The IMViC Tests

The IMViC tests are used to differentiate the enterics (Family Enterobacteriaceae). These
are the Indole test (tryptone broth), the Methyl Red and Voes!"ros#auer tests (MRV"
broth) and the Citrate test (Citrate aar slants). For these IMViC tests use the enterics
E. coli and Enterobacter. $or# in roups of %!& students.
The sinificance of these tests is that 'hen testin drin#in 'ater for the presence of the
se'ae indicator E. coli, one must be able to rule out Enterobacter aerogenes.
E. aerogenes is not al'ays associated 'ith se'ae, and its presence in 'ater 'ould not
necessarily indicate se'ae contamination.

CONTROLS Indole Methyl Red VP Citrate
Escherica. coli + + - -
Enterobacter. aerogenes - - + +
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1. Indole test (tryptone broth
Pro!ed"re# Ino!"late a loop$"l o$ ba!teria into a tryptone broth. In!"bate %& ho"r'.
(e'!ription# Tryptophan hydroly'i' -So)e ba!teria 'plit tryptophan into
indole and pyr"*i! a!id "'in+ the hydrola'e !alled tryptophana'e. Indole
!an be dete!ted ,ith -o*a!.' rea+ent (Indole rea+ent. Thi' te't i' *ery
i)portant in di$$erentiatin+ E. coli (indole po'iti*e $ro) 'o)e !lo'ely
related enteri! ba!teria. It al'o di$$erentiate' Proteus mirabilis (indole
ne+ati*e $ro) all other Proteus 'pe!ie' (indole po'iti*e. Tryptone broth
i' "'ed $or thi' te't a' it !ontain' a lar+e a)o"nt o$ tryptophan.
Interpretation# (fter incubation# The broth )"'t be t"rbid. / !lear broth
indi!ate' that yo"r or+ani') did not +ro, and !annot be te'ted. /dd a $e,
drop' o$ Indole rea+ent to the broth !"lt"re (tryptone broth. (O NOT
S0/-1 T01 T231. / po'iti*e re'"lt ha' a red layer at the top. /
ne+ati*e re'"lt ha' a yello, or bro,n layer.
4. Methyl Red test (MRVP broth
Pro!ed"re# Ino!"late a loop$"l o$ ba!teria into MRVP broth. In!"bate 5 to 6 day'.
(e'!ription# Mi7ed a!id $er)entation - Many +ra)-ne+ati*e inte'tinal
ba!teria !an be di$$erentiated ba'ed on the prod"!t' prod"!ed ,hen they
$er)ent the +l"!o'e in MR-VP )edi"). Escherichia, Salmonella8 and
Proteus $er)ent +l"!o'e to prod"!e la!ti!8 a!eti!8 '"!!ini!8 and $or)i!
a!id' and CO
4
8 0
4
8 and ethanol. The lar+e a)o"nt' o$ a!id' prod"!ed
lo,er' the p0 o$ the )edi") - Methyl red (a p0 indi!ator ,ill t"rn red
,hen added to the )edi") i$ the or+ani') ,a' a )i7ed a!id $er)enter.
Many o$ the'e or+ani')' al'o prod"!e +a'.
Interpretation# (fter incubation# The broth )"'t be t"rbid. / !lear broth
indi!ate' that yo"r or+ani') did not +ro, and !annot be te'ted. Re)o*e 1
)l o$ broth and pla!e into a 'terile t"be be$ore per$or)in+ the )ethyl red
te't i$ yo" are +oin+ to "'e the 'a)e broth $or the VP te't. /dd 5-% drop'
o$ )ethyl red to the ori+inal broth. (O NOT S0/-1 T01 T231. /
po'iti*e re'"lt ha' a di'tin!t red layer at the top o$ the broth. / ne+ati*e
re'"lt ha' a yello, layer.
5. Voes!"ros#auer test
Pro!ed"re# Ino!"late a loop$"l o$ ba!teria into MRVP broth. In!"bate 5 to 6 day'.
(e'!ription# Or+ani')' that are ne+ati*e in the )ethyl red te't )ay be
prod"!in+ 48 5 b"tanediol and ethanol in'tead o$ a!id'. The'e non-a!id
prod"!t' do not lo,er the p0 a' )"!h a' a!id' do. Enterobacter, Serratia
and 'o)e 'pe!ie' o$ Bacillus prod"!e the'e '"b'tan!e'. There i' no
'ati'$a!tory te't $or deter)inin+ prod"!tion o$ 48 5 b"tanediol. / pre!"r'or
o$ 485 b"tanediol !alled a!etoin !an be dete!ted ,ith 3arritt.' rea+ent.
Interpretation# (fter incubation# Read the VP te't ,hen yo" ha*e +ood
t"rbidity. / !lear broth indi!ate' that yo"r or+ani') did not +ro, and
!annot be te'ted. 3arritt.' rea+ent / (VP / !ontain' naphthol and 3arritt.'
3 (VP 3 !ontain' -O0. Te't 1 )l o$ yo"r !"lt"re $ro) the MRVP broth.
I$ yo" ha*e already !ond"!ted the )ethyl red te't8 yo" 'ho"ld ha*e
already pla!ed 1 )l o$ "nte'ted broth in a 'terile t"be. I$ yo" ha*ent done
thi'8 do 'o no,. /dd the entire !ontent' o$ the VP / rea+ent (16 drop'
and 6 drop' o$ the VP 3 rea+ent to the 1 )l o$ yo"r broth !"lt"re. S0/-1
91LL. Thi' rea!tion ,ill ta:e a $e, )in"te' be$ore yo" ,ill 'ee a !olor
!han+e. S0/-1 the t"be e*ery $e, )in"te' $or be't re'"lt'. 9ith a
po'iti*e rea!tion the )edi") ,ill !han+e to pin: or red indi!atin+ that
a!etoin i' pre'ent. 9ith a ne+ati*e rea!tion the broth ,ill not !han+e !olor
or ,ill be !opper !olored. 9ait at lea't 16 )in"te' $or !olor to de*elop
be$ore !allin+ the te't ne+ati*e.
%. Citrate test (Si))on.' Citrate 'lant
Pro!ed"re# Strea: a loop$"l o$ ba!teria onto a !itrate a+ar 'lant8 do not 'tab
the b"tt. In!"bate 4% to %& ho"r'8 lon+er $or Bacillus 'pe!ie'. In!"bate
,ith a loo'e !ap.
(e'!ription# Si))on.' !itrate a+ar te't' $or the ability o$ an or+ani') to
"'e !itrate a' it' 'ole 'o"r!e o$ !arbon. Thi' )edia !ontain' a p0 indi!ator
!alled bro)thy)ol bl"e. The a+ar )edia !han+e' $ro) +reen to bl"e at an
al:aline p0.
Interpretation# (fter incubation# / po'iti*e rea!tion i' indi!ated by a
'lant ,ith a Pr"''ian bl"e !olor. / ne+ati*e 'lant ,ill ha*e no +ro,th o$
ba!teria and ,ill re)ain +reen.
6. )ther media* (not part o$ the IMViC te't
Eosine Methylene +lue (EM+) (ar
Pro!ed"re# Strea: $or i'olation.
(e'!ription# / 'ele!ti*e )edi") $or +ra)-ne+ati*e ba!teria. Le*ine'
1M3 a+ar !ontain' )ethylene bl"e8 ,hi!h inhibit' +ra)-po'iti*e ba!teria.
(i$$erential $or enteri!'# ,ill di$$erentiate la!to'e $er)enter' $ro)
non$er)enter'.
Interpretation# (fter incubation# La!to'e non$er)enter' ,ill ha*e !rea)
!olored !olonie'. La!to'e $er)enter' ,ill ha*e pin:i'h !olonie'8
'o)eti)e' ,ith dar: !enter'. E. coli o$ten ha' a +reeni'h )etalli! 'heen.
Eosine Methylene +lue (EM+) (ar
Thi' )edia 'ele!t' $or +ra)-ne+ati*e
or+ani')'.
La!to'e $er)enter' !an be di$$erentiated $ro)
non$er)enter' by the !olor o$ the !olonie'.
Non$er)enter' ha*e a !rea)i'h ,hite !olony.
;er)enter' ha*e pin: !olonie' o$ten ,ith dar:
!enter'. 1'!heri!hia!oli o$ten ha' a )etalli!
+reen 'heen8 a' 'een on thi' Plate.
Thi' i' a la!to'e $er)enter that doe' not prod"!e
the )etalli! +reen 'heen.
IMViC Tests Results
Indole Test
+acteria 'ere ro'n in tryptone
broth.
The uninoculated tube is on the left.
The positi,e indole test is in the
center.
The neati,e indole test is on the
riht.
Methyl Red (MR) Test
+acteria 'ere ro'n in MRV"
broth.
The uninoculated tube is on the left.
The positi,e MR test is in the center.
The neati,e MR test is on the riht.
Voes!"ras#auer (V") Test
+acteria 'ere ro'n in MRV"
broth.
The uninoculated tube is on the left.
The positi,e V" test in in the center.
The neati,e V" test is on the riht.
Citrate test
Motility Test
The two tubes on the left
contain a nonmotile bacterial
species. Notice the clearly
visible line of growth (streak
line).
The two tubes on the right
contain a motile bacterial
species. Notice the cloudy
media and the less distinct line
of growth.