Depar

tmentofmi
crobi
ology

Shahr
iarshami
m

MostProbableNumber( MPN)isamet hodusedt oesti

matetheconcentr
at i
onofv iabl
e
micr
oorganismsi nasamplebymeansofr epl
i
cateli
qui
dbrothgrowthi
nt en-folddil
uti
ons.It
i
scommonl yusedinesti
mati
ngmicrobialpopulat
ionsi
nsoil
s,water
s,agricult
uralproduct
s
andispart
icular
lyuseful
wit
hsamplest hatcontai
npart
icul
atemater
ialt
hati nterf
ereswith
pl
atecountenumer ati
onmethods.

MPNi smostcommonl yappl iedf orqualit

ytesti
ngofwateri.
etoensurewhet herthewater
i
ssaf eornotinter
msofbact eri
apr esentinit
.Agroupofbacteriacommonl yreferr
edtoas
fecalcoli
for
msactasani ndicatoroff ecalcontami
nati
onofwat er.Thepresenceofv er
yfew
fecalcoli
for
m bacter
iawoul di ndicatethatwaterpr
obablycontai
nsnodi seasecausing
organisms,whil
ethepr esenceofl ar
genumber soffecalcol
if
orm bacteri
awoul dindi
catea
veryhighprobabi
li
tythatthewat ercouldcontaindi
seaseproducingorganismsmaki ngthe
waterunsafeforconsumpt ion.

Cont
ent
s[hi
de]

• 1Pr
inci
ple

• 2Pr
esumpt
ivet
est

o 2.
1Requi
rement
s

o 2.
2Pr
epar
ati
onoft
heMedi
um

o 2.
3Pr
ocedur
eofMPNt
est

2.
3.1Forunt
reat
ed(
pol
l
uted)wat
er

2.
3.2Fort
reat
ed(
unpol
l
uted)wat
er

• 3Conf
ir
mat
oryTest

• 4Compl
etedTest

• 5Adv
ant
agesofMPN

• 6Di
sadv
ant
agesofMPN

Pr
inci
ple

Watertobet estedisdilut
edser i
all
yandi nocul
atedinl act
osebr oth,coli
formsi fpresentin
waterut
il
izest helactosepresentinthemedi um toproduceaci dandgas.Thepr esenceof
aci
disindicatedbyt hecolorchangeoft hemedi um andt hepresenceofgasi sdet ectedas
gasbubblescol l
ectedintheinvertedDurham tubepr esentinthemedi um.Thenumberof
tot
alcol
if
or msi sdeterminedbycount i
ngt henumberoft ubesgivingposi t
ivereaction(i.
e
bothcol
orchangeandgaspr oduct i
on)andcompar ingt hepatternofposi t
iveresults(the
numberoft ubesshowi nggrowt hateachdi l
uti
on)wi thstandardstatisti
caltabl
es.

MPNt
esti
sper
for
medi
n3st
eps

1. Pr
esumpt
ivet
est
2. Conf
ir
mat
oryt
est

3. Compl
etedt
est

Pr
esumpt
ivet
est

Thepresumpt
ivet
esti
sascr
eeni
ngt
estt
osampl
ewat
erf
ort
hepr
esenceofcol
i
for
m
organi
sms.

Ift
hepresumpti
vet
estisnegati
ve,nof
urt
hert
est
ingi
sper
for
med,
andt
hewat
ersour
cei
s
consi
deredmicr
obi
ologi
cal
lysafe.

Ift
hepr esumptivetesti
snegati
ve,nofurt
hertesti
ngisperf
ormed,andthewat
ersourcei
s
consideredmicrobiol
ogi
call
ysafe.I
f,however
,anyt ubei
ntheseri
esshowsacidandgas,
the
waterisconsideredunsafeandtheconfir
medt esti
sperfor
medont hetubedi
spl
ayi
nga
posit
ivereact
ion.

Themet
hodoft
hepr
esumpt
ivet
estv
ari
esf
ort
reat
edandunt
reat
edwat
er.

Requi
rement
s

Medi
um:
Lact
osebr
othorMacConkeybr
othorLaur
ylt
rypt
ose(
lact
ose)br
oth

Gl
asswar
es:
Testt
ubesofv
ari
ouscapaci
ti
es(
20ml
,10ml
,5ml
),Dur
ham t
ube

Ot
her
s:St
eri
l
epi
pet
tes

Pr
epar
ati
onoft
heMedi
um

Prepar
emedium (ei
therMacConkeybr
othorl
act
osebr
oth)i
nsi
ngl
eanddoubl
e
st
rengt
hconcent
rat
ion.

Forunt
reat
edorpol
l
utedwat
er:

Dispensethedoubl
estr
engt
hmedium i
n10t ubes(
10mLineacht
ube)andsingl
e
str
engthmedi um i
n5tubes(10mLineacht
ube)andaddaDurham t
ubei
naninver
ted
posit
ion.

Fort
reat
edwat
er:

Di
spenset
hedoublest
rengthmedi
um i
n5t ubes(10mLineachtube)and50mL
si
ngl
estr
engt
hmedium i
n1bot t
leandaddaDurham tubeinani
nvert
edpositi
on.

Exami
net
het
ubest
omakesur
ethatt
hei
nnerv
ial
isf
ull
ofl
i
qui
dwi
thnoai
rbubbl
es.

St
eri
l
izebyaut
ocl
avi
ngat15l
bspr
essur
e(121°
C)f
or15mi
nut
es.

Pr
ocedur
eofMPNt
est

Forunt
reat
ed(
pol
l
uted)wat
er

MPNWat
erTest
ing

1. Take5tubesofdoubl
est
rengt
hand10t
ubesofsi
ngl
est
rengt
hforeachwat
er
sampl
etobetested.
2. Usi
ngast
eri
l
epi
pet
teadd10mLofwat
ert
o5t
ubescont
aini
ng10mLdoubl
e
st
rengt
hmedi
um.

3. Si
mi l
arl
y,add1mLofwaterto5tubescont
aini
ng10mLsingl
estr
engt
hmedi
um and
0.
1mLwatertotheremai
ning5t
ubescontai
ning10mLsingl
est
rengthmedi
um.

4. I
ncubat
eal
lthet
ubesat37°
Cfor24hr
s.I
fnot
ubesappearposi
ti
ver
e-i
ncubat
eupt
o
48hr
s.

5. Compar
ethenumberoft
ubesgivi
ngaposi
ti
ver
eact
iont
oast
andar
dchar
tand
recor
dthenumberofbact
eri
apr
esenti
nit.

Forexampl
e,awatersampletestedshowsaresul
tof3–2–1(3×10mLposi t
ive,
2×1mL
posi
ti
ve,1×0.
1mLposi ti
ve)givesanMPNv al
ueof17,i.
e.t
hewatersampl
econtainsan
est
imated17col
i
formsper100ml

Fort
reat
ed(
unpol
lut
ed)wat
er

1.
Take1t ubeofsi
nglestr
engt
h(50mL)and5t
ubesofdoubl
est
rengt
h(10mL)f
oreach
watersampletobetest
ed.

2.
Usi
ngast
eri
l
epi
pet
teadd50mLofwat
ert
othet
ubescont
aini
ng50mLsi
ngl
est
rengt
h
medi
um.

3.
Simi
l
arl
y,add10mLofwat
ert
o5t
ubescont
aini
ng10ml
doubl
est
rengt
hmedi
um.

4.I
ncubat
ethet
ubesat37°
Cfor24hr
s.I
fnot
ubesappearposi
ti
ver
e-i
ncubat
eupt
o48hr
s.

5.
Comparethenumberoft
ubesgi
vi
ngaposi
ti
ver
eact
iont
oast
andar
dchar
tandr
ecor
dthe
numberofbact
eri
apr
esenti
nit
.

Forexampl
e,awat
ersampletest
edshowsaresul
tof1-
4(1×50mLposi ti
ve,4×10mL
posi
ti
ve)gi
vesanMPNvalueof16,i
.e.t
hewat
ersamplecont
ainsanest
imated16col
i
for
ms
per100mL.

Conf
ir
mat
oryTest

Somemicr
oorgani
smsothert
hancol
if
ormsal sopr
oduceaci
dandgasfr
om lactose
fer
ment
ati
on.I
nordert
oconfi
rmthepresenceofcol
if
orm,aconf
ir
mator
ytestisdone.

Fr
om eachoft
hef
erment
ati
ont
ubeswi
thposi
ti
ver
esul
tst
ransf
eronel
oopf
ulofmedi
um t
o:

1. 3mLl
act
ose-
brot
horbr
il
li
antgr
eenl
act
osef
erment
ati
ont
ube,

2. t
oanagarsl
antand

3. 3mLt
rypt
onewat
er.

I
ncubatet
heinocul
atedlact
ose-brot
hf er
mentati
ontubesat37°Candi
nspectgasf
ormat
ion
aft
er24±2hours.I
fnogaspr oduct
ionisseen,
furt
herincubat
euptoamaximum of48±3
hourst
ocheckgaspr oduct
ion.

Theagarsl
ant
sshoul
dbei
ncubat
edat37°
Cfor24±2hour
sandGr
am-
stai
nedpr
epar
ati
ons
madef
rom t
hesl
ant
sshoul
dbeexami
nedmi
croscopi
cal
l
y.

Theformat
ionofgasi
nlactosebrot
handthedemonst
rati
onofGram-negat
ive,
non-spore-
formi
ngbacil
l
iint
hecorr
espondingagari
ndi
catest
hepresenceofamemberoft hecoli
for
m
groupi
nthesampleexamined.

Theabsenceofgasf or
mati
oni nlactosebrothorthef
ail
uretodemonstr
ateGram-negat
ive,
non-spor
e-f
ormingbaci
ll
ii
nthecor respondingagarsl
antconst
it
utesanegat
ivetest
(absenceofcol
i
f or
msinthetestedsampl e).

Tr
ypt
oneWat
erTest

1. I
ncubat
ethet
rypt
onewat
erat(
44.
5±0.
2°C)f
or18-
24hour
s

2. Fol
l
owi
ngi
ncubat
ion,
addappr
oxi
mat
ely0.
1mLofKov
acsr
eagentandmi
xgent
ly.

3. Thepresenceofi
ndol
eisindi
cat
edbyar
edcol
ori
ntheKov
acsr
eagent
,for
minga
fi
l
m ov
ertheaqueousphaseofthemedium.

a.Conf
ir
mat or
ytestsposi
ti
vef
ori
ndol
e,gr
owt
h,andgaspr
oduct
ionshowt
hepr
esenceof
ther
motol
erantE.coli
.

b.Gr
owt
handgaspr
oduct
ioni
ntheabsenceofi
ndol
econf
ir
mther
mot
oler
antcol
i
for
ms.

Compl
etedTest

Si
ncesomeoftheposit
iveresul
tsf
rom t
heconfir
matoryt
estmaybefal
se,i
tisdesi
rabl
eto
docomplet
edtest
s.Fort
hisinocul
um f
rom eachposi
ti
vetubeoft
heconf
ir
mat or
ytesti
s
st
reakedonaplat
eofEMBorEndoagar .

Int
hisprocess,aloopf
ulofasampl efrom eachposi
ti
veBGLBtubeisst
reakedont
o
sel
ecti
vemedi um li
keEosi
nMet hyl
eneBlueagarorEndo’smedium.Oneplat
eeachis
i
ncubatedat37°Candanot herat44.5±0.2°Cfor24hour
s.

Hi
ght
emper
atur
eincubat
ion(
44.
5±0.
2)i
sfordet
ect
ionoft
her
mot
oler
antE.
col
i
.

Fol
l
owi
ngi
ncubat
ion,
all
plat
esar
eexami
nedf
ort
hepr
esenceoft
ypi
cal
col
oni
es.

Coli
for
msproducecoloni
eswithagreeni
shmet all
i
csheenwhichdi
ff
erent
iat
esitf
rom non-
coli
for
m col
oni
es(shownosheen).Thepresenceoftypi
calcol
oni
esonhightemperat
ure
(44.
5±0.2)i
ndi
cat
esthepresenceofther
mot ol
erantE.
col
i.

Adv
ant
agesofMPN

1.
Easeofi
nter
pret
ati
on,
eit
herbyobser
vat
ionorgasemi
ssi
on

2.
Sampl
etox
insar
edi
l
uted

3.
Eff
ect
ivemet
hodofanal
yzi
nghi
ghl
ytur
bidsampl
essuchassedi
ment
s,sl
udge,
mud,
etc.

4.
thatcannotbeanal
yzedbymembr
anef
il
tr
ati
on.

Di
sadv
ant
agesofMPN

1.
Itt
akesal
ongt
imet
ogett
her
esul
ts
2.
Resul
tsar
enotv
eryaccur
ate

3.
Requi
resmor
ehar
dwar
e(gl
asswar
e)andmedi
a

4.
Probabi
l
ityoff
alseposi
ti
ves