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$6$ )))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))) 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 L,-./ E0,1. ITEM ELAP E0,1 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 L,-./ E0,1. ITEM ELAP E0,1 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$-001, V.!.2 a* 3alance has readability of /*1 "* %1& %,& b* 3alances sho#ld detect 1// m" (ith 1-/ " load* %1& %,& CC c* 3alance 7,1=->,;64 983;51F #sin" A!T+ type 1. ,. or 2 (ei"hts and the data recorded* %minim#m of 2 traceable (ei"hts that brac@et laboratory>s (ei"hin" needs& D,;, >678>464* %1& J/,/3*2*bM %,& CC d* Non'reference (ei"hts %if #sed& 7,1=->,;64 606>F /=G 983;5/ #sin" reference (ei"hts. and the 4,;, >678>464* %1& %,& CC e* Reference (ei"hts re'certified if dama"ed or corroded* %,& CC f* 3alance maintained thro#"h ser9ice contract or internal maintenance protocol* (annually at minimum& %1& J/,/3*,M %,& +ay ,-. ,//0 Pa"e H of 2, ELAP +CR)3)L)45 C6EC7L!T L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS !. CONDUCTIVIT# METER '1( SM 2510A '2( EPA %15-"-9$-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, ELAP +CR)3)L)45 C6EC7L!T L,-./ E0,1. ITEM ELAP E0,1 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$-001,V.!.5 a* !iFe is s#fficient for (or@load* %1& J/2/3*2 b* Press#re coo@ers are not #sed* %,& c* A#tocla9e has internal heat so#rce. temperat#re "a#"e (ith sensor in exha#st. press#re "a#"e. and operational safety 9al9e* %1& J/2/3*2M %,& d* +aintains steriliFation temperat#re d#rin" cycle and completes entire cycle (ithin 0- min#tes (hen 1, to 1- min#te steriliFation period is #sed* %1& J/-/A*2M %,& CC e* A#tomatic timin" de9ice is chec@ed :#arterly for acc#racy (ith another acc#rate timin" de9ice* %,& CC f* +axim#m temperat#re re"isterin" de9ice #sed at least (ee@ly to chec@ the steriliFation temperat#re of 1,1 G, o C* %1& J/,/3*,*iM %,& "* 6eat indicatin" tape #sed to identify items that ha9e been steriliFed* %1& J/,/3*,*i CC h* !pore strips or amp#les #sed monthly as bioindicators to confirm steriliFation %1& J/,/3*,*iM %,& i* Ro#tine maintenance performed. incl#din" cleanin" of drain screen and the door seals* %,& S* A#tocla9e is ser9iced ann#ally or as needed by :#alified technician* Records maintained* %1& J/,/3*,M %,& +ay ,-. ,//0 Pa"e 1/ of 2, ELAP +CR)3)L)45 C6EC7L!T L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS CC @* A#tocla9e records are maintained* date contents time in and time o#t total expos#reEcycle time steriliFation time steriliFation temperat#re initial of analyst or technician %1& J/,/3*,*iM %,& 9. HOT AIR OVEN '1( SM 9020".2.5D 92!0".2 '2( EPA %15-"-9$-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$-001 a* +F #nits made of stainless steel. "lass. or a#tocla9able plastic and not scratched or corroded and do not lea@* %1& !+ J/2/3*1-M %,& K*2*11*1 b* +embrane filtration e:#ipment a#tocla9ed before be"innin" of first filtration series* %Filtration series ends (hen 2/ min* or lon"er elapse after a sample is filtered*& %1& J/,/3*,*lM J,,,3*-M %,& K*0*1*2 +ay ,-. ,//0 Pa"e 11 of 2, ELAP +CR)3)L)45 C6EC7L!T L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS CC c* 4rad#ations on f#nnel. if #sed to meas#re sample 9ol#me. are chec@ed for acc#racy and ha9e tolerance of T,*-Q* Record of calibration chec@ retained* %,& K*2*11*, d* Filters appro9ed by man#fact#rer for total coliform (ater analysis* Filters made of cell#lose ester. (hite. "rid mar@ed. 0C mm diameter. and /*0- Um pore siFe* Lot n#mber and date recei9ed recorded for membrane filters* %1& J,,,3*1*"M %,& K*2*11*0 e* Absorbent pads. if #sed. of s#fficient thic@ness to absorb 1*H to ,*, mL media #sed* %1& J,,,3*1*hM %,& f* +embrane filters and pads p#rchased$ 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$-001,V.!.12 a* Pre'steriliFed plastic dishes )pened pac@a"es of plastic dishes re'sealed after #se to maintain sterility* %1&%,& +ay ,-. ,//0 Pa"e 1, of 2, ELAP +CR)3)L)45 C6EC7L!T L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS b* Re#sable "lass dishes #sed$ 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, ELAP +CR)3)L)45 C6EC7L!T L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS 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$-001,V.4.5 a* Distilled or deioniFed (ater #sed for final rinse* %1& J/0/M %,& CC b* 3atches of dry "lass(are spot'chec@ed for p6 reaction* %1& J/,/3*2*a*1&M %,& +ay ,-. ,//0 Pa"e 10 of 2, ELAP +CR)3)L)45 C6EC7L!T L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS 5. MEDIA '1( SM 9020", 9050A,C '2( EPA %15-"-9$-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, ELAP +CR)3)L)45 C6EC7L!T 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$6C* +ay ,-. ,//0 Pa"e 1A of 2, ELAP +CR)3)L)45 C6EC7L!T E. MICRO"IOLOGICAL METHODS L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS 1. GENERAL '2( EPA %15-"-9$-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, ELAP +CR)3)L)45 C6EC7L!T 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, ELAP +CR)3)L)45 C6EC7L!T E. !.0 MICRO"IOLOGICAL METHODS FERMENTATION METHODS L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS
!.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 L,-./ E0,1. ITEM ELAP E0,1 REFERENCES COMMENTS !.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,