oven without TEO. After the drying, to assess the survival of E.coli on the bacteria-treated samples, repeat the same procedure of washing the samples in peptone water with one altercation. In this case, after the 10 minutes of shaking, transfer the samples in the bath to an incubator at 37 degrees Celsius for a six-hour pre-enrichment incubation.After the pre-enrichment, make five 10-fold dilutions of the bathwater as before. Then, use the 6x6 drop method to plant the dilutions on the detection plates and incubate the plates for 24 hours. The next day, determine the colony forming units per gram of meat.Examine the plates for the presence of mesophilic aerobic bacteria which appear as white spots on plate count agar, and count the typical E.coli colonies on the MacConkey agar which appear as red to dark pink. To analyze the microbiological data, convert the counts to the log of the CFU per gram and use an analysis of variants to determine the main affects of treatment. During the drying process, not all E.coli is eliminated.Some bacteria enter a viable but nonculturable state. So, when meat is stored improperly under high temperature and/or humidity, these bacteria can become viable again and potentially harm consumers. Treatment of the meat with different doses of TEO during drying could completely or partially eliminate E.coli.The number of mesophilic aerobic bacteria and enteric bacilli on meat samples was determined on PCA and MCA plates. When 1.5 milliliters of TEO was applied, no growth of bacteria was observed even after a six-hour pre-enrichment phase. This dose corresponds to 0.028 milliliter of TEO vapor per liter of air.After watching this video, you should have a good understanding of how to treat meat with vapor phase of essential oils during drying and evaluate the number of viable bacteria in the final product. In addition to this procedure, other methods like sensory evaluation and GC analysis can be performed in order to answer additional questions about consumer acceptability of the final product and amount of thyme essential oil residue. Don't forget that working with a meat cutter and pathogenic bacteria can be hazardous, and precautions such as wearing appropriate protective equipment and use of a Laminar safety cabinet should always be taken.

the-effect-application-thyme-essential-oil-on-microbial-load-during

Research

JoVE Journal

Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7.
T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 &#176;C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the&#160;tomato Solanum lycopersicum)11. Together, these&#160;model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed.

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A multidimensional gas chromatography method for the analysis of dissolved hydrogen sulfide in liquid crude oil samples is presented. A Deans switch is used to heart-cut light sulfur gases for separation on a secondary column and detection on a sulfur chemiluminescence detector.

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The system comprises a high-pressure rheometer which is connected to a circulation loop. The rheometer has a rotational flow-through measurement cell with two alternative geometries: coaxial cylinder and double gap. The circulation loop contains a mixer, to bring the crude oil sample into equilibrium with CO2, and a gear pump that transports the mixture from the mixer to the rheometer and recycles it back to the mixer. The CO2 and crude oil are brought to equilibrium by stirring and circulation and the rheology of the saturated mixture is measured by the rheometer. The system is used to measure the rheological properties of Zuata crude oil (and its toluene dilution) in equilibrium with CO2 at elevated pressures up to 220 bar and a temperature of 50 &#176;C. The results show that CO2 addition changes the oil rheology significantly, initially reducing the viscosity as the CO2 pressure is increased and then increasing the viscosity above a threshold pressure. The non-Newtonian response of the crude is also seen to change with the addition of CO2.

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The ...

Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope facilities. This protocol has been used to effectively evaluate many different bedding materials, environmental variables (temperature, humidity), and potential mitigation treatments that can improve air quality in commercial deep-bedded mono-slope facilities. The model is dynamic and allows researchers to easily collect many chemical and physical measurements from the bedded pack. Weekly measurements, collected over the course of six to seven weeks, allows sufficient time to see changes in air quality measurements over time as the bedded pack matures. The data collected from the simulated bedded packs is within the range of concentrations previously measured in commercial deep-bedded mono-slope facilities. Past studies have demonstrated that 8 - 10 experimental units per treatment are sufficient to detect statistical differences among the simulated bedded packs. The bedded packs are easy to maintain, requiring less than 10 minutes of labor per bedded packs per week to add urine, feces, and bedding. Sample collection using the gas sampling system requires 20 - 30 minutes per bedded pack, depending on the measurements that are being collected. The use of lab-scaled bedded packs allows the researcher to control variables such as temperature, humidity, and bedding source that are difficult or impossible to control in a research or commercial facility. While not a perfect simulation of &#34;real-world&#34; conditions, the simulated bedded packs serve as a good model for researchers to use to examine treatment differences among bedded packs. Several lab-scale studies can be conducted to eliminate possible treatments before trying them in a research or commercial-sized facility.

A protocol has been developed to measure gases, odors, and nutrient composition in lab-scaled bedded manure packs, which can be used to study ways to improve air quality in commercial cattle facilities using deep-bedded manure packs.

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...