Flow Cytometry

Cryptosporidium and Giardia Standards

The inaccuracy and imprecision of standard dilution methods for preparing low level parasite stock suspensions are well known. Therefore, the US EPA has proposed that all parasite quality control standards used for method 1622/1623 under Long Term 2 Enhanced Surface Water Treatment Rule be prepared using flow cytometric methods.

Preparation and distribution of spiking suspensions containing precisely-enumerated Cryptosporidium parvum and Giardia lamblia has been a routine part of the WSLH operation since 1997. From 1998 - 1999, the WSLH served as the referee laboratory for the interlaboratory method 1622/1623 validation studies (Connell et al. Journal AWWA, October 2000, 92(10):30-43) and the Laboratory is currently under contract to prepare spiking suspensions for the EPA-sponsored laboratory approval program (http://www.epa.gov/safewater/disinfection/lt2/lab_home.html).

The Laboratory's Cryptosporidium and Giardia standards have been used as control samples for EPA method 1622/1623 and for researchers developing and optimizing protozoon detection methods.

The WSLH is the sole source of live, flow cytometer-prepared spiking suspensions within the United States. Custom standard orders with varied organism concentration (1 organism - 10 7 /tube), tube type (low volume PCR - 250 mL conical bottom tubes), diluent composition, and final diluent volume are easily accommodated.

Please contact us at 608-224-6260 for more information.

• Standard Preparation

• Standard Specifications

• Shelf Life Study

Standard Preparation

Suspensions are prepared using the Ames isolate (Cryptosporidium parvum) and the CH3 isolate (Giardia lamblia). Concentrated stock suspensions are assessed for general quality and intactness using DIC microscopy prior to use in standard preparations. Additionally, a viability estimate is made using propidium iodide exclusion methods.

Instrument performance checks are also part of the Laboratory's standard operating procedures and include both an initial instrument calibration verification to confirm instrument accuracy (n = 10) and ongoing calibration verification performed before and after generation of each set of 10 standards. The relative standard deviation of the parasite counts associated with each standard set must be less than 2.5%.

The mean and the relative standard deviation along with other relevant lot information are provided to each laboratory. Standards have an expiry of 6 weeks when used the Envirochek filters and 2 weeks when used with FiltaMax filters.

Standard Specifications

NOTE: Custom standard orders with varied organism concentration (1 organism - 10 7 /tube) , tube type (low volume PCR - 250 mL conical bottom tubes), diluent composition, and final diluent volume are easily accommodated, typically at no additional cost. Call (608) 224-6260 for more information.

Shelf Life Study

Assessment of the holding time for live, flow-cytometer sorted spiking suspensions

Method 1623 data analysis was completed by EPA contract staff. Qualitative assessment data analysis was completed by WSLH staff. The EPA has not reviewed this summary.

In June 2003, the US EPA Office of Water tasked Computer Sciences Corporation (CSC, an EPA contract organization) to generate data to determine the holding time for live, flow cytometer-prepared Cryptosporidium parvum and Giardia lamblia spiking suspensions used in method 1623. Recommendations in EPA method 1623 at this time stated live standards must be used within 2 weeks of sample preparation. The study was designed to determine mean method 1623 recovery of cysts and oocysts at times 0, 14, 30, 60 and 120 days.

Methods

Stock suspensions of C. parvum oocysts and G. lamblia cysts were obtained from Sterling Parasitology at the University of AZ and Waterborne Inc. respectively. Spikes containing 100.43 (rsd 1.39) C. parvum oocysts and 100.26 (rsd 1.24) G. lamblia were prepared at the WI State Laboratory of Hygiene according to established protocols. Two laboratories both with LT2 approval status were awarded contracts to perform analyses - one laboratory performed method 1623 with filtration using the Envirochek HV, the other using the FiltaMax filtration system. Immediately prior to each time point (0, 15, 30, 58, 121 days), laboratories were each shipped 15 spiking suspensions for analyses. In addition to the spiking suspensions, the laboratories were shipped 15 tubes containing 0.2 g Tennessee River sediment. Sediment was added to the spiking suspensions immediately prior to seeding into 10 L reagent grade water and processing by EPA method 1623. Laboratories were not aware of the concentration of the organisms in the samples. All raw data was forwarded to CSC for analyses.

Additional samples were retained at the WSLH for quality assessment procedures. Membrane filter enumeration (n=3) was performed at each time point by filtering each sample through 0.8 um polycarbonate track-etched membrane filter, staining with fluoresceinated anti- Cryptosporidium and anti- Giardia antibodies, and enumerating under epifluorescent microscopy. Qualitative assessments described below were made using well slide evaluation procedures of standards concentrated by centrifugation and stained using method 1623 staining methodology. Cells were scored for fluorescence intensity using the 0 - 4 + scale (0 = not visible; 4 = brilliant green), morphology (0=atypical; 1=typical) general intactness (0= nonintact, 1= partially intact, 2=fully intact) and DAPI positivity (+ or -).

Results and Conclusions

Results of the method 1623 analyses in table 1 show that live, flow cytometer-enumerated spiking suspensions were stable throughout the entire 121 day test period in the laboratory using the Envirochek HV filter. Cryptosporidium and Giardia recoveries using the FiltaMax filter were no different from the time 0 point at the 15 day time point but by the 30 day time point recoveries were statistically lower. However, the C. parvum recovery at the 58 day time point was not statistically different from the 0 and 15 day time points using the FiltaMax filter.

Membrane filtration analysis confirms the stability of the Cryptosporidium and Giardia in live spiking suspensions showing recoveries of 97.91% and 99.08% respectively at day 120. Furthermore, these organisms retained their fluorescent intensity up to 120 days. While oocyst morphology began to become less typical at 120 days (dented or burst), most remained DAPI positive and DIC analyses indicated the oocysts were at least partially intact. Giardia, often considered the weaker of the two organisms, maintained its structure and integrity throughout the analysis period.

However, while the results indicated method 1623 recovery using Envirochek filters was not statistically different up to 121 days, WSLH staff chose to assign a shelf life of 6 weeks for live cytometer prepared spiking suspensions. This decision was based morphological observations at the 60 day time point.

Table 1. Summary Results for use of Live Flow-Cytometer Sorted Spiking Suspension with Two Different filtration options for Method 1623

Notes:

• Number of replicates for all tests was 15
• Mean recoveries in red are significantly different than mean recoveries at Time 0 based on Dunnett's Test with overall alpha =0.05
• Spike enumerations were prepared on August 4, 2003
• Analysis of samples at "Time 0" began two days after spike enumeration
• Number of days listed for the 4 holding times is the days from spike enumeration

Table 2. Percent recovery of cysts and oocysts filtered though membrane filter and stained with fluoresceinated antibodies

n=3

Table 3. Summary of cyst and oocyst characteristics during storage.

FITC scored 0-4+ criteria where 0 = not visible; 4 = brilliant green

Morphology scored 0 = atypical; 1=typical

DAPI scored + or -

DIC scored 0 - 2 where 0 = not intact; 1=partially intact; 2= intact

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