The Project described is supported by Award Number 5R01AI116895 from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

1. Generating Samples&#160;
<ol>
	<li>Heat activate C. difficile spores at 65 &#176;C for 30 min and store on ice. 
	Note: C. difficile spores can be produced and purified as described previously7,9,10.</li>
	<li>Prepare the germination solution at X + 1 ml&#160;where X is the number of samples to be taken during the assay. 
	Note: The germination solution is specific to the species of bacteria spore being studied. For assaying C. difficile spore germination, we used a solution of 10 mM Tris (pH 7.5), 150 mM NaCl, 100 mM glycine and 10 mM taurocholic acid in deionized water.</li>
	<li>Before beginning the assay, draw a 1.0 ml sample of the germination solution without spores to serve as a blank for cortex fragment detection.</li>
	<li>Add spores to an OD600 of ~3.0 to the germination solution and vortex quickly. 
	Note: For our germination studies in B. subtilis, we used the same OD600 spores as for C. difficile. 
	Note: This amount of spores worked well for monitoring cortex fragment release during C. difficile spore germination. The amount of spores used in this assay should be determined experimentally for each bacterium or strain.</li>
	<li>Remove a 1.2 ml sample immediately after adding the spores and centrifuge at room temperature for 1 min at 14,000 &#215; g. This is the zero time point.</li>
	<li>Transfer 1.0 ml of the supernatant from the centrifuged sample to a fresh tube for subsequent cortex fragment analysis. 
	Note: Should DPA need to be monitored, remove a separate 100 &#956;l sample for the detection of DPA in the solution using an assay based on terbium fluorescence9,18,19.</li>
	<li>Freeze the collected sample at -80 &#176;C.</li>
	<li>Repeat steps 1.4 and 1.5 at regular intervals until all time points completed. 
	Note: Because of time required for centrifugation and sampling, time points less than 2 min apart were not possible during our procedure, so our sampling intervals were once every 2 min for 14 min.</li>
	<li>As a positive control for reducing sugar detection and quantification, include the following amounts of N-acetylglucosamine (nmol): 0, 12.5, 25, 50, 100, 250, 500, and 5,000. Prepare these samples at the same time as cortex samples and run along with the cortex samples so that the standard curve is relevant to the data generated by germinating spores.</li>
	<li>After all time point samples are collected, lyophilize the samples. 
	Note: We lyophilized our samples in 2 ml screw-cap tubes.</li>
</ol>
2. Measuring Cortex Fragments
<ol>
	<li>Resuspend lyophilized samples in 120 &#181;l of 3 N HCl supplemented with 1% phenol and 0.5% &#946;-mercaptoethanol.</li>
	<li>Transfer the resuspended samples to 2-ml screw-cap tubes. 
	Note: For reproducibility, ensure all the lyophilized sample is resuspended and transferred.</li>
	<li>Incubate the samples in a recirculating water bath at 95 &#176;C for 4 hr.</li>
	<li>After incubation, place the samples on ice until they are cool.</li>
	<li>Neutralize the samples with 120 &#181;l of 3 M NaOH.</li>
	<li>Add 80 &#181;l of a saturated sodium bicarbonate solution and 80 &#181;l of a 5% acetic anhydride solution to each sample and vortex.</li>
	<li>Incubate the samples at room temperature for 10 min.</li>
	<li>Transfer the samples back to the 95 &#176;C water bath for EXACTLY 3 min. 
	Note: This step is time sensitive and longer times can affect downstream signal development.</li>
	<li>Remove samples from the water bath and cool on ice.</li>
	<li>Add 400 &#181;l of 6.54% K2B4O7&#183;4H2O to each tube and vortex.</li>
	<li>Incubate the samples in a recirculating water bath at 95 &#176;C for EXACTLY 7 min. 
	Note: This step is time sensitive and longer times can affect downstream signal development.</li>
	<li>Remove the samples and place on ice for 5 min.</li>
</ol>
3. Color Reagent
<ol>
	<li>While samples are on ice (Step 2.12), prepare the color reagent. Dissolve 0.320 g of p-dimethylaminobenzaldehyde (DMAB; Ehrlich&#39;s reagent) in 1.9 ml of glacial acetic acid. 
	Note: The reagent is time, temperature and light sensitive and should not be made in advance.</li>
	<li>After the DMAB completely dissolves, add 100 &#181;l of 10 N HCl and vortex.</li>
	<li>Add 5 ml of glacial acetic acid and vortex.</li>
</ol>
4. Colorimetric Reaction
<ol>
	<li>Transfer 100 &#181;l of each cooled cortex sample to a new 1.5 ml microcentrifuge tube.</li>
	<li>Add 700 &#181;l of the freshly-prepared color solution to each sample.</li>
	<li>Immediately transfer the samples to a 37 &#176;C water bath and incubate for EXACTLY 20 min.</li>
	<li>Transfer 200 &#181;l of each sample to a clear 96-well plate.</li>
	<li>Quantify the samples at 585 nm on a plate reader. Samples should start at a yellow color and a positive reaction will shift to purple. The more cortex present, the deeper the purple color.</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genetics+of+bacterial+spore+germination.">The Genetics of bacterial spore germination.</a> Annu. Rev. Microbiol. 44, 531-553 (1990).</li><li>Zhang, P., Kong, L., Wang, G., Setlow, P., Li, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+Raman+tweezers+and+quantitative+differential+interference+contrast+microscopy+for+measurement+of+dynamics+and+heterogeneity+during+the+germination+of+individual+bacterial+spores.">Combination of Raman tweezers and quantitative differential interference contrast microscopy for measurement of dynamics and heterogeneity during the germination of individual bacterial spores.</a> Journal of biomedical optics. 15, 056010 (2010).</li><li>Ghuysen, J. -. M., Tipper, D. J., Strominger, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SleC+is+essential+for+cortex+peptidoglycan+hydrolysis+during+germination+of+spores+of+the+pathogenic+bacterium+Clostridium+perfringens.">SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens.</a> J Bacteriol. 191, 2711-2720 (2009).</li><li>Paredes-Sabja, D., Sarker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+cortex-lytic+enzyme+SleC+from+non-food-borne+Clostridium+perfringens+on+the+germination+properties+of+SleC-lacking+spores+of+a+food+poisoning+isolate.">Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate.</a> Can J Microbiol. 56, 952-958 (2010).</li><li>Hindle, A. A., Hall, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dipicolinic+acid+(DPA)+assay+revisited+and+appraised+for+spore+detection.">Dipicolinic acid (DPA) assay revisited and appraised for spore detection.</a> The Analyst. 124, 1599-1604 (1999).</li><li>Kort, 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=Assessment+of+heat+resistance+of+bacterial+spores+from+food+product+isolates+by+fluorescence+monitoring+of+dipicolinic+acid+release.">Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release.</a> Appl Environ Microbiol. 71, 3556-3564 (2005).</li><li>Elson, L. A., Morgan, W. 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Bacteriol. 192, 418-425 (2010).</li><li>Akoachere, 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=Indentification+of+an+in+vivo+inhibitor+of+Bacillus+anthracis+spore+germination.">Indentification of an in vivo inhibitor of Bacillus anthracis spore germination.</a> J. Biol. Chem. 282, 12112-12118 (2007).</li><li>Duncan, C. L., Strong, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+medium+for+sporulation+of+Clostridium+perfringens.">Improved medium for sporulation of Clostridium perfringens.</a> Appl Microbiol. 16, 82-89 (1968).</li><li>Heffron, J. D., Lambert, E. A., Sherry, N., Popham, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacillus+anthracis+SleL+(YaaH)+protein+is+an+N-acetylglucosaminidase+involved+in+spore+cortex+depolymerization.">The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization.</a> J Bacteriol. 190, 7601-7607 (2008).</li><li>Wang, S., Shen, A., Setlow, P., Li, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Dynamic+Germination+of+Individual+Clostridium+difficile+Spores+Using+Raman+Spectroscopy+and+Differential+Interference+Contrast+Microscopy.">Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy.</a> J Bacteriol. 197, 2361-2373 (2015).</li></ol>

The authors have nothing to disclose.

Upon stimulation, spores undergoing the process of germination lose their resistance properties without the need for macromolecular synthesis. When spore germination is triggered, the spore core releases DPA in exchange for water2. Due to the high DPA content of the dormant spore, the spore core is under intense osmotic pressure and the specialized peptidoglycan, cortex, helps prevent the core from swelling by acting, presumably, as a barrier to expansion2.
The method described above uses Ehrlich&#39;s reagent (a yellow solution) to detect the presence of reducing sugars as a measurable shift to a purple tint. During cortex hydrolysis, NAG and NAM sugars are liberated (depending on the extent of hydrolyzing enzymes, the sugars can be liberated as disaccharides or polysaccharides). Hydrolysis of the glycosidic linkages by HCl (Step 2.1) generates monosaccharides with free reducing ends7. After neutralizing the acid with an equal amount of sodium hydroxide (Step 2.5), the alcohols surrounding the NAG and NAM monosaccharides are acylated by acetic anhydride (Step 2.6)13. Subsequently, unreacted acetic anhydride is converted to acetic acid by heating the sample (Step 2.8). As hypothesized by Morgan and Elson20, the terminal aldehyde spontaneously converts to the enol form. Under alkaline conditions (Step 2.10), this enol form can react with the adjacent acetyl to generate an oxazole derivative which reacts with Ehrlich&#39;s reagent (Step 4.3) to generate a purple color15,20.
This method described above yields consistent, reliable results for the detection of cortex fragments from germinating spores, both B. subtilis and C. difficile9,16,17. The assay requires special attention to ensure the reaction times, temperatures and procedures are performed with precision. Without proper attention to detail, it is easy for the reaction conditions to fail or not reproduce between experiments13. However, the inclusion of a NAG standard aids in the application of the assay between laboratory environments. The NAG positive control standard included in every experiment controls for any inter-experimental variability (e.g., slight variations in reaction times or effectiveness of the color reagent). Reactions using only buffer conditions and reactions with spores incapable of hydrolyzing cortex both yielded no change in color.
The most common problem with this method is inter-experimental variability or complete lack of signal. To control for the variability, the spore preparations must be free of any vegetative cell debris. This cell debris will contain peptidoglycan fragments (NAG and NAM) and will result in a high background signal for the germinating spores. To ensure a pure spore preparation (&#62;99.9% spores), refer to the literature for methods to prepare pure spore suspensions in the organism to be studied7,9,21-23.
If the assay is not yielding any detectable signal, we observed that the lyophilizing step can introduce variability. Due to the force with which the vacuum is released, we observed that the dried sample powder can be blown out of the sample tube. To reduce/prevent this from occurring, use a small syringe needle to poke holes in the tops of sample tubes. The small area of the introduced holes will reduce the likelihood of the lyophilized sample (now in powder form) from being disturbed. Finally, as noted in the methods above, the several incubation steps are very time sensitive. Ensure that all timed steps are monitored carefully. An important consideration for this assay is that the cortex fragments must be released from the germinating spore in order to be detected. If the cortex is hydrolyzed into large fragments, these fragments may be too large to be released through the spore coat. Though an important consideration when troubleshooting an experiment that does not yield a signal, we recommend to confirm that the conditions used to germinate most of the spores in the solution and the other troubleshooting options listed here prior to considering cortex fragment size as a potential problem in the assay.
Not all germination buffers/conditions are suitable for this assay. Because this assay detects the presence of reducing sugars liberated during the germination process13, any germination buffer that contains reducing sugars or any germinant that is, itself, a reducing sugar (e.g., glucose) will result in a strong signal in this assay. If this is the case, high pressure liquid chromatography (HPLC) can be used to detect the presence of cortex fragments24-26. However, because not all laboratories are equipped with such instrumentation, the method described herein can be used by most laboratories and offers simple and easily quantifiable results.
Germination by bacterial spores has largely been studied in Bacillus and a few Clostridium species2,8. The process is generally conserved, however differences exist. Importantly, not all organisms germinate using mechanisms of spore germination as observed in the model spore-formers9,27. Because germination is becoming studied in more diverse organisms, the assay described above can be applied to these organisms to detect cortex fragments during spore germination. By applying this method to these newly studied organisms, researchers can determine the role various germination proteins play in cortex hydrolysis.

Endospores are metabolically dormant forms of bacteria that allow bacteria to persist in unfavorable environments. In many spore-forming bacteria, spore formation is induced by nutrient deprivation but this process can be controlled by changes to pH, exposure to oxygen or other stresses1. While in their metabolically dormant spore form, bacteria resist UV radiation, desiccation, higher temperatures, and freezing2. Most of the knowledge on the sporulation process comes from studies in the model organism, Bacillus subtilis. The sporulation process begins with DNA replication and the formation of an axial filament3,4. An asymmetric septum then divides the cell into two unequally sized compartments. The larger compartment, the mother cell, engulfs the smaller compartment, the forespore. Both the mother cell and forespore coordinate gene expression to mature the spore and the mother cell eventually lyses, releasing the dormant spore into the surrounding environment1.
The spore structure and composition is conserved across many bacterial species. The spore core has a lower water content than the vegetative cell and is rich with dipicolinic acid (DPA)1,2. During spore formation, DPA is pumped into the spore core in exchange for water. Surrounding the core is an inner spore membrane where, in most spore forming bacteria, the receptors which recognize the small molecules that stimulate germination (germinants) are located2. Located just outside the spore&#39;s inner membrane is a layer of cell wall peptidoglycan. A specialized peptidoglycan layer (cortex) surrounds the cell wall peptidoglycan and is composed of many of the same components as cell wall peptidoglycan [alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)]. However, approximately 50% of the NAM residues in the cortex have been converted to muramic-&#948;-lactam5,6. During spore germination, these muramic-&#948;-lactam residues are recognized by the spore cortex lytic enzymes (SCLEs), thus allowing the cortex to be degraded (a process essential to complete germination) but not the cell wall. Surrounding the cortex is an outer membrane and several layers of coat proteins 2.
Controlling the process of germination is critically important for spore-forming bacteria. Germination is initiated when germinant receptors interact with their respective germinants2. In many spore-forming bacteria, these germinants are amino acids, sugars, nucleotides, ions or combinations thereof2. C. difficile spore germination is initiated by combinations of certain bile acids, [e.g., taurocholic acid (TA)] and amino acids (e.g., glycine)7-10. Though there are differences between the C. difficile germination pathway and the pathways studied in other spore-forming bacteria, such as B. subtilis, common to all is the absolute requirement to degrade the spore cortex to allow a vegetative cell to grow from the germinated spore2,8. Cortex degradation can be accomplished by the SCLEs CwlJ/SleB (as found in B. subtilis) or SleC (as found in many Clostridia). Cortex hydrolysis reduces the constraint on the cell wall and the spore. This allows for full core rehydration, a necessary step in reactivating many of the proteins necessary for cellular metabolism 2.
When spores germinate, the dormant spore changes from a phase-bright state to a phase-dark state and this process can be measured by a change in optical density (OD) at 600 nm11. A previous report suggests that much of this change in OD is due to the release of DPA from the spore12. During our recent study, we sought to compare the timing of C. difficile spore germination and monitored the release of DPA and cortex fragments9. For this study it was critical to monitor the release of cortex fragments as they began to be released by germinating spores.
The colorimetric assay used here was based upon a method for detecting sugars with reducing ends developed Ghuysen et al.13. Because others have described protocols for the detection of reducing sugars14 or have modified those protocols15, the literature on this subject can be confusing. Here, we detail a step-by-step method for the colorimetric detection of reducing sugars liberated from germinating C. difficile spores. Though this study uses C. difficile spores, the reducing sugars released during the germination of spores from other spore-forming bacteria are able to be detected with this protocol9,16,17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 mL screw cap tube</td>
			<td>USA scientific</td>
			<td>1420-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p20 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-1250uL pipet tips</td>
			<td>VWR</td>
			<td>89079-486</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200uL pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-D-glucosamine</td>
			<td>Sigma</td>
			<td>A3286-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid - 10N</td>
			<td>BDH-Aristar</td>
			<td>BDH3032-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Alfa Aesar</td>
			<td>L04295</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>BDH</td>
			<td>BDH0280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Tetraborate tetrahydrate</td>
			<td>Alfa Aesar</td>
			<td>39435</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>BDH8019-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma</td>
			<td>156477-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturated Phenol</td>
			<td>Fisher</td>
			<td>BP1750-400</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M3</td>
			<td colspan="2">Molecular Devices</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>BDH</td>
			<td>BDH3098-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated, Circulating Water Bath</td>
			<td>VWR Scientific</td>
			<td>Model 1136</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtest 96</td>
			<td>Falcon</td>
			<td>353072</td>
			<td>96 well clear tissue culture plates</td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>VWR</td>
			<td>89000-506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Blocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Table 1 shows typical results using NAG standards. Data are used to generate a standard curve. Table 2 shows typical results from a germination assay in germination buffer supplemented with 100 mM glycine and 10 mM TA (germination-promoting conditions) or 100 mM glycine only (non-germinating conditions). In the absence of TA, C. difficile spores do not germinate and there is little change in the presence of cortex fragments in the solution. However, in the presence of both TA and glycine, C. difficile spores germinate and release cortex fragments into the solution. This is observed as an increase in the OD585 over time of the reacted samples. The total amount of cortex fragments (nmol) released during this assay was calculated using a standard curve similar to Figure 1B. In this assay, most of the cortex is hydrolyzed (or released) by 15 minutes post-germinant exposure, suggesting that most of the spores have germinated in this time frame (Figure 2; consistent with a previous publication7).
After developing the samples in the color reagent, most samples may not show the purple color to the naked eye. Figure 1 shows a representative sample generated from N-acetylglucosamine. Many of the samples do not show a visible purple color. However, the 250 nmol, 500 nmol and 5,000 nmol samples appear visually different from the negative control (0 nmol). When measured at OD585 nm, the signal generated best fits a linear regression (R2 = 0.999).
<img alt="Figure 1" src="/files/ftp_upload/54146/54146fig1.jpg" /> 
Figure 1: Detecting N-acetylglucosamine. (A) N-acetylglucosamine samples (0 nmol, 12.5 nmol, 25 nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, 5,000 nmol) were lyophilized and reacted according to the described procedure. (B) Samples were added to a 96-well plate and the amount of reacted material detected at 585 nm. The signal was used to generate a standard curve. <img alt="Figure 2" src="/files/ftp_upload/54146/54146fig2.jpg" /> 
Figure 2: Graphical Representation of Germination Results. The calculated nmol cortex in Table 2 was plotted vs. time. (&#9679;) 100 mM glycine + 10 mM TA; (&#9632;) 100 mM glycine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54146/54146fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NAG Concentration (nmol)</td>
			<td>OD585</td>
		</tr>
		<tr>
			<td>0</td>
			<td>0.047</td>
		</tr>
		<tr>
			<td>12.5</td>
			<td>0.054</td>
		</tr>
		<tr>
			<td>25</td>
			<td>0.066</td>
		</tr>
		<tr>
			<td>50</td>
			<td>0.084</td>
		</tr>
		<tr>
			<td>100</td>
			<td>0.128</td>
		</tr>
		<tr>
			<td>250</td>
			<td>0.285</td>
		</tr>
		<tr>
			<td>500</td>
			<td>0.556</td>
		</tr>
		<tr>
			<td>5,000</td>
			<td>4.0</td>
		</tr>
		<tr>
			<td>blank =</td>
			<td>0.046</td>
		</tr>
	</tbody>
</table>
Table 1: NAG Standard. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Clostridium difficile Spore Germination</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">Germination Solution (10 mM Tris (pH 7.5), 150 mM NaCl)</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">100 mM Glycine + 10 mM TA</td>
			<td colspan="2">100 mM Glycine&#160;</td>
		</tr>
		<tr>
			<td>Time (minutes)</td>
			<td>OD585</td>
			<td>Calculated (nmol)</td>
			<td>OD585</td>
			<td>Calculated (nmol)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>0.046</td>
			<td>0.00</td>
			<td>0.047</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.048</td>
			<td>2.54</td>
			<td>0.048</td>
			<td>1.27</td>
		</tr>
		<tr>
			<td>4</td>
			<td>0.05</td>
			<td>5.07</td>
			<td>0.049</td>
			<td>2.54</td>
		</tr>
		<tr>
			<td>6</td>
			<td>0.055</td>
			<td>11.41</td>
			<td>0.05</td>
			<td>3.80</td>
		</tr>
		<tr>
			<td>8</td>
			<td>0.059</td>
			<td>16.49</td>
			<td>0.05</td>
			<td>3.80</td>
		</tr>
		<tr>
			<td>10</td>
			<td>0.06</td>
			<td>17.76</td>
			<td>0.051</td>
			<td>5.07</td>
		</tr>
		<tr>
			<td>12</td>
			<td>0.061</td>
			<td>19.02</td>
			<td>0.05</td>
			<td>3.80</td>
		</tr>
		<tr>
			<td>14</td>
			<td>0.063</td>
			<td>21.56</td>
			<td>0.051</td>
			<td>5.07</td>
		</tr>
	</tbody>
</table>
Table 2: Sample Spore Germination Results.

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.3K Views.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Video: Detecting Cortex Fragments During Bacterial Spore Germination

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...