Table 2: Microbial Monitoring under GWDR: Analytical Methods (DRAFT 10/29/97)

Organism	Methods1	Sample volume	Commercial cost/ sample2	Remarks
Bacterial indicators of fecal contamination3
Total Coliforms4	Colilert (Method 9223B) Colisure (Method 9223B) Fermentation Technique (92221A,B,C) Membrane Filter Technique (9222A,B,C) Presence-Absence Coliform Test (9221D) Membrane Filter Method with MI Agar5	100 ml	$18	Several methods have already been approved under the Total Coliform Rule and/or the SWTR
E. coli	Colilert (Method 9223B) Colisure (Method 9223B) Membrane Filter Method with MI Agar5	100 ml (1L)6	$18 ($40)6	Good indicator of fecal contamination in non-tropical waters
Enterococcus	Multiple-Tube Tech. (Method 9230B) Membrane Filter Tech. (Method 9230C) Enterolert7	100 ml (1L)	$20 ($40)
Clostridium perfringens spores	MF Method for Clostridium perfringens in Water and Sediments8 Iron Milk Medium, Modified9	100 ml (1L)	$35 ($50) $20	Requires anaerobic incubation Incubation temperature at 42.5C May survive in environment much longer than most pathogens. Thus may not represent recent fecal contamination. Research underway to determine occurrence of C. perfringens in ground water (Abbaszadegan)
Coliphage indicators of fecal contamination3,10
Somatic phage	Coliphage Detection (Method 9211D), as modified by Sobsey and others	1L (100L)	$20-$100 ($200)	Research underway to evaluate utility of 1L samples, improve overall method, and to determine bacterial host range (Sobsey)
Male-specific phage	Future modification by Sobsey & Yates of existing methods	1L (100L)	$20-$100 ($200)	Research underway to evaluate utility of 1L samples and to improve method (Sobsey) Concern about utility of indicator for very small systems (<5% pop. shed these viruses)
Enteric Viral Pathogens11
Enteric viruses by tissue culture12	Method 9510		$600-$1,000
Enteroviruses by PCR	RT-PCR13	1L (100L-3,000L)	$250-$500 ($1,000)	Research underway to evaluate utility of 1L samples (Abbaszadegan)6 PCR used to amplify virus nucleic acid (regardless of whether from "live" viruses or not). It must be used in conjunction with techniques to collect samples and to visualize PCR results (e.g., tissue cultures).

¹ Unless otherwise specified, all methods are described in Standard Methods for the Examination of Water and Wastewater (19th ed).

² Excludes collection and shipping costs.

³ Fecal contamination is contamination by human or animal feces. A fecally contaminated water may or may not contain pathogens.

⁴ Total coliforms (TC) used to: (1) determine treatment efficiency, (2) assess integrity of the distribution system, and (3) screen for fresh fecal contamination. Thus, total coliforms are used to assess the vulnerability of a system to fecal contamination, not whether the system is contaminated by fresh fecal contamination. Fecal coliforms (FC) and E. coli are used to determine whether the system is contaminated by fresh fecal contamination. They are not used to assess system vulnerability because they are not as sensitive as total coliforms as a monitoring tool. FC are a small subset of the TC group, while E. coli is a large subset of the fecal coliform group.

⁵ Brenner, K.P., C.C. Rankin, Y.R. Roybal, G.N. Stelman, P.V. Scarpino, and A.P. Dufour. 1993. New medium for the simultaneous detection of total coliforms and Escherichia coli in water. Appl. Environ. Microbiol. 59:3534-3544. EPA is in the process of approving this method.^.

⁶ Values in parenthesis is associated with parenthesis in next column, i.e., a larger sample volume costs more. All costs are estimates and prices vary.

⁷Idexx Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092.

⁸EPA/ORD publication (March 1994), based on method of Bisson and Cabelli (1979), as modified by Armon and Payment (1988).

⁹ R.N. Atlas. Handbook of Microbiological Media. P. 458. CRC Press, Boca Raton.

¹⁰Coliphage are viruses that infect the bacterium, E. coli.

¹¹Enteric pathogens are those which cause damage in the alimentary tract, usually the intestines.

¹² A tissue culture for viruses will only allow the propagation of viruses that infect the cells used in the tissue culture. Method 9510 allows the propagation of polioviruses, coxsackieviruses, echoviruses, and some reoviruses, but not rotaviruses, Norwalk virus, and many other pathogenic viruses. Other tissue culture types will allow the propagation of HAV or rotaviruses, etc.

¹³Fout, S. et al. 1997. Water Sample Analysis by RT-PCR. EPA. In preparation.