Senso s 2015, 15, 29635-29647; doi:10.3390/s151129635
senso s
ISSN 1424-8220
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Technical No e
In luence o Cul u e Media on Mic obial Finge p in s Using
Raman Spec oscopy
Ka a ína Mlyná iko á 1, O a Samek 2, Sil ie Be na o á 2, Filip Růžička 1,*, Jan Ježek 2,
And ea Há oniko á 3, Ma in Šile 2, Pa el Zemánek 2 and Ve onika Holá 1
1 Depa men o Mic obiology, Facul y o Medicine and S . Anne’s Facul y Hospi al, B no 65691,
Czech Republic; E-Mails: k.mlyna iko [email protected] (K.M.); [email p o ec ed] (V.H.)
2 Ins i u e o Scien i ic Ins umen s o he Academy o Sciences o he Czech Republic,
. .i., K álo opolská 147, B no 61264, Czech Republic; E-Mails: osamek@isib no.cz (O.S.);
[email p o ec ed] (S.B.); jezek@isib no.cz (J.J.); sile @isib no.cz (M.Š.); pa [email p o ec ed] (P.Z.)
3 Cen e o Ma e ial Resea ch, B no Uni e si y o Technology, Pu kyňo a 118, B no 61200,
Czech Republic; E-Mail: [email p o ec ed]
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +420-543-183-090.
Academic Edi o : Bayden Wood
Recei ed: 3 Sep embe 2015 / Accep ed: 19 No embe 2015 / Published: 24 No embe 2015
Abs ac : Raman spec oscopy has a b oad ange o applica ions ac oss nume ous
scien i ic ields, including mic obiology. Ou wo k he e moni o s he in luence o cul u e
media on he Raman spec a o clinically impo an mic oo ganisms (Esche ichia coli,
S aphylococcus au eus, S aphylococcus epide midis and Candida albicans). Choosing an
adequa e medium may enhance he ep oducibili y o he me hod as well as simpli ying he
da a p ocessing and he e alua ion. We es ed ou di e en media pe o ganism depending
on he nu i ional equi emen s and clinical usage di ec ly on a Pe i dish. Some o he media
ha e a signi ican in luence on he mic obial inge p in (Roos el -Pa k Ins i u e Medium,
CHROMaga ) and should no be used o he acquisi ion o Raman spec a. I was ound ha
he mos sui able medium o mic obiological expe imen s ega ding hese o ganisms was
Muelle -Hin on aga .
Keywo ds: Raman spec oscopy; bac e ia; yeas s; cul u e media
OPEN ACCESS
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1. In oduc ion
Raman spec oscopy is a physical me hod wi h a b oad ange o applica ions—i is sui able o he
iden i ica ion o subs ances, hei s uc u e and composi ion. The me hod is based on a non-des uc i e
measu emen o inelas ic sca e ing o ligh (Raman sca e ing) ha allows iden i ica ion o a b oad
spec um o molecules o cha ac e iza ion o molecula composi ion o a biological/non-biological
sample by p o iding i s spec oscopic inge p in [1,2]. In las ew yea s has been used o iden i ica ion
and desc ip ion o biological sys ems anging om biomolecules o whole cells and o ganisms [3–20].
The mos equen use o he Raman spec oscopy in mic obiology is a pheno ype measu emen
o s ain iden i ica ion (including bac e ia, yeas s and algae) [2,3,11,13], bu he me hod can be used o
de e mine a p esence o i ulence ac o s in a ce ain s ain, oo [10,16,21]. Mic oo ganisms can be analyzed
di ec ly om colonies g own on an aga pla e [10,16], mic ocolonies on solid cul u e media [22] o
single-cells ob ained a e a cul i a ion in liquid media [23]. As he Raman spec oscopy measu es he
molecula composi ion o he cells, spec a migh be in luenced by cul u e condi ions.
Cul u e media di e in hei composi ion o sa is y he nu i i e equi emen s o a pa icula g oup
o o ganisms as well as o se e as a selec i e and/o diagnos ic ool. The aim o ou s udy is o ind
sui able cul u e media ( om commonly used media in he labo a o ies o clinical mic obiology) o
selec ed o ganisms by assessing limi a ions caused by he in luence o cul u e media on he Raman
inge p in o he gi en s ain. Thus ep oducible spec a o mic oo ganisms can be ob ained which,
in u n, simpli ies he da a p ocessing and e alua ion. The in es iga ion p esen ed he e shows he in luence
o commonly used cul u e media on he Raman inge p in o selec ed mic oo ganisms belonging o he
g oups o G am-posi i e bac e ia, G am-nega i e bac e ia and yeas s—namely, S aphylococcus au eus,
S aphylococcus epide midis, Esche ichia coli, and Candida albicans.
S aphylococci a e G am-posi i e (wi h a compac cell wall con aining a hick pep idoglycan laye )
cocci ha end o o m i egula clus e s. Mos o he species belonging o he genus S aphylococcus
colonize he skin plus mucous memb anes o humans [24,25] and unde ce ain condi ions can cause
in ec ions [26]. The mos clinically ele an species o he genus is S aphylococcus au eus, which can
cause in ec ious diseases like impe igo, os ei is, celluli is, boils, scalded skin synd ome, and sep icemia,
as well as a oxin-media ed ood poisoning and a oxic shock synd ome [27]. When S. au eus en e s he
bloods eam, li e- h ea ening in asi e diseases, such as sepsis, os eomyeli is, endoca di is and meningi is
can a ise. These in ec ions o en ha e a nosocomial cha ac e [28] and a e di icul o ea [28,29].
S aphylococcus epide midis, he second mos equen ly isola ed ep esen a i e o s aphylococci,
is an impo an sap ophy ic o ganism ound on he human skin. Howe e , i can signi ican ly con ibu e
o e e -inc easing mo bidi y and mo ali y o hospi al-acqui ed in ec ions [30]. Majo in ec ions caused
by S. epide midis a e bio ilm-associa ed medical-de ice- ela ed in ec ions. These include p os he ic
al e endoca di is, u ina y ac in ec ions, cen al ne ous sys em in ec ions, eye in ec ions as well as
in a ascula -ca he e associa ed in ec ions and sep ic loosening o join p os heses a e o al join
a h oplas y [25,31–37].
Esche ichia coli is a G am-nega i e (ha ing a mo e complica ed cell-wall s uc u e han he
G am-posi i e bac e ia: he pep idoglycan laye is hin and sandwiched be ween he cy oplasmic memb ane
and bac e ial ou e memb ane, and i con ains phospholipids, lipopolysaccha ides and p o eins) od ha
occu s in he in es inal ac o mammals as a common pa o no mal mic o lo a. Howe e , ce ain
Senso s 2015, 15 29637
s ains may cause in ec ion in he human in es inal ac (e.g., oxin-p oducing s ains). Mo eo e ,
E. coli is he mos equen ly isola ed pa hogen om communi y-acqui ed u ina y ac in ec ions as well
as he mos equen ly isola ed G am-nega i e species om bloods eam in ec ions [38].
Candida albicans s ill emains he mos equen ly isola ed yeas om clinical specimens [39,40].
I is a polymo phic ungus ha can swi ch pheno ype be ween he unicellula yeas o m and he
ilamen ous o m (hypha, pseudohypha) [41,42] ha usually inhabi s he mucosal memb anes in
humans. Howe e , when he balance be ween C. albicans and hos is dis up ed, his yeas can cause a ious
in ec ions anging om o al/supe icial candidiasis and in ec ions o he ep oduc i e sys em o se ious
in ec ions—in asi e candidiasis and candidaemia, which a e o en li e- h ea ening [43].
In he p esen s udy, we exploi poin -by-poin eco ding o signi ican pa s o he mic obial colonies
g own on ou di e en cul u e media (di ec ly on he Pe i dish) o each s ain (selec ed media a e
commonly used in a mic obiological labo a o y o cul i a ion o he gi en o ganism). Speci ically, we
es ic ed he spo measu emen s o he cen al, middle and uppe pe iphe y o he mic obial colony
su aces. We engaged he app op ia e e ocusing on a sample o each Raman spec a o he pu pose o
s aying wi hin he ocal dep h o he lase exci a ion and imaging op ics.
2. Resul s and Discussion
As was men ioned abo e, in o de o assess he in luence o ce ain media on Raman spec a
o mic oo ganisms conce ning he epea abili y as well as he quali y o speci ic inge p in s, we
analyzed ou o ganisms (S aphylococcus au eus, S aphylococcus epide midis, Esche ichia coli and
Candida albicans), each on ou cul u e media, as desc ibed u he in Ma e ials and Me hods ion.
All cul u e media con ain biomolecules so hey can p o ide an adequa e nu ien supply o he
mic oo ganisms o g ow and ep oduce. As mic obial colonies a e ela i ely small, a signal acqui ed
om a mic obial colony can also include a signal om cul u e medium when measu ing he edges plus
he molecules om he medium can be ound in he olume o a colony (see Expe imen al Se up).
The Raman spec a o selec ed cul u e media a e shown in Figu e 1. Majo i y o es ed media (Endo
aga —ENDO, blood aga —BA, blood aga wi h 10% NaCl—BA-NaCl, Muelle -Hin on aga —MH)
show weak in ensi y o peaks ( anging up o 2500–3000 a.u.). The o he wo media (CHROMaga
Candida—CHROM and Roos el -Pa k Ins i u e Medium 1640—RPMI) show s ong signals wi h nume ous
de ec able peaks wi h in ensi y up o 5000 a.u. o CHROM and 12,000 a.u. o RPMI. The in ensi y o
he peaks de ec able in mic obial spec a anges om app oxima ely 2000 a.u. up o 20,000 a.u.
As we showed in ou p e ious wo k, he o e all epea abili y o he speci ic Raman spec a associa ed
wi h a ce ain mic obial s ain a e epea ed cul i a ion is e y high, e en o samples p epa ed mo e
han a yea apa [16]. To assess he epea abili y o each o ganism-medium combina ion wi hin he
analysis we an a p incipal componen analysis (PCA) o all measu emen s o one o ganism g own on
all co esponding media (Figu e 2). All o ganisms we e measu ed a e iden ical cul i a ion condi ions using
he same equipmen and exposi ion ime.
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Figu e 1. Raman spec a ob ained om di e en cul u e media (ENDO—Endo aga ,
BA—blood aga ; BA-NaCl—blood aga wi h 10% NaCl; MH—Muelle -Hin on aga ;
CHROM—CHROMaga Candida; RPMI—Roos el -Pa k Ins i u e Medium 1640).
Figu e 2. Repea abili y o Raman spec a o he gi en o ganism a e g ow h on di e en
cul u e media (ENDO—Endo aga , BA—blood aga ; BA-NaCl—blood aga wi h 10% NaCl;
MH—Muelle -Hin on aga ; CHROM—CHROMaga Candida; RPMI—Roos el -Pa k
Ins i u e Medium 1640). Plo s ma ked “Wi hou RPMI” and “Wi hou CHROM” a e displayed
o simpli y he compa ison o a iabili y o Raman spec a measu emen on all o he media.
The lowe he a iabili y wi hin he species is, he smalle he a ea co e ed by an ellipsoid.
Clus e ing o S. au eus spec a indica es ha he mos sui able medium o Raman spec al acquisi ion
o his o ganism is MH medium. BA and BA-NaCl can also be used, bu he epea abili y o he inge p in
is lowe . BA-NaCl is a selec i e medium, which could easily induce a dissocia ion o he s ain
(in a-s ain a ia ions) and he e o e i migh cause he a iabili y in Raman spec a. A chemically
Senso s 2015, 15 29639
well-de ined medium con aining many supplemen s, RPMI, induces a high a iabili y in Raman
spec a o S. au eus and he e o e should no be used o analyses o his o ganism. Resul o
S aphylococcus epide midis show he e y same pa e n.
Repea abili y o C. albicans Raman spec a is high when using MH as well as SAB medium. On he
o he hand, cul i a ion on CHROM aga and RPMI medium induces a high a iabili y o spec a.
This migh by in luenced by di e en colony mo phology induced by nu ien s in a medium.
Fo example, RPMI medium induces he hyphal o m de elopmen leading o domina ion o his o m
o e he yeas o m [44].
The e is a low a iabili y o Raman spec a ob ained om E. coli colonies g own on MH aga , oo.
Cul i a ion on BA aga is shown o be compa able o MH, conce ning he a iabili y o spec a. E. coli
inge p in epea abili y on ENDO aga is, howe e , lowe han on p e iously men ioned media. This
migh be caused by he p esence o a dye ( uchsine) in he medium. Fuchsine is ela i ely oxic o
bac e ia and can induce a dissocia ion o he gi en s ain. Equally as in s aphylococci, RPMI medium
induces he high a iabili y o Raman spec a in E. coli and he e o e he epea abili y o measu emen
on his medium is no su icien . Figu e 3 shows he compa ison o a iabili y in E. coli spec a om
RPMI and MH as an example o bes -case scena io and wo s -case scena io cul i a ion media o Raman
spec al acquisi ion om bac e ial colonies. Fo compa ison, Figu e 3c includes a plo o loading o he
bes -case scena io o all es ed mic oo ganisms—cul i a ion on MH aga .
Mo e de ailed analysis o Raman spec a co esponding o he selec ed o ganisms show ha he
inge p in o RPMI medium (he e RPMI appea ed as a wo s -case scena io medium) is isible in he
spec um o mic obial colonies (de ec able peaks wi h high in ensi y a app ox. 820 cm−1, 1050 cm−1,
1310 cm−1 and 1450 cm−1) (Figu e 4). Consequen ly, such a high in luence o RPMI medium on he Raman
spec a o selec ed mic oo ganisms ansla es o he la ge size o he a ea gi en by he Mahalanobis
dis ance (Figu es 2 and 3). Also high in luence o hese peaks can be a ace in dedica ed PC-loadings
(each PCA can be u he analyzed using he PC-loadings) when using PCA analysis o he selec ed
samples (Figu e 3). This is in con a y o MH aga (see Figu e 3c) which con ibu ion o PCA analysis
o gi en mic oo ganisms is e y small—i is also ansla ed o small a ea o ellipsoids calcula ed using
Mahalanobis dis ance.
This p oblem (as wi h RPMI medium) is also obse ed when using CHROMaga (de ec able peaks
a app ox. 1040 cm−1, 1360 cm−1 and 1600 cm−1).
Thus, de ec ion o medium-induced peaks in Raman spec a o mic oo ganisms impai s he associa ion
o he spec a wi h he ce ain co esponding o ganism as well as he speci ici y o mic obial inge p in
and he e o e should be a oided.
In addi ion, he spec a o each o ganism sligh ly change wi h he used cul u e medium. This migh
be a esul o di e en nu ien s in ake leading o he di e ences in cell and colony composi ion.
Acco ding o he esul s p esen ed abo e, he mos sui able medium o analyses o all es ed bac e ial
s ains is MH aga . Mo eo e , analyses o colonies g own o MH aga show a e y good epea abili y
also o he yeas s ains. This wi hin-analysis epea abili y o MH aga used o yeas s ains a e
consis en wi h ou ecen wo k employing Candida pa apsilosis s ains [16]. In addi ion o MH aga ,
SAB aga is shown o p o ide he compa able epea abili y o he yeas s ain measu emen .

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(a)
(b)
(c)
Figu e 3. (a) Sco es plo o he i s wo p incipal componen s ela ion o E. coli cul u ed
on wo di e en media— op clus e RPMI (down- iangle), bo om clus e MH (up- iangle).
Using wo p inciple componen s, one can clea ly sepa a e he clus e s o spec a ela ed o
cul i a ion o he same mic oo ganism on wo di e en media. This sugges s ha du ing he
g ow h o bac e ia cul i a ed on RPMI aga , some subs ances om aga we e abso bed in o
he olume o he colony ansla ing o he inal bac e ial spec a; (b) Plo o loading o PC2
co esponding o Figu e 3a suppo ing he con ibu ion o spec al bands ela ed
o RPMI medium. Because o ocusing condi ions his is no due o he con ibu ion om
he unde lying aga ; (c) Plo o loading o PC2 co esponding o ou bac e ia included in
ou s udy cul u ed on he Muelle -Hin on aga suppo ing he con ibu ion o spec al bands
ela ed o gi en mic oo ganisms ( h ee bands o be a-ca o ene). This is in con as o
Figu e 3b whe e spec al bands ela ed o RPMI medium we e obse ed and shows ha he
Muelle -Hin on aga should be he medium o choice o mic obiological expe imen s
employing Raman spec oscopy measu emen s.
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Figu e 4. No malized ep esen a i e Raman spec a o he mic oo ganisms on all used
cul u e media (ENDO—Endo aga , BA—blood aga ; BA-NaCl—blood aga wi h 10% NaCl;
MH—Muelle -Hin on aga ; CHROM—CHROMaga Candida; RPMI—Roos el -Pa k Ins i u e
Medium 1640).
3. Ma e ials and Me hods
3.1. Mic oo ganisms and Sample P epa a ion
Selec ed o ganisms ep esen ing G am-posi i e bac e ia (S aphylococcus au eus D 47,
S aphylococcus epide midis, STO 60), G am-nega i e bac e ia (Esche ichia coli, CCM 3988) and yeas s
(Candida albicans, CCM 8261) we e used in he s udy. The CCM s ains we e ob ained om he
Czech Collec ion o Mic oo ganisms. Bo h s aphylococcal s ains we e isola ed om clinical ma e ial
and s o ed in he Cul u e Collec ion o he Depa men o Mic obiology, S . Anne’s Facul y Hospi al in
B no, Czech Republic. All o hese s ains we e s o ed a –70 °C. Be o e he expe imen , he s ains we e
hawed quickly a he oom empe a u e and cul i a ed on he Muelle -Hin on aga (MH, Oxoid,
Basings oke, UK) a 37 °C o 24 h. G own colonies we e consequen ly ans e ed on o ce ain solid
media in Pe i dishes: MH, Blood aga wi h sheep e y h ocy es (BA), Blood aga wi h sheep e y h ocy es
and 10% NaCl (BA-NaCl), Sabou aud aga (SAB, Me ck, Ge many), Roos el -Pa k Ins i u e Medium
1640 wi h L-glu amine (RPMI, Sigma Ald ich, Munich, Ge many) and CHROMaga Candida
(CHROM, CHROMaga Company, Pa is, F ance), Endo aga (ENDO, Oxoid, Basings oke, UK), wi h
each o ganism cul u ed on ou media (see Table 1). Selec ed media a e commonly used in clinical
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labo a o ies. Howe e , selec ed o ganisms ha e di e en nu i i e demands and he e o e no all o he
media a e sui able o all o he o ganisms and can inhibi hei g ow h (Table 1).
The cul i a ion on selec ed media be o e he expe imen an o 24 h (excep o Candida albicans
on CHROMaga , 48 h) a 37 °C.
Table 1. Cul i a ion o mic oo ganisms using di e en cul u e media (x = used o he
gi en o ganism).
Medium/O ganism S aphylococcus au eus S aphylococcus epide midis Esche ichia coli Candida albicans
Muelle -Hin on aga x x x x
Blood aga x x x
Blood aga -NaCl x x
CHROMaga Candida x
Roos el -Pa k Ins i u e Medium
1640 wi h L-glu amine x x x x
Sabou aud aga x
Endo aga x
3.2. Expe imen al Se up
G own mic obial colonies we e examined using a comme cial Renishaw Raman spec ome e
(Renishaw inVia Raman Spec ome e , Renishaw plc., Wo on-unde -Edge, UK), wi h 785 nm single-mode
diode lase as he exci a ion sou ce. A e cul i a ion o 24 h on MH aga he colony size was
app oxima ely 100 µm so ha spec a could be aken easily wi h ocused lase (diame e o lase is
app ox. 2 μm × 10 μm). The s eep dec ease in colony heigh ou side he la cen e o a g owing colony
(he e he diame e is app ox. 40 μm). This is isualized by un ocused colony su ace a he pe iphe y o
he colony (Figu e 5). No e ha he displayed colony is ela i ely small and was chosen jus o demons a e
he shape o a bac e ial colony. Ma u e colonies can be as la ge as a ew mm in diame e wi h es ima ed
hicknesses o hund eds o mic ome e s. We es ima ed he heigh o he displayed colony a he dis ance
o 15 μm om he bo om o he colony by e ocusing lase spo on he sample a 30 μm and he la
cen e on he op o he colony a 110 μm.
Figu e 5. Image o a colony o S aphylococcus epide midis.
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A lase beam was ocused on o a sample by he mic oscope objec i e (Leica, We zla , Ge many, 50×,
NA (Nume ical ape u e) 0.5) wi h he lase spo diame e o app oxima ely 2 μm × 10 μm (no e ha
such lase spo shape is cha ac e is ic o he Renishaw inVia ins umen ), wi h ull axial dep h o
he exci a ion egion a 8 µm [45]. As he lase was ocused on o a su ace o he pa icula colony,
we measu ed he esponse o a small ac ion o a colony di ec ly g own on he cul u e medium in
he pe i dish as ecen ly desc ibed in he wo k o Samek e al. [16]. To eliably apply he Raman
echnique, he lase was no ocused a he a ea nea he pe iphe y whe e he heigh o he colony alls
s eeply. In his way, he signal con ibu ion om he unde lying aga can be minimized— he signal
which o igina es om he cul u e medium is e y low and no in luencing he Raman spec a om
a colony.
O e iew spec a we e acqui ed in he ange o 600–1700 cm−1. As he in e e ing signal om
he cul u e medium may a y depending on ac ual colony p opo ions, each spec um was measu ed o
15 s om di e en pa s o a colony in a o al o 10 measu emen s o one s ain (spec a we e ob ained
om a leas 3 colonies pe s ain).
3.3. Da a Analysis
The Raman spec a we e ea ed wi h he Sa i zky-Golay coupled ad anced olling il e backg ound
emo al ou ine (see [13]), and subsequen ly analyzed using he s anda d mul i a ia e p inciple componen
p og am w i en in-house using Ma Lab so wa e (Ma hWo ks, Na ick, MA, USA). Spec a shown a
Figu e 2 we e no malized o he peak assigned o he amino acid phenylalanine [12] a app oxima ely
1004 cm−1. Phenylalanine is p esen in all o he es ed mic oo ganisms and was used o s anda diza ion
ollowing nume ous es s o use his peak in ensi y. This is no ideal, howe e , his p ocedu e could be
used in ins ances whe e no p ope s anda ds a ailable [12]. The g oups we e ma ked by ellipsoids wi h
Mahalanobis dis ance o 3 [46].
4. Conclusions
We analyzed ou mic oo ganisms (S aphylococcus au eus, S aphylococcus epide midis, Esche ichia coli
and Candida albicans), each g own on ou di e en cul u e media, by Raman spec oscopy o ind
which medium in luences he Raman spec um inge p in o each o ganism less. We showed ha
Muelle -Hin on aga is he mos sui able medium o analyses o all es ed mic oo ganisms (compa ed
wi h blood aga , blood aga wi h 10% NaCl, Roos el -Pa k Ins i u e Medium 1640, Endo aga and
CHROMaga Candida) p o iding a good epea abili y o he mic obial inge p in as well as a low
a iabili y o spec a wi hin he analysis. Con a iwise, he Roos el -Pa k Ins i u e Medium 1640 and
he CHROMaga Candida we e shown o impai he speci ic mic obial inge p in by in oducing he
medium-speci ic peaks in o i and subsequen ly esul ing in a iable wi hin-analysis spec a and low
epea abili y o he measu emen .