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 per 1 cm2 of muscle surface.</li>
	<li>Remove the outer surface of the meat aseptically with a knife to avoid ethanol remaining in the muscle interior. Remove approximately 3 mm of the muscle interior to keep the muscle&#39;s surface uniformity.</li>
	<li>Package the muscle in a plastic sealed bag and transfer it to a freezer.</li>
	<li>Store the muscle at -18 &#176;C for 1 day. Then, thaw the frozen muscle at 4 &#176;C for 6 h. 
	NOTE: For thawing, it is recommended to move the muscle from the freezer to the fridge.</li>
	<li>In a laminar safety cabinet, slice each muscle into 0.5 cm thick slices with a meat cutter. Then, with a knife cut it into small 5 &#215; 2.5 cm2 rectangular samples.</li>
	<li>Package the rectangular meat samples in plastic bags and store them in the freezer at -18 &#176;C for later use.</li>
</ol>
2. Preparation of Standardized Inoculum and Inoculation Procedure in a Laminar Safety Cabinet
<ol>
	<li>Prepare standardized inoculum (1.5 &#215; 108 CFU/mL) of E. coli ATCC 25922 to inoculate the meat samples.
	<ol>
		<li>For the preparation of the stock inoculum, first dispense the lyophilized bacterial culture (delivered by the supplier) into a 15-mL sterilized tube pre-filled with 10 mL of sterilized Buffered Mueller Hinton Broth (BMHB). Cultivate this suspension for 24 h at 37 &#176;C.
		<ol>
			<li>Prepare the bacterial stock solution as follows: Take approximately 0.1 - 0.2 mL of bacterial suspension and dilute into a 20-mL vial closed with a rubber stopper with an aluminum cap pre-filled with 15 mL of sterilized BMHB. Cultivate this suspension for 24 h at 37 &#176;C.</li>
			<li>Store inside the refrigerator at 4 &#176;C for the preparation of the standardized inoculum.</li>
		</ol>
		</li>
		<li>From the stock solution (see step 2.1.1) of E coli take approximately 0.1 - 0.2 mL of bacterial suspension and dilute in a 15-mL plastic sterilized tube pre-filled with 10 mL of sterilized Buffered Mueller Hinton Broth (BMHB). Incubate the tube at 37 &#176;C for 24 h.</li>
		<li>For the preparation of the standardized inoculum (1.5 &#215; 108 CFU/mL), add small amounts of this suspension in a 15-mL sterilized tube pre-filled with 10 mL of sterilized BMHB.</li>
		<li>Thoroughly vortex the mixture and measure the optical density (OD) at 600 nm by a densitometer32.</li>
		<li>Repeat steps 2.1.3 - 2.1.4 until the OD expressed as the McFarland value is increased by 0.5 compared to the value of clean BMHB.</li>
	</ol>
	</li>
	<li>For the inoculation procedure, place the rectangular meat samples in two different aluminum foils (20 cm x 30 cm), one for the control samples and the second for the inoculated meat samples.
	<ol>
		<li>Over the second aluminum foil, inoculate the raw rectangular meat samples with 800 &#181;L of bacteria suspension of the selected strain (this corresponds to 1.2 &#215; 108 CFU per meat sample) by evenly distributing the inoculum on the surface.
		<ol>
			<li>Pipette 400 &#181;L on one side of the sample and gently spread using a sterile cell spreader on the surface. Let them dry for 10 min. Repeat the same procedure for the rest of the suspension on the other side of the sample.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Drying and TEO Application
<ol>
	<li>Transfer both aluminum foils containing the rectangular meat samples from the laminar safety cabinet to the dryer: cover each with aluminum foil, and then place the samples inside the dryer.</li>
	<li>Carry out the drying in a standard laboratory dryer. 
	NOTE: First, preheat the oven to 55 &#176;C. This procedure can last for 20 min.
	<ol>
		<li>Dry the control samples for 6 h at 55 &#176;C, with drying air relative humidity values ranging from 30 - 45%. 
		NOTE: Drying air relative humidity values vary in time depending on the rate of evaporation of liquid from the meat.</li>
	</ol>
	</li>
	<li>Calculate the volume of TEO applied, and express the essential oil concentration as a volume of TEO per dryer volume (mL/L air). For example, the dose of 1.5 mL of TEO in 53 L (volume of the dryer) results in a concentration of 0.028 mL/L air. To determine the MIC of TEO for E. coli, use doses of 1.5 mL (0.028 mL/L air), 1 mL (0.019 mL/L air), and 0.75 mL (0.014 mL/L air).</li>
	<li>Before drying, for the application of the TEO vapors (with thymol as the main compound 79%), soak a filter paper (12 cm x 20 cm) with a 1.5 mL dose of TEO and place into the dryer in front of the fan.</li>
	<li>Dry the TEO treated meat samples using the same procedure as for the control samples (steps 3.1 and 3.2). 
	NOTE: After the drying process ends and samples are removed, switch on the oven for 3 h at 80 &#176;C and set the air valve indication to 100% air vent in order to clean the essential oil residues from the oven.</li>
</ol>
4. Microbial Analysis
<ol>
	<li>Before the meat inoculation with bacteria, examine the meat samples for any adulteration. The appearance of slime and the detection of any strong and pungent smells are indicative of meat spoilage. If the texture feels slimy, the bacteria may have started to multiply on the surface of the meat.</li>
	<li>To assess the inoculation efficiency, test the raw inoculated samples for the presence of E. coli ATCC 25922 and compare them with non-inoculated control samples before the drying procedure. For this purpose:
	<ol>
		<li>Wash each meat sample (2 control samples and 2 inoculated samples). Suspend each meat sample in a sterilized flask with buffered peptone water (8.5 g of NaCl, 1 g of peptone, 5 tablets of phosphate-buffered saline, and 1 g of Polysorbate 80 in 1 L of water) in a ratio of 1:10 (w/v) with a pH range from 7 - 7.3. Shake using a shaker at 140 rpm for 10 min at room temperature. 
		NOTE: Wash immediately after the inoculation procedure.</li>
		<li>Evaluate the number of bacteria by an adjusted 6 &#215; 6 drop plate procedure summarized by Chen, Nace, and Irwin33 on Plate Count Agar (PCA) and MacConkey Agar (MCA). 
		NOTE: The 6 &#215; 6 drop method uses the broth micro dilution method to prepare 10-fold serial dilutions of the investigated sample with a multichannel pipette, which is less labor intensive and more economical compared to the conventional method33,34.</li>
		<li>Cultivate the 10-fold serial sample dilutions by the 6 &#215; 6 drop plate procedure for the evaluation of E. coli. 
		NOTE: Particularly for the 6 &#215; 6 drop method, for cultivation use six 5 &#181;L-drops, from six selected dilutions of the investigated sample with a multichannel pipette. On appropriately dried Petri dishes, the drops absorb quickly into the agar and the planting by this method is very convenient and manageable34.</li>
		<li>Incubate the Petri dishes at 37 &#176;C for 24 h. After the cultivation period, evaluate the number of colonies of E. coli on the Petri dishes (CFU g-1 of dried meat) as described in section 5.</li>
	</ol>
	</li>
	<li>After the drying, take two inoculated dried samples and compare them with two dried non-inoculated control samples for viable E. coli, respectively. To determine the presence or absence of E. coli of these four samples, carry out the pre-enrichment process of each meat sample as follows:
	<ol>
		<li>Suspend each meat sample in a sterilized flask with buffered peptone water (see step 4.2.1) and shake using a shaker at 140 rpm for 10 min at room temperature. Then incubate each flask at 37 &#176;C during 6 h for pre-enrichment.</li>
		<li>For the evaluation and cultivation of the bacteria, follow the same procedure as described in steps 4.2.2 - 4.2.4.</li>
	</ol>
	</li>
</ol>
5. Review Results
<ol>
	<li>After the incubation is complete, remove the Petri dishes from the incubator and review the results as follows:
	<ol>
		<li>To evaluate the total number of colonies, examine the plates for the presence of mesophilic aerobic bacteria on PCA (white spots) and typical E. coli colonies (red to dark pink) on MCA. If the pathogen is absent, both agars present no growth.</li>
		<li>Count the colonies and determine the amount of E. coli (CFU/g-1 of dried meat) present. 
		NOTE: Count the number of colonies (N) at two consecutive dilutions containing 30 or less colonies per drop (Figure 1). The number N of CFU/g-1 of dried meat is determined as follows35 
		<img alt="Equation" src="/files/ftp_upload/57054/57054eq1.jpg" /> 
		where, C is the sum of colonies on all drops counted, v is the volume of sample dilution used per drop (here, 0.05 mL), n1 is the number of drops used at the first dilution, n2 is the number of drops used at the second dilution, and d represents the dilution from which the first counts were captured.</li>
	</ol>
	</li>
	<li>To analyze the microbiological data, convert the number of colonies to log CFU g-1 and subject them to analysis of variance (ANOVA) for the main effects of treatment36.
	<ol>
		<li>Perform the Tukey Honest Significant Difference test (Tukey HSD) for multiple mean comparisons36 and determine the significant differences between treatments.</li>
	</ol>
	</li>
</ol>

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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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activity+of+selected+plant+essential+oils+against+Escherichia+coli+O157:H7.">Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7.</a> Lett. Appl. Microbiol. 36 (3), 162-167 (2003).</li></ol>

The authors have nothing to disclose.

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>&lt;em&gt;Escherichia coli&lt;/em&gt; 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,&amp;nbsp; 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 &amp;mu;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-effect-application-thyme-essential-oil-on-microbial-load-during

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

9.2K Views.  Czech University of Life Sciences Prague. 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.

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

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

Biochemistry

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Immunology and Infection

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized placebo-controlled crossover clinical evaluation, we assessed&#160;the effectiveness of a dedicated dietary supplement to improve chronic halitosis in 32 dogs of different breeds and ages. This protocol describes how to evalute the presence of oral volatile suphur compunds, e.g. methyl mercaptan, hydrogen sulfide and dimethyl sulfide, by means of a portable gas chromatograph device coupled with a syringe, which was used to collect the breath, and a dedicated software, which allows the operator to monitor each compound concentration during each measurement, in a relatively short time (8 min).
A significant modification of halitosis parameters was observed after 30 days since the beginning of treatment (p &#60;0.05), while a long-lasting effect was still observed even 20 days after the suspension of the treatment. Portable gas chromatograph, which is also widely used in clinical practice, can be therefore used to confirm&#160;and control halitosis in humans and animals. Even though human and animal species present some differences,&#160;this innovative and alternative therapy for halitosis management might be extended to human clinical practice as an adjuvant dietary approach.

We describe a simple approach for diagnosis of halitosis in dogs as well as a dietary approach for its management. This protocol may be extended to the management of halitosis in humans in the near future.

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

Medicine

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

Bioengineering

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biology

Antimicrobial Efficacy of Nano-Herbal-Coated Fabrics

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user&#39;s body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.

Author Spotlight: An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils  

Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...

Optimizing Spray-Dried Probiotic Product Quality Through Process Development

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

Author Spotlight: Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality  

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...

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Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality