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

38.4K Views.  Emory University School of Medicine.  Clostridium несговорчивый является патогенным бактерия, которая является строгим анаэробных и вызывает антибиотик диарею, ассоциированную (AAD). Здесь, способы выделения, культивирования и поддержания C. несговорчивый вегетативные клетки и споры описаны....

Video: Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Biochemistry

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Environment

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 ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Biology

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

Cultivation Strategies for Uncultured Microbial Diversity in the Chicken GI Tract

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken&#39;s GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken&#39;s GIT.

Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry

The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance.

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed &#34;extremophiles&#34;, these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.
Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.
Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.
Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).
Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.
One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.
This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Enrichment Cultures, Selective and Differential Media

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, ...