UNIVERSIDAD DE SAN CARLOS DE GUATEMALA

I-ACULTAD DE INGENIERÍA
LABORATORIO
ERIS (Escuela Regional de Ingeniería Sanitaria)

PROCEDIMIENTO PARA EXÁMENES

BACTERIOLÓGICOS DEL AGUA

Guatemala, abril de 2016

9-48 MICROBIOLOGiCAL EXAMIWOTON (9OO0)

9218 B. Membrane Filter Method

1. Apparatus with a ciosure liiat includes a Ihermoroeter, c o n t a i n í n g the same

volmne as tbai of the water sample in tlie test Baska. Place
a. Bottles or erleraneyer flasks with closures, suitable for control flask into the water bath along wjth the saísqjles. W b e n
samplc size choseu and capable of withstanding hot and cold using a water bath w i t h shaker, agítate samples at 8 D to ?D r p m .
lemperatures. I f a s h a ü n g bath is Dot avaüablc, periodically shake samples h y
b. Culture dLihes: See SeciioQ 9222B,le. " • ' manual swirliiLg. M o n i t o r temperature i n p ü o t control flask..
c. FÜTration units: See Section 9 2 2 2 B . ! / When pilot flask reaches desired temperature, b e g í n timing and
d. Water bath, preferably shaking stj'le, capable o f heating hold sample for the specificd time. A l a t e m p e r a t m e o f 75''C h e a í
samples to WC. sampíes for 15 m i n ; at a temperalure of S C C heat samples f o r l O
e. Thermometer. m a n . ' ' ' A t the end o f heat treatment, cool samples iianaediately tn
\ a bath coEiaining a slurry o f wet ice. L e t s a n ó l e s coo) to
2. Materials and Culture Media approximateJy r o o m temperature,
c. FÜtratiort of samples and counríng: Filter samples as de-
a. Nutrient agar plus trypan bíue: scribed i n Sectioc 9222B.5c. Place membrane Sttter on agar
surface and i n c ú b a t e for 24 ± 2 h at 35 ± O-S^C. Coont cnlonies
Peptune ,. 5.0 g
Beefextract 3,0 g with a l o w - p o w c r { 1 0 to 15 magiñlications) binocutarwide-field
Trypan blue 0.015 g dissecüng microscope or other optical device, Oonsider any
Agar 15.0 g bacterial colomes ^ w i n g on the membrane as acmbic spcre-
Reageni'grade water 1.0 L forming bacteria.
d. Jnte^retation:
Heal to near boiling to dissolve agar. Sterílize by autoclaving
íbr 15 mió at 12rC. After autoclaving, coo! to SO^C and
dispense asepticaily Ínto 50- X 9-mm plástic culture piales witb FOT colony countiug procedure see Section 9215iA,8i,
ioose-fitlúíg lids. The pH should be 6.8 ± 0.2. Reftigerated
médium can be beld for 20 d at 4 to S^C.
4. References
3. Cocedura 1. Ricx, E . W . , K J L Pojc, R J . MILTTJER, D . A . L Y I L E & C H . JOHNSON,
1996. Bvaluatíng plant performance with eodosporcs, i Anur. Waier
- a. Sample size: See Section 9222B.5fl. WorisAssoc. 88:122-
b. Heat rreatmem of sample: Distribute samples into botlles or 2. B-íRBEAU, B., L . BouLOE, R. DESJARDINS, J. COALLIER, M . PRBVOST &

erlenmeyer flasks 2a). Prepare a pilot ccmtrol fiask equipped D. DucHESNE. 1997. A laodified method for the Msroeiatioíi of
aerobio Ejiore-fomiing bacteria. Can. J. Microbioi 43:976.

9221 MULTIPLE-TUBE FERMENTATION TECHNiQUE FOR MEMBERS OF THE

COLIFORM GROUP'

9221 A. Introduction

Tbe coiiform groiip eotisisls of several genera o f bacteria
belonging to the fainily Enterobacteriaceae. The historical deíi-
nition of thií group has been based on the m e t i i o d usad for
dctection (laclóse fermentadon) rather than o n the tenets o f
systematic bacteriology. Accordingly, whec the fermentation
technique is used, (bis group is defined as all faciiltative anaer-
obic, gram-ncgative, non-spore-fomung, rod-shaped bacteria
"Af^jiovcd hy SUndard MeOiud! Coniniiítee, A-E, 1999; F, 2001.
loini Tast Grcup: (9221C)-, Eugeae W, lüce Ichuir). Pau! S. Berger, Junes A.
Oark, Slcpben C. Háhag. Waliacc E, Ganbtjglu. Nancy H. HaJl. Shuodar Un;
(9221F); Mark C, Mecfces Icbaii), Paiii S. Berger, James A. ClaiK Waliacc E.
Gnduiglil, Nuicy H. Hall, Sbundai Lin.
thal ferment lactose w i t h gas and acid formation w i i b i n 48 h at
The standard test for tite colifonn group may be carried out
cither b y the multiple-tube fermentation technique o r presence-
absence procedure (íhrongh the presumptive-confinned phases
or completed test) descnbed herein, by the membrane fiiter ( M F j
technique (Section 9222) or by the enzymatic subsirale coiiform
test (Section 9223). Each technique is a p p í i c a b l c w i t h i n the
litDitationE specified and with due consideratíon c í the puipose
o f the examination. Production of valid results rcquires strici
adheretice to quaiity control procedures. Quality ctHitrol guide-
lines are ouiUned i n Section 9020.
When m ú l t i p l e Cubes are used i n the fermentation technique,
results o f the examination o f replicare tubes and d ü u t i o o s are
MULTIPLE-TUBE FERMBn"ATION TECHMQUE (9221VStancIani Total CoSform Fermentation Tedinique
reported in icrnis of üie Mosl Probable Number (MPH) of
orgamsms prcsenl. This number, bascd on certain probability
formulas, is an e s t í m a l e o f Ibe mean dcnsity of coliíorais in the
sample. CoUforro daisity, togcther with other information ob-
tained by engincering or sanitary surveys, provides the besl
assessment of water treatment effeciiveness and the sanitary
quality of source water.
TTie precisión of each tes! dcpcnds on the nomber of tubes
used. The mosi saiisfactory infonnation wiU be oblaincd when
Ihe iargest sample inoculum e^umined shows gas in somc or all
of the mbes and the smallesl sample inoculum shows no gas in
all or a majority of the mbes. Bacterial density can be estimated
by I h e formula given or í r o m the table using the number of
posilive tubes in the múltiple dilutions (922IC.2). The number of
sample portioos selected w i l i be g o v e m e d by the desired preci-
sión of the resull. M P N tables are based on the assumption of a
PoissoD distribution (tandom dispersión). However, i f the sam-
ple is not adequately sbaken before the portions are removed or
if dumping of bacleiial cells occurs, the M P N valué will be an
underestimale of Úie acmal bacterial density.
1. Water of Drinklng Water Quaiity
When diinking water is analyzcd to dcwnnine i f the qnality
raects the standards of Ihc U.S. Eavironmcnial Protecdon
Agency (EPA), use the fermentation technique with 1 (1 replícate
tubes each containing 10 m L , 5 leplicate mbes each CMitaining
20 m L , or a single bottle containing a 100-mL sample portion.
When examining drinking water by the framcntation technique,
process all tubes or bottles demonstraling growth with or without
•a posilive acid or gas reaction lo the confiimed phase (9221B.2).
Apply the completed test (9221 B.3) to not less tban 10% o f all
coliform-positivc samples per quaiter. Obtain at least one posi-
live sample per quarter. A posilive BC broth (9221E) or a
posilive EC M U G broth (9221F) test lesult is considered an
altemative to the posilive completed test phase.
FOT the routine e x a m i n a ü o D of public water supplies the object
of the total coiiform test is to determine the efíiciency of treat-
ment plañí operatioa and the integrity of the distribuüon sysiem.
It is aiso used as a scrcen f o i the preseoce of fecal contaminadon.
9221 B. Standard Total Coiiform Fermentation Technique
1. Presumptive Phase
Use lauiyl üyptose broth in Ihe presumptive poitiOD o f the
mulüple-lube test. I f the médium has been refngerated afler
sterihzalion, iiKiibate ovemight at room tempxrature (20"C)
before use, Discard tubes showing growtb and/or bubbles.
a, Reagems and culture medúon:
1) Lauryl tryplóse broth:
Tryptose 20.0 g
Lactose 5.0 g
Dipotassiuin hydrogea pbosphalc, K2HPO4 2-75 g
Potassium dihydiogeD phospbaie, KHjPO^ 2,75 g
»48
A high proportion of colifonn occurrences i n a distribution
system may be attributcd not to treatment failure at the plant or
the w c ü source, but to bacterial rcgrowth m the mains. Bccause
it is difficuli lo thstinguish between cohform regrowih and new
contamication, aisume all colifonn occurrences to be new con-
tamination unless otbcrwise dcmonstrated.
2. Water of Other than Drinking Water Quality
I D the examination of uonpotable waters inocúlate a series o f
tubes with appropriate decimal dilutions of the water (múltiples
and submultiplcs of 10 m L ) , based on the probable coiiform
densit>*. Use the presumptive-confinaed phase of the multiple-
tube procedure. Use the more labbr-inteosivc completed test
(922)B.3) as a quaüty control measure nn at least 10% of
coliform-positive nonpoiable water s a n ó l e s on a seasonal basis,
The object of the examination of nonpotable water geoerally is to
estímate the density of bacterial conlaminatíon, determine a
source of pollution, enforce water quality standards. or trace the
survivai of micmorganisms, The multiple-tube fermentation
technique may be used l o obtain siatistically valid M P N esti-
mates o f colifonn density. Examine a sufficienl number of sam-
ples to yield lepresentalive results for the sampling slaúon.
Generaliy, Ibc gcometiic mean or median valué of the results of
a number of samples w i l l yield a valué in which the cífect o f
sample-lo-sample variation is minimized.
3. Ottier Samples .
Tbc mulliple-tubc íwmcntation technique is af^licable lo the
analysis of salt or brackisfa waters as wcll as muds, scdimenls,
and sludges. Follow the precautions given ^ x i v c on portion sizes
and numbers of tubes per dilution.
To prepare solid or semisolid samples weigh the .sample and
add diluent to make a 1 0 ' ' dilution. For exan^ile, place 50 g
sample i n steiiJe blender jar, add 450 m L sieiile phosphate buffer
or 0 1 % pcptone dilution water, and blend for I to 2 min at l o w .
specd (8000 rpm). Prepare the appropriate decimal dilutions of
the homogenized slurry as quickiy as possible to nünimize
seltüng.
Sodium chJoride. NaQ 5.0 g
Sodium lauryl sulfate 0.1 g
Reageai-grade wue- 1 L •
Add dehydrated ingredients to waícr, mix fijoroughly, and heal to
dissolve. pH should be 6.8 ± 0.2 after sierilization. Before sieril-
izatím, dispense sufhcient médium, in fermmtation tubes \\iih an
invciied vial, to i:over ñiveited vial ai least one-half to iwo-thitds
afler sterilizatioc. Altemaíively, canil inverted vial and add 0,01 g/L
bromcresol purpie to presumptive médium to determine acid pro-
duction, tbc indicator of a posilive result in fliis part of the colifram
test Qose mbes with metal or heal-iesistant plástic c^s.
9-50
TABLE 9 2 2 1 : 1 . PREPARATION OF LAURYL TRYPTOSE BROTH
Amount of
Inoculum Médium in Tube
mL mL
l 10 or more
10 10
10 20
20 10
100 50
100 35
100 20
Make lauryl tryptose broth o f such strength that adding 100-
m L , 20-mL. or 10-roL portions o f sample to m é d i u m w i l l not
reduce ingredient concentra ti ons below those «f the standard
mediura. Prepare in accordance with Table 9221:1.
b. Procedure:
1) AiTange fermentation tubes i n r o w s of five or ten tubes each
i n a test lube rack. The number of rows and the sample volumes
selected depend upoD the quality and character o f the water to be
examined. For potable water use five 2 0 - I R L portions, ten 10-mL
portions, o r a single botüe of 100 m L portion: for nonpotable
water use five tubes per dilution (of 10, 1, 0.1 r u L , etc.),
In inaking dilutions and mea.^tring dilutcd sample volumes,
follow (be precautions given i n Section 9215B.2. Use Figure
9215:1 as a guide to preparing dilutions. Sbake sample and
diluliocs vigorously about 25 times. Inocúlate each tube i n a set
,of five with replicare sample volumes {in increasing decimal
dilutions. i f decimal quantities o f tbe sample are used), M i x test
portions i n Ihe m é d i u m by gentle agitation.
2) Incúbate inoculaied tubes o r bottles at 35 ± 0,5°C, After 24
± 2 h swirl each lube or bottle genfiy and examine i t for growth,
gas, and acidic reaction (shades of yellow color) and, i f no gas or
acidic reaction is evident. reincubate and reexamine at tbc end of
48 ± 3 h. Record presence o r absence o f g r o w t h , gas, and acid
production. I f the inner vial is ocoittcd, g r o w t h with a d d i t y
signiñes a positive piesuraplivc reaction.
c. In'erpreiution: Production o f an acidic reactioD or gas i n the
tubes o r bottles within 48 ± 3 h constitutes a positive presump-
tive reaction. .Submit tubes w i t h a posilive prestimptive reaction
to the confirmed phase (9221B.2).
Tbe absence of acidic reaction or gas formation at the end of
48 1: 3 h of incubation constimtes a negative test. Submit
drinldng water samples demonstraling growth w i t h o u t a positive
gas or acid reaction to the confirmed phase (9221B.2). A n
arbitrary 48-h hmit for observation doubtless exeludes occa-
sional members of tbe coiiform group that grow very slowly (see
Section 9212).
2, Confirmed Phase
a. Culture médium: Use brilliant green lactose b ü e broth
fermentation tubes for tbe confirmed phase.
Brilliant green lactose bile broth:
Peptone 10.0 g mbes i n w h i c h gas o í acidic g r o w i h is produced o a l y after
Lactose lO.O g 48 h .
WICROBiOLOGICAL E X A M I N A T I O N (9000)
Dehydrated Lauryl
Volume of Médium Tryptose Broth
+ Inoculum Required
aiL £A
11 or more 35.6
20 112
30 534
30 m x
150 I06.B
135 137.1
120 213.6
Oigan 20-0 g
Brilliant green 0.0133 g
Reagent-grade water I L
. A d d dehydrated ingredients to water, mix ihorougbly, and heat
ta dissolve- p H should be 7.2 3 0.2 after steriiization, Before
steiiLization, dispense, m fermentation tubes w i t h a n inverted
vial, safficient m é d i u m lo cover inverted vial at least one-half to
two-thirds after steriiization. Glose tubes with m e t a l or hcai-
resistant plástic caps.
b. Procedure: Submit a l l presumptive tubes o r bottles
s h o w í n g growth, any amount of gas, or acidic reaction w i t h i n
24 ± 2 h o f incubation to the confirmed phase. i f activte
fermentation or acidic reaction appears i n the p r e s u m p t i v e
tube e a r ü e r than 24 ± 2 h , transfer to the c o n f i r m a i o r y
m é d i u m ; preferably examine tubes at 18 x 1 h . I f a d d i t i o n a l
presumptive tubes or bottles show active fermentation or
acidic reaction a i the end o f a 48 ± 3 h i n c u b a i i o n p e r i o d ,
subirrrt these to the coofiimed phase.
G e a ü y shake or roíate presumptive tubes or bottles showing
gas or acidic growth to resuspend the organisms. W i t h a sterile
lo<^ 3.0 to 3.5 m m in diametcr, tran.sfer one or more l o o p f o h o f
culture 10 a fermentation tube containing brilliant green lactose
bile brotb or inserí a sierüe wooden appbcator at least 2.5 cm
into the culture, promptly remove, aud plunge jqjpücator to
bottom o f fenneotation tube containing brilliant green lacío.se
bile broth. Remove and discard applicator. Repeat f o r a ü other
positive presumptive tubes.
Incnbate the inoculated brilliant grera Jactóse b i l e b r o t h tobe •
at 35 ± 0.5''C. Formation of gas in any amount i n tbe inverted
vial of tbc brilliant green lactose bile broth fermentation tube at
any tiioe (e.g., 6 ± 1 h, 24 H: 2 h) within 48 ± 3 h constitutes
a positive confirmed phase. Calcúlate the M P N v a l u é from ihe
numbra o f positive brilliant green lactose bile mbes as descnbed
in Section 9 2 2 I C .
c. Aiiematrve procedure: Use this altemative only for poOuied
water or wastewater known to produce positive results c»nsisteniiy,
I f a i l presumptive oibcs are positive i n two o r m o r e r o n -
secutive dilutions w i t h i n 24 h . submit lo the c o n f i r m e d phase
only the mbes o f the highest dilution (smallesl sample inoc-
u l u m ) i n w h i c h a l l mbes are positive and any p o s i t i v a mbes
i n stiÜ higher dilutions, Submit to ihe confirmed phase a l l
MULTIPLE-TUBE FERMEtíTATlON TECHNIQUE (9Z21)/StatK)Erd Total Coiiform Fermentattor, Technique 9-51

Inocúlale lauryl tryptose broth of presence^bsence broth lermentation tubes or bottles and iricubate 24 + 2 (i al 35 ± 0.5''C,

(1) (2)
Gas and/or acidic growth produced. TTansfer to No gas or acid produced. Incúbate additlorial
confirmatofy tiriliiafit green lactose bile. incúbate 48 24 h (total 48 ± 3 h).
±3hal35±0,5'C.*

(a) (c)
Gas (n acid No gas ur acid produced.
(b)
produced. Continué Negative test. Colitorm
Gas produced. Transferí to LES No gas produced. as in (la).
Negative test group absent
Endo or MacConkey. Incúbale
24±2hBt35±0.5'C. Coiiform group
absent.

Acidic growth.
Confirm as in (1).
(1.1) {12)
Typteal OR atypical cotiform colonies. Negative colonies.
Transfer to agar slant and lauryt tryptose Coiiform group
broth fermentation tube. Incúbate agar absent.
slant 18 tcr 24 h and lauryl üyptose bf oBi
24 ± 2 h to 48 ± 3 h at 35 ± 0.5''C.

Gas produced. fio gas produced.

Grem-stain portion
of agar slant growth
íl-ll)
GtanHtegsIíve rods ptesent, no spores
present. Completed test: coiiform group
present Grarrvposítrve and -negative rods
txrth present, Repeat procedure
beginning at 1.1.
Negative test.
Cotiform group absent
* If gas or acid powtb occurs tiefore máximum
incubation time (ex. 6 ± 1 h), transfer to next
(1,12) appropriate médium.
Spores or giam-posrtlve
t Attemately use EC test (Section 9221E).
rods and spores present.
Completed test: coiiform I Optional for drinking water samples.
group absent

Figorc 9221:1. Sáatmatíc outliiie of prcstimptm, confirmed, taá compkted phasef for total colifonn dettctiiHi.

3. Completed Phnse A d d ingredients to water, mix thoroughly, and heat to boiling

to dissolve. Sterihze by autoclaving for 15 min at 121°C. Temper
To establish the presence of coiiform bacteria and to provide agar after stedlization and pour into petri plates (ItX) X 15 m m ) .
quality control data, use the completed test on at least 10% of p H should be 7.1 ± 0.2 after strailization.
positive confitmed tubes (see Figure 9221:1). Súnultaneous i n - 3) Nutrienl agar:
oculation into brüliant green lactose bile broth for total coliforms
and EC broüi for fecal coliforms (see Section 9221E below) or PeptoDC .• 5.0 g
E C - M U G broth for Escherichia coii may be used. Consíder Beef exlract 3,0 g
Agar 15,0 g
positive EC and EC-MUG broths elevated temperature (44,5°C)
Reagent-grade water 1 L
results as a positive con^eted test response. Parallel positive bril-
liant gicen lactose bile broth cultures with negative EC or EC-MUG
A d d ingredients to water, mix thoroughly, and heat to dis-
feroth cultures indicate tbe presMice of nonfecal colifonns.
solve. p H should be 6.8 - 0.2 anca- steriiization. Before sterü-
a. Culture media and reagents: izalion, dispense i n screw-capped tubes. After steriUzation, i m -
1) LES Endo agar: See Section 9222B. Use ICO- X I5-mm mediately place tubes i n an inclined posilion so that the agar w i l l
petri plates. solidil^ wilh a sloped surface. Tighten screw caps after cooling
2) MacConkey agar: and store in a protected, cool siorage área.
4) Gram-stain reagents:
PeptoDc. 17 g a) Ammonium oxalaie-crystal vioiet (Hucker's): Dissolve 2 g
Proteose pqtfone 3 g oystal violet (90% dye content) ra 20 m L 95% ethyl alcohol;
Lactose ¡O g dissolve 0,8 g (NHsljC^O^-HjO in 80 m L reagent-grade water;
Bilesalts 1.5 g mix the two solutions and age for 24 h before use; filter through
Sodiinn chloride, NaO 5 g
paper into a siaining bottle.
Agar 13-5 g
Neutral red 0.03 g b) Lugol's xolution, Gram's modificalion: Grijid 1 g iodine
Crysial violet Ú.OOl g ctystals and 2 g K I i n a mortar. Add reagent-grade water, a few
Reagcni-grade water 1 L milliliters at a time, and grind thoroughly after each additioD
9-5?
untjl soiution is complete. Rinse solution into an amber glass
bottle with the rüraaining water (using a total o f 300 mL).
cj Cour.iers!íiinj Dissolve 2.5 g safraniu dye i n 100 mL 95%
ethyl alcohol. Add 10 m L to 100 m L reagent-grade water.
á) Acetone alcohol: equsl volumes of ethyl alcohol (95%)
with acetone. . , . drain o f f excess; apply L u g o l ' s solution for 1 náa.
b, Procedure:
1) Using aseptic technique, strcák o n e LES Endo agar (Section
9222B.2) or MacConJsey agar piate from each tube of brilliant
green lactose b ü e broth showing gas, as soon as possible after the
observaiioE of gas. Streak plates i n a manncr to insure presence
of some discrcie coionies separaled b y at least 0.5 c m . Observe
the following precautions when stieaking plates to obtain a high
proportion of snccessfu! isolations i f coiiform organisms are
present: (a) Use a sterile 3-nun-diam loop or an inoculating
needle slightly curved al the tip; (fc) tap aad incline the fermcD-
tation tube to avoid p i c k i n g up any membrane or scum on the
needle; (cj insert end o f loop o r n e e d l e into the liquid in the tube
to a depth of appro.ximately 0.5 c m ; and (d) .streak píate for
isolation witb curved section o f the needle i n contact witb the
agar to avoid a scratched or toro surface. F í a m e loop between
second and third quadrants to improve colony isolation.
Incúbate plates (inverted) at 35 ± O-S^C for 24 ± 2 h.
2) The colonies developing on L E S Endo agar are defined as
Typicat ( p i u t to dark red w i t h a greco metalllc siirface sheen) or
aíypical (pink, red. while, o r colorless colonies without sheen)
after 24 h incubation. Typical laciose-feamenting colonies de-
veloping o n MacConkey agar are red and may be surroimded by
an opaque zone of precipitated bile. From each piale pick one or
more typicat, well-isolated coiiform colonies or, i f no typical
colonies are present, pick. two or more colonies considerad most
líkely to consist of organisms o f the coiiform group. and transfer
growth from each isolate to a single-strcngth lauryl tryptose
broth fermentation tube and onto a nutrient agar slant. (The laiter
is unnecessary for drinking water samples.)
I f needed, use a colony raagnifying device to provide optimum
magnification w h e n coioities are picked from the LES Endo or
MacConkey agar plates. When transferring colonies, choose
well-isolated enes and barely toucb the surface o f the colony
with a flame-sterilized, air-cooled transfer n e e d l e to minimíze
the danger o f transferring a mixed culture.
I n c ú b a t e secondaiy broth tubes Gauiyl tryptose broth with
inverted fermentation vials inserted) at 35 ± 0 , 5 ° C for 24 :r 2 h;
i f gas i s not produced within 24 ± 2 h reincubate and examine
agaic al 48 ± 3 h. Micro stopi cali y examine Gram-stained prep-
aratiüQs from those 24-h nutrient agar slant cultures correspond-
ing to the secondary tubes that show gas.
3) Grara stain technique—Thé Gram stain raay b e o:mitted
from the completed test for potable water samples, oniy because
the occurrences « f gram-positive bacteria and spore-fonning
organisnis surviving this selective screening procedure are i n -
trequEnl i n drinking water.
Various modifications o f the Gram stain technique exist. Use
tfae following modificanon b y Hucker for staining smears o f p u r é
c,u!ture; intlude a g r a m - p o s i ü v e and a gram-negative culture as
controls.
M I C R O B I O L O G I C A L E X A M I N A T I O N (90D0)
Prepare sepárate light emulsions of the test hactcriai g r o w t h
and positive and negative control cult>ires on the same slide
using drops of distilled water on the slide. A i r - d r y and (ix b y
passing slide through a flame and stain for 1 m i n w i t h anuno-
n i u m oxalate-crystal violet solution, Rinse slide i n water aud
Rinse slained slide in tap water. Decolorize for ^ j p r o x i m a t e l y
15 to 30 s with acetone alcohol by holding slide between the
fingers and letfing acetone alcohol fiow across the slained smeai
until the soJvent fiows colorlessly from the slide. D o n o t o v a -
decolorize, Coimterstain with safranin for 15 s, l i n s e w i t h tap
water, b l o t dry w i t h absorbent paper or air dry, « n d examine
nticroscopicaily, Gram-positive organisnis are b l u ^ gram-nega-
tive organisms are red. Results are acceptable o n l y ^ e n controls
have given proper reactions.
c. Interpretation: Formation o f gas in the secoodary tube o f
lauryl tryptose broth within 48 ± 3 h and d e m o n s t r a ñ o n o f
gram-negative, nonspore-forming, rod-shaped b a c t m a from the
agar culture constitute a positive rcsult for the ccanpleted test,
demonstraling the presence o f a m c m b w of the coJBfcroi group.
4. Bibliography
MEYER, E M . 1918. Aa aerobic spore-foiming baciUus: ^ v i n g ga.": i n
lactose broth isoktcd i n routiite water examinatioa. J. Bacte.rioi
3:9.
HiKXER, G J , & H J . CONN. 1923. Metbods of Gram S t a i m g . N.Y, Stare
AgT- Eip. Sta, Tech. Bu!l. No. 93. '
N08TCW, J.F. & J.J. WincaíT. 1924. Aerobic spore-fonuiog lactose fer-
meníÍEg orgaaisms and Iheir significancc in water a n a l y á s . Amer. J.
Pub. Health 14:1019.
HUCKER, G J , & H J . CONN, 1927. Fuither Studies on * c Methods of
Gram Staining. N.Y. Smte Agr. Exp, Sta. Tech. No. 128.
PORTE», R , . C.S. MCCLESKEY & M . LEVIKE. i937. Hje facaltaiive .•¡¡«sni-
lating bacteria producing gas from lactose. / . BacitricL 33:163.
Cowixs, i ' . B . 1939, A modiíied fermentation mbe, J. BaOeriol. 38:677,
SHEKMAH, V . B . D . 1967. A Guide 1 0 the Identification o f Ihe Genera of
Baaena. WiUiams & WiUdns, Balümore, M d .
GELPUBICH. E.E. 1975. Haodbook for Evaluaáng WaterBactedological
Laboratories, 2nd ed. EPA-570/9-75-006, U . S . Envíroraníiita] Pro-
tection Agency, Cinciimati, Ohio.
EvANS, T - M . , C E , WAABVICK, R . J , SEroias & M . W . LECHEVALLIEK,
198J. Failure of the most-probable number lechnéqne to deieci
colifomis in drinking water and raw water supplies, AppL Environ.
Microbio!. 41:130.
SEnsjK, R J „ T.M, EVANS. J , R -KAUFMAN, C E . W A A E V K X SL M . W . "
LEQ!EVAIJ,T:-!R, 1981. Limitations of standard colifomi ennmcration
techmques. J. Amer. Water Works Assoc. 73:538.
GERHAITDS, ?., ed. 1981. Manual of Methods for Genertó Bacteiioiogy,
American Soc, Microbiology, Washingt«i. D.C.
KMEG. N - R . &. J . G . HOLT. eds, 1984, Bergcy's Manud o f Systeniatic
Bacterinlogy, Vol 1. Wilhams & W Í M D S , BaltimoiE, Md.
GREENBERG, A . E , & D . A . HUKT, eds. 1985. Laboratoiy Procedures foi
the Examination of Seawater and SheUfisb, 5tb ed. American Public
Health Assoc., Washington, D.C.
U . S . ENVIRQNMEOTAL PROTECTION AGENCY. 1989. National primary drink-
. ing water regulations: analyrical techniques; coliforuE bacteria; final
míe. Federal Register 54(135):29998 (July 17, 1989).
MULTÍPLE-T>JBE FERMENTATION TECHNIQUE {9221)/Estima!¡on d Bactwial Density 9-53

9221 C. Estimation of Bacterial Density

1. Precisión of the MPN Fermentation Tube Test

Volume

Unless a large number of sampie portions is examincd, the Combüiation M P N Index

precisión of the most probable number (MPK) fermentatiOD tube Eiainple 10 1 0.1 0.01 0,001 o í Positives NoJlOO'mL
test is rather low. For example, Table 9221:17 shows ihal the
a 5 5 1 0 5-1-0 330
95% confidcTice limits are often onc-thírd times and Chree times 4 5 0 0 4-5-1 48
b 1
Ihe estímate. Consequently, use caution w h e n inlerpreiing the c 0 0 1 0 0 0-0-1 1,8
sanitary significancc of auy single cohform r c s u l t . When severa! á 5 4 4 1 0 4-4-1 400
samples from a given sanyiling point are estimated separatcly f. 5 4 4 e I 4-4-1 400
and tbe results combined in their geomctric mean, the prccisioo f 5 5 5 5 2 5-5-2 54 000
is greatly improved.
Select highest dilution that givce posilive results i n all five
2. Table Reading and Recordtng of MPN portions tested (no lower dilution giving any negative results)
and the two next succeeding higher dilutions (Example a), I f the
RecOTd coiiform concentraüon as the Most Probable Number lowcst dilution tested has less than five portions w i t h positive
(MPN)/100 m L . The M P N vaiucs, for a variety o f posilive aad rcsuits, sclcct i t and the two next succeeding higher dilutions
negative tube combinations, are given iu Tables 9221:11, I I I , and (Examples b, c).
I V , Tbe sample volumes indicated in Tables 9221:11 and ID are When a positive result occurs i n a dilution higher than the
choscn especially f o i examining fiiiished waters. Table 9221 ;IV thrcc selected according to the foregoing rules, change the se-
illustrates M P N valúes for combinad ons of positive and negative lection to the lowest dilution that has less than five positive
results when five 10-mL, five 1.0-mL, and five 0.1-mL sample rcsuits and the next two higher dilutions (Example d). When all
portion volumes are tested. I f tbe sample portion volumes used the foregoing selecüon rules have lefl unselected any higher
are those found i n tbe tables, r ^ r t the valué coircspooding to dilutions with positive rcsuits, add those highcr-dilution positive
tfae number of positive and negative results i n tbe series as the results to the results for the highest selected dilution (Exampie
MPN/100 m L , or, when appropriate, as presence or absence. c). I f there were not enough higher dUutions tested to select three
When the series of decimal dilutions is different from that in the dilutions, thcn select the next lowcr dilution (Example f ) ,
table, select the M P N valué from Table 9221:IV for the combi- When it is desired lo sumroarize witb a single M P N valué the
natioL of positive results and calcúlate according to the follow- rcsuits from several sanqiles, use Ihc geomctric mean or tbe median.
ing formulas: The geometric mean is conputed by averaging the logaritiunic
Vflhtcs; c,g„ the geometric mean of A , B, and C is lO'" wherc

MPN/lDOmL = (Table MPN/l 00 mL) X 10/V

L --^ nogjoA + logiofl + logjoO / 3

wbcrc: Mean valúes are reponed as the antilog o f L.

V = volume of one wiqile portion ai tbe lowest selected
dilatioa.
When more Üian three dilutions are used i n a decimal series'
of dilutions, select the three most appropnate dilutions and refer
to Table 9221 :r\'. Several examples (a through fj ilhislratc
correct selection.
Table 9221 :IV shows all but the very improbable positive tube
combinations for a thrce-dilution series. I n tesling 10 di^crcnt
samples, there is a 99% chance of finding all the results among
TABI.H 9221 J n . M P N b.-DEx AND 9 5 % CONFIDENCE LIMTTS FOR A U ,
COMBIKATIONS OF POSmVE AND NEGATT-T RfiStXTS WHE.V Tíü 10-ML
PORTIONS ARE USED

No. of Tubes 9 5 % Confidcnce

Giving Positive MPN L Í ^ U (Exaci)
TABLE 9 2 2 I : I L M P N INDEX AND 9 5 % COWEEN-CE LÍMTTS mu ALL
RcatlioD Out of Index/ —
CoMBKATtoNS OF PosmvE AND NÉGATTITÍ KESULTS WHEN FlVE 20-ML
mL Each) 100 mL Lowcr Lppa
Poiinow ARE USED

0 <I.l 3,4
No. of Tubes 9 5 % Confldence Limitt
Giving Positive MPN (Exact)
1 1.1 0.ÚS1 3.9
2 12 037 •3
Rcíction Out of Index/
5 (20 m L Each) 100 mL I^ower Upper 3 16 MX- M

»
4 5J U
0 <l.í — 3.5 5 6S 23 15
1 1.1 0,051 5.4 6 9.2 3,3 19
2 2.6 0.40 8.4 7 12 4.8 24
3 4.6 1,0 13 8 16 5,8 34
4 8.0 2.1 23 9 23 S.l 53
5 >8.0 3.4 ~ 10 >23 13 —
9-54 MICROBIOLOGICAL DCAMIhíATION { 9 0 0 0 )

T A B Ú 9221:1V. M P N bioEX AND 9 5 * CONFEDBUCE LIMITS K W VARIOUS COMBINATIONS ot= PosmvE RESULTS WHEN FIVE TUBES ARE U S E » PER Dn.unoN
( 1 0 M L 1.0 M L , 0.1 M L ) »

Confidence LirniK COEfiidcDce Limits

CombÍEisiíüii M P N Index/ ComtanatiOD MPN Index/
oí Positives 100 mL Low High oí Poñtives iOO mL Low líigh

0-0-0 <1,8 6.8 4-(V3 25 9.8 70

(H)-l 1.S a09D 6.8 4-1-0 17 6,0 40
ta .U90 6.9 4-1-1 21 6.8 42
0-1-1 M a n 10 4-1-2 26 9.8 70
O H 17- a70 10 4-1-3 31 10 70
0^1 M 15 4-2-0 22 6J 50
094 - üS" u 15 4-2-1 26 9,8 70
1<00 zo oio 10 4-2-2 - 32 10 70
14-1 AS¡ a70 ' 10 4-2-3 38 14 100
1-03 &0 u 15 4-3-0 27 9.9 70

>I - l«- l 4.0 0.71 12 4-3-1 33 10 70

6.1 1.8 15 4-3-2 39 14 100
M-2 S.1 3.4 22 4-4-0 34 14 100
1-2-0 6.1 1.8 15 4-4-1 40 14 100
. 1-M 8.2 3^ 22 4-4-2 47 15 las
8J 3^ 22 4-5-0 41 14 100
U-l 10 3.5 22 4-5-1 48 15 120
M 10 3J 22 5-0-0 23 6.8 70
MM> AS OL7» 15 5-0-1 31 , 10 70
3*1'i'.. k* 15 5-0-2 43 14 100
. -'• ' . . •: MI iAy. - 22 5-0-3 58 22 150
6J U 17 5-1-0 33 10 100
2rl.l 9,2 3.4 22 5-1-1 46 14 120
M-2 12 41 26 5-1-2 63 22 150
» 0 93 3.4 22 5-1-3 84 34 229
34-1 12 4.1 26 5-2-0 49 15 . 130
14 5.9 36 5-2-1 70 22 170
. ^^-o 12 4.1 26 5-2-2 94 34 230
2-3-1 14 19 36 5-1-3 120 36 230
344 15 19 36 5-2-4 150 58 400
-•OO - • . U ti 22 5-3-0 79 22 220
11 . 15 23 5-3-1 110 34 230
»M ». 16 35 5-3-2 140 52 . 400
M-O U 3S 26 5-3-3 170 70 400
M-1 14 5JS 36 5-3-4 210 70 400
3-1-2 1? 10 36 5-4-0
- ^ 36 . 400
3-2-0 14 36 5-4-1 ñtf n 400
3-2P1 17 40 5-4-2 230 70 440
S3-2 18 40 5-4-3
3ff 100 710
»»« n 6.8 40 3-4-4 « 0 100 710
M-1 21 18 40 5-4-5 430 150 1100
24 9J 70 5-5-0 340 70 710
21 18 40 S-5-1 350 100 1100
>*•
M-t . . 34 9JÍ 70 5-5-2 5W 150 1700
J94MI . . 25 9.8 70 5-5-3 •J20 •-• 220 2600
13 , 4.1 35 5-5-4 1600 400 4600
S i 17 5.9 36 5-5-5 >1600 700
4J3-2 21 6.8 40
* Ruulu 10 ovo sigúñcuil figures.

these 9 5 outcomes. l í untabulatcd combinatíons occur w t h a
frequeíicy greater than 0.1?c, i t indicates that the t e c h i ñ q u e is
faulty or that the statisticai aBSumptions underlying the M P N
estimatc are not hcing fulfiUed (e.g., growth inhibition at l o w
dilutions).
The M P N fot combinations not appearing i n the table, or for
other combinations of tubes or dilutions. may be estimated as
follows: R r s t , select the lowcst dilution that does not have aJl
positive rcsuits. Second, select the highest d i l u t i o n w i t h at least
one posilive result. Finally, select all the dilutions between them.
For example, from (5/5, 'lO/lO, 4/10, 1/10, 0/5) use o n l y (-, - ,
4/10,1/10, - ) , 4/10 @ 0.1 m L sample/mbe and l / I O @ 0.01 m L
s a m p í e t o b e ; from (5/5, 10/10,10/10,0/10, 0/5) sctect only (_-. - .
10/10, 0/10. - ) , 10/10 @ 0.1 m L sample/tube ancS O/IO @ 0.0!
MÜLTIFIÍ-TUBE FERMEMTATION TECHNIQUE (922ll/Presance-Absence f-A) Coiiform Test 9-55

m L sample/tube. Use oniy the selccied dilutions i n tbe following example tiiscussed above was (5/5. 10/10, 10/10, O/IO. 0/5),
formula o í Thomas^; with portions 10, 1,0.1, 0 . 0 1 , and 0.001. Tbe tbird d i l u i i o u is
the highest with positive ponions, so V = 0 . 1 . Tlie M P N for
MPN/100 mL (approx.) = 100 X P / ( ^ - X d " ^ Ihc third and fourth dilution would be c x a c ü y

where:
P - nomber of positive rcsuits, MPN/100 roL = (1/0.1) X [ m 3 logm (1.1/0.1)]= 2400/100 m L
N - volume of sample in all the negadve portions combined,
m L , and
3, Reference
T - total volume of sample in tbe selected düutioiis, mL,
L T^QMAS. H.A., JR. 1942. Bacterial deosiiies fiom famraitatjon tube
In the first example above, tests. J . Amer. Water Works Assoc. 34:572.

MPWIOO mL (ípprox.) = 100 X 5/ (0.69 x 1.1)'°=

50Crt).87 = 57W100mL '
In the second example above,
MPN/100 mL(approx.) - 100 X 10/(0.1 X 1,1)"^=
nmo^il = 3000/100 mL
The two examples compare weU with the truc MPNs, 590/100
m i , and 2400/100 mL, respeciively. The second example is a
special case for which an exaet solution can be calculated d i -
rectly for the two selected dilutjons.
Although M P N tables and calculations are descnbed for use i n
tbe coiiform test, ihcy also dctcimiiie the M P N of any other
organisms providcd that suitable test media are available.
When all Ihe results at the lowcr dilutions are positive and all
the results at higher dilutions are negative, i t is possible to
calcúlate an exact MPN for lwo selected dilutions as follows:
When V is tbe voliune of each individual sample portion at the
highest dilution wilh all positive portions,
MPN/100 mL = (i/V) [230.3 log.o (TIN)}
where T and N are defined as for Thomas's formula- The lasi
4. Bibliography
MCCRADY, M , H . 1915. Tbe numérica] interpretation of fenncntuioD
tube results. J . Irg'eei. Dis. 12:183.
MCCRADY, M . H . 1918. Tables for rapid iiitetprctation of fermentation-
Cübe results. Pub. Heallk J. 9:201.
HosKiKs, J . K , 1933. The most probable numbers of B. cali in water
analysis. / . Amer. Water Works Assoc. 25:867.
HosKiNS, J . K . 1934. Most Probable Numbers for evaluatjon of coli~
a^rogenes tefili by fennentation mbe method. Pub, Heahh Rep.
49:393.
HALVOBSON, H . 0 . & N.R. ZJEOLEX. 1933-35. Appücaiion of stadstics to
problems in bacteriolog>'. Bacicriol. 25:101; 26:331, 559; 29:
609.
FJ.'IBÍHART. C . & P.W, WiLSON. 1943. Statiitical methods and control to
bacteriology. Baeteriol. Rev. 1:51. ,
COCKRAN, w . G . 1950. EstimalioD of bacterial den^ties by tneans of &e
"Most Probable Niunbw." Biometrics 6;!05,
WoMiWARD. R.L 1957. How probable ia ihe Most Probable Nurabcr?
J. Amer. Water Works Assoc. 49:1060.
DFMAN, J.C. 1983. MPN tables. cotrected, Evr J. AppL Bioiechnol.
17:301.
GARTHRIGHT. W . E 1998. Appendix 2. Mosi probable number from serial
diluúons. FI)A Badcriological Analytical ManuaL 8th ed.. Rev, A .
A O A C lutenuUicm], Gaithersburg, Md.

9221 D. Presence-Absence (P-A) Coiiform Test

The pres«ice-absencc (P-A) test for the c o l i f o n n group is a The P-A test is iniended for use on routí;ne samples coUccted
simple modification of the multiplc-fiibc procedure. Simplifica- fiom distribution sysiems or water treatment plants. When sam-
tion, by use of one large test portion ( l O O m L ) i n a single ctfiture ple locatíons produce a positive P-A result for coliforms, i i may
bottle to obtain quahtative itiformation on the presence or ab- be advisable to determine coiiform densities i n repeat samples.
sence of coliforms, is justified on the theory that no coliforms Quantitative information may indicate the magnimde o f a con-
should be prescmt in ¡ 0 0 m L o í a drinking water sample. The taminating cvcnL
P-A test also provides the optional opportuuity for funhcr
screening o f the culture to isolate other indicators (fecal coii- 1, Presumptive Phase '-
form, Aeromonas, Stapkylococcus, Pseudomonas. fecal strepto-
coccus, and Clostridium) on the same q u a ü t a t i v e basis. Addi- a. Culture media:
tional advantages include the possibility of exainining a larger 3) P-A broth: This médium is comroercially available i n
ntmiber o f samples per unil of time. Comparative studies w i l h dehydrated and in sterile conceotrated form,
the membrane filter procedure indicate that the P-A test may
maximize coiiform dctection in samples containing many organ- Beefextract 3.0 g
isms that could overgrow coiiform colonies and cause problems PeptoDc 5.0 g
Laclóse 7.46 g
in dctection.
Tryptose 9.83 g
9-56
Dipotassium hydrogen phosphate, KjHPOj L35
Potassium dihydrogen phosphate, KHjPO, 1.35
Sodium chioride, NaCi 2,46
Sodium lauiyl sulfate 0.05
Biomcresol purpie 0.0085
Beageut-grade water 1 L
Make this formulation triple ( 3 X ) strengfh when examining
lOO-tol. samples, Dissolve the P-A broth m é d i u m i n water
without heating, using a s t i n i n g device. Dispense 50 m i . pre-
pared m é d i u m into a screw-cap 250-mL n ü l k diluiion bottle. A
fercoentation tube insert is not necessary. Autoclave for 12 m i n
at ! 2 r C w i l h the tota] time i n üie autoclave limited to 30 m i n or
less. p H should be 6.8 ± 0.2 after steriiization. W h e n the P A
mediiun is straili2ed by filtration a 6 X strength m é d i u m may be
used. Asepticaily dispense 20 m L o f the 6 X m é d i u m into a
sterile 250-mL dilution bottle or equivalent container.
2) Lauryl tryptose broth: See Section 9 2 2 1 B . L
b. Procedure: Shake sample vigorously for 5 s (approximately
25 times) and inocúlate 100 m L into a P-A culture bottle. M i x
thoroughly by inverting bottle once or twice to achieve even
distribution of the triple-strength m é d i u m throughout the sample.
Incúbate at 35 ± 0.5''C and inspect after 24 and 48 h for acid
reactions.
c. Interpretation: A d i s ñ n c t yellow color forms i n tbc m é d i u m
when acid conditions exist f o l l o w i n g lactose fermentation. I f gas
also is being produced, g e n ü y shaking the bottle w i l l result m a
foaming reaction. A n y amount o f gas and/or acid constitutes a
positive presumptive test requiring confinnation.
2, Confirmed Phase
The confirmed phase is outJined i n Figure 9221:1.
a. Culture médium: Use brilliant green lactose bile fermenta-
tion tubes (see 9221B.2).
9221 E. Fecal
Eevatcd-temperature tests for distinguishing organisms of ihc
total coiiform group that also bciong to the fetal coiiform group are
described herein, Modifications i n lechmcal jMT>ccdtucs, standard-
ization of methods, and delailed studies of the fecal coiiform group
have esiablished the valué o f this procedure. ' l ^ e test can be per-
fonued by one of tbe raulüple-mbe procedures described here or by
niembiane filta: methods as described i n Section 9222. Tbe proce-
dure u.'áng A - 1 broth is a stngle-step method.
The fecal coiiform test {using EC médium) is applicable to
investígatioQS o f diiniáog water, stream poUutioii, raw v/ater
sourccs, wastewater treatment systems, bathing waters, seawatcrs,
and general water-quality monitoring. 5'rior enricbrnent i n presump-
tive media is required for optimum lecovery of fecal coliforms
when using E C m é d i u m . The le^t using A - 1 m é d i u m is appücable
to source water, seawaier, and treated wastewater.
1. Fecal Coiiform Test (EC M é d i u m )
The fecal c o i i f o r m test is used to d i s t i n g u i s h those total
c o l i f o i m organisms that are fecal c o l i f o n n s . Use E C m é d i u m
MICROBIOLOGICAL EXAMINATION (9D0Q)
g b. Procedure: Transfer all cultures that show a d d reaction or
g acid and gas r c a c ü o n to fariUiaot green lactose bÜe ( B G L B ) broth
g for incubation at 35 ± 0.5''C (see S e c ü o n 9221B.2).
g
c. Interpretation: Gas production i n the B G L B ts'oth culture
g
w i t h i n 48 ± 3 h confinns the presence o f colifoniii bacteria.
Rcport result as presence-abscnce test positive or negative for
total coliforms in 100 m L o f sample.
3. C o m p l e t e d Phase
T h e completed phase is outlined i n Section 9221B,3 aud
Figure 9221:1.
4, Bibliography \
WEISS, J.E. & C . A . HwTOR. 1939. Simpliüed bacteriologicaj examina-
tion of waiei. / . Amer. Water Works Assoc. 31:707.
CLARK, JJÍ. 1969. The detection of variouí bacteria indícative of water
poflution by a presence-absence (P-A) procedure. Con. J. Micro-
bioi 15:771.
CLARK, J A . &, L.T. VLASSOFF. 1973. Rdaiiojjships among pollution
iodicaior bacteria isolaled from raw water and dismT»tion systems
by the presente-absence (P-A) tesL Health Lab. ScL 10:163.
CLARK, 1.A. 19fiO. The inñuMice of increasing numtieTS o f uoiündicaioT
organisms upon the dctection of indicator organisms by the mem-
brane filter and preseoce-abseiice tests. Can. J. MiaobioL 26:827,
Qjuuc, J.A., C.A. BuRGEH & L . E . SABAUNOE. 1982. Chaiacterization of
iodicaior bacteria in municipal raw water, d r i n ü n g water aud new
Ttmi" water samples. Can. J. MicrobioL 2*:1002. ,
JACOBS, N J . , Wi, ZHGLER, F . C . R£ED, T.A. SVJSEL &. E - W . RICE. 1986.
Comparison of membrane filter, multiple~fenneraation-tabe, and
preseíDce-ahseace techniques for dcteciing total coliforms ia sinall
comraunity water systems. AppL Environ Microbioi. 51:1007,
RICE, E . W . , E . E . GELDREICH & E J . READ. 1989. The ¡msencc-absence
coHfoim test for monitoring drinking water quali^. Pub. Heahh
Rep. 104:54.
üform Procedure ^ >
or, for a more rapid test o f the quality o f shelffish waters,
treated wastewaters, or source waters, use A - 1 m é d i u m i n a
d i r e c i test.
a. EC médium:
Tryptose or trypticasc .- 20.0 g
Lactose 5,0 g
Bile salts raixtore or bile salEs No. 3 1.5 g
Dipotassium hydrogcn phosphate, KjHPO, 4.0 g
Potassium dihydrogen phosphate, K H 3 P O 4 1.5 g
Sodmro chloride, NaCl 5.0 g
Rcagcnt-gradc water 1 L
A d d dehydrated ingredients t o water, m i x t h o r o u ^ y , and heat
to dissolve. p H should be 6.9 ± 0,2 after steriiization. Before
steriiization, dispense i n fermentation tubes. each w i t h un i n -
verted v i a l , sufficient m é d i u m to cover the inverted v i a l at least
panially after steriiization. C i ó s e mbes w i t h metal o r heat-resis-
tant plástic caps.
MULTIPLE-TUBE FERMENTATION T E C H N I Q U E (9221)/Esc/)ertc/M co// Procedure
b. Procedure: Submit all presumptive fermentation mbes or
botües showing any amount of gas, growth, or acidity within
48 b of incubation to the fecal coiiform test.
1) Gcotiy shake or rotatc presumptive fermentation tubes or
botües showng gas, growth, or acidity. Using a sterile 3- or
3.5-iniii-diaio íoop or sterile wooden applicator stick, transfer
growth from each presumptive fermentation tube or bottie to EC
broth (see Section 9221B.2).
2) Incúbate inoculated EC broth tubes i n a water bath at 44.5
:!; 0.2°C for 24 ± 2 h.
Place all EC mbes in water bath wiúiin 30 m i n after ínocula-
tion. Maintain a sufficient water depth in water bath incubator to
immerse tubes to upper Icvel of the médium.
c. Imerpretat'wn: Gas ppxiuction wilh growth i n an EC broth
culture within 24 ± 2 h or less is c^msidered a positive fecal
coiiform reaction. Failure to produce gas (with ü t ü e or no
growth) constitutes a negative reaction. I f múltiple tubes are
used, calcúlale M P N from tbe number of positive EC broth tubes
as described i n Section 9221C. When using only one tube for
subculturing from a single presumptive bottle, rcport as presence
or absence of fecal colifonns.
2. Fecal Cdiforni Direct Test (A-1 Médium)
(LA'} broth: This mediummay be used for the direct isolation
of fecal colifoims from water. Prior carichment i n a p r c s u n ^ v e
m é d i u m is not required
Lactose 5.0 g
Tryptcme 20.0 g
Sodium chloride, N t Q 5.0 g
Salícm 0.5 g
' Folyetbylaie glyc^ ^isoocty^dienyl ObeT* 1.0 mL
Reagent-grade wstcr 1 L
Heat to dissolve solid ingredients, add polyethylene glycol
p-isooclylpbenyl ether, and adjust to pH 5.9 ± 0.1. Brforc
steriiization dispense in fermentation tubes with an inverted vial
* TríloQ X-lOO, Rohm md H u s Co.. ix eqmvileiit.
9221 F. Escheríchia coli ProcecJure (PROPOSED)
Escherichiú coli is a membex o f the fecal coiiform group of
bacteria. Tlus organism in water indicates fecal conlamination.
Enzymatic and biochenücal assays have been devetoped that
allow for the Identification of tiñs organism. Assays for p-glu-
curonidasc, giutamate decarboxylase, or Indole may be used W
determine the presence o f E. coli. For the E coli test with
E C - M U G médium, % I below, £ coli is defined as the species o f
coiiform bacteria possessing the enzyme ^-glucuionidasc and
capable o f cleaving the ñuorogenic subsirate 4-methyIumbel-
hferyl-p-D-glucuroiñde ( M U G ) with tbc corrcsponding reléase
of the fiuoiogeo when ^ w n in EC-MUG m é d i u m at 44.5°C
sufficient médium l o c o v e r the inverted vial at least partially
after steriiization. Cióse with metal or heat-resistant plástic caps.
Sterilize by autoclaving at 121°C for 10 min Store i n dark at
room temperanne for not longer tban 7 d. Ignore formation of
precipítate,
Make A - ! broth o f such strength that adding 10-mL sample
portions 10 mediiun wiJ] not reduce ingredient concentratíons
below those o f the standard m é d i u m . Por lO-mL samples prepare
double-strength mcdiimL
b. Procedure: Inocúlate tubes o f A - I broth as direcied i n
Section 9221B.lfcl). Incúbate for 3 h al 35 ± 0.5°C. Transfer
tubes 10 a water bath at 44.5 ± 0,2''C and incúbate for an
additional 21 ± 2 h.
c. Interpretation: Gas production i n any A - 1 broth culture
within 24 h or less is a positive reaction indicating the presence
of fecal coliforms. Calcúlate M P N from tbc number o f positive
A-1 broth tubes as described m Section 9221C.
3. Brbliogra^rfiy
PERRV, C A . & AJí. llMNA. 1933. A modíficd Eijkman médium. J.
Bacterial. 26:419.
PERRY, C A . & A-A. HiUNA. 1944. Futther evahiaiion of E C médium for
the isolation of colifuim bacteria and Escheríchia cali. Amer. J.
Pub. HealA 34:735.
OEUWEÍCH, EE., ILF. CLABK. P.W. KABLEX, C.B. Hutr & RH. BCWDNEU.
1958. Tbe cdifonn gronp. 11. Reactions in EC médium at 45°C.
AppL Microbioi 6:347.
G E i D m c H . E.E., RJl. BQKONER, C.B. HUFF. H P . C i ^ & P.W, KABIER,'
1962. T ^ diíttibution of colifomi bacteria in the feces of wami-
bJooded annnals. J. Water PoUut Control Fed. 34:295.
GELDRHCH, E.E. 1966. Sanitary significancc of fecal coliforms iu the
environment FWPCA PubL WP-20-3 (Nov.). U . S . Dep. Interior,
Washington. D.C.
AKtstEws. W Ü , & M . W . PnEstíHj., 1972, Rapid tocovciy oíEscherickía
coU from esniarine waiCT, Appl Microbioi 23:521.
OLSON, 6 , H , 1978. Enhanced accniacy of coiiform testing in seawater by
a modiScBticBi of tbc most-probable-Eunüjer method. Appt Micro-
bioi 36:438.
STRANDWDOE, J-H. & J.J. DEUINO, 1981. A - l Mednim: Altemative
technique for fecal colifomi organism enumeratioD in chlorijiated
wastewaicrs. Appl Environ. Microbioi 42:918.
within 24 ± 2 h or less. For Ihe E.coli test with G A D reagent, 1
2 below, ihe E. coli species is defined as coiiform bacteria
possessing the enzymc gíuiamate decarboxylase ( G A D ) and
capable o f producing an alkaiine reaction within 4 h i n a reagent
containing a lytic agcnl and glutamic acid. For the £ . coli test
involving Índole production, 1 3 below, the E. coli species is
defined as colifoim bacteria that can produce mdolc within 24 ±
2 h or less when g r o w n ' i n nyptone water al 44.5''C . Tríese
procedures are used to confirm tbc presence of E. coli after prior
cmichment in a presumptive m é d i u m for total coiiform bacteria.
These tests are performed by the tube procedure as described
8-5S M(CH0B)OLOQiCAL EXAMINATION (9O00)

hwe or by the m c n i r a n c filter method as described in Section L-glotamic acid 1.0 g

9222. The chromogenic substrate procedure (Section 9223) can Sodium chloride, NaQ 90.0 g
Bromocresol green 0.05 g
be used for direct detectiou of E. colL
Polyethylene glycol octylphenyl ether* 3.0 mL
Tests for E. coli are applicable to tiae analysis of drinking Reagent-grade water 1 L
water, siuface water, groimdwater, a n d wastewater. E. coli is a
membcr of the indigenous fecal flora of warm-blooded animáis. A d d ingredients lo water and m i x thoroughly u n ü j a t l ingre-
The occurrencc of E. coli is considercd a spccific indicator of dients are dissolved. p H should be 3.4 ± 0.2. The icageutTs
fecal contamiaation and the possible presence o f cnteric palbo- stahlc for 2 monihs when storcd at 5" C. I t can be filter-sterilized
gens. (0.2-;im ñlter) and treated as a sterile solution.
b. Procedure: Submit all presumptive fermentation tubes or
1. Escheríchia coli Test ( E C - M U G M e d k i m )
botües (9221B, 9221D) showing growth, gas, or a c í d i ^ w i t h i n
48 ± 3 h of incubation to Ihe E. coli test.
Use E C - M U G mediura for the c o n ñ i m a D o n o f £ coli.
1) Gently shake or rotate pre.'amiptive tubes or botlles show-
a. EC-MUG médium:
ing growth, gas, or acidity. Using a graíluated pipet. transfer 5
m L broth from tbe fennentation mbe or bottle to 15-inL c e n i ñ -
Tryptose oí trypticase 20,0 g
Laclóse 5-0 g fuge tube.
Bile salts mixture at büe salts No. 3 U g 2) Conccnlrate the bacterial cells from the broth b y ccntrifti-
Dipotassium hydrogen phos^rfjMe, KjHPO, 4.0 g gation a l 2500 to 3000 X £ for 10 m í n . Discard supematant and
Potassium dihydrogen phosphate, KH2PO4 U g resuspend cells i n 5 m L phosphate buffer. R e c o n c é n t r a t e ceDs b y
Sodium chioride, NaQ 5.0 g
centriñigatíon ( 2 5 0 0 to 3000 X g, 10 m i n ) . Discard snpenvataot
4-methylumbelliferyl-P-i>-glucuronide (MUG) 0.05 g
and add 1.0 m L G A D reagent. Vigorously swirl tbc cube l o
Reagem-grade water 1 L
resuspend cells i n G A D rtiagent.
3) Incúbate mbes at SS^C and observe after 1 h . Tubes may be
A d d dehydrated ingredients to w a t M , m i x thoroughly, and heat
lo dissolve. p H should be 6.9 ± 0.2 after stCTilization for 15 min iacubaled for a maTJmiim o f 4 h .
at 12rC. Before steribzation, dispense in tubos that do not c. Interpretation: Examine all tubes for a distinct c o l a r change
fluorcsce imder long-wavclength (366 n m ) ultraviolct ( U V ) lighl. from yellow to bluc. The prc.<ience o f a blue color is considered
A n inverted mbe is not nccessary. C i ó s e tubes w i l h metal or a positive responso for £ . ccli. A positive control consisting of ^
heat-resistant plástic caps. known E. coli (GAD-positive) culture, a negative c o n t r o l con-
b. Procedure: Submit all presumptive fermentation mbes or sisting of a k n o w n tota! c o l i f o n n organism, e,g., Enferobacter
bottles (9221B, 9221D) showing growüi, gas, o r acidity within cloacae (GAD-negative), and an uninoculated G A D reagent
48 ± 3 h o f incubation to the K coli test. control may be incoiporated i n the assay to assist i n interpreia-
1) Gently shake or rotatc presumptive fermentation tubes or tioQ of results. I f múltiple mbes are used, calcúlate M P N from
bottles showing growth, gas, or acidity, U s i n g a s l e t ü e or the numbCT o f positive G A D tubes as described i n Section
3.5-mm-diam metal loop or sterile wooden applicator stick, 922IC. When using only one mbe or a single presun^itiTe bottie,
transfer growth from presumptive fermentation tube or b o t ú e to repon as presence or absence o f E. cali.
E C - M U G broth.
2) I n c ú b a t e inoculated E C - M U G tubes i n a water b a ± main-
tained at 44.5 í : 0.2°C for 24 ± 2 h . Place all E C - M U G tubes i n 3. Escherfcft/a co//Test (Índole Production)
water bath w i t h i n 30 m i n after inoculation. M a i n t a i n a sufficient
water depth i n tbe watei-bath incubator to immerse tubes to í j s e tiyptone water and Kovacs' reagent for confirmatioD o f R
upper Icvel o f m é d i u m . coli.
c. Interpretation: Examine all tubes exhibiting growth for a. Reagents:
fiuorescence using a long-wavclenglh l a m p (preferably 6 1) Tryptone water:
W ) . The presence o f bright blue fiuorescence is considered a
positive response for E. coli. A positive control consisting of a Tryplone ., 20 g
kuown K coli (MUG-positive) culture, a negative control con- Sodium chloride, NaQ 5 g
sisting o f a thcimotolerant Klebsie^la pneumoniae (ML'G-nega- Reagcnl-giade water -,. 1 L
tivc) culture, antl an iminoculated m é d i u m control may be nec-
essary to interprei the results and to avoid c o n f u s i ó n of weak Add ingrecdeuts to water and m i z thoroughly u n t i l dissolved.
aulofiuorescence o f the m é d i u m as a positive response. I f múl- Adjust p H to 7.5, Dispense 5 - m L portions into mbes, cap, and
tiple tubes are used, calcúlate M P N from ihc number o f positive sterilize for 10 m i n al ! 1 5 ° C .
E C - M U G broth wbcs as described i n Section 9221C. When 2) K o v a c i ' reagent:
using only one tube or subculluring from a single prcsimiptive
bottle. rcport as presence or absence of E. coli. p-Dimetiiylairiinoben/aldehyde 5 g
Amj-l alcohol (analítica! grade) 75 mL
2. Escheríchia coli Test (GAO Procedure) HydrocMoric acid, conc 25 mL

Use G A D reagent for Ihc confirmation o f E. coli.

a. OAD reagent: ' Tritón X-100, Union Ciibidc Co,, or equivalimt.
M a e R A N E FU-TER 7EOIN1QUE f9222^V.troduction
Dissolve aldehyde i n alcohol. Cautiously add acid to alde-
fcyde-alcohol m i i t u r e and swirl to m i x . Store i n the dark at 4 ° C .
This reageai should be palé yeUow to hght browo in color. Use
o f low-q-jality amyl alcohol may produce a dark-colored reagent;
do not use such a rcagenL
b. Procedure: Submit aU presumptive fermentalion tubes OT
bottles (922IB) showing growth, gas. or acidity within 48 ± 3 h
of incubation to the E. coli test.
Gently shake or rotatc presumptive tubes or botlles showing
growth, gas, or acidity. Using a sterile 3- or 3.5-iran-diam meta!
loop or sterile wooden appbcator stick, transfer growth ñxMU
presumptive fennentation tube or bottie to a Uibe containing 5
mJ- tiyptone water. Incúbate inoculated tryptone wato' tubes in
a wat^r bath or incubator maintained at 44.5°C for 24 ± 2 h.
After incubation, add0.2to ü,3 m L Kovacs' reagent to each tube
of tryptone water.
c. Interpretation: Examine all mbes for ^pearancc of a deep
red color in the upper layer. The presence of a red color is
considered a positive response for E. coli. A positive control
consisting o f a known E. coli (indole-positive) culture, a nega-
tive control consisting of a known total coiiform organism, e.g.,
Enterobacier cloacae (indolc-ncgative), and an uitinoculated
reagent control may be incoiporated i u tbe assay to assist in
interpretation o f results. I f múltiple mbes are used, calcúlate
MPN from the number of indole-positive mbes as described in
Section 9221C. When using only one Uibe or a single presump-
tive bottle, report as presence or absence o f E. coli.
9222 MEMBRANE FILTER TECHNIQUE FOR MEMBERS OF THE COLIFORM GROUP*
9222 A.
The membrane filter (MF) technique is highly rcproduciblc,
can be iised lo test rdatively large sample volumes, and usually
yields numerical results more rapidly than the multiple-tube
fermentation procederé. The M F tecfuúque is cxtremcly useful in
moititoring drinldng water and a variety o f natura] waters. H o w -
ever, the M F technique has limitations, particularly when tesling
waters with high turbidity or large numbers o f noncoliform
(background) bacteria. When the M F technique has not been
used prpviously, it is desirable lo conducl parallel Icsts with the
meüiod Ihe laboratory is using currentiy to dcmonsuaie ^pUca-
bihty and comparabüity.
1. Definitíon
As rclated to the M F technique, the coiiform group is defined
as those facullative anaerohic, gram-negative, non-spore-form-
ing, rod-shaped bacteíia thal develop red colonies with a metaÜic
• Appmvei! by Standard Meibod» Coraniinee. 1997.
Joinl Task Group: 20lh EdiüOD—-Nancy H. Hall (chaii), l'aul S. Berger, IJnda R.
Bliih, Robot t i . Bordnci, John K, Brokaw, Hlen P. Ranafian, Edwiu E. Geld-
reich, Paul J. Hickey, Mnk W. l.eCheval!¡ei, ShundarLin, Goidrai A, McFetos,
Báaa A. Nüfca, Pc¿gy A- Rykfr, tynn P. Smiih, Jody A. Suvcevidt, Ori A. ZieL
9-59
4. Bibliography
FENO, P.C.S. & p j \ HABTWAN. 1982. Fluorogcnic assays for immcdiate
confinnation of Eicherichia coix. Appl Ercñron Microbioi. 43:
1320.
HARTMAN, P A . 1989. Tbc MTJG (Ehicuronidase) test for £. coli in food
and water. In A. Balows et al., eds., Rapid Meójods and Automation
in Microbiology and Imnnniotogy, Proc. 5th Int!. Symp. on Rapid
Methods and Automation b Microbiology & Immunology, Flo-
rencc, Iialy, Nov. 4-6. 1987.
'P^ssixs-, 1. & J. RmXE. 1990. Gtuuminsauredecarboxylase-schnelltcst
zur idenrifikaiion vcm Escherichta colL Z Ges. Hyg. Crenzgeb.
36:620.
SHADIX, L . C . & E.W. Rx£. 1991. Evahution of ^-ghicuronidatie assay
for the dctection of Escheríchia coli from environmental waters.
Can. J. MicrobioL 37:908.
RICE. E.W., CU. JOHNSON, M . E . EVONNIGAN &. D J . RI^ONER. 1993.
R ^ d gluiamaie decaiboxyUse asssy for dctection of Escheríchia
cali Appl Environ. MicrobioL 59:4347. Erala. 1995. AppL Envi-
ron. Microbioi. 61:847.
RICE, E.W., C H , laasson & D.J. REASONBK. 1996. Dctection of Esche-
ríchia coli 0157:H7 in water liom coiiform cnrichment cultures.
Leo. AppL Microbioi. 23:179.
STANDING CcwMrrrEE OF AÍJALYSTS. 1994. Report on Public Health and
Medical Snbjecu No. 71. Metbods for tbc Examinaücm of Watas
and Associated Materials, Tbe Microbiology of Watci Part
l-Drinldiig Waici, HMSO Books, Lonóon. U.K.
Introduction
(golden) sbecn within 24 h at SS'C on an Endo-type médium
coctairúng laclóse. Some members of the total coiiform group
may produce dark red, mucoid, or nucleated colonies without a
mctallic sheen, When verified these are classificd as atypical
coiiform colonies. When purified cultures of colifonn bacteria
are tested, they produce negative cytochrome oxidase and posi-
tive p-galactosidase test rcactions.t Generaliy, pink (non-mu-
coid), bluc, white, or colorless colonies lacking sheen are con-
sidered noncoliforms by this technique.
2. A p p J i c ^ o n s
Turbidity caused by the presence of algae, particulates, or
other interfering material may not permit lesting of a sample
volume sufficient lo yield significanl rcstüts, L o w coiiform es-
tímales may be caused by tbe presence of high numbers of
noncoliforms or of toric substanccs, The M F techuique is appli-
cable to the examination of saline wateis, but nol wastewaters
Üiat have rcceived only primary treatment followed by chlorina-
tioa because o f turbidity ip high volume samples or wastc'í'aters
t ONPG i i a tubaoMc fot OK p-gÚMcumiue tetL
9-60
containiag toxit metáis or toxic organic compounds such as
phenols. For the detectiou o f stressed total coliforms i n treated
drinking water and chlorinated secondaiy or tertiaiy wastewater
cffluenLs, use a method designed for stressed orgamsm rccovety
(sec Section 92I2B.1). A modified M F technique for fecal coÜ-
fnrms {Section 9212) in cíJorinated wastewater may be used i f
parallel testing over a 3-month period with the multiple-mhe
fermentation lechtiique shows comparabihty for each site-spe-
cific type of sample.
The standard volmne to be filtered for drinking water samples
is 100 m L . This may be distributed among m ú l t i p l e membranes
i f necessary. However, for special monitoring purposes, such as
troubleshootitig water quality pioblcms or idcntification of coli-
f o i m breakthrough i n low conccntrations from treatment barri-
crs, i t may be desirable to test 1-L samples. I f particulates
prevent filtcring a 1-L sample through a single filter, divide
sample into four portions of 250 m L for analysis. Total the
coiiform coimts on each membrane to report the number of
colifoims per üter. Smailer sampie volumes w i l l be oecessary for
source or rccrcational waters and wastewater effluents thal have
much higher colifoim densities.
9222 B. Standard Total Coiiform Membrane Filter Procedure
1. Laboratory Apparatus
For M F analyscs use glassware and other apparatus composcd
o f matwiaJl firee from agents that may affect baaerial growth.
a. Sample bottles: See Section 9030B.18.
b. Dilution botlles: See Section 90308,13.
c. Pipéis and graduated cylinders: See Section 9030B.9. Be-
fore sterihzation, loosely cover opening of graduated cylinders
w i t h metal foil or a suitable heavy wrapping-paper substitute.
Tmmediately after sterifization secute cover to prevent conlam-
ination,
d. Container! for culture médium: Use clean borosihcate
glass fiasks. Any size or shape o f flask may be used, but erlen-
meyer flasks witb metal caps, metal foil covers, or screw caps
provide for adequate itiixing of the m é d i u m contained and are
conveifient for siorage,
e. Culture dishes: Use sterile borosilicatc glass or disposable,
presterilized plástic petri dishes, 60 X 15 m í e , 5 0 X 9 mm, o t
other appropriate size. Wrap convcnient numbers of clean, glass
culture dishes in metal foil ¡f slerilized by dry heat, or suitable
heavy wrapping paper when autoclaved. I n c ú b a t e loose-Iidded
glass and disposable plástic culture dishes i n tightly closed
containers with wet paper or cloth to prevent moisture cvapora-
tion with resultant drying o f m é d i u m and to maintain a humid
environment for optimum colony dcvelopmcnt,
Presterilized disposable plástic dishes with tíght-fitting üds
that meet the .specifications above are available coriunercially
and are tiscd widely. Reseal opened packages o f disposable dish
supplies for stoiage.
/ Filtration units: The filter-holding assembly (constiucted of
glass, auloclavable plástic, porcelain, or slainless steel) consísts
M I C R O B I O L O G I C A L E X A M I N A T I O N (900D¡
Statistital comparisons of results obíained by the m t ü t i p l e -
tube method and the M F technique show that the M F is more
precise (compare Tables 9221:0 and B I with Table 9222;U),
Data from each test yield approximately the same water quality
information, although niuuerical results are not identical ( s e e
Section 9010 for drinking water).
3. Bibliography
CiABK. H J . , B£. GHUSRHCH, H . L Jifint & P.W. K M L E R . 1951. The
mciobraiie filter in sanitary bacteriology. Pub. Health Rep. 66:951.
KABLBR, P.W. 1954. Water examinalions by membrane filter and M P N
procedures. Amer. J. Pub. Heahh 44:3'79.
THCMA.'!, ñJí. & R . L . WooDWARD. 1956. Use of molecular filter mem-
branes for water p4tability control. J . Am^r. Water Worics Assoc.
48:1391.
MCCARTHY, L A . , J £ . DEIJVNEY & R . I . GRASSO. 1961. Measuring coli-
fomis in water. Water Sewage Works 108:238.
LIN, S. ]I973. P.valuaiioii of colifonn test for chloricated sccraidaty
cffiuents, J. Water Follul. Control Fed 45:498.
MAJJDB, J. & L P . NANKI. 197K. Measureroeni cvaluatJOB, In S.L.
hibom, cd. Quality Assurance Piactíces for Health Laboratoiie&, p.
209. American Pubhc Health Assoc., Washington. D . C .
o f a seamiess funne] fastened to a base by a locking device or by
magnetic forcé, TTie design should permit the membrane filter to
be held securely on the pcrous píate of tbe receptacle w i t h o u t
mechanical damage and allow aU finid to pass t h r o u g h the
membrane during filtration. Discard plástic fuunels w i t h dccp
scratches on imier surface or glass funuels with chipped surfaces.
Wrap the assembly (as a whole or sepárate parts) i n heavy
wrapping paper or alumlnuro foil, sterilize by autoclaving, and
store until use, Altematively expose al! surfaces o f the p r c v i -
oufily cleaned assembly to ultraviolct radiation (2 m i n exposure)
for the initial sanitization before use i n the test procedure, or
before reusíng units between successive filtration series. R e í d
tmits may be sanitized by dipping or spraying with alcohol and
then igniting or immersing i n boiling water for 2 m i n . After
submerging unit i n boiling water, coo! i t to room temperature
before rcuse. D o not ignitc plástic parts. Sterile, disposable ficld
tmits may be used.
For filtration, mount receptacle o f filter-hoidíng assembly ón a
1-L filtering fiask with a side mbe or other suitable device
(manifoid to bold three to six filter as.iemblies) such that a
pressure differential (34 to 51 kPa) can be exerted o n the filter
membrane. Coimcct flask to a vacuum line. an clectric vacuum
pump, a filter ptmip operating on water pressure, a hand aspira-
lor, or Other mcans of securing a pressure differential (138 to 207
kPa). Connect a flask of approximately the same capacity be-
tween filtering flask and vacuum source to trap carry-ovcr water.
g, Membrane filter: Use membrane fiJters (for additional
specifications, see Section 9020) with a ratcd pore diametcr such
that there is complete retention o f cohform bacteria. U s e only
those filter mcmbrancs that have been found. through adequate
quality control testing and certification by the rrumufacturer, lo
V~MBPLAN£ H L T B I T E C H N I Q U E p222).T'otal Cofifofms 9-61

exMbit; fuO rsteotion of lite organisnis to be cuitivated. stability Test each new médium lot agaiost a previously acceptable l o l
in use, freedom from chemica! exíiactables that may inhibit for satisfactory performance as described in Section 9020B.
bacteria] growth and devclopment, a satisfactory speed o f filtra- With each new l o t of Endo-type médium, verify a m í n i m u m 10%
don (within 5 min), no sigmficant influcnce on medicm p H of coiiform colonies, obtained from natural samples or samples
(beyoDd r 0.2 tmits), and no increase i n number o f confiuent with known additions, lo establish the comparative recovery of
colonies or spreaders compared to control membrane filters. Use the medimn l o t
membranes grid-marked in such a roanner that bacterial growth Before use, test each batch oflaboratory-preparedMF m é d i u m
:s ncither inhibiled ñor slimulated along the grid línes when the for performance with positive and negative culture controls.
mcmliranes with cntiapped bactaia are incubated o n a suitable
Check for coiiform conlamination al the beginning and end o f
médium. Preferably use fresh stocks of membrane filters and i f
each filtration series by filtering 20 to 30 m L of dilution or rinse
necessary store them in an enviromaent without extremes o f
water through the filter. I f confrols indicate coutamination, reject
temperature and humidity. Obtain no more than a year's supply
all data fiom affected samples and request resamplc.
at any one time.
a. LES Endo agar:'
Preferably líse prestcri!Í7ed membrane filters for which the
manufacturcr has certified that the steriiization technique has Yeast extraer 1.2 g
neiiber induced toiicity ñor altered tbe chemica) or physical Casilonc or liypticaíc 3.7 g
propwties of tfae membrane. I f mcmbrancs are sterilized iu the ThiopqMooe or tbioloDe 3.7 g
laboratory, autoclave for 10 m m at !21°C. A t the end o f the T^ose 7,5 g
Lactose 9.4 g
steriiization period, let the stcam escape rapidly to minimize
Dipotassium hydrogen fdjosphate, KjHPO, 3.3 g
acctmiulation of water of condensation on filters.
Potassium iHtydrogen phosphate, KH^PO, LO g
k. Absorben! pads consist o f disks o f filter paper or olhea- Sodium chloride, NaCl 3,7 g
material ceitiflcd for each lot by the manufacturcr to be of high Sodium descxycbolate 0.1 g
quaüty and fi^ee of sulfiles or other substanccs o f a concentration Sodium lauryl sulfate 0,05 g
that could inhibit bacterial growth. Use pads a p p r o x i m ^ l y 48 Sodium sulfile, Na^SOj 1.6 g
mm i n diametcr and o f sufficient thickness to absoib 1.8 to 2.2 Basic facbsia 0.8 g
m L o f mediimi. Presterihzed absorbent pads or pads subse- Agar 15.0 g
quently sterilized i n the laboratory should reléase less tban 1 mg Reagenl-grade water 1 L
total addity (calculated as CaC03) when titrated to the phenol-
Rchydrate product i n I L water containing 20 m L 95% etha- •
phliialein end point, p H 8.3, using 0.02A' NaOH and produce p H
nol. Do not use tícnatured etha^ol, which reduces background
levéis of 7 ± 0.2. Sterihze pads simultaoeously w i l h membrane
growth and coiiform i'obn;. sus. Brlng to a near boíl lo dissolve
filters available in resealablc kraft envelopcs, or separatcly i n
agar, then p r o n ^ ü y remove from i:ea: and cool lo 45 to 50°C. D o
other suitable containers. Dry pads so they are free o f visible
not sterijze by autoclaviLg. Rnaj p>i 7.2 ± 0.2. Dispense i u ' 5 -
moisture before use. See steriiization proccdtnrc described for
to 7-mL quantities into ¡ o * e r section of 60-imn glass o r trfastic
memteane filters above and Section 9020 for additiona] specifi-
catíons on absorbent pads. petri dishes. I f dishes o í acy ciaer sae are used, adjust quantitv
lo give an equivalen! depth of J te 5 mm. D o not expose p o u r ^ "
f. Fórceps: Smooth flat fórceps, without corrugations on the plates lo direct sunhght; refr.tcrate in the dark, preferably in
inner sides of the tips, Sterilize before use by dipping i n 95% scaled plástic bags or other containers t o reduce moisture loss,
ethyl or absolute methyl alcohol and fiaming, Discard unused médium afier 1 weeks or sooner i f there is
j . Incubaiors: Use incubators to provide a temperature of 35 evidence o f moisture loss, mec;üii! ccntamination, dr médium
± 0.5°C and to maintain a humid envinmment ( 6 0 * relabve detcrioration (darkeaing of the ^JeciT;^l^
humidity).
b. M'Endo meáiutn:'\
it, Microscope and light source: To determine colony coimts
on manbrane filters, use a magnification o f 10 to 15 diametsrs Tryptose or polypeptone 30.0 g
and a cool white fluorcscent ü g h l source adjustcd to give m á x - Thicpcptone or thioione 5.0 g
imum sheen discemmcnt, Optimaljy use a binocular wide-field Casitone or trypticase 5.G g
dissecting microscope Do not use a microscope illuminator w i l h YcasI cxtract IJ g
óptica] system for light concentration from an incandescent light Lsciose 12 J g
source for disceming cohform colonies on Endo-type media. Sodium chloride, NaCl 5,0 g
Uipolassimn hydrogen phosphate, KjHPO, 4375 g
Potassium dihydrogen phosphate, KHjPOi 1315 g
Sodium iaury! sulfate 0.05 g
2 Materials and Culture Media Sodium desoxychoiate 0.10 g
Sodium sulfile, NajSO, 2.10 g
The need for tauformity díctales the use of commercial dehy- Basic tuchsin 1.05 g
drated media. Never prepare media from basic ingredients when Agar (optíowd) 15,0 g
suitable dehydrated media are available. Follow manufacturcr's Reagent-grade water 1 L
directions for rchydratíon. Store opened supplies o f dehydrated
media in a desiccaior. Comraercially prepared media i n liquid
fonn (sterile ampule or other) may he used i f fcnown l o give • l>hydrited Difco M-Eodo A|ar LES (No. 0736). debyitrtud BBL M-Endo
equivalent results. See Section 9020 for media quality control Agar LES (No. 11203), or enuivaltm.
t Dthydrated DiÍMi M-Endo Broth KtF (No. 0749), dehydraled BRL m-CoMonn
specifications. Bfoth (No. 11119), orKjUivílem msy b« u»ed if absorbent psdí ME asta.
9-«S
TABLE 9 2 2 2 : L SUOOESTED SJMFÍM VM,UMES POR MHMBRANE FILTER TOTAL COLITORM T K T
Waler Souree 100 50
Eirinlring wsier X
SwimmÍQg pools X
Wells, springB X X
LakM, rescrvoiis X X
Water supply intake
Bathing beacbes
River water
Cblorinated sew age
Raw sewage
1) Agar preparatioD—^Rchydrate product k 1 L water c o n -
taining 20 m L 95% cthanol. Heat to near boiling t o di.ssolve agar,
then promptly remove from heat and cool to between 45 and
SO'C. Dispense 5- lo 7-niL quantities into 60-mjQ sterile glass or
plástic petri d i ^ s . I f dishes of any other úze are used, adjust
quantity to give an equivalent dcp&. D o not steriUze by auto-
claving. Final p H should be 7.2 ± 0.2, A precipitate is normal in
Endo-tyiie media.
Refrigérate íinished m é d i u m i n the dark and discqrd unused
agar after 2 weeks.
2) Broth preparation—Prepare as above. omitting agar. Dis-
pense liqttid m é d i u m (at least 2.0 m L per píate) onto absorbent
pads (sec abswbeni pad specifications, Section 9222B.I) and
carcfully rranove excess m é d i u m by decanting the píate. The
broth may have a precipítate b u l this docs not intcrferc with
mediimi performance i f pads are certified firee o f sulfile or other
toxic agents at a couccnti^tion that could inhibit bacterial
growth. Refrigerated broth may be stored for u p to 4 d.
c. Buffered dilution rinse water: See Section 9050C.I.
3. Samples
Collect samples as directed i n Sectíons 9 0 6 0 A and B .
4. Cofiform Definition
Bacteria that produce a red colony w i t h a metalJic (golden)
sheen within 24 h incubation at 35''C^ on an Endo-type mcdium
are considered members o f tbe coiiform group. The sheen may
cover the entire colony or may appcar only in a central arca or on
tbe pcriphery. The colifonn group thus defined is bascd on tbe
fjroduction o f aldehydcs from lermentation o f lactose. While this
biocbenúcal charactcristic is pan of tbe metabotic pathway of
gas production in the multiple-m'be test, some variations in
degrec of meialhc sheen dcvelopment may be observcd among
coiiform sirains. However, this slight difference i n indicator
definition is not considered critical to change its pubhc healtli
significance, particularly i f suitable smdies have been conductcd
to estabhsh the relationship between results obtained by the M F
and those obtained by the standard muItiplc-tube fermentation
procedure.
5. Procedures
a. Selection of sample size: Size o f sample w i l l be govemed
by expectcd bacterial density. In drinking water analyscs, sample
MICROBIOLOGICAL E X A M I N A T I O N (9000)
Volume (X) To Be Filtered
mi
10 1 0.1 0.01 0.001 0.0001
X
X
X X X
X X X
X X X X
X X X
X X X X
1
size wiU be limited only by the degree o f mrbidity or b y the
noncolifoim g r o w t h on the m é d i u m (Table 9222:1). For regula-
tioD puiposes, 100 m L is Ihe official sample size.
A n ideal sample volimie will yield 20 to 80 colifonn colotucs
and not more than 200 colonies of all types on a m e m l w a n o f i l i e r
surface. Analyze drinking wau^s by filtering 100 to IQOO m L , or
by filtering replicate smailer sample volumes such as duplicate
50-inL or fom^ repticates of 25-nrd, portions. A n a l y z e other
waters by filtering three düferent volumes ( d ü u t e d or u n d i l u t e d ) .
dcpending on the expected bacterial density. See Section
9215B.2 for preparation o f dilutions. When less than 10 n í L o f
sample (tiilutcd or tmdiluted) is to be filtered, add approximately
10 m L sterile dilution water lo the ñ m n e l before filtration c>r
pipet the sample volume into a sterile dilution bottle, I h c o filter
the entire dilution. This increase i n water volume aids i n urtiform
dispersión o f the bacterial suspensión over the entire effectivc
filtering surface.
b. Sterile filtration units: Use sterile filtration i m i l s at the
beginning o f each filtration series as a m i n i m i m i precaution TO
avoid accidental coutamination, A filtration series is considered
to be intcnuptcd when an interval of 30 m i u or longer ciapses
between sample fiitrations, After such inicrruption, t r e a i any
fuither sample filtration as a new filtration series and sterilize all
membrane filter holders i n use. See Section 9 2 2 2 B . l / f o r sterii-
ization procedures and Section 9020B.3Í and m for U V cleaning
and safety guidclines.
c. Filtration of sample: Using sterile fórceps, place a steiile
membrane filter (grid side up) over porous píate o f receptacle,
CarefuUy place matched funnel unit over receptacle and l o c k i t
in place. Filter sample tmder partial vacuum. W i t h filter stiU i n
place, rinse the interior surface of the funnel by filtering thrcc K i -
lo 3 0 - m L portions of sterile dilution water. Alteruativcly, rinse
funnel by a flow o f sterile dilution waler from a squeeze bottle,
This is satisfactory only i f the squeeze bottle and its coctents do
not become contaminaied during use, Rinsing betweeu samples
prevenís carryover conlamination. Upon complction o f final
rinse and the filtration process disengage vacuum, u n l o c k and
remove ftinnel, jmmediatcly remove membrane fiiter w i l h sterile
fórceps, and place i t on selected m é d i u m w i t h a roUing m o t i o u to
avoid entrapmeni o f air. I f the agar-based m é d i u m is used, place
prepared filter directly on agar, inven dish, and i n c ú b a t e for 22
to 24 h at 35 ± 0.5''C.
I f liquid m é d i u m is used, place a pad i n the culmre dish and
satúrate with at least 2,0 m L M-Endo mediura and carcfully
remove excess m é d i u m by decanting the píate. Place prepared
MEMBRANE HLTTR TCCHNIQUE (9222VTotal Coliforms
íiltcr direciiv on pad, lEVCrt dish, acd incúbate for 22 to 24 h a
35 ± 0.5 ° C
Differeatiaticin o f some colonieü from ei&er agar or liquid
mediuin substrates may be lost i f cnloires are iacubated beyond
24 h.
Inserí a sterile rinsc water samplc (100 m L ) after filtiBtioa of
a series of 10 sampies lo chcck f o i possibk cross-contaminatioc
or contaminated rinse water. Incúbate thc rinsc water control
membrane culture undei the same conditions as the sample.
For noEpotable water sampies, prefcrably dccontaminaie filter
unil after each samplc (as described above) because o f Che high
numbcr of coliform bacteria picscul i n thcse sampies. Altema-
tively, use an addiüonal buffcr linse of the filter nnii after the
fiiter is rcnaoved lo prcvenl carryover between sampies.
á. Altemative enrichmeni íechnique: Place a steaile absor-
bcnt pad i n the Hd of a sterile culture dish and pipet at leust 2.0
mL lauryl tryptose broth, prepared as direcled i n 9 2 2 1 B . l a l ) , to
satúrate pad. Carefully removc any excess liquid frora absorbeni
pad by decanting píate, AsepticaUy place fiUer through which ihe
sample has been passed on pad. Incúbate filter, without invertiiig
dish, for 1.5 to 2 h at 35 ± 0.5''C i n an atmosphcrc of at leasi
60% relative bumidity.
I f Ihe agar-based Endo-typc médium is used, rcmove enrich-
mcnt culture from iacubator, l i f l filter from ennchment pad, and
l o l l it onto Ihe agar surface, which has been aÜowed lo equih-
brate to room temperatura Incorrecl filter placement is al once
obvious, because parches of unsiained m c m l s T u i c indícale en-
trapment of air. Where such patcbes occur, carefiilly reseat filter
on agar s u r ^ c c . I f the liquid médium is used, prepare final
culture by removing emichmcnt culture from iacubator and
scparating the dish halvcs. Place a íiresb sterile pad in botcom half
of dish and satúrate with at Icast 2.0 m L of M-Endo médium and
carefully remove cxcess liquid from absorbent pad by dccaniÍBg
píate. Transfer filter, with same precautions as above. to new
pad. Discard used ennchment pad.
W i t h either the agar or the liquid médium, invert dish and
incúbate ÍOT 20 to 22 h ai 35 * OJ*'C. Proceed to 1 e beíoi*.
e. Couniing: To detcnmnc colony connís OQ merntraae f i -
lers, use a low-power (10 to i 5 magnificanons) binocular »Tde-
field dissecting microscope or other opdcal device, with a cool
while fluorescea ligfal sourcc dirccted to pro^^ide opamal ^iew-
ing of sheen, Thc typical c o l i f w m colony ha* a p s n i lo dark-red
C O I O T with a mctaüic suiface sheen. Coimi both rypical and
aiypical coliform colonies. The sheen arca may vary i n size from
a small piahead to complete coverage of the colony surface.
Atypical coliform colonies can be dark red, mucoid, or nuclcated
without sheen, GcueraÜy pink, bluc, whitc, or colorless colonies
lacking sheen are considered noncolifonns. H i e total count o f
colonies (coliform and n o t K x i l i f o r m ) on Endo-type médium has
no consistcnt relaiionship lo thc total nurober of bacteria prescnt
in the original sample. A high couni of noncoliform colonies
may intcrfere with the máximum deveiopment o f coliforms.
Refrigcialiiig cultures (after 22 h incubation) w i t h bigh densities
of noncoliform coloiúes for 0.5 to I h befare coimting may deter
Spread of confluence while aiding sheen discemment.
Sampies o f disinfetted water or wastcwaier effiuent may i n -
dude sircssed organisms that grow rclatively slowly and produce
máximum sheen i n 22 lo 24 h. Orgauisnis f r o m imdisinfected
sources may produce shccn at 16 to 18 h, and the sheen subse-
qucnlly may fade aftei 24 to 30 h.
/ Coliform verificaiion: Occasionally, typical íheen colonies
may be produced by noncoliform organisms and atypical colo-
nies (daric red or nucleatcd colonies without sheen) may be
coliforais. Preferably vwify all typical and atypical colony typcs.
For drinldng w a t e r , veiify all suspeci colonies b y swabbing the
cutiré membrane or pick al leas! five typical colonies and fivc
atypical colonies from a given mctnbrane ñitei culture. For
waters olher than drinking water, al a m i n i m u m , vcrify at leasl
10 sheen colonies (and rcprcscnlaljvc atypical colonies o f
difíercnl morphological typcs) from a positive water sample
monthly. See Section 9O20B.8, Based on need and samplc
typc, laboratories may i n c o r p o T a t e more stringent q u a l i t y
confrol measures (e,g., vcrify at leasl o n e colony frora each
lypical or atypical colony typc f r o m a given membrane filter
culture, vcrify 1 0 % o f the positive sampies). Adjust counts on
[he basis of verifrcatios resulta. Verification tests are Usted
below.
1) Lactose fermentalion—Transfer growlh from each colony
or s w a h the entire membrane w i t h a sterile cotton swab (for
presence-abseacc results in drinking water sampies) and place so
lauryl tryptose broth; incúbate Ihe lauryl tryptose broth al 35 ±
O J ' C for 48 h. Gas formed in lauryl tryptose broth and con-
firmed in brilliant green lactose brolh (Section 9221B.2 for
médium ptqiaration) within 48 h vcrifics tbe colony as a c o l i -
form. Simuttaneons inoculation o f both media for gas production
is acceptable. Inclusión of EC broih inoculation for 44.5 ± 0.2°C
incubation w i l l provide infonnaiion on the presencc of fecal
cohforms. Use o f EC-MUG w i t h incubaáon ai 44.5 ± 0.2''C for
24 h w i l l provide information on presence o f E. cali. See Section
9222G for M F partition proccdmrs.
2} Altemative coliform verifications—Apply this altemative
coliform verification procedure lo isolated colonies on the mem-
brane filter culture, I f a mixed culture is suspcctcd or i f colony
separatioD is less than 2 m m . sireak the growth to M-Endo
mcd:^ S Í Í : C Tücey agar :J. a.<«jr; r j l r . r T parity or submit
i i R¿7 ' nr : -r-.r-iz.- cc.omes utJiics lesi
reacíd ' " . ' •--j.z- . i - . - í COi and p-galactosidase.
C : Í - Í ¿ - , - . Z Í ÍTÍ CO ncgauve and /í-galactosidascpositive
" - r. ".-übütion of tube culture or mJCTo (spot) test proce-
deré.
b) Commcrcia] multi-test systcms—Verify the colony b y
streaiing it for purification, selecling a well-isolated colony, and
inocnlating into a multi-tcst identíficatíon system for Enterobac-
tcriaccae that includes laclóse fermcnlaóon and/or p-galactosi-
dase and CO test reactions.
6. Calculation of Coüform Densrty
C o n s t e the count. using membrane fillcrs w i t h 20 to 80
cohfoim colonies and nol more than 200 colonies o f all types per
membrane, by tbe foUowing equaüon:
c o l i f o r m colonius counted X 100
(Total) coUforms/iOO mL =
mL sample filtered
I f no coliform colonies are obscrved, r ^ i o r i the coliform
colorjes counted as " < 1 coliform/lOO m L . "
 9-6A M I C T I O B I O L O G I C A L EXAMINATTON (9000)

F O T \erified coKfom counts, adjust ibc initial count based Similarly, i f 50-, 25-, and 10-ml- portions were exammed and
upon the positive verificatioc pcrcemage and repoit as "verified the counts wcre 15, 6, and <l coliform colonies, respectively,
cohfomi count/100 m L . " leport tbe count as 25/100 m L , i.e..

Percentage verified colifomis

[(i5 -f 6 ->- Q) X 100]
number o f verí£ed colomes = 25 coliforms/lOO m L
(50 + 25 -f- 10)
XlOO
t o i d Dumber o f caliícnn colonies subjected t o v o i & c a d o n
On the other hand, i f 10-, 1.0-, and 0.1-mL porücms were
a. Water of drinking water quality; While the EPA Total examined with counts o f 40, 9, and < 1 c o l i f o r m colonies,
Coliform Rule for public water supply sampies requires only a respectivcly. select the 10-mL portion only f o r calculating Üie
record o f coliform presence or absence in lOO-mL sampies, it coliform density because diis filter had a coliform couoi falling
may be advisable lo determine coliform densities i n repeat m thc ideal range. The result is 400/100 m L , i.e..
sampling situations, This is of particular importancc whcn a
coliform biofilm problem is suspected in thc distribution system,
(40 X 100)
Quantilatíve information may provide an indication ot the mag- = 400 colifonns/lOO m L
nimde of a coniaminating e\'ent. 10

With water of good quahty. the occunence o f coliforms gen-

In thJ! lasi example, i f Üie membrane w i t h 4 0 c o l i f o n n colo-
eraUy w i l l be minimal. Therefore, count all colifonn colonies
nies also had a total bacterial colony count greater than 2(K),
(disregarding thc lower limit of 20 cited above) and use tbe
i«pon thc cohform count as ^400/1 (XJ m i . .
formula given above t o oblain colifonn density.
Report confluent growth or membranes w i t h coltmics too
If confluent growth occurs, covcring either the entire filtration
numerous to count as described in a above. Request a new
arca of the membrane or a portion thereof, and cxjionies are not
samplc and select more appropriatc volumes for filtration or
discrete, repon results as "confluent growth w i t h (or without)
utilize the multiple-tubc fcnnentation technique.
cohforms," I f the total munber o f bacterial colonies, coliforms
plus noncolifonns. exceeds 2Ü0 per membrane, o r i f the colonies c. Statistical reliabiliiy of membrane filter results: A l t b o u g h
are uot distinct enough for accurate counting, report results as the precisión o f thc M F technique is greater than that o f t h c M P N
"too numerous to count" (TNTC) o r "confluent," r e s p e c t i v c l y . procedure, membrane counts may underestimatc the nomber of
For drinking water, the presence o f c o l i f o r m s i n such cultures \Table coliform bacteria. Table 9222:11 Ülustrates same 9 5 %
showing no sheen may be confirmed by either t r a n s f e r r i n g a fcw confidence limits. These valúes are based on thc a s s u n ^ o n that
colonies or placing t h e entire memlxane filter culture into a bacteria are distributed randomly and follow a Poisson d j s n i b u -
, sterile tube o f brilliant green lactose bile broth. A s an altotiative, lion. For results with counts, c, greater Ihan 20 organisms,
bnish the entire filter surface with a sterile loop, a p p l i c a t o r stick, calcúlate the approximate 95% confidence l i m i t s using the foU
or cotton s w ^ and inocúlate this growth to the tube o f brilliant lowing normal distribution equations:
green lactose hile broih. I f gas is produced froto the brilliant
green bile broth tube within 48 h at 35 ± 0.5''C, c o l i f o r m s are
prescnt. For compüance with the EPA Total Coliform Rule, T A B L E 9222:11. CosFmencs, LTMTTS FOR M I M B R A N E F Q - T B I C o i p c i f t u
repon confluent growlh or T N T C with al least one detectóle R E S U L T S USTNO Í O O - M L S A M P U E
coliform colony (which is verified) as a total colifonn positive
sample. Report confluent growth or T N T C without dctcctable 95% Confidence Limits
Number of Colifonn
colifotms as invalid. For invalid sampies, reqnest a new samplc Colonies Counted LOWCT Uppcr
i r o m the same location within 24 h and select more appropriate
volumes to be filtered per membrane, obser\'ing the r c q u i r e m M i t 0 0.0 3.7
1 0.1 5.6
that the standard drinking water poition is 100 m L . or cboosc
2 02 72
another colifonn meihod that is less subjecl to hetcrotrophic
3 OA M
bacterial inlcrfercnces. Thus. to reduce intcrfcrence frora over-
4 1.0 102
crowding, inslcad of filtering 100 m L per membrane, filter
5 1.6 11.7
50-mL portíons ihrough two sepárate membranes, 25-mL por- 6 12 13.1
tions through each of four membranes, etc. Total t h e coliform 7 2J 14.4
counts observed on all membranes and icport as number per 100 8 3A 15.8
mL. 9 4Jt 17.1
10 4.7 18.4
b. Water of other than drinking water quality: As with potable 11 5,4 19.7
water sampies, i f no filter has a colifonn count falling i n ihe ideal 12 42 21.0
range, total the coliform counts on all filters and report as 13 6S 22.3
number per 100 m i . . Fot cxample, i f duplícale 5 0 - m L portíons 14 7.7 23.5
wcre examined and the two membranes had five ar)d three 15 8.4 24.8
coliform colonies, r e s p e c t i v e l y , report the count as cighi coü- 16 9.2 26.0
17 9.9 . 27.2
form colonies per 100 mL, i.e..
18 10.7 28.4

»
U5 -t- 3) X 100] IIJ 29.6
= í colifonns/lOOmL 20 12.2 30.8
(50 + 50)
Inlroducción
Lor, dispcííttivos Qtjanli-Tray'/2000 de IDEXX están diseñíidos para producir recuentos bacterianos cuantificados de
muestras de 100 mi, al ser utilizadas con productos de reactivos de la Tecnología de! Sustrato Definido(Cefined
Substrate Technology") de IDEXX. Agregue la nnezcla de reactivo y muestra a un dispositivo Quanti-Tray/2000,
séllelo en el Quanti-Tray Sealer (Sellador) e incúbela según las instrucciones del reactivo. Luego cuenta el número
de celdas positivas y utilice la tabla de MMP adjunta para deternninar el Número Más Probable (WMP).

Contenido

Este paquete contiene 100 dispositivos Qijanti-Tray/2000 esíérites.

insifucciones para el usuario

1. Sostenga en una mano ei 2. Apriete la parte superior de el 3. Abra el dispositivo Quanti-Tray

dispositivo Quanti-Tray en dispositivo Quanti-Tray de desprendiendo la lengüeta
posición vertical, con el lado de modo de doblarla hacia ia metálica del lado que contiene
las celdas orientado hacia la palma. las celdas. Evite tocar el interior
palma. def metal o del dispositivo.

6. Selle el dispositivo según las

instrucciones sellador,

7. Incube de acue'co con las

instrucciones dei reactivo.

8. Cuente las celdas positivas.

4. Vierta la mezcla de reactivo y la
muestra directamente dentro de
e! dispositivo Quanti-Tray,
evitando tocar la lengüeta
metálica. Golpear los pequeños
pocilios 2 o 3 veces para
eliminar posibles burbujas de
aire. Deja reposar la espuma.
Para determinar el número más ,
probable, recurra a la tabla NIVIP
al dorso de esta hoja de
instrucciones.
5. Coloque el dispositivo Quanti-
Tray lleno de muestra sobre el 9. Verter el medio de cultivo
portadispositivo de goma de! conforme a Buenas Prácticas
sellador Quanti-Tray, orientando del Laboratorio.
el lado de las celdas (plásticas)
del dispositivo hacia abajo de
manera que quepa en el
portadispositivo.

•QuanH-TiJí y Oelined SulKltatc Tedmology son marcas O maisss .-egisinirias (le lüEXX Laüoraicries, Inc. en los Estados Unidos de ftmerica ya en
otros países. Protegida por las paier,tfls noneaniericanas número 4.925,789 ; 5,439,933 ; 5,518.892. Otras patentes pendinmes úe concesión.
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 lUEiAA Uuanti-rray72000 Tabla, número más probable
^ Grandes it Poc !Ios f eque ños ,F 0SÍtÍ\ OS
Pcsit'voa 25 2E 27 2» 29 30 31 32 23 34 35 36 38 39 40 41 42 43 44 45 46 Al 4J1
0 25.3 26.4 27.4 ZS.A 29,5 30.5 31.5 3Z6 33.6 34.7 35.7 36.8 37.8 38.9 40.0 41.0 42.1 43.1 44.2 4S,3 46.3 47.4 •i» 4'i r.
1 26,6 27,7 28.7 29 6 30,B 31.9 32.9 34,0 35.0 36.1 37.2 3B>2 39.3 40.4 41.4 42.5 43.6 44,7 4S.7 46.8 47,9 49.0 LO 1
2 27,9 Í9,0 30.0 31.1 32,2 33.2 34,3 35.4 • 36.5 .37,5 38.6 39.7 40.8 41,9 43.0 44.0 45,1 46.2 473 48.4 49.5 50,5 61,7 .12 8
3 39.3 30,4 31.4 32,5 33 5 34.7 35.8 36,8 37.9 39 0 40.1 41.2 42.3 43.4 44,5 45.6 46.7 47.8 48.9 5C.0 51.2 52.3 53 4 549
* 30,7 31,8 32.8 33.3 35.0 . 35,1 37.2 38.3 39,4 40.5 41.6 42.3 43.9 45.0 46.1 472 48,3 43.5 50,5 51.7 52.9 54,0 5'j.1 .36,3
5 32,1 33.Z 34.3 35.4 36.5 376 38.7 39.9 41.C 42,1 43.2 44,4 45.5 4E,S 47.7 48,9 50,0 51,2 5Z.3 53.5 54.6 55, B 56.9 5H.1
6 33,5 34.7 35.B 36.9 38.0 39.2 40.3 41.4 42.5 43.7 44.8 46.0 47.1 4B.3 49.4 5D,fi 51.7 52.9 54,1 55.2 56.4 57.6 58.7 59,9
7 35.0 38.Z 37.3 38.4 39.S -40.7 41.9 43.0 41.2 45.3 48.S 47.7 48.8 50.0 51.2 52.3 S3.S 54.7 55.9 57.1 58,3 53,4 60.6 61,8
3 36,G 37.7 38,9 40.0 41.2 42.3 43.5 44.7 45.9 47.0 4B.2 46.4 50.6 51.8 53.0 S4,l 55 3 56.5 57.7 59.0 60.2 61.4 62.6 63.8
9 38.1 3B.3 40.5 41.6 42.8 44.0 45.2 46.4 47.6 4B.B 50,0 S1.2 52.4 53,6 54.8 56.0 E7.2 58.4 59 7 60,9 62.1 83.4 S4.6 65.8
10 33.7 40.9 -42.1 43.3 44.5 45.7 46.9 48.1 49.3 50.6 51.8 53.0 54-2 55.5 5S.7 57.9 S9.2 50,4 61,7 62.9 64.2 65.4 56.7 87.9
11 41.4 ^2,6 43.8 45,0 46.3 47.5 48.7 49.9 S'.2 52.4 53.7 54.9 56.1 57.4 58,3 59,9 61.2 62.4 63.7 65.0 66.3 67.5 68.B 70.1
12 •13.1 14.3 4F.6 46,8 48 1 49.3 S0,6 61,3 53.1 54.3 55.6 56.8 58.1 53,4 S0.7 6a 0 63,2 64,5 65.8 67.1 68.4 69,7 71.0 72..1
13 •14.9 46.1 47,4 43.6 49.3 51.2 52.5 S3.7 55.0 56.3 57.6 S8.0 eo.2 Sl.S 62.8 54.1 05.4 BS,7 68.0 69.3 70,7 72.0 73.3 74. í
14 43,0 43.3 50.5 51.8 53.1 E4.4 55.7 57.0 SB,3 59.6 60.9 62.3 63,6 64.S 66.3 6/,G 6E.9 70.3 71.6 73.0 74 4 7,=;.7 77.1
1S 48.6 49.9 51.2 52.5 53.8 55,1 56.4 57.B 53A 60.4 61.G 63.1 64.5 85,3 67.2 68.5 69,9 71,3 72.6 74.0 75,4 76.3 73.2 79.8
16 50,5 S1.8 53,2 54,5 S5.B 57.2 56.5 59.9 61.2 62.6 64.0 6S.3 66.7 68.1 69.5 7Q.9 72.3 73.7 75.1 75.5 77.9 .79.3 80,8 32.2
17 S2.5 53.9 55.2 56,E 58.0 £9.3 60.7 63.1 63.5 64.9 66.3 87,7 69.1 70.5 71.9 73.3 74.B 76.2 77.6 79.1 B0,5 B2.D 83.5 94.9
18 S4.6 56.0 57.4 58,8 60.2 61.6 63.0 64.4 65.B 67.2 68.6 70.1 71,5 73.0 74.4 75,9 77.3 78,8 80,3 81.B B3.3 84,8 66.3 B7.B
13 S6.B 58.2 59.6 61.0 62.4 63.9 65.3 66.E 68.2 69.7 71,1 72.6 74.1 75.5 77.0 78.5 BOO 81.5' Q3.1 E4.6 B6.1 87,5 39.2 90,7
ao sa.o 30.4 E1.9 63.3 64.8 55,3 67.7 69.2 70.7 72,2 73.7 75.2 76.7 78.2 79,B 81.3 32,8 84,4 35.9 87.5 89.1 90.7 92,2 93.8
21 61.3 62.8 B4.3 55.8 67,3 se.a 70.3 71.9 73.3 74,9 • 76,4 779 7S.5 81,1 84.2 85.8 37.4 89.0 90.5 92.2 93.3 95.4 97.1
22 63.a 65.3 BS.a 3S.3 63.8 71,4 72.9 74.5 76.1 77.6 79.2 80.8 B2.4 84.0 85.6 B7.2 88.9 eo.s 82.1 93.8 9S.5 97.1 93.S 100.5
Z3 B6.3 676 69.4 71.0 72.5 74.1 75.7 77.3 7B.9 80.5 82.2 S3.S 85.4 37.1 88.7 S0.4 92.1 B3.B 95.5 S7.2 9B.S 100.6 102,4 104.1
Z4 se, 9 70.5 72,1 73,7 75.3 77.0 78.6 83.3 81.B 83.6 85,2 86.9 88.6 90.3 92,0 93.8 95.5 97.2 98.0 100.7 102.5 104.3 106.1 107.3
25 '17 73.3 75.0 76.6 78.3 BO.O 81.7 83.3 65.1 86,8 aa,s 90.2 92.0 93.7 95,5 97.3 93.1 100.9 102.7 104.5 106,3 108.2 IIO.D 111.9
2E 74 e 76.3 78.0 79.7 81,4 83.1 84.8 66.8 83.4 90.1 SI.9 93.7 95.5 97.3 99.2 101,0 102,9 104.7 106.6 10B.5 110.4 112.3 114,2 116.2
zr 77,B 79,4 81.1 82-9 84.6 86.4 38.2 90.0 91.9 93.7 95.5 97.4 99.3 101.2 103.1 105,0 105.9 108.B 110.8 112.7 114.7 116.7 118.7 120.7
28 30.3 02.6 84,4 86,3 S3.1 89.9 91.8 93,7 05.6 97.5 99.4 101.3 103.3 105.2 107.2 109.2 111.2 1!3.2 115,2 117J •119.3 121,4 123.5 125.6
28 04.2 B6.1 B7.9 89.8 S1.7 S3,7 S5.6 97.5 99. S 101,5 103.5 105.5 107.5 109.5 111.6 113.7 115.7 117.8 120.0 1Z2.1 124.2 126 4 128.6 130.B
30 87.8 S9.7 91.7 93 6 95,6 97.6 99.6 101.6 103.7 105.7 107.8 109.9 112.0 114,2 116,3 118.5 120.6 122.8 125.1 127.3 129.5 131.B 134,1 136,4
31 81.6 93.6 95.6 977 99.7 101.8 1C3.9 106.0 108.2 110.3 112.5 114.7 116.9 119.1 121,4 123.6 125.9 128.2 130.5 132,9 13S.3 137.7 140.1 142.5
31 95.7 S7,fl 99.B 102.0 104.2 106.3 108.5 110.7 113.0 115.2 117.6 119.B 12Z.1 124,5 126.8 129,2 131.B 134.0 135.5 139.0 141.5 144.0 146.6 149,1
33 lOO.O 102.2 104.4 '.af>3 108.9 111.2 113.5 115.8 118,2 120.5 122.9 125.4 1278 130,3 132.8 135.3 137.8 140.4 143:o 145.6 148,3 150.9 153.7 15S.4
34 104.7 107.0 109.3 111.7 114.0 116.4 113.9 121.3 123.8 126.3 123.8 131.4 134.0 136.S 139.2 141.9 1+4.6 147.4 150.1 152^9 155.7 158,8 161.5 164.4
35 1M.7 m 2 114,6 1171 119,6 122.2 124.7 127.3 123.9 1 3 ? .6 135.3 136.0 140.B 143.6 146.4 149.2 152.1' 155.0 153.0 161.0 164.0 167.1 170.2 '73,3

36 115.2 117.8 120.4 123,0 125.7 123.4 131.1 133.9 136,7 139.5 142.4 145.3 148.3 151.3 154.3 157,.^ 160,5 163,6 166,8 170.0 173.3 175.6 179.9 183.3
37 121.3 134.0 126.B 129.6 135.3 138.2 141.3 1442 147.3 160.3 153.5 156.7 15S.9 163.1 165.5 159.8 173.2 176.7 130.2 183.7 1873 19!.Ü 1S4.7

38 1279 130.B 133.8 136,B 133.9 143.0 146.2 149,4 152,6 155.9 159.2 15Z6 166.1 169.6 173.2 176,8 . 180.4 134,2 188.0 1S1.8 195.7 199.7 Z03.7 207.7
39 135.3 138,5 141.7 145.0 148,3 151.7 1S5.1 158 6 182.1 165.7 169,4 173.1 176.9 180.7 184,7 188.7 192.7 196,8 201.0 206.3 209.6 214.0 218.5 223.0
40 143.7 147.1 150.6 1542 157,8 151,S 165.3 169.1 173.0 177.0 181.1 135.2 139.4 193.7 193.1 202.5 207,1 211.7 216 4 221.1 226.0 231 0 235 0 241.1
41 153.2 1570 160.S 164.3 163.S 173.0 177.2 ,!B1.5 165.8 190.3 194,8 199.5 204.2 209.1 214.0 219,1 224.2 229.4 234.S 240.2 245.8 251 5 257,2 263.1
42 1&4.3 168.6 172.9 177.3 181.9 156,5 191.3 196.1 201.1 236.2 211.4 216.7 222.2 227.7 233.4 239.2 245.2 251.3 2575 263,8 270.3 276.9 263,6 290.5
43 177.5 162.3 1B73 192-4 197,6 202.9 208.4 214.0 219.8 225.H 23!.B 238.1 244,5 251,0 257.7 264.6 271.7 278.9 2BS.3 293.8 301.5 309.4 317,4 325.7
i4 1B3.5 1B9.3 205.1 211.0 2172 Z23.5 230.0 236,7 243.6. 250.8 258.1 265.6 273.3 231.2 289.4 297.8 306.3 315.1 324,1 333.3 342,8 3S2.4 362.3 372.4
45 214.1 220 9 227.9 235.2 242.7 250.4 258 i 266 7 275 3 2E4 1 293.3 3016 312.3 322,3 332.5 343,0 353.8 364.9 376.2 387.9 399.8 412.0 424,5 437.4
4G 241,5 2M.0 2.18.3 2682 277.S 287.8 258,1 .308.6 3199 331.4 343.3 355,5 368.1 38L1 394.5 408,3 422.5 437.1 452.0 467,4 483.3 499.6' 61S.3 533.5
47 ?eo.9 292.4 304.4 316.9 330,0 343.6 357.8 37Z5 387.7 403,4 419.8 436.6 454,1 472.1 490.7 509.9 529,9 550,4 571.7 593 8 616,7 54C.5 665.3 691.0
4a 344.1 360,9 378.4 396.8 416.0 436.0 455.9 478.6 501.2 524.7 649.3 574.8 601.S 629.4 658.6 889.3 721.5 755.6 791.5 829.7 1370 4 913.9 B60.S 1011.2
49 461,1 468.4 6172 547.5 579.4 613,1 648,8 686.7 7270 770.1 B1B.4 86S.4 920.8 980 4 1045.2 1119.9 1203,3 1299.7 1413.S 1553.1 1732.9 1386.3 2419.6 >2419.6