Method 1623 IMS Procedure Supplemental Information July 10, 2007

* £1 l

%PRo^°

Method 1623 IMS Procedure - Supplemental Information

July 10, 2007

The immunomagnetic separation (IMS) procedure of EPA Method 16231 is designed to separate
Cryptosporidium oocysts and Gictrdict cysts from the debris in the sample and eliminate the transfer of the
debris to the microscope slide. When problems with the IMS process are identified, review the IMS
techniques and standardized protocols carefully. It is important to standardize each IMS processing step
to maximize recovery and precision. This supplemental document does not replace Method 1623, but
provides processing tips, special techniques, and quality control recommendations that laboratories may
implement to improve recovery and precision in problematic samples. Before processing field samples
with any of the modifications described in Section II of this document, laboratories should demonstrate
acceptable performance of the modified method (see Section III).

PLEASE NOTE:

• All Method 1623 section re ferences are from the 2005 version.

• Inclusion of individual vendor or product/equipment does not indicate an endorsement by
EPA, other vendors and equivalent products/equipment may be used.

• The MPC^-M is no longer available for purchase and the MPC®-S has a stronger magnet.
Accordingly, only the MPC®-S is referred to in this document. Product information for
beads and magnets can be obtained from the manufacturer2' \

I. Processing Tips

Pellet Transfer to Flat-sided Tube - Method 1623 Section 13.3.2.1

Completely homogenize resuspended pellet immediately prior to transfer. Vortex vigorously for 10-
15 seconds and/or pipette mix, then visually inspect to ensure complete homogenization and lack of
debris aggregates. This is particularly important for samples with high clay content.

The volume of the transferred sample plus 2 reagent water rinses should total 10 mL. The addition
of 2 mL of buffers brings the total in the flat-sided tube to 12 mL.

Measure the volume being transferred using a serological or graduated transfer pipette to determine
the maximum volume that can be used for the centrifuge tube rinses.

Do not rinse the pellet prior to transfer, as oocysts and cysts may be lost.

Be sure IMS reagents are at room temperature before use.

Do not refrigerate samples between the completion of centrifugation and the start of IMS.

Decanting Supernatant from Flat-sided Tube - Method 1623 Sections 13.3.2.10 -13.3.2.11

Do not let the flat-sided tubes sit motionless before decanting; continuous movement of the tubes
prevents binding of low-mass, magnetic, or magnetizable material.

Use a clean, lint-free tissue to blot the end of each flat-sided tube after decanting to remove more
matrix debris.

Office of Water (MS-140)

EPA 815-B-07-003

-------
Allow the flat-sided tube to sit undisturbed in the MPC for 30-60 seconds after decanting; then
aspirate residual liquid and debris from the bottom of the tube.

If using MPC -6. rock each side through a 90° angle 3 times before decanting the remaining tubes.

With the MPC -6. the supernatant may be decanted from 1 to 3 flat-sided tubes at one time;
maximum 3 tubes at once.

Transfer to Microcentrifuge Tube - Method 1623 Section 13.3,2.12 -13.3.2.13

Add 0.5 mL of IX SL Buffer A directly to the flat side of the tube to resuspend the bead pellet, avoid
any debris present on the round side of the tube.

Pipette mix gently to completely resuspend the bead pellet prior to initial transfer and each rinse
transfer.

During each pipette mix step, release the liquid down the flat side of the tube to further rinse the
tube.

Allow the flat-sided tube to sit undisturbed for 60 seconds after the last rinse to collect residual liquid
for transfer to the microcentrifuge tube.

Microcentrifuge tubes should be in the MPC®-S with the magnet in the vertical position during
transfer to produce an immediate bead pellet which aids in the separation of the debris.

o The MPC "-S magnet has two notch positions for the placement of the magnet (Figure 1).
o The vertical position is used for producing the bead-organism pellet prior to dissociation
(Figure 2 top).

Figure 1 Figure 2

Aspiration and Dissociation in Microcentrifuge Tube - Method 1623 Sections 13.3.2.16
and 13.3.3.6

Perform the aspiration described in Section 13.3.2.16 with the magnet in the vertical position,
aspirate the waste liquid to just below the bead pellet and discard; without disturbing the bead pellet
gently pipette mix the remaining liquid and debris to resuspend the debris and improve the removal.

The slanted magnet position (Figure 1 and Figure 2 bottom) should be used in Section 13.3.3.6
when collecting the bead pellet during acid dissociation.

Vertical

Slanted

-------
II.

Special Techniques: Potential Method Modifications

NOTE: These protocols are to be used only by experienced analysts with successful OPRs and
requisite quality control for using method modifications (Section III)!

IMS may not perform consistently when applied to certain source waters and special techniques may be
necessary when matrix spike recoveries are low, pellet sizes are large, and/or extraneous debris carries
over to successive steps. Modifications to Method 1623 may be used to increase recovery (Section 9.1.2).
Some special techniques are not routinely used, and not firmly established, but may be beneficial for
some source waters. It is often helpful to discuss technique modifications with other experienced analysts
before trying them. Always practice and test modified techniques prior to use on field samples.
Performing the appropriate QC validates the use of the modification (see III. Quality Control
Recommendations). The proposed protocols included in this document address:

• Removing magnetic minerals
Adjusting pH

Additional rinse of flat-sided tube
Pellet wash in microcentrifuge tube
Heat dissociation

Modified sample application to microscope slide

Implementation of any individual special technique should be performed independently from other
modifications; do not attempt to incorporate multiple changes at once. Application of any special
technique should be based on laboratory experience analyzing samples from a particular source.

1) Removing Magnetic Materials4 - This modification to Method 1623 Section 13.3
must meet QC requirements (See Section III below).

Some source water samples can contain high concentrations of iron and other magnetic material that may
interfere with IMS. Some laboratories have determined the removal of extraneous magnetic material
from the sample, prior to the addition of beads, can improve recoveries. The sample is processed through
filtration, elution, and concentration then extraneous magnetic material is removed. The remaining
sample is carried through the IMS process.

Process sample(s) according to Method 1623 through Section 13.2.4.2; then proceed at 13.3.1 with the
following substitutions and additions:

13.3.1 Preparation and addition of reagents

13.3.1.1 Same as method

13.3.1.2 Same as method

13.3.1.3 Same as method with following steps added:

13.3.1.3.1 Use a graduated, 10-mL pipette that has been pre-rinsed with
elution buffer to transfer the water sample concentrate from
Section 13.2 to another flat-sided tube(s) not containing IMS
buffers. If all of the concentrate is used, rinse the centrifuge tube
twice with reagent water and add the rinsate to the flat-sided tube
containing the concentrate (or to the tube containing the first
subsample, if multiple subsamples will be processed). Each of
the two rinses should be half the volume needed to bring the total
volume in the flat-sided sample tube to 8 mL. (For example, if 5
mL of sample was transferred after resuspension of the pellet,
the centrifuge tube would be rinsed twice with 1.5 mL of reagent
water to bring the total volume in the flat-sided tube to 8 mL.)
Visually inspect the centrifuge tube after completing the transfer

-------
to ensure that no concentrate remains. If multiple subsamples
will be processed, bring the volume in the remaining flat-sided
tubes to 8 mL with reagent water. Label the flat-sided tube(s)
with the sample number (and subsample letters).

13.3.1.3.2 Place the flat-sided tube in the MPC®-1 or MPC®-6; check the
tube is tight against the magnet.

13.3.1.3.3 Rock the magnet and tube gently and smoothly through a 90°
angle for 2 minutes with approximately one 90° rock per second.
Ensure the tilting action is continued throughout the 2 minute
period.

13.3.1.3.4 If the sample is allowed to stand motionless for more than 10
seconds, remove the tube from the magnet, shake to resuspend
all materials, replace the sample tube in the magnet, and repeat
the 2 minute rocking.

13.3.1.3.5 Return tube to upright position and immediately remove the cap.

13.3.1.3.6 Keeping the flat side of tube on top, pour off supernatant into
another flat-sided tube containing Buffers A and B. Without
removing the tube from the MPC, rinse the round side of tube
twice with 1 mL of reagent water. This tube now contains 12
mL of the sample and buffers ready to continue through the IMS
process. Label this flat-sided tube(s) with the sample number
(and subsample letters).

13.3.2 Oocyst and cyst capture

13.3.2.1 Omit this step

13.3.2.2 Same as method, to completion of the method

NOTE: The flat-sided tube remaining in the magnet should contain extraneous iron and other
magnetic material removed by the magnet. This extraneous material may be discarded as waste
and the tube either discarded or cleaned for reuse per laboratory standard operating procedure.

2) Adjusting pH4 - This modification to Method 1623 Section 13.3.2.1 must meet QC
requirements (See Section III below).

Some source water samples may produce a pellet with non-neutral pH characteristics. Low recoveries
could result if acidity or alkalinity of the pellet is not adequately buffered during the IMS process. Some
laboratories have determined the addition of HC1 or NaOH to neutralize the sample, after buffers have
been added, can improve recoveries.

Process sample(s) according to Method 1623 through Section 13.3.2.1, transferring all sample(s) to flat-
sided tube(s) with buffers; then proceed with the following additions:

13.3.2.1 Same as method

13.3.2.1.1 Gently mix the buffers with the transferred sample by inverting
the flat-sided tube 3 times.

13.3.2.1.2 Read and record the pH of the suspension.

13.3.2.1.3 Adjust the pH of the suspension with INHClor INNaOHas
needed to establish pH = 7.

13.3.2.2 Same as method, to completion of the method.

NOTE: The pH of the sample could also be checked and adjusted before buffers are added
(Section 13.2.2), and after the 1-hour rotation (Section 13.3.2.7) to ensure the pH is stable. Some
laboratories have used IQ Scientific Instruments-handheld pH meter with Micro Probe (PHI 7-
SS) to check the pH in the flat-sided tube. Alternative pH measurement techniques may be used.

-------
3) Additional Rinse of Flat-sided Tube- This modification to Method 1623 Section
13.3.2.11 must meet QC requirements (See Section III below).

When source water pellets contain visible excess debris after aspiration of the supernatant (Section
13.3.2.11), debris carryover may interfere with recoveries and an additional rinse of the flat-sided tube
may be performed. If this additional rinse of the flat-sided tube is performed, then the additional
modification of a pellet wash in the microcentrifuge tube (described later) should not be performed, to
prevent over-rinsing and loss of organisms.

Process sample(s) according to Method 1623 through Section 13.3.2.11, remove supernatant from flat-
sided tube; then proceed with the following additions:

13.3.2.11 Same as method with following steps added:

13.3.2.11.1 Orient the tube horizontally with bead pellet and magnet on top.

13.3.2.11.2 Gently add 10 mL of PBS or PBST (0.05% Tween 20) to the
rounded side of the tube opposite the beads; do not disturb the
bead pellet.

13.3.2.11.3 Gently tip the rinse solution over the side of the tube opposite of
the bead pellet, three times.

13.3.2.11.4 Decant while continuing to keep magnet and flat-side of tube up.

13.3.2.12 Same as method, to completion of the method.

4) Pellet Wash in Microcentrifuge Tube- This modification to Method 1623 Section
13.3.2.16 must meet QC requirements (See Section III below).

Do not perform the pellet wash in the microcentrifuge tube if an additional rinse at the flat-sided tube was
performed to prevent over-rinsing and loss of organisms.

Recoveries for source water matrices that produce debris carryover are usually improved by additional
rinse of the flat-sided tube (see above). However, some source water matrices are better improved by a
pellet wash in the microcentrifuge tube. This wash may be used with difficult matrices that exhibit visible
debris trapped in the bead pellet at the back or bottom of the microcentrifuge tube after aspiration of the
supernatant.

Remember: Any debris remaining in the microcentrifuge tube will end up on the slide!

Process sample(s) according to Method 1623 through Section 13.3.2.16, remove supernatant from
microcentrifuge tube; then proceed with the following additions:

13.3.2.16 Same as method with following steps added:

13.3.2.16.1 Remove the magnetic strip from the MPC®-S after aspiration of
supernatant.

13.3.2.16.2 Gently add 1 mL of PBS or PBST (0.05% Tween 20) to the bead
suspension.

13.3.2.16.3 Gently invert the tube 5-10 times to completely resuspend the
bead pellet.

13.3.2.16.4 Replace the magnetic strip in the MPC®-S and repeat Sections
13.3.2.15-13.3.2.16

13.3.2.16.5 After aspirating the wash, allow the microcentrifuge tube to sit
undisturbed for 30-60 seconds before aspirating any remaining
liquid.

13.3.3 Dissociation of beads/oocyst/cyst complex; same as method, to completion of the
method.

-------
5) Heat Dissociation5 - This modification to Method 1623 Section 13.3.3 must meet
QC requirements (See Section III below).

The addition of acid to some source water matrices for dissociation may drive chemical reactions that
interfere with the method. Heat can be used instead of acid to inhibit reformation of the bead oo/cyst
complexes and potentially improve recoveries.

Process sample(s) according to Method 1623 through Section 13.3.3.1; then revise 13.3.3.2 through
13.3.3.10 as follows:

13.3.3.2 Add 50 |_iL of reagent water [instead of HC1], then vortex at the highest
setting for approximately 50 seconds.

13.3.3.3 Place tube(s) in heat block stabilized at 80°C for 10 minutes.

13.3.3.4 Remove tube(s) from heat block, and vortex at the highest setting for
approximately 30 seconds.

13.3.3.5 Same as method

13.3.3.6 Same as method

13.3.3.7 Same as method

13.3.3.8 DELETE this step in the method.

13.3.3.9 Same as method except omit "with the NaOH".

13.3.3.10 Replace "acid" with "heat"; (second dissociation may not be necessary)

13.3.3.11 Same as method, to completion of the method.

NOTE: Some laboratories have used aMulti Block Heater, Model 2050 or Grant UBD1 heat
block. The Grant UBD1 heat block has options of various block sizes to accommodate different
plasticware including a 1.5 mL microtube interchangeable block BB-E1 that can hold 24 tubes at
once.

6) Increasing Surface Area for Sample Application to Slide - This modification to

Method 1623 Section 13.3.3.7 must meet QC requirements (See Section III below).

Matrix debris may result in loss of organisms during the staining process and visual obstruction of
oo/cysts on the slide. Increasing the surface area for sample application to slides can reduce interference
from debris.

Use slides with larger diameter wells to spread the debris and organisms over a larger surface area.
Commercially available microscope slides have well diameters ranging from 9 mm to 15 mm.

Split the dissociation volumes from each sample evenly between two slide wells.

To split each dissociation volume, process sample(s) according to Method 1623 through Section 13.3.3.6
(sample is in the microcentrifuge tube in the MPC®-S); then proceed at 13.3.3.7 with the following
substitutions and additions:

13.3.3.7

13.3.3.8

13.3.3.9

13.3.3.10

13.3.3.11

13.3.3.12

Prepare two separate wells for each sample.

OMIT this step

Same as method, except apply half of the dissociation volume, 25 |_iL. to one
well and the second half to a second well (See Figure 3; apply 25 |_iL to Well
A and 25 (iL to Well B)

Same as method, except apply splits of the dissociation volume to the two
slide wells as before (modified 13.3.3.9 above, See Figure 3).

Add 5 (iL of 1.0 N NaOH to each of the two wells after applying the samples
Same as method, to completion of the method.

-------
1st dissociation

25 nL 25 nL

I I

Well A Well B

t t

25 jiL 25 jiL

2nd dissociation

Figure 3

III. Quality Control Recommendations

Method 1623 is a performance-based method and modifications are allowed if the laboratory can meet
applicable QC criteria (Section 9.1.2). Before modifications with the special techniques described above
are incorporated for processing field samples, initial and on-going QC samples (described below) should
be analyzed to demonstrate acceptable performance of the modified method. Laboratories should
repeatedly perform the modified techniques and demonstrate acceptable organism recovery using these
modified procedures with reagent and matrix samples prior to use on field samples.

Initial Demonstration of Acceptable Performance:

Required, Section 9.4: Initial Precision and Recovery (IPR) samples consisting of 4 reagent water
organism-spiked samples and one method blank (Section 9.6).

Strongly Recommended, Section 9.5: Matrix spike, matrix spike duplicate, and unspiked field
sample analyses.

On-going Demonstration of Acceptable Performance, Section 9.7:

Process matrix spike sample using the same modification as the associated field sample
Process On-going Precision and Recovery (OPR) samples using the special techniques at the same
percentage as the modification is used on field samples

Example: If 25% of the field samples are processed with an additional rinse, every 4th OPR
would be analyzed using the same type of additional rinse.

Processing IMS controls helps to monitor performance of modifications, new IMS kits, spike organisms
and staining kits, as well as troubleshoot low recoveries in OPR and proficiency test samples. An IMS
control is a 10-mL reagent water sample that is spiked with a known concentration of organisms and
processed through IMS and staining to determine recoveries. Percent recovery should be at least 70% and
typically is >80%. If the sample matrix is suspected of interfering with recovery, an IMS control may be
analyzed using a 10-mL concentrate from the suspect source to help pinpoint the interference. The IMS
control should be run weekly or with every 20 samples. An IMS control may also be run before
processing field samples with a new lot of reagents (IMS kit, spike organisms, or stain). An IMS control
may be used as a quality control check when processed with each batch of samples, if desired.

Control charts may be used to determine if problems are related to processing, reagent lots, equipment,
and/or analysts. Control charts should include reagent lot numbers, analyst names, and method
modifications. See Figure 4 for an example of an IMS Control Chart. Further guidance is available in
Sections 3.3.5, 3.3.12.3 and 3.3.13 of the Laboratory Guidance Manual6.

-------
Example Control Chart
Cryptosporidium IMS Control with No Modifications

Date

—IMS % Recovery —©—Mean % Recovery — — Lower Control Limit — — Upper Control Limit



















Spiking











Total

Estimated

IMS %

Mean %

Standard

Lower

Upper



Suspension

IMS Kit

Stain

HCI

NaOH

Date

Count

# Spiked

Recovery

Recovery

Deviation

Control Limit

Control Limit

Analyst

Lot#

Lot#

Lot#

Lot#

Lot#

1/8/07

108

150

72.0

77.64

5.14

67.35

87.93

TK

061220

48473

250050.111

H4056

NH0789

1/15/07

104

149

69.8

77.64

5.14

67.35

87.93

TK

070108

48473

250050.111

H4056

NH0789

1/22/07

125

149

83.9

77.64

5.14

67.35

87.93

TK

070108

48473

250050.111

H4056

NH0789

1/29/07

121

148

81.8

77.64

5.14

67.35

87.93

JS

070122

48473

250050.111

H4056

NH0789

2/5/07

117

148

79.1

77.64

5.14

67.35

87.93

JS

070122

48473

250050.111

H4056

NH0789

2/12/07

117

149

78.5

77.64

5.14

67.35

87.93

JS

070205

48473

250050.111

H4056

NH0789

2/19/07

125

149

83.9

77.64

5.14

67.35

87.93

JS

070205

48473

250050.111

H4056

NH0789

2/26/07

124

150

82.7

77.64

5.14

67.35

87.93

TK

070219

48473

250050.111

H4056

NH0789

3/5/07

123

150

82.0

77.64

5.14

67.35

87.93

TK

070219

48473

250050.111

H4056

NH0789

3/12/07

120

150

80.0

77.64

5.14

67.35

87.93

TK

070219

48474

250050.111

H4056

NH0789

3/19/07

119

150

76.1

77.64

5.14

67.35

87.93

JS

070219

48474

250050.111

H4056

NH0789

3/26/07

112

148

74.7

77.64

5.14

67.35

87.93

JS

070319

48474

250050.112

H4056

NH0789

4/2/07

109

148

73.6

77.64

5.14

67.35

87.93

JS

070319

48474

250050.112

H4056

NH0789

4/9/07

102

148

68.9

77.64

5.14

67.35

87.93

TK

070319

48474

250050.112

H4056

NH0789

Figure 4

8

-------
IV. References

1.	Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA, Office of Water,
Office of Ground Water and Drinking Water Technical Support Center, U.S. Environmental
Protection Agency, Cincinnati, OH (2005) EPA 815-R-05-002
http://www.epa.gov/microbes/1623de05.pdf

2.	Invitrogen Water and Environmental Testing Product Selection Guide;
http://\\\\\\.invitrogen.com/content.cfm'.)pageid= 11110.

3.	Aureon Biosystems or Immtech, Inc. (US distributor);

htto://www.aureonbio.com/ProductList.htm. http://www.immtech.net/html/imskr.html

4.	Kuhn, R.C., C. M. Rock, and K. H. Oshima. Effects of pH and Magnetic Material on
Immunomagnetic Separation of Cryptosporidium Oocysts from Concentrated Water Samples.
Appl Environ Microbiol. 2002 April; 68(4): 2066-2070. doi: 10.1128/AEM.68.4.2066-
2070.2002. Copyright © 2002, American Society for Microbiology.

http://www.pubmedcentral.nih.gov/picrender.fcgi?tool=pmcentrez&artid=123889&blobtvpe=pdf

5.	Ware, M. W., L. Wymer, H. D. A. Lindquist, and F. W. Schaefer III, 2003. Evaluation of an
Alternative IMS Dissociation Procedure for Use with Method 1622: Detection of
Cryptosporidium in Water. Journal of Microbiological Methods 55:575-583.

6.	Microbial Laboratory Guidance Manual for the Lorn Term 2 Enhanced Surface Water Treatment
Rule (LT2 Rule). Office of Water, Office of Ground Water and Drinking Water Technical Support
Center, U.S. Environmental Protection Agency, Cincinnati, OH (2006) EPA 815-R06-006
http://www.epa.gov/safewater/disinfection/lt2/pdfs/guide lt2 microbialguidancemanual.pdf

9

-------