Name: a modified epa method 1623 that uses tangential flow hollow-fiber ultrafiltration and heat dissociation steps to detect waterborne cryptosporidium and giardia spp.
Uploaded: 2012-07-09T13:00:00
Duration: 12 min 11 s
Description: Watch this Scientific Journal Video about A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia

We would like to thank Ann Grimm and Michael Zimmerman for critical review of this manuscript and Doug Hamilton for his technical support.

1. Tangential Flow Hollow-fiber Ultrafiltration Procedure
<ol>
 <li>Preparation of buffers and solutions:
 Elution Solution (1 L): 
 To 1 L of reagent grade water add 0.1 g sodium polyphosphate, 0.1 ml Tween-80 and 0.01 ml Y-30 antifoam. </li>
 <li>Prepare filter assembly (Figure 1) in a biological safety cabinet:
 <ol>
 <li>Insert Masterflex I/P 73 lab tubing through a Masterflex I/P Easy-Load pump head connected to a Masterflex I/P precision brushless drive.</li>
 <li>Secure the tubing to a 0-60 psi, glycerin-filled pressure gauge fitted with a NPT branch T-connector upstream of the pump head with a screw clamp, and to a HDPE T-connector downstream of the pump head.</li>
 <li>Assemble the retentate bottle by fitting a Nalgene 53-B filling/venting cap with Masterflex L/S 15 lab tubing and a T-connector and securing it to a 1 L Nalgene heavy duty polypropylene bottle.</li>
 <li>Fit Masterflex L/S 24 tubing with a pinch clamp (pinch clamp 2) and connect it from the retentate bottle to the HDPE T-connector.</li>
 <li>Connect the Masterflex L/S 24 lab tubing from the pressure gauge to the Asahi Kasei Rexeed 25S high-flux dialyzer with a screw clamp, and from the dialyzer to the retentate bottle. Use custom-made DIN adapters designed to accommodate &frac14;" ID tubing to connect the tubing to the hollow-fiber ultrafilter.</li>
 <li>Fit Masterflex L/S 24 lab tubing with a pinch clamp (pinch clamp 1) and connect it to a 10 ml plastic pipette with the tip and cotton plug removed to act as the sample uptake line and then attach the tubing to the HDPE T-connector.</li>
 <li>Fit one end of Masterflex L/S-36 lab tubing with a ramp clamp and connect the tubing to the effluent port located near the exit end of the filter. Place the other end in the waste container.</li>
 </ol>
 </li>
 <li>Add 0.01 % (w/v) sodium polyphosphate to the 10 L water sample and mix for 3 min.</li>
 <li>Close pinch clamp 2 and remove the vent cap from the retentate bottle. All other clamps should be open.</li>
 <li>Set the pump direction to move the fluid from the T-connector to the pressure gauge (right to left following Figure 1). Adjust the pump speed to 25% of the maximum speed and turn on the pump.</li>
 <li>When the retentate bottle is 2/3 full, open pinch clamp 2 and quickly replace the vent cap to the retentate bottle. Check all of the lines and fittings to ensure there are no leaks.</li>
 <li>Slowly increase the pump speed to the desired filtration rate (approximately 1.5 L/min), checking for leaks. Using a graduated cylinder and a timer or flow meter, check the filtrate rate of the water exiting from the effluent line (blue L/S 36 tubing in Figure 1). Air bubbles can typically form at the exit end of the filter making it difficult to achieve a stable pressure and filtration rate. This is corrected by pinching the effluent line by hand for a moment. This action will generally coax the air bubble to the filtrate port and to exit via the effluent line. Repeat as needed, keeping in mind that pea size air bubbles are often unavoidable.</li>
 <li>Monitor the filtration process. Measure and record the pressure and the filtration rate as needed. The pressure should never exceed 20 psi. It is recommended that the filtration rate should not exceed 2.0 L/min. It is important that the volume of water in the retentate bottle be monitored to ensure that it never empties. It is normal for the volume to increase or decrease slightly. If the water volume in the retentate bottle falls below 1/3 full, then remove the vent cap and close pinch clamp 2 on the retentate bottle line. Bring the volume back to about 2/3 full, open the clamp and quickly replace the vent cap, ensuring a tight seal. If the volume in the retentate bottle falls quickly and continuously, then ensure the retentate bottle lid is tight and the vent cap is securely in place, and that the tubing is still in contact with the water sample. If these issues are not occurring, then it is likely that the seal in the retentate bottle lid is bad and should be replaced.</li>
 <li>When the sample container is empty, immediately close pinch clamp 1, reduce the pump speed to 20% of its maximum, remove the vent cap from the retentate bottle and close the ramp clamp.</li>
 <li>Adjust the volume of sample in the retentate bottle to approximately 200 ml by tightening or loosening the ramp clamp. After the volume is approximately 200 ml, tighten the ramp clamp for the elution steps (1.11-1.12).</li>
 <li>Add 500 ml of elution solution to the sample container and rinse the inside of the container. Place the 10 ml pipette connected to the sample uptake line into the container that contains the elution solution. Ensure that the ramp clamp is closed. Open pinch clamp 1 and close pinch clamp 2 momentarily to draw up the elution solution.</li>
 <li>After the 500 ml of elution solution is drawn up, close pinch clamp 1 and open pinch clamp 2. Allow the elution solution to circulate for 5 minutes with a pump speed of 20% of its maximum.</li>
 <li>Adjust the volume of sample in the retentate bottle to about 100 ml by tightening or loosening the ramp clamp on the effluent line. Tighten the ramp clamp and allow the sample to circulate for 1 minute. Avoid pulling air into the tubing by ensuring the volume of the sample in the retentate bottle is high enough to cover the L/S 15 tubing entering the retentate bottle.</li>
 <li>Reverse the direction of the pump which forces the sample into the retentate bottle. Allow the pump to run in reverse for 20 seconds resulting in a total of ~225 ml in the retentate bottle. Turn off the pump.</li>
 <li>Remove the Masterflex I/P 73 tubing from the pump head and disconnect the pressure gauge. Disconnect the Masterflex L/S 24 tubing exiting the hollow-fiber ultrafilter. Hold the tubing above the retentate bottle to force any remaining sample into the retentate bottle.</li>
 <li>Disconnect all tubing from the bottle and replace the venting cap with a non-venting cap.</li>
 <li>Continue to the IMS/IFA procedure with the ~225 ml retentate.</li>
</ol>
2. Immunomagnetic Separation Procedure
<ol>
 <li>Preparation of buffers and solutions:
 <ol>
 <li>Allow the buffers included in the Dynabeads: Cryptosporidium/Giardia combo kit to reach room temperature.</li>
 <li>1X SL-buffer A: Add 1 ml of 10X SL-buffer A to 9 ml reagent grade water.</li>
 </ol>
 </li>
 <li>Transfer the ~225 ml of liquid from the retentate bottle to a labeled 250 ml conical centrifuge tube. Rinse the retentate bottle twice with 10 ml of reagent water, and add the rinses to the conical centrifuge tube. Centrifuge the suspension at 1500 x g for 15 min at 4 &deg;C with no brake.</li>
 <li>Carefully aspirate the supernatant from the air-water interface to 5 ml above the packed pellet for every 0.5 ml of pellet volume (i.e. aspirate to 15 ml above a pellet volume of 1.3 ml, and aspirate to 5 ml for a pellet of 0.5 ml or less).</li>
 <li>Thoroughly resuspend the pellet into the supernatant by vortexing and/or pipette mixing. Transfer each 5 ml volume of the liquid to the flat-sided Dynal L10 tube containing 1 ml each of 10X SL-buffer A and 10X SL-buffer B. Rinse the conical centrifuge tube twice with 2.5 ml of reagent water and add the rinse to the L10 tube, bringing the total volume in the L10 tube to 12 ml, including the buffers.</li>
 <li>Add 100 &mu;l each of well-mixed resuspended anti-Cryptosporidium and anti-Giardia Dynabeads to the L10 tube. Rotate the L10 tube at 18 rpm for 1 hour at room temperature on a rotator mixer.</li>
 <li>Place the flat side of the L10 tube against the MPC-6 magnet and gently hand rock the tube end-to-end, 180&deg; for 2 minutes.</li>
 <li>Keeping the L10 tube in the MPC-6 magnet with the magnet side up, decant the supernatant away from the bead/(oo)cyst complexes bound to the magnet. Remove the L10 tube from the magnet and add 0.5 ml of 1X SL-buffer A to the tube. Transfer the suspension using two additional rinses of 0.5 ml of 1X SL-buffer A into a 1.5 ml microcentrifuge tube held in the MPC-S with the magnet in the vertical position.</li>
 <li>Gently rock the tube in the MPC-S magnet 180&deg; for 1 minute. With the magnet in place, aspirate the supernatant using a Pasteur pipette directed to the bottom of the microcentrifuge tube.</li>
 <li>Add 1 ml of 1X PBS to the front side of the microcentrifuge tube, remove the magnet and rock the tube gently just until the beads are resuspended. Replace the magnet in the vertical position and gently rock the tube 180&deg; for 1 minute. Aspirate the PBS rinse, without disturbing the bead pellet, using a Pasteur pipette to remove as much debris as possible.</li>
 <li>Remove the magnet and add 50 &mu;l of reagent water to the back side of the microcentrifuge tube. Vortex the tube at full speed for 50 seconds, then incubate the tube at 80 &deg;C for 10 minutes followed by a 30 second vortex. Replace the magnet into the MPC-S in the slanted position, binding the beads to the magnet and leaving the (oo)cysts in the liquid. Apply the (oo)cyst suspension to a SingleSpot well slide.</li>
 <li>Repeat step 2.10, applying the liquid to the same well containing the first dissociation. Place slide on a 37 &deg;C slide warmer for 1 hour to dry the suspension to the slide well.</li>
</ol>
3. Staining and Examination
<ol>
 <li>Preparation of buffers and solutions: 
 Working DAPI solution: Add 25 &mu;l of DAPI stock solution (2 mg/ml in methanol) to 25 ml of 1X PBS. Store stock and working solutions between 1 &deg;C and 10 &deg;C in the dark.</li>
 <li>Apply 50 &mu;l of methanol to the slide well and allow it to dry at room temperature.</li>
 <li>Add 50 &mu;l of the working DAPI solution to the slide well and incubate for 2 minutes at room temperature.</li>
 <li>Use a Kimwipe to wick the DAPI from the well. Apply 50 &mu;l of EasyStain. Incubate at 35 &deg;C for 30 minutes.</li>
 <li>Wick the stain off the well with a Kimwipe, and then slowly add 300 &mu;l of cold EasyStain fixing buffer, allowing it to flow over the well edge. Incubate for 2 minutes at room temperature.</li>
 <li>Use a Kimwipe to wick the buffer from the well and apply 10 &mu;l of EasyStain Mounting Medium.</li>
 <li>Carefully apply a cover slip, removing any bubbles that occur. Seal the cover slip with clear nail polish.</li>
 <li>Scan the entire slide using the FITC filter, at 200X total magnification, for ovoid or spherical apple-green fluorescent objects that resemble an oocyst or cyst. Examine all such objects with the DAPI filter at 1000X total magnification and then with DIC, also at 1000X total magnification. Record the size using a calibrated ocular micrometer and morphological characteristics.</li>
 <li>Document results.</li>
</ol>
Note: Additional information about the original procedure can be found in the December 2005 version of EPA Method 162312. The tangential flow hollow-fiber ultrafiltration procedure described is used in place of Section 12.0 of EPA Method 1623. The heat dissociation modifies Section 13.3.3 of EPA Method 1623. The procedure also describes an additional PBS rinse during the IMS process which can be inserted into the December 2005 version of Method 1623 after section 13.3.2.16. The complete list of consumables, reagents and equipment used for EPA Method 1623 including these modifications is listed in the equipment list.
4. Representative Results
Cryptosporidium oocysts and Giardia cysts recovered through the processes of filtration and immunomagnetic separation are detected by microscopic analysis. At 200X total magnification, each organism exhibiting a typical staining pattern, size, and shape as shown in Figure 2 should be further observed using oil immersion at 1000X total magnification. This will allow for measurement and identification of either typical defining features or atypical features that would rule out positive identification. Cryptosporidium is an ovoid to spherical object 4 to 6 &mu;m in diameter which exhibits brilliant apple-green FITC fluorescence with brightly highlighted edges (Figure 3A). With DAPI UV, an oocyst will exhibit one of the following typical feature categories: light blue internal staining with a green rim and no distinct nuclei (DAPI negative), intense blue internal staining, or up to four distinct, sky-blue nuclei (DAPI positive - Figure 3B). Atypical features include deviations in color, structure, or DAPI fluorescence (e.g., too many stained nuclei, red fluorescing internal structures). If the fluorescent object has met criteria for typical FITC and DAPI staining, it is examined using differential interference contrast (DIC). The object is examined for atypical external or internal morphological characteristics such as cell wall ornamentation, or one or two large nuclei filling the cell. If atypical structures are not observed, the object is recorded in the total IFA count and categorized as an empty amorphous structure or with one to four sporozoites present (Figure 3C). Similarly, Giardia-like objects are examined with regard to FITC and DAPI staining as well as DIC characteristics, like axonemes, median bodies, and nuclei. Giardia cysts are round to ovoid brilliant apple-green objects, 8 - 18 &mu;m long by 5 - 15 &mu;m wide with brightly highlighted edges (Figure 3D). With DAPI UV, the Giardia cyst will exhibit DAPI-negative staining, or DAPI-positive characteristics (Figure 3E). The fluorescent object is examined by DIC for typical and atypical features in the same manner as described for Cryptosporidium. If atypical features are not observed, the object is recorded in the total IFA count and categorized as empty containing amorphous structure, or with one or more type of internal structures present (Figure 3F).
Any organism that is observed to have atypical features should not be counted as an (oo)cyst. Microscopic analysis of environmental samples can be challenging as there are organisms that may auto-fluoresce or cross-react with the FITC-conjugated anti-Cryptosporidium and/or anti-Giardia antibodies 1. It is recommended that an analyst be familiar with aquatic microbes and review dozens of slides to gain experience identifying Cryptosporidium and Giardia. At least three (oo)cysts on the positive staining control slide should be characterized prior to every session at the microscope.
Quality control samples may be spiked with (oo)cysts to determine the percent recovery for each protozoan using the calculation: 
(Oo)cyst Percent Recovery = ((QC Sample Count - Count from Unspiked Sample) / Spike) x 100.
<img alt="Figure 1" src="/files/ftp_upload/4177/4177fig1.jpg" /> 
Figure 1. Graphic representation of the tangential flow hollow-fiber ultrafiltration system. The tubing is color coded to aid is assembly of the system.
<img alt="Figure 2" src="/files/ftp_upload/4177/4177fig2.jpg" /> 
Figure 2. Representative fluorescence image of Cryptosporidium and Giardia (oo)cysts. Cryptosporidium oocysts and Giardia cysts were stained with FITC labeled anti-Cryptosporidium/Giardia antibodies. Arrows, Giardia cysts; arrowheads, Cryptosporidium oocysts. A total of four Cryptosporidium oocysts and six Giardia cysts were found in the plane of focus. Samples observed under 200X magnification.
<img alt="Figure 3" src="/files/ftp_upload/4177/4177fig3.jpg" /> 
Figure 3. Representative microscopic images of Cryptosporidium oocysts and Giaridia cysts used for characterization. Cryptosporidium oocysts (A - C). Brilliant apple-green FITC fluorescence of spherical objects 4 to 6 &mu;m in diameter with brightly highlighted edges (A) containing up to four distinct, sky-blue DAPI nuclei (B) and one to four sporozoites (S) per oocyst (C). Giardia cysts (D - F). Brilliant apple-green FITC fluorescence of round to ovoid objects 8 - 18 &mu;m long by 5 - 15 &mu;m wide with brightly highlighted edges (D) containing up to four sky-blue DAPI nuclei (E) and with one or more discernable internal structure such as nuclei (N), median body (M) and or axonemes(A) (F). White arrows, brilliant apple green fluorescence staining Cryptosporidium oocysts and Giardia cysts walls; white arrowheads, DAPI positive nuclei. Samples observed under 1000X magnification.

<ol>
	<li>DiGiorgio, C. L., Gonzalez, D. A., Huitt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptosporidium+and+Giardia+recoveries+in+natural+waters+by+using+Environmental+Protection+Agency+Method+1623.">Cryptosporidium and Giardia recoveries in natural waters by using Environmental Protection Agency Method 1623.</a> Appl. Environ. Microbiol. 68, 5952 (2002).</li><li>Hill, V. R., Kahler, A. M., Jothikumar, N., Johnson, T. B., Hahn, D., Cromeans, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistate+evaluation+of+an+ultrafiltration-based+procedure+for+simultaneous+recovery+of+enteric+microbes+in+100-liter+tap+water+samples.">Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples.</a> Appl. Environ. Microbiol. 73, 4218-4225 (2007).</li><li>Hill, V. R., Polaczyk, A. L., Hahn, D., Narayanan, J., Cromeans, T. L., Roberts, J. M., Amburgey, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rapid+method+for+simultaneous+recovery+of+diverse+microbes+in+drinking+water+by+ultrafiltration+with+sodium+polyphosphate+and+surfactants.">Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants.</a> Appl. Environ. Microbiol. 71, 6878-6884 (2005).</li><li>Hill, V. R., Polaczyk, A. L., Kahler, A. M., Cromeans, T. L., Hahn, D., Amburgey, J. E. <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+hollow-fiber+ultrafiltration+to+the+USEPA+VIRADEL+technique+and+USEPA+method+1623.">Comparison of hollow-fiber ultrafiltration to the USEPA VIRADEL technique and USEPA method 1623.</a> J. Environ. Qual. 38, 822-825 (2009).</li><li>Holowecky, P. M., James, R. R., Lorch, D. P., Straka, S. E., Lindquist, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+ultrafiltration+cartridges+for+a+water+sampling+apparatus.">Evaluation of ultrafiltration cartridges for a water sampling apparatus.</a> J. Appl. Microbiol. 106, 738-7347 (2009).</li><li>Lindquist, H. D., Harris, S., Lucas, S., Hartzel, M., Riner, D., Rochele, P., Deleon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+ultrafiltration+to+concentrate+and+detect+Bacillus+anthracis,+Bacillus+atrophaeus+subspecies+globigii,+and+Cryptosporidium+parvum+in+100-liter+water+samples.">Using ultrafiltration to concentrate and detect Bacillus anthracis, Bacillus atrophaeus subspecies globigii, and Cryptosporidium parvum in 100-liter water samples.</a> J. Microbiol. Methods. 70, 484-492 (2007).</li><li>Polaczyk, A. L., Roberts, J. M., Hill, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+1MDS+electropositive+microfilters+for+simultaneous+recovery+of+multiple+microbe+classes+from+tap+water.">Evaluation of 1MDS electropositive microfilters for simultaneous recovery of multiple microbe classes from tap water.</a> J. Microbiol. Methods. 68, 260-266 (2007).</li><li>Rhodes, E. R., Hamilton, D. W., See, M. J., Wymer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hollow-fiber+ultrafiltration+primary+concentration+of+pathogens+and+secondary+concentration+of+viruses+from.">Evaluation of hollow-fiber ultrafiltration primary concentration of pathogens and secondary concentration of viruses from.</a> 176, 38-45 (2011).</li><li>Shaw, N. J., Villegas, L. F., Eldred, B. J., Gaynor, D. H., Warden, P. S., Pepich, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+to+EPA+Method+1623+to+address+a+unique+seasonal+matrix+effect+encountered+in+some+U.S.+source+waters.">Modification to EPA Method 1623 to address a unique seasonal matrix effect encountered in some U.S. source waters.</a> J. Microbiol. Methods. 75, 445-448 (2008).</li><li>Simmons, O. D., Sobsey, M. D., Heaney, C. D., Schaefer, F. W., Francy, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+detection+of+Cryptosporidium+oocysts+in+surface+water+samples+by+method+1622+using+ultrafiltration+and+capsule+filtration.">Concentration and detection of Cryptosporidium oocysts in surface water samples by method 1622 using ultrafiltration and capsule filtration.</a> Appl. Environ. Microbiol. 67, 1123-1127 (2001).</li><li>Sobsey, M. D., Glass, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+water+quality+on+enteric+virus+concentration+by+microporous+filter+methods.">Influence of water quality on enteric virus concentration by microporous filter methods.</a> Appl. Environ. Microbiol. 47, 956-9560 (1984).</li><li>USEPA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+1623:+Cryptosporidium+and+Giardia+in+Water+by+Filtration/IMS/FA.">Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA.</a> Office of Water. 815-R-05-002, (2005).</li><li>Ware, M. W., Wymer, L., Lindquist, H. D., Schaefer, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+alternative+IMS+dissociation+procedure+for+use+with+Method+1622:+detection+of+Cryptosporidium+in+water.">Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water.</a> J. Microbiol. Methods. 55, 575-583 (2003).</li><li>Zuckerman, U., Tzipori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portable+continuous+flow+centrifugation+and+method+1623+for+monitoring+of+waterborne+protozoa+from+large+volumes+of+various+water+matrices.">Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices.</a> J. Appl. Microbiol. 100, 1220-1227 (2006).</li></ol>

The United States Environmental Protection Agency through its Office of Research and Development in collaboration with the Office of Ground Water and Drinking Water's Technical Support Center funded the research described here. All work was supported on-site at the US EPA, Cincinnati, Ohio. Although the information described in this article has been funded wholly or in part by the United States Environmental Protection Agency under contract (Contract EP-C-06-031) to Shaw Environmental and Infrastructure, Inc, it does not necessarily reflect the views of the Agency and no official endorsement should be inferred. It has been subjected to Agency review and approved for publication.

Tangential flow hollow-fiber ultrafiltration is an alternative and effective technique for the initial concentration of Cryptosporidium oocysts and Giardia cysts from water. Hollow-fiber ultrafiltration is less expensive than traditional filters. Since it has the ability to concentrate Cryptosporidium oocysts and Giardia cysts from a variety of different water matrices it is a useful alternative to the current filtration techniques used for EPA Method 1623. As with most other filtration methods, hollow-fiber ultrafiltration is prone to fouling with extremely turbid samples. High water pressure would result from the filter fouling; therefore it is recommended to monitor the pressure during the filtration run. In addition to Cryptosporidium oocysts and Giardia cysts, hollow-fiber ultrafiltration has been shown to be capable of concentrating bacteria and viruses1-3,5,8 . Hollow-fiber ultrafiltration outlined in this method can be used to concentrate multiple organisms in a single sample. It is noteworthy that obtaining a final volume between 200 and 250 ml is the critical final step in the concentration procedure so that extra centrifugation steps, that may result in (oo)cyst loss, are avoided (step 2.2). However, allowing the volume in the bottle to drop too low can have unfavorable effects on the recoveries since there will not be enough liquid volume to force all the oocysts or cysts into the retentate bottle. Therefore it is recommended to maintain a final volume between 200 and 250 ml.
Heat dissociation is an alternative to the acid dissociation step in Method 1623. This alternative step has been shown to improve Cryptosporidium oocyst recovery and reduce the method variation when isolated from either river or reagent water9. A side-by-side comparison of acid and heat dissociation methods demonstrated that using heat to dissociate the organisms from the immunomagnetic beads produced higher mean recoveries for both Cryptosporidium and Giardia. In addition, the precision of Cryptosporidium and Giardia recoveries was better in samples processed with heat dissociation compared with acid dissociation9.
The incorporation of HFUF as the concentration step allows more flexibility by providing the ability to concentrate multiple organisms. In addition it is a less expensive alternative to current Method 1623 filtration options.

<table border="1">
 <tbody>
 <tr>
 <td>Equipment/Reagent</td>
 <td>Vendor</td>
 <td>Catalog #</td>
 </tr>
 <tr>
 <td>Asahi Kasei Rexeed 25 S/R wet hollow-fiber ultrafilters</td>
 <td>Dial Medical</td>
 <td>REXEED25S/R</td>
 </tr>
 <tr>
 <td>I/P 73 (Masterflex R-3603), or equivalent</td>
 <td>Cole Parmer</td>
 <td>EW-06408-73</td>
 </tr>
 <tr>
 <td>L/S 24 (Masterflex Platinum-Cured), or equivalent</td>
 <td>Cole Parmer</td>
 <td>EW-96410-24</td>
 </tr>
 <tr>
 <td>L/S 15 (Masterflex Platinum-Cured), or equivalent</td>
 <td>Cole Parmer</td>
 <td>EW-96410-15</td>
 </tr>
 <tr>
 <td>L/S 36 (Masterflex Platinum-Cured), or equivalent</td>
 <td>Cole Parmer</td>
 <td>EW-96410-36</td>
 </tr>
 <tr>
 <td>I/P Precision Brushless Drive</td>
 <td>Cole Parmer</td>
 <td>EW-77410-10</td>
 </tr>
 <tr>
 <td>I/P Easy Load Pump Head</td>
 <td>Cole Parmer</td>
 <td>EW-77601-10</td>
 </tr>
 <tr>
 <td>Black HDPE Tee, 1/4"x 3/8" x 3/8"</td>
 <td>US Plastics</td>
 <td>62064</td>
 </tr>
 <tr>
 <td>Masterflex T-connector L/S 15-25</td>
 <td>Cole Parmer</td>
 <td>EG-30613-12</td>
 </tr>
 <tr>
 <td>Nalgene heavy-duty pp 1 L bottle</td>
 <td>Cole Parmer</td>
 <td>EW-06257-10</td>
 </tr>
 <tr>
 <td>10 ml pipettes</td>
 <td>Fisher Scientific</td>
 <td>13-678-11C</td>
 </tr>
 <tr>
 <td>Nalgene filling/venting cap for 1/4" tubing, 53B</td>
 <td>Cole Parmer</td>
 <td>EW-06258-10</td>
 </tr>
 <tr>
 <td>Pressure gauge</td>
 <td>Cole Parmer</td>
 <td>A-680-46-10</td>
 </tr>
 <tr>
 <td>Straight coupling, NPT(F), 1/4" </td>
 <td>Cole Parmer</td>
 <td>EW-06469-18</td>
 </tr>
 <tr>
 <td>NPT branch tee, natural pp</td>
 <td>Cole Parmer</td>
 <td>A-30610-75</td>
 </tr>
 <tr>
 <td>Pinch clamps, 1/2"</td>
 <td>Cole Parmer</td>
 <td>EW-06833-00</td>
 </tr>
 <tr>
 <td>Custom fit DIN adapters</td>
 <td>Molded Products Corp</td>
 <td>MPC-855NS.250</td>
 </tr>
 <tr>
 <td>Ring stand</td>
 <td>Fisher Scientific</td>
 <td>14-670B</td>
 </tr>
 <tr>
 <td>Ring stand clamps</td>
 <td>Fisher Scientific</td>
 <td>05-769-6Q</td>
 </tr>
 <tr>
 <td>Keck ramp clamp, 14mm</td>
 <td>Cole Parmer</td>
 <td>EW-06835-10</td>
 </tr>
 <tr>
 <td>Sodium polyphosphate</td>
 <td>Sigma Aldrich</td>
 <td>305553</td>
 </tr>
 <tr>
 <td>Sodium thiosulfate pentahydrate</td>
 <td>Sigma Aldrich</td>
 <td>72050</td>
 </tr>
 <tr>
 <td>Antifoam Y-30 emulsion</td>
 <td>Sigma Aldrich</td>
 <td>A5758</td>
 </tr>
 <tr>
 <td>Tween-80</td>
 <td>Sigma Aldrich</td>
 <td>P1754</td>
 </tr>
 <tr>
 <td>10 L Collapsible high-density polyethylene cubitainer</td>
 <td>VWR</td>
 <td>IR314-0025</td>
 </tr>
 <tr>
 <td>Centrifuge bottle rack</td>
 <td>Fisher Scientific</td>
 <td>05-663-103</td>
 </tr>
 <tr>
 <td>250 ml conical centrifuge tubes</td>
 <td>Corning</td>
 <td>430776</td>
 </tr>
 <tr>
 <td>Disposable funnel</td>
 <td>Cole Parmer</td>
 <td>U-6122-10</td>
 </tr>
 <tr>
 <td>Wash bottle</td>
 <td>Cole Parmer</td>
 <td>U-06252-40</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Allegra X-15R</td>
 </tr>
 <tr>
 <td>Swinging bucket rotor</td>
 <td>Beckman Coulter</td>
 <td>ARIES SX4750</td>
 </tr>
 <tr>
 <td>Centrifuge bucket adapters for 250 ml conical tubes</td>
 <td>Beckman Coulter</td>
 <td>349849</td>
 </tr>
 <tr>
 <td>200 &mu;l large bore pipette tips</td>
 <td>Fisher Scientific</td>
 <td>02-707-134</td>
 </tr>
 <tr>
 <td>VacuShield Filter</td>
 <td>Gelman</td>
 <td>629-4402</td>
 </tr>
 <tr>
 <td>5 ml pipettes</td>
 <td>Fisher Scientific</td>
 <td>13-678-11D</td>
 </tr>
 <tr>
 <td>Dynabeads: Cryptosporidium/Giardia combo kit </td>
 <td>IDEXX</td>
 <td>73002</td>
 </tr>
 <tr>
 <td>50 ml conical centrifuge tubes</td>
 <td>Falcon</td>
 <td>352098</td>
 </tr>
 <tr>
 <td>Dynal L10 flat sided tubes</td>
 <td>IDEXX</td>
 <td>74003</td>
 </tr>
 <tr>
 <td>Timer</td>
 <td>VWR</td>
 <td>23609-202</td>
 </tr>
 <tr>
 <td>Dynal MPC-6 magnet</td>
 <td>IDEXX</td>
 <td>12002D</td>
 </tr>
 <tr>
 <td>1 ml pipettes</td>
 <td>VWR</td>
 <td>53283-700</td>
 </tr>
 <tr>
 <td>1.5 ml low adhesion microcentrifuge tubes</td>
 <td>Fisher Scientific</td>
 <td>02-681-320</td>
 </tr>
 <tr>
 <td>1000 &mu;l pipette &amp; corresponding barrier tips</td>
 <td>Gilson</td>
 <td>P1000/DF1000ST</td>
 </tr>
 <tr>
 <td>100 &mu;l pipette &amp; corresponding barrier tips</td>
 <td>Gilson</td>
 <td>P100/DF100ST</td>
 </tr>
 <tr>
 <td>9 inch Pasteur pipettes</td>
 <td>VWR</td>
 <td>14672-412</td>
 </tr>
 <tr>
 <td>Dynal MPC-S magnet</td>
 <td>IDEXX</td>
 <td>12020D</td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>VWR</td>
 <td>14216-188</td>
 </tr>
 <tr>
 <td>Dynabeads rotator mixer</td>
 <td>IDEXX</td>
 <td>94701</td>
 </tr>
 <tr>
 <td>Heat block</td>
 <td>Fisher Scientific</td>
 <td>11-718-2</td>
 </tr>
 <tr>
 <td>Lab Armor Beads</td>
 <td>Lab Armor</td>
 <td>42370-750</td>
 </tr>
 <tr>
 <td>Digital thermometer</td>
 <td>Fisher Scientific</td>
 <td>15-077-60</td>
 </tr>
 <tr>
 <td>Phosphate-buffer saline 1X pH 7.4 (1X PBS)</td>
 <td>Sigma</td>
 <td>P4417</td>
 </tr>
 <tr>
 <td>Single Spot slides</td>
 <td>IDEXX</td>
 <td>30201</td>
 </tr>
 <tr>
 <td>Cover glass</td>
 <td>Corning</td>
 <td>287018</td>
 </tr>
 <tr>
 <td>EasyStain direct kit</td>
 <td>BTF</td>
 <td>-</td>
 </tr>
 <tr>
 <td>10 &mu;l pipette &amp; corresponding barrier tips</td>
 <td>Gilson</td>
 <td>P10 &amp; DF10ST</td>
 </tr>
 <tr>
 <td>4',6'-Diamidino-2-phenyl indole dihydrochloride (DAPI)</td>
 <td>Sigma</td>
 <td>D9542</td>
 </tr>
 <tr>
 <td>Clear nail polish</td>
 <td>Fisher Scientific</td>
 <td>S30697</td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher Scientific</td>
 <td>L6815</td>
 </tr>
 <tr>
 <td>Kimwipes</td>
 <td>Kimberly Clark</td>
 <td>34155</td>
 </tr>
 <tr>
 <td>Incubator</td>
 <td>Boekel Scientific</td>
 <td>133000</td>
 </tr>
 <tr>
 <td>slide warmer</td>
 <td>Fisher Scientific</td>
 <td>11-474-521</td>
 </tr>
 <tr>
 <td>Immersion oil, Type A ND= 1.515 </td>
 <td>Nikon</td>
 <td>MXA20234</td>
 </tr>
 <tr>
 <td>Nikon 90i microscope with DIC capabilities</td>
 <td>Nikon</td>
 <td>MBA 77000</td>
 </tr>
 <tr>
 <td>Plan APO 100X oil objective</td>
 <td>Nikon</td>
 <td>MRD01901</td>
 </tr>
 <tr>
 <td>Plan Achro 20X </td>
 <td>Nikon</td>
 <td>MRL00202</td>
 </tr>
 <tr>
 <td>FITC filter</td>
 <td>Nikon</td>
 <td>96302</td>
 </tr>
 <tr>
 <td>DAPI filter</td>
 <td>Nikon</td>
 <td>96301</td>
 </tr>
 <tr>
 <td>X-cite fluorescence illuminator</td>
 <td>Nikon</td>
 <td>87540</td>
 </tr>
 <tr>
 <td>Lens paper</td>
 <td>Nikon</td>
 <td>76997</td>
 </tr>
 <tr>
 <td>Biohazard disposable bag</td>
 <td>Fisher Scientific</td>
 <td>01-829D</td>
 </tr>
 <tr>
 <td>Biohazard sharps container</td>
 <td>Fisher Scientific</td>
 <td>14-827-117</td>
 </tr>
 <tr>
 <td>3 % hydrogen peroxide</td>
 <td>VWR</td>
 <td>BDH3540-2</td>
 </tr>
 <tr>
 <td>Bleach</td>
 <td>Fisher Scientific</td>
 <td>1952030</td>
 </tr>
 <tr>
 <td>Wypall</td>
 <td>Kimberly Clark</td>
 <td>34790</td>
 </tr>
 </tbody>
</table>

a modified epa method 1623 that uses tangential flow hollow-fiber ultrafiltration and heat dissociation steps to detect waterborne cryptosporidium and giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

Watch this Scientific Journal Video about A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

a-modified-epa-method-1623-that-uses-tangential-flow-hollow-fiber

Research

JoVE Journal

Immunology and Infection

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...