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Membrane Filtration Regulations and Determination of Log Removal Value 157
Table 4.4
Potential microbiological surrogates for Cryptosporidium
Microorganism Size range (mm) Target organism Enumeration method
Micrococcus l. 7–12 Giardia Standard Methods (24)
Bacillus subtilis 1 Cryptosporidium Barbeau et al. (25)
Escherichia coli 1–4 Cryptosporidium Standard Methods (24)
Pseudomonas diminuta 0.3 Cryptosporidium Standard Methods (24)
Serratia marcescens 0.5 Cryptosporidium Standard Methods (24)
MS2 bacteriophage 0.01 Enteric virus Adams (26)
the size range cited in Table 4.4, B. subtilis could potentially be considered an ideal surrogate for
Cryptosporidium, pending a rigorous comparison of other characteristics (e.g., shape, surface
charge, etc.) between these two organisms. However, because of this same size range overlap,
B. subtilis could not be considered a conservative surrogate for Cryptosporidium.
The primary advantage of many microbial surrogates is that enumeration is fairly simple and
inexpensive, typically involving culturing the test organisms present in the feed and filtrate
samples. The ease with which these organisms can be cultured allows many to be grown in a
laboratory to produce a stock for use in challenge testing. Bacteria can be cultured to yield stock
concentrations in the range of 10 5 to 10 9 organisms per 100 mL, while MS2 bacteriophage can be
grown at concentrations in the range of 10 7 –10 12 organisms per 100 mL. Any microbial stock used
for the purpose of seeding during a challenge test should be enumerated prior to conducting the
challenge test to facilitate seeding at the target level.
2. Inert particles: Inert particles may also be used as a surrogate for Cryptosporidium under the
LT2ESWTR. For example, polystyrene latex microspheres (i.e., latex beads) have been used as a
surrogate for Cryptosporidium in a number of studies. Historically, microspheres have been used
in the calibration of particle counters and similar optical equipment in which a challenge particle
of a known size and geometry is required by the investigator. Microspheres can be manufactured
with very high particle uniformity and a smooth surface, both of which are important considerations
when selecting a conservative surrogate. Microspheres are chemically inert, easy to handle,
and relatively inexpensive. Furthermore, microspheres without a significant surface charge can be
produced to minimize the potential for adsorption and interaction with either other particles or the
membrane surface. Microspheres are also readily available with particle concentrations ranging
from 10 7 to 10 9 particles per mL.
The primary difficulty associated with the use of microspheres is particulate enumeration.
Although particle counting is a simple means of enumeration, this technique may not meet the rule
requirement that the challenge particulate be discretely quantified as a result of the potential for
background particles other than the microspheres to affect the results. Furthermore, other problems
such as coincidence error and the dynamic range of most particle counting instruments may
also skew the results. Any clumping of microspheres may also complicate particulate enumeration.
A more reliable, albeit more expensive, means of enumerating microspheres is through
capture (normally on a laboratory-grade membrane filter) and direct examination. The use of
fluorescent microspheres is recommended to facilitate particulate identification. Methods for
microscopic analysis of fluorescent microspheres are reported in the literature (27, 28).
The appropriateness of microspheres as a surrogate for Cryptosporidium could be directly
verified through a comparative study; however, microspheres that meet certain criteria might be
deemed conservative surrogates that would not require direct verification. For example, neutral,
spherical-shaped microspheres with a maximum diameter of 1 mm and which are completely