ELAP-MAIN OFFICE ELAP LOS ANGELES FIELD OFFICE ELAP SACRAMENTO OFFICE

DEPARTMENT OF HEALTH SERVICES DEPARTMENT OF HEALTH SERVICES DEPARTMENT OF HEALTH SERVICES

1625 SHATTUCK AVENUE, RM. 101 1449 EST TEMPLE STREET, RM 2!1601 NORTH SEVENTH, MS 92
"ERKELE#, CA 94$09-1611 LOS ANGELES, CA 90026-569% PO "O& 942$!2
TEL '510( 540-2%00 TEL '21!( 5%0-5$!1 SACRAMENTO, CA 942!4
FA& '510( %49-5106 FA& '21!( 5%0-5$06 TEL '916( !2!-4$6$
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ENVIRONMENTAL LA"ORATOR# ACCREDITATION PROGRAM
CHECKLIST FOR LA"ORATOR# ON-SITE INSPECTION
LA"ORATOR# INFORMATION
Laboratory Name ____________________________________________________________________________
ELAP Reference No ___________________ Certificate No __________________ Expiration Date ____________
Telephone No ____________________________________ FAX No ___________________________________
Laboratory Director __________________________________________________________________________
Contact Person and Title ______________________________________________________________________
DEPARTMENT INFORMATION
nspector Name _____________________________________________________________________________
!i"nat#re of nspector _______________________________________________ Date ____________________
nspection Type$ % & Pre'certification nspection
% & nitial Certification nspection
% & Rene(al Certification nspection
% & Post'Certification nspection
% & Follo('#p nspection
% & )ther %specify& ________________________________________
LA"ORATOR# CONFIRMATION OF INSPECTION
This is to confirm that an on'site inspection (as cond#cted on this date at the laboratory* !i"nat#re does not
necessarily constit#te a"reement (ith the findin"s of the inspector*
________________________________________________________________ ________________________
!i"nat#re of Contact Person Date
+ay ,-. ,//0 Pa"e 1 of 2,
ELAP +CR)3)L)45 C6EC7L!T
INSTRUCTIONS FOR FILLING OUT ELAP MICRO"IOLOG# LA"ORATOR# EVALUATION CHECKLIST
A. DESCRIPTION OF COLUMNS*
1* +L,-./ E0,1.2 8 Laboratory #ses this col#mn for self'e9al#ation and to indicate (hether or not it is in
compliance (ith the re:#irement #nder ;TE+*<
,* +ITEM2 describes the re:#irement*
2* +ELAP E0,1.2 8 ELAP #ses this col#mn for its e9al#ation*
0* +REFERENCES ,34 COMMENTS2 col#mn lists the citation%s& for the re:#irements* The space for
+COMMENTS2 is reser9ed for ELAP a#ditors*
========================================================================================
". INSTRUCTIONS FOR THE LA"ORATOR#*
1* The laboratory records its self'e9al#ation in the ;L,-./ E0,1*< col#mn* This is done prior to ELAP>s site 9isit*
T56 789:16;64 7567<1=/; =/ >6;?>364 ;8 ;56 ELAP ,?4=;8>.
,* ?nder ;ITEMS.< the laboratory ;chec@s< applicable items or fills in information (here instructed in italics*
C. MARKING THE CHECKLIST*
1* ?se +C567<9,><< to indicate that laboratory is in compliance*
,* ?se +&2 to indicate that laboratory is not in compliance*
2* ?se +NA2 to indicate that the re:#irement is not applicable to the laboratory*
D. SECTIONS TO COMPLETE*
Pa"e -$ Laboratory E:#ipment
Pa"e A$ Rea"ent 4rade Bater. +icrobiolo"ical Bater !#itability Test. nhibitory Resid#eTest %lab deter"ent&
Pa"es C to 1C %(here applicable&$ D#ality Ass#ranceED#ality Control and 4eneral +icro +ethods
M=7>8-=818@=7,1 M6;584/ 8 Complete only those microbiolo"ical methods for (hich lab is certified or see@in"
certification
E* ,*/ 6eterotrophic Plate Co#nt
E* 2*/ Fermentation +ethods for Total Coliforms %+#ltiple T#be Fermentation. Presence'Absence&
E* 0*/ Fermentation +ethods for Fecal Coliforms %EC. A'1&
E* -*/ EnFyme !#bstrate +ethods for Coliforms %ECG+?4. Colilert. Colilert 1H. Colis#re.EIColite&
E* A*/ +embrane Filtration %mEndoEmEndo LE!. mFC. NAG+?4&
E* C*/ +embrane Filtration %m'Colibl#e,0. +&
E* H*/ +embrane Filtration for Enterococcal +ethods %m'Enterococc#s. mE. mE&
E* J*/ +ost Probable N#mbers for Enterococcal +ethods %AFide Dextrose. Enterolert&
========================================================================================
E* REFERENCE L!T AND T)TAL C)LF)R+ R?LE$
Pa"e 2 lists the references cited in the Chec@list
Pa"e 0 describes the re:#irements of the Total Coliform R#le*
+ay ,-. ,//0 Pa"e , of 2,
ELAP +CR)3)L)45 C6EC7L!T
REFERENCES A8> CITATIONS$
1& !tandard +ethods for the Analysis of Bater and Baste(ater. 1H
th
. 1J
th
. and ,/
th
ed*. AP6A. ABBA. BEF*
,& +an#al for the Certification of Laboratories AnalyFin" Drin@in" Bater. EPA H1-'3'JC'//1. +arch 1JJC*
2& Code of Federal Re"#lations. Kol* 0/. ,//1l
0& A3 011 8 Recommended +ethods for the Analysis of Recreational +arine Bater
%(((*dhs*ca*"o9EpsEdd(emEbeachesEab011_methods*htm&*
-& +an#al for the Certification of Laboratories AnalyFin" Drin@in" Bater. Critical Elements for +icrobiolo"y %Draft
1,E/1E/1&
A& Prod#ct nserts 8
DEXX Laboratories. nc*. )ne DEXX Dri9e. Bestbroo@. +aine /0/J, %Colilert
L
. Colilert
L
1H. Colis#re.
Enterolert&
6ach Company. 1// Dayton A9en#e. Ames. A -//1/ %m'Coli3l#e,0
L
&
Charm !ciences. nc*. 2A Fran@lin !treet. +alden. +A /,10H'01,/ %EIColite
L
&
C& EPA +embrane Filter +ethod for the !im#ltaneo#s Detection of Total Coliforms and Escherichia coli in Drin@in"
Bater. EPA A//'R'//'/12. Febr#ary ,///*
H& California Code of Re"#lations. Title ,,. Di9ision 0. Chapter 1J
+ay ,-. ,//0 Pa"e 2 of 2,
ELAP +CR)3)L)45 C6EC7L!T
TOTAL COLIFORM RULE 'TCR( A8> DRINKING ATER
REFERENCES
R6B?=>6963;/ , A7;=83, R6/:83/6
'2( EPA %15-"-9$-001
'!( CFR 141.21
1* )nly analytical methods specified in Total Coliform R#le %0CFR 101*,1%f&
and !#rface Bater Treatment R#le %0/CFR 101*C0%a& #sed for compliance
p#rposes (ith drin@in" (ater*
%,& K*-*1*1
,* Laboratory is certified for all analytical methods it #ses for compliance
p#rposes*
%,& K*-*1*,
2* All total coliform positi9e drin@in" (ater samples tested for presence of either
fecal coliforms or E. coli*
%,& K*J*1
0* Total Coliform positi9e res#lts are based on confirmed phase for +TF
techni:#e and P'A 3roth coliform tests*
%,& K*J*,
-* Total Coliform positi9e res#lt for +F techni:#e based on 9erification test*
%,& K*J*,
A* Laboratory promptly notifies proper a#thority of positi9e total coliform. fecal
coliform. or E. coli res#lts* Notification record @ept*
%,& K*J*,
C* n9alidation of Total Coliform 8 Positi9e !ample
Applies only if conditions listed in CFR 101*,1%c&%1&%i&.%ii&. or %iii& are met*
%2& CFR 101*,1%c&%1&
H* n9alidation of Total Coliform 8 Ne"ati9e !ample
Applies to Fermentation broths %LT3 and P'A& that are t#rbid (itho#t "as or acid
or membrane filters that sho( confl#ent "ro(th or are TNTC (ith no coliforms
detected*
Notify s#pplier promptly*
Replacement sample (ithin ,0 ho#rs*
%,& K*J*2M %2& CFR
101*,1%c&%,&
+ay ,-. ,//0 Pa"e 0 of 2,
ELAP +CR)3)L)45 C6EC7L!T
Laboratories are to complete this page before site visit$
LA"ORATOR# ECUIPMENT
ECUIPMENT MANUFACTURER MODEL
DATE LAST
MAINTENANCE
p6 +eter
3alance%s&
Cond#cti9ity +eter
nc#bator%s&
Bater 3ath%s&
Refri"erator%s&
A#tocla9e%s&
6ot Air )9en
Colony Co#nter
?ltra9iolet Li"ht ,-0 nm
?ltra9iolet Li"ht 2A-'2AA nm
1/'1-x !tereo +icroscope %+F&
Li"ht +icroscope
REFERENCE THERMOMETERS %Recommend re-calibration every 3 years EPA %15-"-9$-001, V.!.!.!(
M,3?A,7;?>6> ,34 I463;=A=7,;=83 N8. R,3@6 ,34 C,1=->,;=83 P8=3;/
D,;6 8A L,/;
C6>;=A=7,;=83
+ay ,-. ,//0 Pa"e - of 2,
ELAP +CR)3)L)45 C6EC7L!T
REAGENT GRADE ATER
ELAP
E0,1
1( SM 9020".!.7D 9050".1
'2( EPA %15-"-9$-001,V.4.!
S8?>76 8A >6,@63; @>,46 E,;6>*
Lab prepared$_________________________________________
Commercially prepared %mf"&$ ____________________________
CC
C?,1=;F 8A >6,@63; E,;6> =/ ;6/;64 ,34 966;/ ;56 A8118E=3@ 7>=;6>=,. '1,- 8>
78996>7=,11F :>6:,>64(
T6/;64 983;51F:
Cond#cti9ityNNNNO,micromhosEcm %microsiemensEcm& at ,-
o
C*
Total Chlorine resid#al NNNNNNNNO/*1 m"EL %DPD method&
6eterotrophic plate co#ntNNNNN**O-//Eml %po#r plate method&
T6/;64 ,33?,11F:
Pb. Cd. Cr. C#. Ni. PnNNNNN***Not "reater than /*/- m"EL per
contaminant. and collecti9ely no "reater than /*1 m"EL total*
3acteriolo"ical :#ality of rea"ent (aterNNNN*Nratio /*H to 2*/
(Test not required for ASTM Types 1 and Type 2 reagent water SM
1080)


%1& Table J/,/$M %,&
M=7>8-=818@=7,1 ,;6> S?=;,-=1=;F T6/;
Tested by %lab&$ _________________________________________
Analyst$ ______________________________________________
Date Tested$_______________________
Res#lts$ R,;=8 of Lab>s Bater %colony co#ntEml& = _____________
Control Bater %colony co#ntEml&
INHI"ITOR# RESIDUE TEST 'A8> E,/5=3@ 789:8?34(
%1& !+ J/,/3
%,& EPA H1-'3'JC'//1.K*0*-
CC
4lass(are inhibitory resid#e test is performed on initial #se of (ashin"
compo#nd. (hen there is a chan"e in the form#lation #sed. or a chan"e in the
method of (ashin" of re#sable c#lt#re t#bes. pipets. flas@s. "rad#ated cylinders.
bea@ers. blenders. f#nnels. etc*
Compo#nd _______________ +f"* _________________ Lot _____________

Tested by %lab& _____________________ Analyst ______________________
Date _____________ and Res#lts of most recent inhibitory resid#e test$
3 8 A x 1// = __________Q %Q difference bet(een ro#tinely rinsed petri
3 dishes and controls&
3 8 C x 1// = __________Q %Q difference bet(een #nrinsed petri dishes
3 and controls*&
3 8 D x 1// = __________Q %Q difference bet(een presteriliFed
3 disposable petri dishes and controls&
A 8 C x 1// = __________Q %Q difference bet(een #nrinsed petri dishes
A and ro#tinely rinsed petri dishes&
%1& J/,/3*2*a*1&M %,&
+ay ,-. ,//0 Pa"e A of 2,
ELAP +CR)3)L)45 C6EC7L!T
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ITEM
ELAP
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REFERENCES
COMMENTS
A. LA"ORATOR# RECORDS
'1( SM 9020"
'2( EPA %15-"-9$-001
'%( CCR T=;16 22, D=0.4, C5 19
1. C?,1=;F A//?>,376 'CA( P1,3
%,& EPA H1-'3'JC'//1.K*C
%H& Art*H. A0H1-
a* Britten DA Plan prepared. follo(ed. and a9ailable for inspection*
%,&
2. R678>4/ ,34 D,;, R6:8>;=3@
%,& EPA H1-'3'JC'//1.K*H
a* !ample Collection nformation
dentification %n#mber&
!o#rce %location collected&
Name of (ater system
Date collected
Time collected
Name of collector %initial&
Chlorine resid#al %if any&
!ample type 8 DB. BB. s#rface. process. ro#tine. repeat. etc*
%1& J/,/3*-
%,& K*A*-
b* !ample Transport and Condition
Name of person transportin" sample %if not sampler&
Name of person recei9in" sample
Date recei9ed
Time Recei9ed
6oldin" time met %collection to testin"&
2/ hrs* drin@in" (ater
H hrs* s#rface (ater. recreational (ater and 6PC %O1/C for
s#rface (ater&
A hrs* (aste (ater
De9iations noted and recorded*
%1& J/,/3%-&
%,& K*H*2
c* Analytical Records
!ample identification
Analytical method #sed
Date testin" started
Time testin" started
Analytical res#lts recorded
Name of analyst
Date reported
%1& J/,/3%-&
%,& K*H*0
d* +aintenance of Records$
Records of microbiolo"ical analyses @ept by or accessible to
laboratory for at least - years*
Client (ater system notified before disposal of records*
Pre9enti9e maintenance and repair records @ept for - years*
%1& J/,/3*-
%,& K*H*,. K*H*-
%H& Art* C. A0H12
e* All data recorded in in@ (ith chan"es lined thro#"h s#ch that ori"inal
entry 9isible* Chan"es initialed and dated*
%,& K*H*2. K.H*0
+ay ,-. ,//0 Pa"e C of 2,
ELAP +CR)3)L)45 C6EC7L!T
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REFERENCES
COMMENTS
". FACILITIES AND ORK AREA
'1( SM 9020"
'2( EPA %15-"-9$-001,V.2
'$( CCR T=;16 22, D=0.4, C5 19
1* Facilities
Clean
Controlled temperat#re and h#midity
Ade:#ate li"htin" and 9entilation
3ench top is non'poro#s. easily cleaned and disinfected
%1& J/,/3*1*aM J/,/3M %,&
%C& Art* C. A0H12
,* Bor@ Area
Ade:#ate bench space for pea@ (or@ periods %, metersEanalyst&
!#fficient space 8 e:#ipment. sample processin". media prep.
steriliFation. "lass(are (ashin". and stora"e of media and
s#pplies
%1& J/,/3*1*cM J/,/3*1*bM %,&
2* Pro9isions are made for disposal of microbiolo"ical (astes*
1/J/D*,*b*,&M %,&
C. LA"ORATOR# ECUIPMENT
1. :H METER
'1( SM 9020", 90!0"
'2( EPA %15-"-9$-001 ,V.!.1
a* Acc#racy and scale "rad#ations (ithin /*1#nit*
%1& J/2/3*AM %,&
b* Electrodes maintained as per man#fact#rer>s instr#ctions*
%,&
CC
c* !tandardiFed each day of #se (ith t(o b#ffers %0 R C or C R 1/& that
brac@et p6 meas#red* D,;6 ,34 -?AA6>/ ?/64 >678>464*
%1& J/,/3*,*cM %,&
CC
d* Commercial b#ffer sol#tions labeled (ith 4,;6 8A >676=:; and 4,;6
8:6364* !ol#tions discarded (hen expired*
%1& J/,/3*,*cM %,&
e* 3#ffer ali:#ots #sed only once*
%,&
2. "ALANCE ';8: 18,46> 8> :,3(
'1( SM 9020".2.-D 90!0".$
'2( EPA %15-"-9$-001, V.!.2
a* 3alance has readability of /*1 "*
%1& %,&
b* 3alances sho#ld detect 1// m" (ith 1-/ " load*
%1& %,&
CC
c* 3alance 7,1=->,;64 983;51F #sin" A!T+ type 1. ,. or 2 (ei"hts
and the data recorded* %minim#m of 2 traceable (ei"hts that brac@et
laboratory>s (ei"hin" needs& D,;, >678>464*
%1& J/,/3*2*bM %,&
CC
d* Non'reference (ei"hts %if #sed& 7,1=->,;64 606>F /=G 983;5/ #sin"
reference (ei"hts. and the 4,;, >678>464*
%1& %,&
CC
e* Reference (ei"hts re'certified if dama"ed or corroded*
%,&
CC
f* 3alance maintained thro#"h ser9ice contract or internal
maintenance protocol* (annually at minimum&
%1& J/,/3*,M %,&
+ay ,-. ,//0 Pa"e H of 2,
ELAP +CR)3)L)45 C6EC7L!T
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REFERENCES
COMMENTS
!. CONDUCTIVIT# METER
'1( SM 2510A
'2( EPA %15-"-9$-001, V.!.%
a* !#itable for chec@in" laboratory rea"ent (ater and readable in
micromhosEcm or microsiemensEcm
%1& %,&
CC
b* Cell constant determined monthly #sin" a method indicated in !+
,-1/ or by #sin" a lo( le9el standard* Data recorded*
%1& %,&
c* f an in'line #nit cannot be calibrated. it sho#ld not be #sed to chec@
rea"ent "rade (ater*
%,&
4. TEMPERATURE MEASURING DEVICES
'1( SM 9020".2.,
'2( EPA %15-"-9$-001,V.!.!D
V.!.9
a* Temperat#re monitorin" de9ices "rad#ated in increments
appropriate for #se and immersed in li:#id %except for electronic
thermometers&
/*,
o
C for tests inc#bated at 00*-

/*,
o
C
/*-
o
C for tests inc#bated at 2-*/ /*-
o
C
1*/
o
C for refri"erators
%1& %,&
%,& K*2*J
b* No separation in the fl#id col#mn of thermometer*
%,&
c* Dial thermometers that cannot be adS#sted are not #sed*
%,&
CC
d* Thermometers are calibrated a"ainst reference N!T thermometer
or one meetin" the re:#irements of N3! +ono"raph !P ,-/',2*
Thermometer is calibrated at the temperatures used.
4lass %ann#ally&
Electronic %ann#ally&
Dial %:#arterly&
%1& %,&
CC
e* Thermometers labeled (ith date of calibration and applicable
correction factor* D,;, >678>464*
%1& %,&
f* Thermometer discarded if off more than 1
o
C from reference
thermometer*
%,&
"* Contin#o#s recordin" de9ices that are #sed to monitor inc#bator
temperat#re re'calibrated ann#ally as abo9e*
%,&
5. INCU"ATOR'S(
'1( SM 9020".2.:D 92!0".1
'2( EPA %15-"-9$-001,V.!.4
a* !#fficient siFe for (or@load*
EPA DB Lab Cert* for +icro
Co#rse +an#al 2EJC
b* nc#bator #nit has an internal temperat#re monitorin" de9ice and
maintains temperat#re at 2- /*-C*
%1& J/2/3*1M %,&

c* Lar"e inc#bators ha9e thermometers placed on top and bottom
shelf*
%1& J/2/3*1M %,&
CC
d* Corrected temperat#re recorded t(ice each day of #se. (ith
readin"s at least 0 ho#rs apart*
%1& %,&
+ay ,-. ,//0 Pa"e J of 2,
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REFERENCES
COMMENTS
e* Al#min#m bloc@ inc#bators 8 t#bes fit sn#"ly and co9er le9el of
li:#id in t#be*
%,&
6. ATER "ATH
'1( SM 9020".2.:D 92!0".1
'2( EPA %15-"-9$-001,V.!.4
a* !#fficient siFe for (or@load*
EPA DB Lab Cert* for +icro
Co#rse +an#al 2EJC
b* E:#ipped (ith (ater circ#lator and a "abled co9er*
%1& J/2/3*1M %,&
c* Bater bath has an internal temperat#re monitorin" de9ice and
maintains temperat#re at 00*- /*,C*
%1& J/2/3*1M %,&
CC
d* Corrected temperat#re recorded t(ice each day of #se. (ith
readin"s at least 0 ho#rs apart*
%1& %,&
$. REFRIGERATOR
'1( SM 9020".2.HD 92!0".11
'2( EPA %15-"-9$-001,V.!.9
a* Refri"erator temperat#re maintained at 1
o
to -
o
C*
%1& J/2/3*11M %,&
CC
b* Temperat#re recorded at least once for days in #se* %(ee@ends
excepted&
%1& J/,/3*,*SM %,&
%. AUTOCLAVE
'1( SM 9020"D 90!0"D 9050A
'2( EPA %15-"-9$-001,V.!.5
a* !iFe is s#fficient for (or@load*
%1& J/2/3*2
b* Press#re coo@ers are not #sed*
%,&
c* A#tocla9e has internal heat so#rce. temperat#re "a#"e (ith sensor
in exha#st. press#re "a#"e. and operational safety 9al9e*
%1& J/2/3*2M %,&
d* +aintains steriliFation temperat#re d#rin" cycle and completes
entire cycle (ithin 0- min#tes (hen 1, to 1- min#te steriliFation
period is #sed*
%1& J/-/A*2M %,&
CC
e* A#tomatic timin" de9ice is chec@ed :#arterly for acc#racy (ith
another acc#rate timin" de9ice*
%,&
CC
f* +axim#m temperat#re re"isterin" de9ice #sed at least (ee@ly to
chec@ the steriliFation temperat#re of 1,1 G,
o
C*
%1& J/,/3*,*iM %,&
"* 6eat indicatin" tape #sed to identify items that ha9e been steriliFed*
%1& J/,/3*,*i
CC
h* !pore strips or amp#les #sed monthly as bioindicators to confirm
steriliFation
%1& J/,/3*,*iM %,&
i* Ro#tine maintenance performed. incl#din" cleanin" of drain screen
and the door seals*
%,&
S* A#tocla9e is ser9iced ann#ally or as needed by :#alified technician*
Records maintained*
%1& J/,/3*,M %,&
+ay ,-. ,//0 Pa"e 1/ of 2,
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REFERENCES
COMMENTS
CC
@* A#tocla9e records are maintained*
date
contents
time in and time o#t
total expos#reEcycle time
steriliFation time
steriliFation temperat#re
initial of analyst or technician
%1& J/,/3*,*iM %,&
9. HOT AIR OVEN
'1( SM 9020".2.5D 92!0".2
'2( EPA %15-"-9$-01,V.!.6
a* +aintains steriliFation temperat#re of 1C/
o
to 1H/
o
C at least , hrs*
%1& %,&
b* )9en thermometer "rad#ated in 1/
o
C increments or less (ith b#lb
immersed in sand d#rin" #se*
%,&
CC
c* !pore strip #sed monthly to chec@ effecti9eness of the steriliFation
process*
%1& J/,/3*,*hM %,&
CC
d* 6ot air o9en record maintained$
date
contents
steriliFation time
steriliFation temperat#re
initial of analyst or technician
%,&
10. COLON# COUNTER
%1& !+ J,1-A*H
%,& EPA H1-'3'JC'//1.K*2*C
a* A dar@ field colony co#nter #sed to co#nt 6eterotrophic Plate Co#nt
colonies*
%1&%,&
11. ULTRAVIOLET LAMPS
'1( SM 9020".2.9
'2( EPA %15-"-9$-001,V.!.1$
CC
a* ?K Lamp 8 ,-0 nm %membrane filtration&
?nit is disconnected monthly and cleaned by (ipin" (ith soft
cloth moistened (ith ethanol*
f #sed for sanitiFation. #nit is tested :#arterly (ith ?K li"ht
meter or by a"ar spread plate method*
%1& %,&
b* ?K lamp %,-0 nm& replaced if o#tp#t %meter& is OC/Q of initial o#tp#t
or if , min#tes expos#re of plates containin" ,// to ,-/
microor"anisms to ?K do not sho( co#nt red#ction of JJQ*
%1& %,&

12. MEM"RANE FILTRATION 'MF( ECUIPMENT
'1( SM 9020"D 90!0"D 9222"
'2( EPA %15-"-9$-001
a* +F #nits made of stainless steel. "lass. or a#tocla9able plastic and
not scratched or corroded and do not lea@*
%1& !+ J/2/3*1-M %,& K*2*11*1
b* +embrane filtration e:#ipment a#tocla9ed before be"innin" of first
filtration series* %Filtration series ends (hen 2/ min* or lon"er elapse
after a sample is filtered*&
%1& J/,/3*,*lM J,,,3*-M %,&
K*0*1*2
+ay ,-. ,//0 Pa"e 11 of 2,
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c* 4rad#ations on f#nnel. if #sed to meas#re sample 9ol#me. are
chec@ed for acc#racy and ha9e tolerance of T,*-Q* Record of
calibration chec@ retained*
%,& K*2*11*,
d* Filters appro9ed by man#fact#rer for total coliform (ater analysis*
Filters made of cell#lose ester. (hite. "rid mar@ed. 0C mm
diameter. and /*0- Um pore siFe*
Lot n#mber and date recei9ed recorded for membrane filters*
%1& J,,,3*1*"M %,& K*2*11*0
e* Absorbent pads. if #sed. of s#fficient thic@ness to absorb 1*H to ,*,
mL media #sed*
%1& J,,,3*1*hM %,&
f* +embrane filters and pads p#rchased$
pre'steriliFed
a#tocla9ed 1/ min#tes at 1,1C before #se*
%1& J,,,3*1*"M %,& K*2*11*0
"* 1/x to 1-x stereo microscope (ith fl#orescent li"ht so#rce m#st be
#sed to co#nt sheen colonies*
%1& !+ J/2/3*-*bM %,& K*2*11*2
1!. INOCULATING ECUIPMENT
'1( SM 90!0".1$
'2( EPA %15-"-9$-001,V.!.10
a* Chec inocu!ating equip"ent used#
% & +etal loops or needles of nic@el alloy or platin#m*
Nicel loops!needles not used for o"idase test.
% & Plastic loops or needles*
% & Bood applicator stic@s steriliFed by dry heat*
% & !(ab %membrane filtration&
% & Disposable plastic pipet tips*
14. PIPETS
'1( SM 90!0".9
'2( EPA %15-"-9$-001,V.!.1!
a* Disposable "lass or plastic pipets #sed$
)pen pac@s resealed bet(een #se periods*
%1& %,&
b* Re#sable pipets #sed$
!tainless steel or al#min#m canisters #sed to steriliFe and
maintain sterility of "lass re#sable pipets*
ndi9id#al pipets (rapped in char'resistant paper or al#min#m
foil*
%,&
c* Pipets m#st ha9e le"ible mar@in"s. not chipped or etched*
%1&%,&
d* Pipets deli9erin" 9ol#mes of 1/ ml acc#rate (ithin ,*-Q*
%1&%,&
e* +icropipettors #sed (ith sterile tips. calibrated ann#ally and
replaced if tolerance "reater than ,*-Q
%,&
15. PETRI DISHES
'1( SM 90!0".14
'2( EPA %15-"-9$-001,V.!.12
a* Pre'steriliFed plastic dishes
)pened pac@a"es of plastic dishes re'sealed after #se to
maintain sterility*
%1&%,&
+ay ,-. ,//0 Pa"e 1, of 2,
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b* Re#sable "lass dishes #sed$
4lass dishes stored in stainless or al#min#m canisters
Brapped in hea9y al#min#m foil or char'resistant paper
%1&%,&
16. CULTURE TU"ES
'1( SM 90!0".1!
'2( EPA %15-"-9$-001,V.!.14
a* T#bes made of borosilicate "lass or other corrosion'resistant "lass
or plastic*
%1&%,&
b* T#be clos#res of stainless steel. al#min#m. or plastic slip caps or
scre('caps (ith non'toxic liners* Cotton pl#"s not #sed*
%1&%,&
c* C#lt#re t#bes and containers of s#fficient siFe to contain medi#m
pl#s sample (itho#t bein" more than V f#ll*
%,&
1$. MISCELLANEOUS GLASSARE I PLASTICARE
'1( SM 90!0".1!
'2( EPA %15-"-9$-001,V.!.16
a* +iscellaneo#s "lass(are made of borosilicate "lass or other
corrosi9e'resistant "lass. free of chips and crac@s. (ith mar@in"s
le"ible*
%1&%,&
b* Plastic items clear and non'toxic to microor"anisms*
%,&
CC
c* Kol#me of "rad#ated cylinders 9alidated to ha9e T,*-Q tolerance
%1&%,&
CC
d* Ne( lots of pre'calibrated containers 9alidated to ha9e ,*-Q
tolerance* %e*"* one sample container selected randomly&
%,&
1%. SAMPLE CONTAINERS
'1( SM 90!0".1%
'2( EPA %15-"-9$-001,V.!.15
,. Chec type of container used*
% & Bide'mo#thed plastic bottlesM caps (ith non'toxic liners*
% & Non'corrosi9e "lass bottlesM caps (ith non'toxic liners or non'
lea@in" "lass stoppers* 4lass stoppers co9ered (ith al#min#m foil
or char'resistant paper for steriliFation*
% & !terile lea@'proof ba"s
%1&%,&
-. !#fficient sodi#m thios#lfate added to sample containers before
steriliFation. if laboratory analyFes chlorinated (ater*
%1& J/A/A*,M %,&
7. Capacity of container at least 1,/ ml %0 oF& (ith ample air space
%1 inch or ,*- cm& to facilitate mixin"*
%1&%,&
4. Empty containers moistened (ith se9eral drops of (ater before
a#tocla9in" to pre9ent ;air loc@< steriliFation fail#re*
%,&
CC
6. At least one bottle from each batch or lot of sample containers is
tested for sterility by addin" ,- ml of a sterile non'selecti9e broth.
inc#batin" at 2- /*-C for ,0 hrs and chec@in" for "ro(th*
%,& EPA H1-'3'JC'//1. K*0*,
+ay ,-. ,//0 Pa"e 12 of 2,
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D. GENERAL LA"ORATOR# PRACTICES
1. STERILIJATION TIMES AND PROCEDURES
'1( SM T,-16 9020*III, 9222E.2.,
'2( EPA %15-"-9$-001, V.4.1
a* Re:#ired times for a#tocla9in" material at 1,1
o
C bein" obser9ed*
+embrane filters and pads NNN1/ min*
Carbohydrate containin" media *** 1, 8 1- min*
P'A brothNN*1, min*
A'1 broth NN*1/ min*
%1& !+ Table J/,/$. J,,,E*,*a.
J,,1D*1*aM %,&
b* Total a#tocla9e expos#re time bein" obser9ed*
A#tocla9ed media. filters. and pads remo9ed immediately after
completion of steriliFation cycle*
0- min#tes for carbohydrate containin" media*
2/ min#tes for Presence'Absence broth*
%1& !+ J,,1D*1aM %,&
c* +inim#m times for a#tocla9in" material at 1,1
o
C* Expos#re time at
this temperat#re may re:#ire adS#stment dependin" #pon 9ol#mes.
containers. and loads*
Contaminated test materials NNN2/ min*
+embrane filter assemblies NNN*1- min*
!ample collection bottles NNNN**1- min*
ndi9id#al "lass(are NNNNNN**1- min*
Dil#tion (ater blan@ NNNNNNN1- min*
Rinse (ater %/*- 8 1 L NNNNNN1-'2/ min*
%1&%,&
2. REAGENT GRADE ATER
'1( SM 9020".!.7D 9050".1
'2( EPA %15-"-9$-001,V.4.!
a* )nly satisfactorily tested rea"ent (ater from stills or deioniFation
#nits #sed to prepare media. rea"ents and dil#tionErinse (ater*
%1& J/-/3*1M %,&
b* 6PC and cond#cti9ity performed monthly on commercially prepared
(ater* %as is re:#ired for (ater prepared by laboratory&
%1& %,&
!. DILUTIONKRINSE ATER
'1( SM 9020"
'2( EPA %15-"-9$-001,V.4.4
a* !toc@ b#ffered (ater or peptone (ater 8 prepared as specified in
!tandard +ethods. !ection J/-/C*
%1& J/-/C*1M %,&
b* !toc@ b#ffers a#tocla9ed or filter'steriliFed and the containers
labeled. dated and refri"erated*
%,&
c* !tored stoc@ b#ffer free of t#rbidity*
%,&
CC
d* Each batch of dil#tionErinse (ater chec@ed for sterility by addin" -/
ml of (ater to -/ ml do#ble stren"th non'selecti9e broth. inc#batin"
at 2- G /*-
o
C for ,0 hrs. and chec@in" for absence of "ro(th*
%1& J/,/3*0*a*,&M %,&
4. GLASSARE ASHING
'1( SM 9020", 9040
'2( EPA %15-"-9$-001,V.4.5
a* Distilled or deioniFed (ater #sed for final rinse*
%1& J/0/M %,&
CC
b* 3atches of dry "lass(are spot'chec@ed for p6 reaction*
%1& J/,/3*2*a*1&M %,&
+ay ,-. ,//0 Pa"e 10 of 2,
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5. MEDIA
'1( SM 9020", 9050A,C
'2( EPA %15-"-9$-001,V.5.1.$
a* Dehydrated or prepared media man#fact#red commercially #sed*
strongly recommended
%1& J/-/CM %,&
b* Date recei9ed and date opened clearly mar@ed on dehydrated and
commercially prepared media*
%1& J/,/3*2*hM %,&
7. Dehydrated media in cool dry location at O2/
o
* Ca@ed or discolored
dehydrated media discarded*
%1& J/,/3*2*hM %,&
CC
d* Records for laboratory prepared media incl#de$
Date of preparation
7ind of medi#m
+an#fact#rer and lot n#mber
!teriliFation time and temperat#re
Final p6
Technician>s initial
%1& J/,/3*2*h*-&M %,&
CC
e* Records for li:#id media prepared commercially incl#de$
Date recei9ed
7ind of medi#m
+an#fact#rer and lot n#mber
p6 9erification for each batch
+edia discarded by man#fact#rer>s expiration date
%1& J/,/3*2*hM %,&
CC
f* Each ne( lot of dehydrated and prepared commercial medi#m
chec@ed before #se (ith positi9e and ne"ati9e control c#lt#res*
Res#lts recorded*
%1& J/,/3*2*h*-M 0*a*%2&
%,&
CC
"* Each ne( batch of laboratory prepared media chec@ed for sterility
and (ith positi9e and ne"ati9e controls* Res#lts recorded*
%1& J/,/3*2*h*-
h* Prepared media labeled (ith identification and preparation or
expiration date*
%,& K*-*1*C*-
i* +edi#m discarded if e9aporation exceeds 1/Q of ori"inal 9ol#me*
%,& K*-*2*,*0
H. Fermentation media. if refri"erated. is inc#bated at room
temperat#re o9erni"ht before #se* +edia (ith "ro(th or "as b#bbles
discarded* Refrigeration of fermentation media not recommended.
%1& J/,/3*2*h*0M %,&
5.1 STORAGE AND HOLDING TIMES OF PREPARED MEDIA
'1( SM 9050A
'2( EPA %15-"-9$-001,V.5.1.$
a* 3roth media in loose'cap t#be stored in dar@ at O2/C and held no
lon"er than , (ee@s*
%1& %,& K*-*1*CM K*-*2*,M K*-*-*2
b* 3roth media in scre('cap t#bes stored in dar@ at O2/C and held no
lon"er than 2 months*
%1&M %,& K*-*1*CM K*-*2*,M K*-*-*2M
K*-*0M %2&101*C0%a&%1& A'1 broth
c* Prepared plated media %a"ar& stored in plastic ba"s or ti"htly sealed
containers in dar@. refri"erated and held no lon"er than , (ee@s*
%1& Table J/,/$KM J,,,3*,M
J,,,D*1M %,& K*-*1*C
d* +'EndoE+'Endo LE! broth held no lon"er than JA ho#rs*
%1& Table J/,/$KM J,,,D*1
+ay ,-. ,//0 Pa"e 1- of 2,
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MICRO"IOLOGICAL MEDIA SPECIFICATIONS S;,34,>4 M6;584/, 1%
;5
I 19
;5
64.
Chec the "edia used in your !a$oratory%
% & La#ryl Tryptose 3roth %LT3&
2-*A "EL %!!&
p6 = A*H /*,
% & 3rilliant 4reen Lactose 3ile 3roth
%34L33&
0/*/ "EL
p6 = C*, /*,
% & Presence'Absence 3roth %P'A&
J1*- "EL %2x E1// mL sample&.
p6 = A*H /*,
% & EC 3roth
2C "EL
p6 =A*J /*,
% & EC G +?4 3roth
2C "EL
A*J /*,
% & A'1 3roth
21 "El G 1*/ mL PE4 p'
isooctylphenyl ether
p6 = A*J /*1
% & +acCon@ey A"ar
-/ "EL.
p6 = C*1 /*,
% & m'Endo A"ar
A2 "EL %3roth = 0H "EL&
p6 = C*, /*,
% & m'Endo LE! A"ar
-1 "EL %3roth = 2A "EL&
p6 = C*, /*,
% & N#trient A"ar E NA G +?4
,2 "EL G /*1 "EL +?4
p6 = A*H /*,
% & m'FC A"ar
-, "EL %3roth = 2C "EL&
p6 = C*0 /*,
% & + 3roth or A"ar
p6 = A*J- /*,
p6 = C*/, /*,
% & m'Coli3l#e,0
p6 = C*/ /*,
% & Plate Co#nt A"ar %Tryptose glucose
yeast agar#
,2*- "EL
p6 = C*/ /*,
% & AFide Dextrose 3roth
20*C "EL
p6 = C*, /*,
% & 3ile Esc#lin AFide A"ar %in place of
P!E A"ar

% & mE A"ar %enterococci&

C/*, "EL G Nalidixic acid and
,.2.-'triphenyl tetraFoli#m
chloride
p6 = C*1 /*,
% & EA !#bstrate %Esc#lin ron A"ar&
1A*A "EL
p6 = C*1 /*,
% & m'Enterococc#s A"ar %fecal
streptococci&
01*- "EL
p6 = C*1 /*,
% & mE a"ar
p6 = C*1 /*,
% & R,A A"ar
1H*1- "EL
p6 = C*,
SUGGESTED CONTROL CULTURES FOR MICRO"IOLOGICAL TESTS
Chec the contro! cu!tures used in your !a$oratory%
GROUP POSITIVE CONTROL NEGATIVE CONTROL
TOTAL COLIFORMS
ferment lactose
Escherichia coli
Enterobacter aero"enes
!taphylococc#s a#re#s
Prote#s 9#l"aris I
Pse#domonas aer#"inosa
FECAL COLIFORMS
thermotolerant %00*-C&
Escherichia coli
7lebsiella pne#moniae %thermotolerant
strain&
Enterobacter aero"enes
E. COLI
+?4 positi9eII
Escherichia coli %+?4 positi9e strain& Enterobacter aero"enes
7lebsiella pne#moniae %thermotolerant&.
+?4 %'&
ENTEROCOCCI Enterococc#s faecalis
Enterococc#s faeci#m
!taphylococc#s a#re#s %nalidixic acid
sensiti9e&
Escherichia coli %sodi#m aFide sensiti9e&
!erratia marcescens %fl#oro"enic
compo#nd not hydrolyFed&
!tandard +ethods. 1H
th
R 1J
th
ed* and EPA Certification +an#al -
th
ed* draft
I P* 9#l"aris #ses hydrolyFed lactose indicatin" ;o9ercoo@ed< medi#m*
II Not all strains of E* coli are +?4 %G&. e*"* E*coli )1-C$6C*
+ay ,-. ,//0 Pa"e 1A of 2,
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E. MICRO"IOLOGICAL METHODS
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1. GENERAL
'2( EPA %15-"-9$-001
'!( CFR 141.21
a* For drin@in" (ater. the sample 9ol#me analyFed is 1// ,*- ml*
%,& K*-*1*A
b* Bater samples are sha@en 9i"oro#sly %abo#t ,- times& before
analysis*
%,& K*-*1*0
CC
c* f no total coliform positi9e res#lts occ#r d#rin" a :#arter. the
laboratory spi@es and analyFes a sample (ith a @no(n total coliform.
fecal coliform andEor E* coli positi9e control or"anism*
%,& K*-*1*-
CC
d* Parallel testin" performed bet(een a ne(ly appro9ed test proced#re
and another EPA'appro9ed proced#re for se9eral months andEor
se9eral seasons* %Recommended&
%,& K*-*1*H
+ay ,-. ,//0 Pa"e 1C of 2,
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E. 2.0 MICRO"IOLOGICAL METHODS PLATE COUNT METHODS
2.1 HETEROTROPHIC PLATE COUNT
'1( SM 9215A,"
'2( EPA %15-"-9$-001,V.5.9
'!( CFR 141.$4
a* Po#r plate method #sed for 6PC testin" of (aters for compliance
(ith !#rface Bater Treatment R#le*
%,&%2&
b* For most potable (ater samples. co#ntable plates are obtained by
platin" 1*/ and /*1 ml 9ol#me of #ndil#ted sample*
%1& J,1-3*,*aM %,& K*-*J*2
c* At least d#plicate plates per dil#tion are #sed*
%1& J,1-3*,*cM %,& K*-*J*2
d* For po#r plate method. melted a"ar is tempered at 00'0AC in (ater
bath before po#rin". held not lon"er than 2 ho#rs. and melted only
once*
%1& J,1-3*2*aM ,& K*-*J*0
e* 1, to 1- mL of melted a"ar added to sample that is aseptically
po#red to bottom of petri dish %1// mm x 1- mm&*
%,& K*-*J*0
f* !ample mixed ade:#ately (ith melted medi#m (itho#t spilla"e and
allo(ed to solidify on le9el s#rface*
%1& J,1-3*2*bM %,&
"* Plates are in9erted and inc#bated at 2- /*-C for 0H 2 ho#rs*
%3ottled (ater inc#bated C, ho#rs&
%1& J,1-A*CM %,& K*-*J*0
h* Plates are stac@ed no more than 0 hi"h and arran"ed to allo(
circ#lation bet(een stac@s*
%,& K*-*J*0
i* A"ar medi#m sterility control incl#ded* Data reSected if control
sho(s any contamination*
%1& J,1-3*2*cM %,& K*-*J*H
S* !terility control for dil#tion (ater %if #sed& is incl#ded*
%1& J,1-3*2*c
@* Room air control is incl#ded* %O1- cf#Eplate for 1- min* expos#re&
%1& J,1-3*2*cM J/,/3*1*e
l* Colonies are co#nted man#ally #sin" a dar@ field colony co#nter*
%1& J,1-A*HM %,&
m* )nly plates (ith 2/ to 2// colonies co#nted. except for plates
inoc#lated (ith 1*/ ml of #ndil#ted sample* %drin@in" (ater and
so#rceEs#rface (ater&
%1& J,1-A*HM %,& K*-*J*C
n* F#lly a#tomated colony co#nter is not #sed for drin@in" (ater or
so#rceEs#rface (ater*
%,& K*-*J*C
o* Colony co#nts ro#nded off to , si"nificant fi"#res*
%1& J,1-A*J
p* For systems "ranted a 9ariance from Total Coliform R#le< maxim#m
contaminant le9el. any method in !tandard methods #sed (ith R,A
medi#m for en#meratin" heterotrophic bacteria in drin@in" (ater*
%,&
+ay ,-. ,//0 Pa"e 1H of 2,
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!.1 MULTIPLE TU"E FERMENTATION 'MTF(
GENERAL T8;,1 C81=A8>9/ 'TC( A::1=6/ ;8 D>=3<=3@,
S8?>76KS?>A,76, ,/;6, R67>6,;=83,1 ,;6>
'1( SM 9221A,",C
'2( EPA %15-"-9$-001,V.5.!
'!( CFR40*141.21D141.$4D1!6.!
'4( A"411
a. La#ryl Tryptose 3roth %LT3& #sed for pres#mpti9e test and 3rilliant
4reen Lactose 3ile broth %34L3& #sed for confirmed test for total
coliform*
%1& J,,13M %,& K*-*2*,
b. f lactose broth %L3& is #sed. system cond#cts at least ,- parallel
tests bet(een this medi#m and LT3 and demonstrates false'positi9e
rate and false'ne"ati9e rate for total coliforms of less than 1/Q. (ith
comparison doc#mented and records retained*
%,& K*-*2*,M %2& 101*,1%f&%2&
c. Concentration of test medi#m adS#sted so that sample addition does
not red#ce in"redient concentrations belo( that of standard
medi#m* %LT3. L3&
%1& !+ Table J,,1$ %,& K*-*2*,
d. n9erted 9ials in sterile medi#m are free of b#bbles and at least one
half to t(o thirds co9ered after (ater sample is added*
%1& J,,13*1M %2& 101*,1%f&%2&
e. noc#lated LT3EL3 medi#m is inc#bated at 2- /*-C and obser9ed
for "as %or acid& and "ro(th at ,0 G , hrs and 0H G 2 hrs*
%1& J,,13M %,& K*-*2*2
f. All ,0' and 0H'ho#r "as'positi9e or acid positi9e t#bes confirmed in
34L3 broth*
%1& J,,13*,M %,& K*-*2*,
!.1.1 MULTIPLE TU"E FERMENTATION 'TC( D>=3<=3@ ,;6>
%2& CFR 0/$101*,1
g. 1// ml of drin@in" (ater tested* $hec set-up used:
1// ml (inverted vial replaced %ith acid indicator#
1/ t#be x 1/ ml
- t#be x ,/ ml
%1& J,,1A*1M %2& 101*,1%f&
h. Completed test not re:#ired for drin@in" (ater*
%2& 101*,1%f&%2&
i. Ne"ati9e n9alidation$ All samples sho(in" a t#rbid c#lt#re %i*e*.
hea9y "ro(th. opa:#e& in the absence of "asEacid prod#ction are
in9alidated and another sample collected from the same location* %f
lab performs confirmed test on t#rbid c#lt#re and confirmed test is
total coliform positi9e. sample reported as s#ch. b#t if total coliform'
ne"ati9e. sample is in9alidated*
%,&.%2&101*,1%c&%,&
&. Bhen +TF test is #sed on (ater s#pplies that ha9e a history of
confl#ent "ro(th or TNTC by the +F proced#re. all pres#mpti9e
t#bes (ith hea9y "ro(th (itho#t "asEacid prod#ction s#bmitted to
confirmed test and fecal coliformEE.coli test to chec@ for coliform
s#ppression*
%,& K*-*2*C
+ay ,-. ,//0 Pa"e 1J of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. !.0 MICRO"IOLOGICAL METHODS FERMENTATION METHODS FOR TOTAL COLIFORMS
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!.1.2 MULTIPLE TU"E FERMENTATION 'TC(
S8?>76KS?>A,76, ,/;6, ,34 R67>6,;=83,1 ,;6>
%,& CFR 0/$101*C0
%0& A3 011
. At least 2 appropriate sample dil#tions of - t#bes each are #sed*
%1& J,,1A*,M J,,13*1
%,& K*-*11*1
l. Completed test not re:#ired for /8?>76K/?>A,76 E,;6>*
%2& 101*C0%a&%1&
m. Completed test is performed on E,/;6E,;6> if lab is not certified for
fecal coliform testin"* %1/Q of positi9e samples or minim#m of once
per :#arter&
%1& J,,13*2

n. S8?>76K/?>A,76 E,;6> samples that prod#ce t#rbid "ro(th in the
absence of "asEacid prod#ction in LT3 or L3 are in9alidated and
another sample obtained. (hich may be tested (ith another
method*
%,& K*-*11*2
o. Alternati9ely. a confirmed test is performed on t#rbid c#lt#re in the
absence of "asEacid prod#ction and. if total coliform positi9e. most
probable n#mber reported. or if total coliform'ne"ati9e. sample is
in9alidated and another re:#ested* %/8?>76K/?>A,76 E,;6>&
%,& K*-*11*2
p. S8?>76K/?>A,76 E,;6>. E,/;6E,;6> and >67>6,;=83,1 E,;6> res#lts
reported as +PNE1// mL #sin" the table !+ J,,1*K*
%1& J,,1C
!.2 PRESENCE-A"SENCE 'CLARKS( T8;,1 C81=A8>9/
D>=3<=3@ ,;6>
'1( SM 9221D.1
'2( EPA %15-"-9$-001,V.5.4
'!( CFR 141.21'7('2(
a* f six'times %Ax& form#lation stren"th medi#m #sed. medi#m is filter
steriliFed. not a#tocla9ed*
%1&%,&
b* 1// ml sample inoc#lated into bottle of P'A medi#m*
%1&%,&
c* +edi#m inc#bated at 2- G /*-
o
C and obser9ed for yello( color
%acid& and "ro(th at ,0 , and 0H 2 ho#rs*
%1&%,&
d* 5ello( broths (ith "ro(th are confirmed for total coliforms in 34L3
broth*
%1&%,&
e* Ne"ati9e n9alidation$ Non'yello(. t#rbid P'A medi#m is in9alidated
and another sample obtained from the same location* %f confirmed
test performed and sample is total coliform'positi9e. sample is
reported as s#ch. b#t if confirmed test is ne"ati9e. sample is
in9alidated*&
%,&%2&
+ay ,-. ,//0 Pa"e ,/ of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 4.0 MICRO"IOLOGICAL METHODS FERMENTATION METHODS FOR FECAL COLIFORMS
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
4.1 EC MEDIUM F67,1 C81=A8>9/ =3 D>=3<=3@, S8?>76K S?>A,76,
,/;6 ,34 R67>6,;=83,1 ,;6>/
'U/64 E=;5 LT"KL", P-A, 9-ENDOK9-ENDO LES(

'1( SM 9221A,",C,E
'2( EPA %15-"-9$-001
'!( CFR 141.21, 141.$4, 1!6.!
a. EC medi#m is #sed to determine presence of fecal coliforms in total
coliform positi9e test* (chec applicable media#
% & LT3EL3. % & P'A broth. andEor % & m'EndoEm'Endo LE!*
%2& 101*,1%f&%-&M 101*C0%a&M 12A*2
%0& A3 011
b. n9erted 9ials free of b#bbles and at least one'half to t(o'thirds
co9ered after sample added*
%,& K*-*2*,*2M %2& 101*,1%f&%2&
MF-D
c. Filter from m'Endo may be c#rled and inserted into EC medi#m
after first remo9in" some of the selected colonies for total coliform
9erification* (not recommended#
CFR 101*,1%f&%-&
d. EC +edi#m inc#bated at 00*- G /*,
o
C for ,0 G , hrs*
%1& J,,1E*1M %,& K*-*-*-M %2&
101*,1%f&%-&
e. Bater le9el in (ater bath abo9e #pper le9el of medi#m in c#lt#re
t#bes*
%1& J,,1E*1M %,& K*-*-*0
f. Any amo#nt of "as detected in in9erted 9ial of t#be (ith "ro(th is
read as a fecal coliform positi9e*
%1& J,,1E*1M %,& K*-*-*A
g. En#meration res#lts reported #sin" +PN table in !+ J,,1C*
%1& J,,1C
4.2 A-1 MEDIUM F67,1 C81=A8>9
S8?>76KS?>A,76 ,34 ,/;6 ,;6>/ 831F
'1( SM 9221A,",C,E
'2( EPA %15-"-9$-001
'!( CFR 141.$4, 1!6.!
a* Do#ble stren"th medi#m #sed for 1/ mL sample 9ol#mes*
%1& J,,1E*,
b* Test is set #p as direct fecal coliform test* Three dil#tions (ith - or
1/ t#bes per sample dil#tion %9ol#me& are #sed*
%,& K*-*-*2

c* n9erted 9ials free of b#bbles and at least one'half to t(o'thirds
co9ered after sample added*
%,& K*-*-*2
d* A'1 medi#m inc#bated at 2- G /*-
o
C for 2 ho#rs and then at 00*- G
/*,
o
C for ,1 G , ho#rs*
%1& J,,1E*,M %,& K*-*-*-
e* Bater le9el in (ater bath abo9e #pper le9el of medi#m in c#lt#re
t#bes*
%,& K*-*-*0
f* Any amo#nt of "as detected in in9erted 9ial of t#be (ith "ro(th is
read as a fecal coliform positi9e*
%1& J,,1E*,M %,& K*-*-*A
"* Res#lts reported #sin" +PN table in !+ J,,1C*
%1& J,,1C
+ay ,-. ,//0 Pa"e ,1 of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 5.0 MICRO"IOLOGICAL METHODS ENJ#ME SU"STRATE METHODS FOR COLIFORMS
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
5.1 EC L MUG MEDIUM E. 781= =3 D>=3<=3@ ,;6>
'U/64 E=;5 LT"KL", P-A, 9-E348K9-E348 LES(
'1( SM 9221E
'2( EPA %15-"-9$-001,V.5.$
'!( CFR 141.21'A('5(D 'A( '6('=(
a* Final +?4 concentration of -/ " per ml added to EC before
a#tocla9in". or commercially a9ailable EC G +?4 #sed*
%,&%2&
b* At least 1/ ml of EC medi#m s#pplemented (ith +?4 m#st be
#sed*
%2&
c* n9erted 9ial omitted %optional&
%,&%2&
d* Test t#bes and a#tocla9ed media chec@ed for a#to'fl#orescence
(ith 2AA nm ?K li"ht before they are #sed*
'() positive and negative (uninoculated# controls may be
necessary to interpret results* especially if there is %ea auto-
fluorescence of the tube or media.
%,&
MF
e* Filter may be c#rled and inserted into EC G +?4 medi#m. after first
remo9in" some of the selected colonies for total coliform
9erification* (not recommended#
CFR 101*,1%f&%-&
f* EC G +?4 +edi#m inc#bated at 00*-
o
C G /*,
o
C for ,0 G , hrs*
%,&%2&
"* Bater le9el in (ater bath is abo9e #pper le9el of medi#m in c#lt#re
t#bes*
%,&
h* Fl#orescence is chec@ed (ith A (att. 2A-'2AA nm ?K lamp in
dar@ened room*
%,&%2&
i* 3ri"ht bl#e fl#orescence is considered a positi9e test*
%,&%2&

5.2 COLILERT COLILERT 1% COLISURE
T8;,1 C81=A8>9 I E. 781=

GENERAL A::1=6/ ;8 ,11 8A ;56 ,-806 964=,K96;584/
'1( SM 922!
'2( EPA %15-"-9$-001,V.5.6
'!( CFR 141.21D 141.$4
'4( A" 411
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;, V.5.!
a* +edia p#rchased from commercially a9ailable so#rce only*
%,&
b* +edia protected from li"ht and stored accordin" to man#fact#rers
recommendations
%,&
c* Each lot of medi#m chec@ed for a#to'fl#orescence before #se (ith A
(att. 2A-'2AA nm ?K lamp*
%,&
d* +edia exhibitin" faint fl#orescence discarded and another lot #sed*
%,&
e* +edi#m exhibitin" color chan"e after sample added. b#t before
inc#bation. is discarded and another batchElot #sed*
%,&
+ay ,-. ,//0 Pa"e ,, of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 5.2 MICRO"IOLOGICAL METHODS COLILERT, COLILERT 1%, COLISURE 'G636>,1(
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
f* 4lass bottles not #sed for test if they fl#oresce (hen chec@ed (ith
?K li"ht*
%,&
CC
"* Each lot of medi#m chec@ed (ith a +?4'positi9e E. coli strain. a
+?4'ne"ati9e coliform and a non'coliform* Read and record
res#lts*
%,&
h* Dil#tion (ater for ColilertEColilert 1H and Colis#re is sterile
dechlorinated tap (ater. deioniFed (ater. or distilled (ater*
%,&
i* D#anti'Tray sealer chec@ed monthly by addin" dye %e"* bromcresol
p#rple& to (ater* f dye is obser9ed o#tside of (ells. there may be a
problem (ith the sealer or (ith the lot of trays*
%,&
S* ColilertEColilert 1HEColis#re Tests not #sed to confirm total coliforms
in +TF. P'A tests or membrane filters*
%,&
@* !amples #sin" C81=16>;
M
medi#m inc#bated at 2- /*-C for ,0
ho#rs*
%1& %,&
l* !amples #sin" C81=16>; 1% medi#m inc#bated for 1H ho#rs*
%2& 101*C0 %a&%1& footnote C.
DEXX instr#ctions
m* !amples (ith yello( color reference comparator indicate presence
of total coliform*
%,&
n* !amples (ith yello( color less than comparator inc#bated additional
0 ho#rs %total of ,H ho#rs Colilert and ,, ho#rs for Colilert 1H&*
%,& %2& 101*C0 %a&%1& footnote C.
DEXX instr#ctions
o* !amples (ith yello( color less that is than comparator after ,H
ho#rs %,, hrs* for Colilert 1H& of inc#bation recorded as ne"ati9e*
%1&%,&
o* !amples that are yello( and fl#oresce (hen exposed to 2A-'2AA
nm ?K
lamp are positi9e for E. coli*
%1&%,&
p* Colilert color comparator not #sed beyond expiration date*
%,& K*-*1*C*2
CC
:* f air type inc#bator #sed (ith cold samples. it may be necessary to
determine time needed to brin" sample to 2-C. to ens#re specified
inc#bation time at that temperat#re is follo(ed*
%,&
+ay ,-. ,//0 Pa"e ,2 of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 5.2 MICRO"IOLOGICAL METHODS- COLILERT, COLILERT 1%, COLISURE 'S:67=A=7(
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
5.2.1 COLILERTKCOLILERT 1% T8;,1 C81=A8>9/ ,34 E. 781=
D>=3<=3@ ,;6>, S8?>76KS?>A,76 ,;6>, R67>6,;=83,1 ,;6>
a* D>=3<=3@ ,;6> - Chec for"at used$
1/ mL sample into each of 1/ t#bes*
1// mL sample into 1 bottle*
1// mL sample into 1 D#anti'Tray
%,&M %2& 101*,1%f&%2&M101*C0%a&%1&M
%0&
b* S8?>76KS?>A,76 ,;6> '63?96>,;=83 831F( - Chec for"at used$
- t#be 8 2 dil#tion set'#p
D#anti'Tray1//
%1& %,& K*-*A*0M K*-*A*1*-
c* f C81=16>; 1% is #sed for total and ;fecal coliforms< in >67>6,;=83,1
9,>=36 E,;6>. parallelEsplit sample testin" (ith m#ltiple t#be
fermentation or membrane filtration method is performed to
determine that the total coliform res#lts are comparable and that the
E. coli res#lts (ill be comparable to fecal res#lts*
+aintain records*
%0&
d* R67>6,;=83,1 9,>=36 E,;6> is dil#ted 1*10 (ith sterile (ater before
addin" C81=16>; 1% medi#m and sealin" in one D#anti'Tray or
D#anti'Tray ,/// per dil#tion*
%2& 101*C0 %a&%1& footnoteC.
DEXX instr#ctions
5.2.2 COLISURE T8;,1 C81=A8>9/ ,34 E. 781= - D>=3<=3@ ,;6>
e* 1// mL sample is tested in a bottle*
%,&
f* !ample #sin" Colis#re medi#m is inc#bated at 2- /*-C for ,0 to
0H ho#rs*
%,& %2& 101*,1%f&%2& footnote J
"* Color chan"e from yello( to ma"enta indicates presence of total
coliforms*
%,&
h* !amples that fl#oresce %dar@ened room& (hen exposed to 2A-'2AA
nm ?K lamp are positi9e for E. coli*
%,&
5.! ENCOLITE MEDIUM T8;,1 C81=A8>9 ,34 E.781=
D>=3<=3@ ,;6>
'!( CFR 141.21'A('!( A88;38;6 10
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;, V.5.!.2.5
a* !ample inc#bated in ba" at 2-C /*-C for ,H ho#rs*
%2&%-&
b* Total coliforms are present if medi#m chan"es from yello( color to
bl#e or bl#e'"reen. or a bl#e color in the corners of the ba"*
%2&%-&
c* E* coli is present if medi#m fl#oresces (hen exposed to 2A-'2AA nm
?K lamp*
%2&%-&
d* f no fl#orescence is obser9ed at ,H ho#rs. medi#m is inc#bated an
additional ,/ ho#rs %0H hrs*&*
%2&%-&
e* f medi#m becomes red. 4=/7,>4 /,9:16* Ass#me lea@a"e of
bactericide from third compartment of ba" into medi#m*
%-&
+ay ,-. ,//0 Pa"e ,0 of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 6.0 MICRO"IOLOGICAL METHODS MEM"RANE FILTRATION 9E348K9E348 LES, 9FC, NALMUG
L,-./
E0,1. ITEM
ELAP
E0,1
REFERENCES
COMMENTS
6.1 GENERAL A11 MEM"RANE FILTRATION 'MF( METHODS
T5=/ /67;=83 ,34 /67;=83 C.12 'MF EB?=:963;( ,::1=6/ ;8 ,11
M69->,36 F=1;>,;=83 M6;584/
'1( SM 9222A,",C
'2( EPA %15-"-9$-001
CC
a !terility chec@ cond#cted on each f#nnel in #se at be"innin" and
end of each filtration series* %Filtration series ends (hen 2/ min#tes
or more elapse bet(een sample filtrations*&
%1&M %,& K*-*,*1*2M K*-*1,
CC
b* f sterility control indicates contamination. all data for that f#nnel is
reSected and another sample is re:#ested*
%,& K*-*,*1*2
c* F#nnels are rinsed (ith t(o or three ,/ 8 2/ mL portions of sterile
rinse (ater after each sample filtration to pre9ent carry'o9er*
%1& J,,,3*-*cM %,& K*-*,*,M K*-*1,
d* As an alternati9e to ;c.< a ,-0 nm ?K li"ht is bein" #sed to sanitiFe
the filtration #nits d#rin" filtration series*
%1& J,,,3*-*bM %,& K*0*1*0
e* nner s#rface of forceps smooth and (itho#t rid"es*
%1& J,,,3*1*i
6.2 M-ENDOKM-ENDO LES '->8;5 8> ,@,>( T8;,1 C81=A8>9/

A::1=6/ ;8 D>=3<=3@, S8?>76KS?>A,76, ,/;6, R67>6,;=83,1 ,;6>/
'1( SM 9222A,",C
'2( EPA %15-"-9$-001
'!( CFR 141.21'A('5(, 141.$4,
1!6.!
a* mEndo E m'Endo LE! #sed in sin"le step or enrichment techni:#e*
%1& !+ J,,,3*-M %,& K*-*,
b* Ethanol not denat#red*
%1& !+ J,,,3*,M %,& K*-*,
c* +edi#m prepared in sterile flas@ and dissol9ed #sin" boilin" (ater
bath or hot plate (ith stir bar* +edi#m not boiled*
%1& !+ J,,,3*,M %,& K*-*,
d* ?ninoc#lated media discarded if "ro(th or s#rface sheen obser9ed*
%,& K*-*,*1
e* For total coliforms dil#tions or 9ol#mes that yield ,/ to H/ total
coliform colonies for at least one dil#tion are filtered*
%1& !+ J,,,3*-*a
f* noc#lated medi#m inc#bated at 2-
o
G /*-
o
C for ,, to ,0 ho#rs in
h#mid en9ironment* (+,- relative humidity#
%1& !+ J,,,3*1
"* All sheen colonies %incl#des :#estionable sheen colonies and non'
sheen red colonies&. #p to maxim#m of fi9e. are 9erified #sin" sin"le
stren"th LT3 or L3 and 34L33 or an EPA'appro9ed cytochrome
oxidase and beta'"alactosidase rapid test proced#re&
%,& K*-*,*-
h* For 4>=3<=3@ E,;6>. entire membrane s#rface is bein" s(abbed for
9erification of coliform* )rder of inoc#lation is EC. LT3. 34L3*
%1& J,,,3*A*aM %,& K*-*,*-
i* For 4>=3<=3@ E,;6>. sample not in9alidated if membrane filter
contains at least one sheen colony* %drin@in" (ater&
%,& K*-*,*0
+ay ,-. ,//0 Pa"e ,- of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 6.0 MICRO"IOLOGICAL METHODS MEM"RANE FILTRATION - 9E348K9E348 LES, 9FC, NALMUG
6.2 M-ENDOKM-ENDO LES '->8;5 8> ,@,>( T8;,1 C81=A8>9/

A::1=6/ ;8 D>=3<=3@, S8?>76KS?>A,76, ,/;6, R67>6,;=83,1 ,;6>/
'1( SM 9222A,",C
'2( EPA %15-"-9$-001
'!( CFR 141.21'A('5(, 141.$4,
1!6.!
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
S* N6@,;=06 I30,1=4,;=83 A8> D>=3<=3@ ,;6>$ !amples res#ltin" in
confl#ent or too n#mero#s to co#nt %TNTC& "ro(th are in9alidated
#nless total coliforms are detected* TNTC is defined as W,//
colonies on membrane in absence of detectable coliforms*
%f 9erification test is performed before in9alidation and test is total
coliform'positi9e. sample is reported as s#ch. b#t if test is total coliform'
ne"ati9e. sample is in9alidated*&
%,& K*-*,*0M %2& 101*,1%c&%,&
@* For /8?>76K/?>A,76, initial co#nts are adS#sted based #pon 9erified
data as accordin" to !+ J,,,3* Report 9erified co#nt per 1// ml*
%1& J,,,3*AM %,& K*-*1/
l* For E,/;6E,;6> ,34 >67>6,;=83,1 E,;6>. 9erify 1/ colonies from
positi9e sample monthly. incl#din" both sheen and atypical colonies*
%1& !+ %,/
th
& J,,,3*-*f
CC
m* f t(o or more analysts are performin" the method. each co#nts total
coliform colonies on same membrane monthly and a"ree (ithin
1/Q
%1& J/,/3*0*dM %,& K*-*1/

6.! M-FC '->8;5 8> ,@,>( F67,1 C81=A8>9/
S8?>76KS?>A,76, ,/;6, R67>6,;=83,1 ,;6>
'1( SM 9222A,",C,D
'2( EPA %15-"-9$-001
'!( CFR 141.$4. 1!6.!
a* 3ro#"ht to boilin" point to li:#efy medi#m* Not a#tocla9ed*
%1& J,,,D*1M %,& K*-*1,
b* ?ninoc#lated medi#m discarded if "ro(th obser9ed*
%,& K*-*1,
c* For fecal coliforms filter dil#tions or 9ol#mes that yield ,/ to A/ fecal
coliform colonies for at least one dil#tion*
%1& J,,,D*1M %,& K*-*1,
d* noc#lated medi#m is inc#bated at 00*- /*,C for ,0 , hrs*
%1& J,,,D*,M %,& K*-*1,
CC
e* f t(o or more analysts are performin" the method. each co#nts
fecal coliform colonies on the same membrane monthly and the
co#nts a"ree (ithin 1/Q*
%1& J/,/3*0*dM %,& K*-*1,
+ay ,-. ,//0 Pa"e ,A of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 6.0 MICRO"IOLOGICAL METHODS MEM"RANE FILTRATION NALMUG
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
6.4 NUTRIENT AGAR L MUG 'MF( &% co!i A8> D>=3<=3@ ,;6>
'2( EPA %15-"-9$-001,V.5.%
'!( CFR 141.21
a* Final +?4 concentration m#st be 1// "EmL
%,& %2& 101*,1%f&%A&%ii&
CC
b* D#ality of lot or batch of medi#m e9al#ated by filterin" or spot
inoc#latin" positi9e and ne"ati9e control c#lt#res onto membrane
filter on m'Endo medi#m. inc#batin" at 2-C for ,0 ho#rs. then
transferrin" filter to NAG+?4 and f#rther inc#batin" at 2-C for 0
ho#rs* Res#lts are read and recorded*
%,&
c* Filter (ith total coliform colonies transferred to s#rface of N#trient
A"ar G +?4*
%,&
d* 3efore inc#bation. indicate location of each sheen colony on petri
dish lid (ith permanent mar@erM dish and lid are mar@ed to reali"n
the lid after bein" separated*
%,&
e* For the total coliform 9erification test. a portion of the colony is
transferred (ith needle before or after NA G +?4 inc#bation*
%,&
f* As an alternati9e to ;e.< the membrane filter s#rface is s(abbed (ith
sterile cotton s(ab after 0'ho#r inc#bation and transferred to total
coliform 9erification media*
%,&
"* noc#lated NA G +?4 medi#m inc#bated at 2- /*-C for 0 ho#rs*
%,&
h* Any fl#orescence in halo aro#nd sheen colony (hen chec@ed (ith A
(att. 2A-'2AA nm ?K lamp in a dar@ened room is reported positi9e
for E. coli*
%,&
+ay ,-. ,//0 Pa"e ,C of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. $.0 MICRO"IOLOGICAL METHODS MEM"RANE FILTRATION TOTAL AND E. COLI
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
$.1 GENERAL A11 MEM"RANE FILTRATION 'MF( METHODS
T5=/ /67;=83 ,34 /67;=83 C.12 'MF EB?=:963;( ,::1=6/ ;8 ,11
M69->,36 F=1;>,;=83 M6;584/ 'E.$.1 =/ /,96 ,/ E.6.1(
'1( SM 9222A,",C
'2( EPA %15-"-9$-001
CC
a !terility chec@ cond#cted on each f#nnel in #se at be"innin" and
end of each filtration series* %Filtration series ends (hen 2/ min#tes
or more elapse bet(een sample filtrations*&
%1&M %,& K*-*,*1*2M K*-*1,
CC
b* f sterility control indicates contamination. all data for that f#nnel is
reSected and another sample is re:#ested*
%,& K*-*,*1*2
d* F#nnels are rinsed (ith t(o or three ,/ 8 2/ mL portions of sterile
rinse (ater after each sample filtration to pre9ent carry'o9er*
%1& J,,,3*-*cM %,& K*-*,*,M K*-*1,
e* As an alternati9e to ;c.< a ,-0 nm ?K li"ht is bein" #sed to sanitiFe
the filtration #nits d#rin" filtration series*
%1& J,,,3*-*bM %,& K*0*1*0
f* nner s#rface of forceps smooth and (itho#t rid"es*
%1& J,,,3*1*i
$.2 M-C81="1?624 MEDIUM 'MF( T8;,1 C81=A8>9/ ,34 &% co!i
D>=3<=3@ ,;6>
'!( CFR 141.21'A( A88;38;6 11
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;,V.5.4.2.1.2

a* Amp#les in9erted ,'2 times to mix contents before brea@in"*
%-&
b* ?nopened refri"erated amp#les may be stored in the dar@ for one
year. b#t discarded earlier if "ro(th is obser9ed*
%-&
c* noc#lated medi#m inc#bated 2-C /*-C for ,0 ho#rs*
%2&
d* Total coliform colonies %non E. coli& are red*
E. coli colonies are bl#e to p#rple*
%2&
$.! MI MEDIUM 'MF( T8;,1 C81=A8>9/ ,34 &% co!i
D>=3<=3@ ,;6> ,34 T8;,1 C81=A8>9/ =3 S8?>76KS?>A,76 ,;6>
'1( SM 9020*IV
'2( EPA %15-"-9$-001
'!( CFR 141.21'A( A88;38;6 6
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;,V.5.4.2.1.!
'$( EPA 600-R-00-01!
a* noc#lated medi#m inc#bated 2- /*-C for ,0 ho#rs*
%2& %C&
*
b* 3l#e colonies #nder 38>9,1 1=@5; are co#nted as &% co!i*
%2&%C&
c* F1?8>6/763; 78183=6/ #nder 2AAnm ?K lamp co#nted as ;8;,1
781=A8>9/*
3l#eE(hite fl#orescent colonies #nder 2AA nm ?K lamp are co#nted
as ;8;,1 781=A8>9*
3l#eE"reen fl#orescent colonies are &% co!i*
3l#eE"reen (ith fl#orescent ed"es co#nted as &% co!i*
Non'fl#orescent bl#e colonies %E. coli& are also co#nted as ;8;,1
781=A8>9.
%2&%C&
d* S8?>76K/?>A,76 E,;6> reported as total coliform per 1// ml*
%2& %C&
+ay ,-. ,//0 Pa"e ,H of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. %.0 MICRO"IOLOGICAL METHODS MEM"RANE FILTRATION ENTEROCOCCAL METHODS
%.1 A11 MEM"RANE FILTRATION 'MF( METHODS - GENERAL
T5=/ /67;=83 ,34 /67;=83 C.12 'MF EB?=:963;( ,::1=6/ ;8 ,11
M69->,36 F=1;>,;=83 M6;584/ 'E.%.1 =/ /,96 ,/ E.6.1I E.$.1(
'1( SM 9222A,",C
'2( EPA %15-"-9$-001
CC
a !terility chec@ cond#cted on each f#nnel in #se at be"innin" and
end of each filtration series* %Filtration series ends (hen 2/ min#tes
or more elapse bet(een sample filtrations*&
%1&M %,& K*-*,*1*2M K*-*1,
CC
b* f sterility control indicates contamination. all data for that f#nnel is
reSected and another sample is re:#ested*
%,& K*-*,*1*2
"* F#nnels are rinsed (ith t(o or three ,/ 8 2/ mL portions of sterile
rinse (ater after each sample filtration to pre9ent carry'o9er*
%1& J,,,3*-*cM %,& K*-*,*,M K*-*1,
h* As an alternati9e to ;c.< a ,-0 nm ?K li"ht is bein" #sed to sanitiFe
the filtration #nits d#rin" filtration series*
%1& J,,,3*-*bM %,& K*0*1*0
i* nner s#rface of forceps smooth and (itho#t rid"es*
%1& J,,,3*1*i
%.2 M-ENTEROCOCCUS 'MF( F67,1 S;>6:;87877= =3 ,/;6E,;6>

'1( SM 92!0C.!
'!( CFR 1!6.!
'5( '5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;, V.5.4.4
a* +edi#m not a#tocla9ed*
%1& J,2/C*,M %-&
b* Appropriate 9ol#mes filtered to yield ,/ to A/ colonies on
membrane*
%1&
c* Plates stand for 2/ min#tes before in9ertin" and inc#batin" at 2- G
/*-
o
C for 0H ho#rs*
%1&
d* All li"ht and dar@ red colonies co#nted as fecal streptococci*
%1&
e* Fl#orescent li"ht and ma"nifyin" lens #sed to co#nt colonies*
%1&
f* Densities reported as fecal streptococci per 1// ml
%1& J,2/C*0
"* Kerification recommended*
%1& J,2/C*-
%.! 9E AGAR 'MF( E3;6>87877= =3 ,/;6 ,34 R67>6,;=83,1
,;6>
'1( SM 92!0C
'!( CFR 1!6.!
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;, V.5.4.4
S,A6;F
a* Proper handlin" of the toxic nalidixic acid %or its salt& is obser9ed*
%-&
b* Appropriate 9ol#mes filtered to yield ,/ to A/ colonies on
membrane*
%1& J,2/C*2
c* Plates in9erted and inc#bated at 01
o
C G /*-
o
C for 0H ho#rs*
%1& J,2/C*2M %-&
d* +embrane filter transferred to Esc#lin ron A"ar %EA& medi#m and
inc#bated at 01
o
C G /*-
o
C for ,/ min#tes*
%1& J,2/C*2M %-&
+ay ,-. ,//0 Pa"e ,J of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. %.0 MICRO"IOLOGICAL METHODS FECAL STREPTOCOCCI AND ENTEROCOCCI
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
e* Pin@ to red colonies (ith blac@ or reddish bro(n precipitate on the
#nderside of filter co#nted as enterococci*
%1& J,2/C*2
f* Density reported as enterococci per 1// ml
%1& J,2/C*0
"* Fl#orescent li"ht and ma"nifyin" lens #sed to co#nt colonies*
%1& J,2/C*2
%.4 9EI AGAR 'MF( E3;6>87877= =3 R67>6,;=83,1 ,;6>

'1( SM 92!0C
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;, V.5.4.4.1
'6( EPA-%21-R-9$-004, M6;584
1600
S,A6;F
a* Proper handlin" of the toxic nalidixic acid %or its salt& is obser9ed*
%-&
b* n9ert plates and inc#bate at 01 /*- C for ,0 ho#rs*
%-& %A&
c* !mall fl#orescent lamp and ma"nification #sed to examine both top
and bottom of plate for colonies (ith bl#e halo*
%-& %A&
d* Colonies (ith bl#e halo. re"ardless of color. co#nted as enterococci*
%-& %A&
e* Density reported as enterococci per 1// ml
%A&
+ay ,-. ,//0 Pa"e 2/ of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 9.0 MICRO"IOLOGICAL METHODS MPN - ENTEROCOCCAL
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
9.1 AJIDE DE&TROSE MEDIUM F67,1 S;>6:;87877= '9?1;=:16 ;?-6(
,/;6 ,34 R67>6,;=83,1 ,;6>
'1( SM 92!0", '!( 7A> 1!6.!
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;, V.5.2.5
a* +#ltiple dil#tions %e*"* 1/. 1. /*1&. and - t#bes for each dil#tion*
%1&
b* Do#ble stren"th broth #sed for 1/ ml 9ol#mes*
%1&
c* T#bes inc#bated at 2- G /*-
o
C and obser9ed for "ro(th %t#rbidity& at
,0 G , ho#r and at 0H G 2 ho#rs*
%1& %-&
d* The confirmed test is performed on all t#bes that become t#rbid at
,0 and 0H ho#rs*
%1& %-&
e* T#rbid broth s#spensions are strea@ed onto 3ile Esc#lin AFide
%3EA& a"ar plate and inc#bated at 2- G /*-
o
C for ,0 G , ho#rs*
%1& %-&
f* 3ro(nish'blac@ colonies (ith bro(n halos %on 3EA a"ar& read as
confirmation of fecal streptococci*
%1& %-&
"* 4ro(th in 36 broth at 0- G /*-
o
C in 0H hrs* and in 36 (ith A*-Q
NaCl at 2- G /*-
o
C in 0H hrs* 9erifies that the colony belon"s to
enterococc#s "ro#p*
%1&
h* Res#lts reported #sin" +PN table in !+ J,,1C*
%1&
+ay ,-. ,//0 Pa"e 21 of 2,
ELAP +CR)3)L)45 C6EC7L!T
E. 9.0 MICRO"IOLOGICAL METHODS MPN - ENTEROCOCCAL
L,-./
E0,1.
ITEM
ELAP
E0,1
REFERENCES
COMMENTS
9.2 ENTEROLERT- E3;6>87877= =3 R67>6,;=83,1 ,;6>
'5( EPA C6>;. M,3?,1, 5
;5
64.
4>,A;, V.5.!.
'6( P>84?7; =3/6>; - IDE&&
a* +edia p#rchased from commercially a9ailable so#rce only*
%-&
b* +edi#m stored in dar@ at 0'2/C #ntil #se*
%-& %A&
c* Each lot of medi#m chec@ed for a#to'fl#orescence (ith A (att. 2A-'
2AA nm ?K lamp before #se*
%-&
d* +edia exhibitin" faint fl#orescence discarded and another lot #sed*
%-&
e* R67>6,;=83,1 9,>=36 E,;6> is dil#ted 1$1/ (ith sterile (ater %e*"*
1/ ml sample G J/ ml (ater& before po(dered medi#m is added*
%A&
f* Dil#tion (ater for Enterolert is sterile dechlorinated tap (ater.
deioniFed (ater. or distilled (ater*
%A&
"* Enterococci are en#merated #sin" D#anti'Tray or D#anti'Tray ,///*
%A&
h* D#anti'Tray sealer chec@ed monthly by addin" dye to (ater sealed
in tray and obser9in" for lea@a"e*
%-&
i* Enterolert tests inc#bated at 01 /*-C for ,0 to ,H ho#rs and are
examined for fl#orescence %positi9e&*
%-& %A&
S* Bells that are positi9e before ,0 ho#rs and (ells that are ne"ati9e
after ,H ho#rs are also 9alid*
%A&
@* Reported as Enterococci per 1// ml. #sin" +PN table pro9ided by
man#fact#rer*
%A&
+ay ,-. ,//0 Pa"e 2, of 2,