We would like to thank Coy Laboratories for kindly providing pictures of the anaerobic chamber. This work was supported by National Institutes of Health grant DK087763 (S.M. M.) and a STEP/HHMI Curriculum Development Fellowship (A.N. E.).

Note: C. difficile is a human and animal pathogen that can cause gastrointestinal disease. Experiments involving C. difficile must be performed with appropriate biosafety precautions (BSL-2).
1. Anaerobic Chamber Use and Maintenance
C. difficile is a strict anaerobe and is extremely sensitive to even low concentrations of oxygen in the atmosphere. Therefore, a controlled, anaerobic environment is needed for its successful manipulation. The use of an anaerobic chamber (Figure 1A) provides the most stable environment and the ideal conditions for effective cultivation of C. difficile and other anaerobic bacteria14. Here, an atmosphere containing a gas mixture (5% CO2, 10% H2, 85% N2) can be stably maintained.
To introduce any items into the chamber without significant oxygen contamination, an airlock must be used (Figure 1B). This device functions as a gas interchange and works automatically, semi-manually or manually. The airlock has two doors: one providing access to the outside of the airlock and the other providing access to the interior of the anaerobic chamber. Depending on the model, the airlock may have an additional door, which can offer access to an adjoining anaerobic chamber, thus saving the cost of a separate airlock. Unless actively moving items in or out of the chamber, both doors should remain closed at all times to avoid oxygen contamination. The airlock possesses an on/off switch on the front and a panel containing four buttons. The gas lines and vacuum pump hose are connected in the rear of the airlock, near the panel where the switches for full manual operation of the unit are located. It is recommended that two purge cycles, using nitrogen gas (N2), are used before filling the interchange with gas mix (5% CO2, 10% H2, 85% N2) to reduce the amount of oxygen introduced into the chamber. It is also important to never leave either door open for longer than the amount time required to move items in and out of the interchange and to carefully plan experiments to reduce the frequency of moving items in and out of the chamber. The different operating procedures for vinyl anaerobic chambers are outlined below. Before following these protocols, ensure that these are compatible with the manufacturer&#8217;s instructions provided with the chamber.
<ol>
	<li>Automatic Mode 
	This is a programmable and customizable mode and is performed entirely by the airlock. To program the airlock, refer to the manufacturer&#8217;s instructions. A recommended program for typical entry into the chamber involves two purge cycles using nitrogen gas followed by refilling the airlock with a gas mix that closely matches the atmosphere maintained within the chamber. During each vacuum cycle, the standard factory procedures suggest pulling a vacuum to 20 inHg and purging to 1 inHg.
	<ol>
		<li>Ensure that the inner airlock door is fully closed.</li>
		<li>Open the outer airlock door.</li>
		<li>Place items into airlock and close the outer airlock door. If introducing liquids, unscrew the caps halfway to ensure efficient gas exchange. Sealed packages and containers should be opened prior to beginning the cycle; refraining from doing so may warp and damage plasticware and containers. To maintain sterility, open the packages enough to only allow efficient gas exchange.</li>
		<li>Press the Start button.</li>
		<li>Wait until the airlock has cycled through the program and the display reads &#34;Anaerobic.&#34;</li>
		<li>Open inner airlock door.</li>
		<li>Move items into the chamber.</li>
		<li>Close inner door.</li>
	</ol>
	</li>
	<li>Semi-manual Mode 
	This mode may be used when moving items in or out of the chamber and is necessary when cycling the gas within the chamber is required.
	<ol>
		<li>Ensure that the inner airlock door is fully closed.</li>
		<li>Open the outer airlock door.</li>
		<li>Place items into airlock and close the outer airlock door. If introducing liquids, unscrew the caps halfway to ensure efficient gas exchange. Sealed packages, such as inoculating loops and 96-well plates, must be opened prior to beginning the cycle.</li>
		<li>Press Menu.</li>
		<li>Press Start. The airlock will display the function of each button and will not respond until this is completed:
		<ul>
			<li>Up arrow: activates the vacuum pump.</li>
			<li>Down arrow: initiates the nitrogen (purge) gas flow.</li>
			<li>Start: initiates the gas mix flow.</li>
			<li>Menu: return to the default display.</li>
		</ul>
		</li>
		<li>Press &#34;Up,&#34; removing gas from the airlock, until the display reads 20 inHg.</li>
		<li>Press &#34;Down,&#34; filling the airlock with nitrogen, until the display reads less than 1 inHg. Do not overshoot, as this will create positive pressure within the airlock, which may affect its integrity.</li>
		<li>Repeat steps 6 and 7 to perform an additional purge cycle.</li>
		<li>Press &#34;Up&#34; until the display reads 20 inHg.</li>
		<li>Press &#34;Start,&#34; filling the interchange with gas mix, until the display reads less than 1 inHg.</li>
		<li>Open inner airlock door.</li>
		<li>Move items into the chamber.</li>
		<li>Close inner door.</li>
	</ol>
	</li>
	<li>Manual Mode 
	This mode may be used whenever the automatic or semi-manual modes are not working or there is no power to the airlock. This mode does not function when the airlock is on; therefore, it is difficult to determine the pressure within the airlock. Because the vacuum pull and gas purge times is dependent upon vacuum and gas flow rates, respectively, the use of a pressure gauge within the interchange is required to monitor the pressure and reduce the risk of personal injury and damage to the airlock.
	<ol>
		<li>Ensure that the inner airlock door is fully closed.</li>
		<li>Open the outer airlock door.</li>
		<li>Place items, including the pressure gauge, into airlock and close the outer airlock door. If introducing liquids, unscrew the caps halfway to ensure efficient gas exchange. Sealed packages, such as inoculating loops and 96-well plates, must be opened prior to beginning the cycle.</li>
		<li>Locate the three switches on the back of the airlock labeled &#34;Manual Control Switches.&#34; These are individually labeled as:
		<ul>
			<li>To Vacuum</li>
			<li>Nitrogen</li>
			<li>Gas Mix</li>
		</ul>
		</li>
		<li>Flip the To Vacuum toggle switch until the pressure gauge reads approximately 20 inHg.</li>
		<li>Flip the Nitrogen toggle switch until the pressure gauge reads approximately 1 inHg.</li>
		<li>Flip the To Vacuum toggle switch until the pressure gauge reads approximately 20 inHg.</li>
		<li>Flip the Nitrogen toggle switch until the pressure gauge reads approximately 1 inHg.</li>
		<li>Flip the To Vacuum toggle switch until the pressure gauge reads approximately 20 inHg.</li>
		<li>Flip the Gas Mix toggle switch until the pressure gauge reads approximately 1 inHg.</li>
		<li>Open inner airlock door.</li>
		<li>Move items into the chamber.</li>
		<li>Close inner door.</li>
	</ol>
	</li>
</ol>
2. Culturing, Enumerating and Storing C. difficile from Stool Samples
This procedure is designed to recover C. difficile from fecal samples containing spores and subsequently maintain isolated colonies as vegetative cells or spores in long-term storage as glycerol stocks. Alternatively, this procedure may be used to enumerate the number of C. difficile present in stool samples (e.g. from animal studies). To selectively and differentially enrich for C. difficile, taurocholate-cefoxitin-cycloserine-fructose agar (TCCFA) is used to inhibit growth of normal fecal flora15-16. Cycloserine is bacteriostatic for Gram-negative bacteria, while cefoxitin more broadly inhibits growth of both Gram-negative and -positive bacteria, with the exception of C. difficile and most enter cocci strains. A pH indicator, neutral red, can be included in the medium, as the fermentation of fructose will result in a decrease in pH and a subsequent color change from red/orange to yellow. To efficiently recover spores of C. difficile as vegetative bacteria, the bile salt sodium taurocholate is used to induce germination17-18. Because C. difficile forms spores, alcohol or heat treatment of samples may be used to reduce or eliminate vegetative cells, limiting the growth of contaminating flora, which may increase the efficiency of C. difficile recovery19. As mentioned above, it is critical to pre-reduce all plates for at least 1-2 hr in the anaerobic chamber prior to use to ensure the removal of residual oxygen. Air drying the plates prior to use in the chamber can reduce condensation. Liquid medium may need up to 24 hr to reduce depending on the volume and the surface-to-air ratio of the container used.
Before beginning, the following items should be placed into the anaerobic chamber:
<ul>
 <li>Stool sample</li>
	<li>Sterile swabs</li>
	<li>Sterile inoculating loops</li>
	<li>1x PBS (see Materials)</li>
	<li>TCCFA plates (see Materials)</li>
	<li>BHIS (Brain Heart Infusion medium with yeast extract) plates and broth (see Materials)</li>
	<li>50% glycerol (sterilized)</li>
	<li>10% L-cysteine (sterilized)</li>
	<li>Cryogenic storage vials</li>
</ul>
*Alternatively, isolated colonies can be spread onto BHIS agar plates, and subsequently scraped off and resuspended in BHIS liquid medium with 15% glycerol for long-term storage at -80&#176;C. **It is important to note that Gram-staining is not an effective strategy to identify C. difficile directly from stool samples23; C. difficile must first be isolated from the other flora present in the stool.
<ol>
	<li>Resuspend the stool sample in 1x PBS (this may be performed in aerobic conditions), and ensure that the stool sample is fully resuspended in 1x PBS by vortexing. If enumerating C. difficile from the stool, weigh the stool sample prior to the addition of 1x PBS.</li>
	<li>Make serial dilutions of the resuspended stool sample in 1x PBS for appropriate isolation or enumeration of colony forming units (CFU) per gram of stool.</li>
	<li>Using aseptic technique, apply 100 &#956;l of each serial dilution to TCCFA plates.</li>
	<li>Using a series of sterile inoculating loops, streak the applied culture for isolated colonies or evenly spread the applied culture on the surface of the TCCFA plate for enumeration of colonies.</li>
	<li>Incubate the plate anaerobically for 48 hr at 37 &#176;C. It is possible to detect and identify C. difficile colonies within 24 hr based on the flat, irregular, ground-glass appearance of the colonies15, although more accurate identification and enumeration is achieved after 48 hr.</li>
	<li>Using sterile inoculating loops, subculture any colonies that appear to be C. difficile onto pre-reduced BHIS agar plates, supplemented with 0.03% L-cysteine20. Pick an individual colony, and streak onto the plate in quadrants, using a new sterile inoculating loop for each quadrant, to obtain isolated colonies. Alternatively, count the C. difficile colonies of each dilution (this may be performed in aerobic conditions), and calculate the number of colony forming units per gram of stool sample.</li>
	<li>Confirm C. difficile identification via Gram-staining. After Gram-staining, C. difficile will appear as purple rods and some cells may contain terminal endospores. Polymerase chain reaction (PCR) identification of the genes that encode toxin B may provide further confirmation of toxigenic strains of C. difficile21 while multilocus sequence typing (MLST) is successful for identification and accurate typing of unknown and nontoxigenic strains of C. difficile22.</li>
	<li>To maintain a stock of C. difficile at -80 &#176;C, pick an individual, isolated colony from the BHIS agar plate using a sterile inoculating loop and resuspend the colony in 10 ml of pre-reduced BHIS liquid medium supplemented with 0.03% L-cysteine.*</li>
	<li>Incubate overnight anaerobically at 37 &#176;C, or until the culture becomes turbid.</li>
	<li>Add 333 &#956;l of 50% glycerol and 666 &#956;l of the C. difficile culture to a 1.8 ml cryogenic tube to create a 15% glycerol stock of the isolated strain.</li>
	<li>Tightly cap the cryogenic tube, mix well and immediately remove the stock from the anaerobic chamber and place in a -80 &#176;C freezer for long-term storage.</li>
</ol>
3. Culturing C. difficile from Frozen Glycerol Stocks
This procedure provides for recovery of C. difficile from glycerol stocks stored at -80 &#176;C. Because repeated freeze-thaw cycles may kill vegetative cells, it is important to keep the glycerol stocks frozen at all times. We do not recommend the use of dry ice for transferring strains in and out of the chamber as the evaporation of dry ice can change the environment within the chamber. Instead, we recommend using frozen cooling racks to keep glycerol stocks frozen during transport. For various selective and differential purposes, three media are commonly used for culturing C. difficile. TCCFA, as discussed above, is selective for C. difficile and contains sodium taurocholate, a germinant. Brain heart infusion supplemented with yeast extract (BHIS) is a commonly used, enriched, non-selective medium which allows for the growth of a wide variety of organisms (Figure 2A)24. Frequently, L-cysteine is added to BHIS as a reducing agent20. Finally, the addition of blood to the medium (Figure 2B) allows for more efficient sporulation than on TCCFA and provides for detection of the unique greenish or chartreuse fluorescence exhibited by C. difficile under long-wave ultraviolet (UV) light15 (Figure 2C). When culturing C. difficile from spore stocks, it is critical to remember that sodium taurocholate must be added to the medium to ensure germination.
Before beginning, the following items should be placed into the anaerobic chamber:
<ul>
	<li>Frozen glycerol stock (in cooling rack)</li>
	<li>Sterile inoculating loops</li>
	<li>TCCFA or BHIS plates (see Materials)</li>
	<li>BHIS broth (see Materials)</li>
	<li>50% glycerol (sterilized)</li>
	<li>Cryogenic storage vials</li>
</ul>
<ol>
	<li>Place the frozen glycerol stock of C. difficile in a cooling rack that has been stored at -80 &#176;C prior to use to prevent thawing.</li>
	<li>Bring the frozen glycerol stock on dry ice into the anaerobic chamber.</li>
	<li>Using aseptic technique and a sterile inoculating loop, place a small amount of the stock onto the plate and streak across one quadrant of the plate.</li>
	<li>Rotate the plate 90&#176; and, using a new sterile inoculating loop, continue to streak across the second quadrant.</li>
	<li>Repeat for the third and fourth quadrants to ensure the isolation of individual colonies.</li>
	<li>Immediately remove the frozen glycerol stock from the anaerobic chamber and return to the -80 &#176;C.</li>
	<li>Incubate the plate anaerobically overnight at 37 &#176;C. Individual isolated colonies should be observed after overnight growth.</li>
</ol>
4. Purifying Spores from C. difficile
As sporulation is required for survival in oxygen-rich environments and for efficient transmission of disease8, the preparation of spore stocks is often necessary for downstream applications, not limited to microscopy and animals studies. It is important to note that enumerating spores requires repetition to ensure reproducibility of counts. Pipetting up and down several times between dilutions also reduces loss since spores adhere to plastic well.
Sporulation of C. difficile is not as rapid or homogeneous as other sporogenic species. To optimize spore production and recovery, either sporulation medium (SMC)17,25 or 70:30 medium26 is recommended. Other media commonly used are BHIS, which requires 4-5 days of growth before efficient sporulation is seen27, and Clospore, a liquid medium that produces high titers of spores (107 - 108 spores per milliliter) after 72 hr of growth. Other protocols use ice-cold water rather than 1x PBS20; however, the use of an isotonic solution can reduce spores from sticking to each other and plastic surfaces. Alternatively, some investigators further purify their spores using a sucrose gradient to fully remove vegetative cells and debris29.
Before beginning, the following items should be placed into the anaerobic chamber:
<ul>
	<li>Sterile inoculating loops</li>
	<li>BHIS, SMC and/or 70:30 plates (see Materials)</li>
	<li>BHIS broth (see Materials)</li>
	<li>1x PBS, filter-sterilized (see Materials)</li>
</ul>
<ol>
	<li>Culture strains from frozen glycerol stock onto pre-reduced BHIS plates and incubate anaerobically overnight at 37 &#176;C.</li>
	<li>Restreak onto several pre-reduced SMC or 70:30 plates and incubate anaerobically at 37 &#176;C for 24-48 hours. Spore formation can be followed via phase contrast microscopy. Spores will appear phase bright while vegetative and mother cells will appear phase dark. *As an alternative, spores can be purified from 70:30 liquid medium after 24-48 hours, which yields 105-106 spores per milliliter, depending on the strain used.</li>
	<li>Using a sterile inoculating loop, scrape plates and resuspend the cells in 10 ml sterile 1x PBS.</li>
	<li>Discard plates and remove spore suspension from the anaerobic chamber. Pellet the cells at 3,000 x g for 15 minutes. Wash the cells twice in 1x PBS, fully resuspending the cell pellet each time.</li>
	<li>Incubate overnight at 4 &#176;C to aid in lysis of vegetative and mother cells.</li>
	<li>Incubate at 70 &#176;C for 20 min to kill any residual vegetative cells.</li>
	<li>To determine colony forming units (CFU) per milliliter, serially dilute each spore preparation in 1x PBS and plate on BHIS + 0.1% sodium taurocholate. Incubate plates for a minimum of 24 hr before enumerating colonies.</li>
	<li>Spores can be stored in 1x PBS at either room temperature or 4&#176;C for long-term storage. If stored at 4 &#176;C, it may be useful to reheat the spore preparation at 55 &#176;C for 15 min to restore efficient germination.</li>
</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=Genetic+manipulation+of+Clostridium+difficile.">Genetic manipulation of Clostridium difficile.</a> Curr. Protoc. Microbiol. Chapter 9, Unit 9A 2 (2011).</li><li>Lemee, L., Pons, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilocus+sequence+typing+for+Clostridium+difficile.">Multilocus sequence typing for Clostridium difficile.</a> Methods. Mol. Biol. 646, 77-90 (2010).</li><li>Shanholtzer, C. J., Peterson, L. R., Olson, M. N., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+study+of+gram-stained+stool+smears+in+diagnosis+of+Clostridium+difficile+colitis.">Prospective study of gram-stained stool smears in diagnosis of Clostridium difficile colitis.</a> J. Clin. Microbiol. 17, 906-908 (1983).</li><li>Smith, C. J., Markowitz, S. M., Macrina, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferable+tetracycline+resistance+in+Clostridium+difficile.">Transferable tetracycline resistance in Clostridium difficile.</a> Antimicrob. Agents Chemother. 19, 997-1003 (1981).</li><li>Permpoonpattana, P., 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=Surface+layers+of+Clostridium+difficile+endospores.">Surface layers of Clostridium difficile endospores.</a> J. Bacteriol. 193, 6461-6470 (2011).</li><li>Putnam, E. E., Nock, A. M., Lawley, T. D., Shen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpoIVA+and+SipL+are+Clostridium+difficile+spore+morphogenetic+proteins.">SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins.</a> J. Bacteriol. , (2013).</li><li>Burns, D. A., Minton, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+studies+in+Clostridium+difficile.">Sporulation studies in Clostridium difficile.</a> J. Microbiol. Methods. 87, 133-138 (2011).</li><li>Perez, J., Springthorpe, V. S., Sattar, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clospore:+a+liquid+medium+for+producing+high+titers+of+semi-purified+spores+of+Clostridium+difficile.">Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile.</a> J. AOAC Int. 94, 618-626 (2011).</li><li>Sorg, J. A., Sonenshein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+the+initiation+of+Clostridium+difficile+spore+germination+using+analogs+of+chenodeoxycholic+acid,+a+bile+acid.">Inhibiting the initiation of Clostridium difficile spore germination using analogs of chenodeoxycholic acid, a bile acid.</a> J. Bacteriol. 192, 4983-4990 (2010).</li><li>O'Connor, J. 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=Construction+and+analysis+of+chromosomal+Clostridium+difficile+mutants.">Construction and analysis of chromosomal Clostridium difficile mutants.</a> Mol. Microbiol. 61, 1335-1351 (2006).</li><li>Buggy, B. P., Wilson, K. H., Fekety, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methods+for+recovery+of+Clostridium+difficile+from+an+environmental+surface.">Comparison of methods for recovery of Clostridium difficile from an environmental surface.</a> J. Clin. Microbiol. 18, 348-352 (1983).</li><li>Koch, C. J., Kruuv, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+release+of+oxygen+from+polystyrene+Petri+dishes.">The release of oxygen from polystyrene Petri dishes.</a> Br. J. Radiol. 45, 787-788 (1972).</li><li>Ethapa, T., 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=Multiple+factors+modulate+biofilm+formation+by+the+anaerobic+pathogen+Clostridium+difficile.">Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile.</a> J. Bacteriol. , (2012).</li><li>Speers, A. M., Cologgi, D. L., Reguera, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+cell+culture.">Anaerobic cell culture.</a> Curr. Protoc. Microbiol. Appendix 4, Appendix 4F (2009).</li><li>Lyras, D., 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=Toxin+B+is+essential+for+virulence+of+Clostridium+difficile.">Toxin B is essential for virulence of Clostridium difficile.</a> Nature. 458, 1176-1179 (2009).</li></ol>

No conflicts of interest declared.

The methods described here allow for simple and quick recovery of C. difficile from a variety of fecal samples, including humans, mice and hamsters, as well as the long-term storage of C. difficile as glycerol or spore stocks. C. difficile can be a difficult organism to cultivate, but careful maintenance of an anaerobic environment and the application of aseptic techniques can provide for robust growth and a reduction in contamination.
Anaerobic chambers: Considerations...

Clostridium difficile is a Gram-positive, spore-forming bacterium that is an obligate anaerobe and a potentially fatal gastrointestinal pathogen of humans and animals. Initially described in 1935 as a commensal organism found in fecal samples from newborns1, C. difficile was later demonstrated to be the causative agent of pseudomembranous colitis associated with antibiotic treatment2. C. difficile infections (CDI) are typically preceded by antibiotic treatment which results in the disruption of the normal colonic flora, creating a niche for C. difficile to thrive2. C. difficile is transmitted as a dormant spore via the fecal-oral route and subsequently germinates within the gastrointestinal tract, producing vegetative cells capable of generating several toxins and causing severe disease and colitis3. CDI are often refractory to conventional treatments and these infections are frequently recurrent4. As a result, CDI are responsible for up to $4.8 billion in health care costs in the United States5-7.	
	C. difficile is very sensitive to even low levels of oxygen in the environment. For C. difficile to persist in the environment and be efficiently transmitted from host to host, the formation of a metabolically inactive spore is critical8. Because the laboratory maintenance and manipulation of C. difficile requires a controlled, anaerobic environment, these techniques necessitate the use of an anaerobic chamber. Use of anaerobic chambers has resulted in increased recovery and isolation of obligate anaerobes9-11, and has allowed a number of molecular techniques to be performed in an anaerobic atmosphere.	
	In addition to C. difficile, the anaerobic chamber use and maintenance described here are applicable to other obligate anaerobes such as other Clostridial species (e.g. C. perfringens), other gastrointestinal species (e.g. Bacteroides species12) and periodontal pathogens (e.g. Peptostreptococcus species13).

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Proteose Peptone no. 2</td>
			<td>BD</td>
			<td>212120</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher</td>
			<td>S373</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>BP362</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S27</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>&#7429;-Fructose</td>
			<td>Fisher</td>
			<td>L96</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma</td>
			<td>T4009</td>
			<td></td>
		</tr>
		<tr>
			<td>&#7429;-cycloserine</td>
			<td>Sigma</td>
			<td>C6880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefoxitin</td>
			<td>Fluka</td>
			<td>C4786</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion medium</td>
			<td>BD</td>
			<td>237300</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose Peptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sigma</td>
			<td>A5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7755</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoPeptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbian sheep blood agar</td>
			<td>Fisher</td>
			<td>L21928</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S27</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP2291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile inoculating loops</td>
			<td>Fisher</td>
			<td>22363596</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile swabs</td>
			<td>Fisher</td>
			<td>1495990</td>
			<td></td>
		</tr>
		<tr>
			<td>Coy Vinyl Anaerobic Chamber and Accessories</td>
			<td>Coy Laboratory Products, Inc</td>
			<td>Customer Specified</td>
			<td>These items are custom ordered per laboratory needs</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TCCFA agar 
			 
			Proteose peptone no. 2 (Difco) 40 g 
			Na2HPO4 5 g 
			KH2PO4 1 g 
			NaCl 2 g 
			MgSO4 (anhydrous) 0.1 g 
			Fructose 6 g 
			Agar 20 g 
			 
			Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			After autoclaving, add: 
			10 ml of 10% (w/v) sodium taurocholate, filter-sterilized (dissolve in water; final concentration, 0.1%) 
			25 ml of 10 mg/ml &#7429;-cycloserine, filter-sterilized (dissolve in water; final concentration, 250 &#956;g/ml) 
			1.6 ml of 10 mg/ml cefoxitin, filter-sterilized (dissolve in water; final concentration, 16 &#956;g/ml) 
			 
			BHIS Medium 
			 
			Brain heart infusion 37 g 
			Yeast extract 5 g 
			 
			For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			Optional (add after autoclaving): 
			 
			3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 
			10 ml of 10% (w/v) sodium taurocholate (dissolve in water; final concentration, 0.1%) 
			 
			SMC Sporulation Medium 
			 
			BactoPeptone 90 g 
			Protease peptone 5 g 
			(NH4)2SO4 1 g 
			Tris base 1.5 g 
			Agar 15 g 
			 
			Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			Optional (add after autoclaving): 
			3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 
			 
			70:30 Medium 
			 
			BactoPeptone 63 g 
			Protease peptone 3.5 g 
			Brain heart infusion 11.1 g 
			Yeast extract 1.5 g 
			(NH4)2SO4 0.7 g 
			Tris base 1.06 g 
			 
			For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. After autoclaving, add 3 ml of 10% (w/v) L-cysteine (final concentration, 0.03%). 
			 
			Blood agar 
			 
			The use of premade Columbia anaerobic sheep blood agar plates (Fisher Scientific, L21928)35 is recommended. 
			 
			1X Phosphate buffered saline (PBS) 
			 
			NaCl 8.01 g 
			KCl 0.2 g 
			Na2HPO4 1.44 g 
			KH2PO4 0.27 g 
			 
			Bring to 1 L with deionized water and adjust pH to 7.4 with HCl. Filter sterilize before use.</td>
		</tr>
	</tbody>
</table>

culturing and maintaining clostridium difficile in an anaerobic environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

An example of C. difficile grown on BHIS and Columbia anaerobic sheep blood agar media can be seen in Figure 2. C. difficile forms irregular colonies that are flat and possess a ground-glass appearance which is evident on both media. Here, an erythromycin-sensitive clinical isolate of C. difficile, 630E30, is grown on BHIS agar, an enriched, non-selective medium, for 24 hr at 37 &#176;C (Figure 2A). Colonies on Columbia anaerobic sheep blood agar app...

Watch this Scientific Journal Video about Culturing and Maintaining Clostridium difficile in an Anaerobic Environment at JoVE.com

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

culturing-maintaining-clostridium-difficile-an-anaerobic

Research

JoVE Journal

Immunology and Infection

38.3K Views.  Emory University School of Medicine. Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Video: Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...