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Zbl. Bakt. H yg. , I. Abr, Orig. A 254, 109-117 (1983)
Dep ar tm ent of Microbiology, Biot est-Serurn-Institut GmbH, 6000 Fra nkfurt /Main 73
Unsuitability ofBlood Culture Media Containing Sodium
Polyanetholesulfonate for the Detection ofFastidious
Microorganism in CSF and Other Blood-free Body Fluids
Untauglichkeit der natriurnpolyanetholsulfonathaltigen Blutkulturmedien zum Nachweis anspruchsvoller Mikroorganisrnen in Liquor undanderen blutfreien Korperfltissigkeiten
M.A.-F. ABDOU and H. STOCKEL
Received September 9, 1982
Abstract
The sui ta bility of well known conventio na l blood culture media for th e detection ofmicro organ isms in CSF and other blood -free body fluids has been tested . It was demonstrated th at such media are uns uitable for the cultiva tion of fastidious and /or an ticoagulant sensitive microorgani sms whic h are frequently isolated from CSF and other bloo d-freebody-fluids.
On the contrary, the recently-developed M OPS electro lyte broth A which pro ved to bea sui table cultivation an d back-up medium for aero bic and facu ltative anaerobic microorganisms in CSF and other blood-free body fluids is not suitable for the detection of microorganisms in blood, because it is free of anticoagulants.
Zusammenfassung
Konvent ionelle Blurkulturrnedien wurden auf deren T auglichkeit zum Nachweis vonErregern in Korperflussigkeiten, auger Blut und Urin, ilberpriift. Es wu rde festgestellt, dagdiese Medien ungeeignet sind zur Anzlichtung anspruchsvo ller und/oder anti koag ulansempfindliche r Mi kroorgani smen, die in Aspiraten und Punkt aten relativ haufig vorkommen.
Dagegen erwies sich M OPS-Electrolyte-Bouillon A, ein neu entwic keltes Medium, einerseits als gut geeignet zur Anziichtu ng und Rlickhaltung (back -up) von Aerobiern undfak ulta tiven Anaerobiern aus Liquor und anderen blutfreien Korperflussigkeiten, andererseits als ungeeignet zum Erregernachweis in Blutprob en, da das Me dium kein Antikoagulans enthalt ,
110 M.A.-F.Abdou and HiStockel
Introduction
A large proportion of specimens are from patients on antibiotic therapy. As aresult, these specimens do not contain a sufficient number of microorganisms, andthose which are present are stressed. This applies above all to fastidious and sensitive microorganisms, which occur relatively frequently in cerebrospinal fluid (CSF)and other blood-free body fluids (1).
Until quite recently, conventional blood culture media were also used for theenrichment of microorganisms from CSF and other blood-free body fluids.
An anticoagulant is one of the essential ingredients of all blood culture media.Sodium polyanetholesulfonate (SPS), has been the most commonly used anticoagulant for more than 40 years (14). SPSis, however, not only an anticoagulant, but alsohas anticomplementary and antiphagocyric properties (8, 12). It also inactivatesserum lysozyme and residues of aminoglycoside and polypeptide antibiotics (3, 13).However, as CSF and other body fluids contain hardly any blood, it is doubtfulwhether the enrichment media used need to contain SPS at all.
The purpose of this study is to determine to what extent the following proceduresa), b) and c) affect the cultivation of microorganisms which occur frequently in CSFand other body fluids :
a) The use of conventional blood culture media not containing blood or serum;b) The use of a recently-developed SPS-free broth for the recovery of microorganisms
in CSF and other body fluids (2), with the addition of 10% human blood;c) The use of the broth mentioned above in point b), with 0.025% SPS but without
the addition of blood.
Materials and Methods
Test strains
1. Aerobes1.1. Yeasts: Candida albicans DSM 70014, Cryptococcus neoformans DSM 702191.2. Gram-positive coccobacilli: Nocardia asteroides ATCC 331181.3. Gram -negative CO2-dependent cocci: Neisseria gonorrhoeae ATCC 19424, N. gonor
rhoeae M. U. Wiesbaden " N. meningitidis M. U. Wiesbaden
2. Facultative anaerobes2.1. Gram-negative bacilli: Haemophilus in{luenzae ATCC 19418, H. in{luenzae M. U.
Wiesbaden2.2. Gram-positive cocci : Staphylococcus aureus ATCC 29213, S. epidermidis ATCC 14990,
Streptococcus pneumoniae ATCC E 6303, S. pneumoniae ATCC 9163
3. MicroaerophilesGram-negative spiral bacilli : Campylobacter fetus ATCC 15296.
Culture Media
- Brain Heart Dipeptone Broth with 0.025% SPS, without blood- Supplemented Peptone Broth with 0.03% SPS, without blood- MOPS Electrolyte Broth A with and without 0.025% Liquoid, with and without 10%
human blood respectively.
1 Medizinal-Untersuchungsstelle Wiesbaden (Director : Prof. Dr. med. H.-H.Schassan) .
Blood Culture Media 111
Or iginally manufactured bottles each containing 45-60 ml broth have been used.T able 1 provides information on the compos itio n of the culture media.
Inoculation
The number of colony for ming units (CFU) to be inoculated was determined in prel iminary experiments. On average, it was app roximately 10' CFU per bottl e of culture media.In addi tion, the initial CFU was checked for each bottle. The venti latio n of the bottlesof culture media was carried out und er ster ile conditions immediate ly afte r inocul at ion .
Subcultures
After 8 h and after 1, 2, 3, 4 and 7 days' incubation, double subcultures were made.Using a calibrated ino culat ion loop , one loopful was taken from each bottle of culturemedium and, with the exceptio n of H. influenzae, spread onto blood agar, so that 3 dilutions were achieved. H. influenzae was subcultured on chocolate agar. Th e Neisseria wereincubated in an atm osph ere containing appro ximately 5% COt . C. fetus was incubated inGas Pak" without a cat alyser. Th e incubation temperature was 35 °C.
Visual Examination
Parallel to the subcultures, the media were checked for turbidity, aggregate and /or scumformation and changes in colour.
pH Measurement
After 1, 2, 3, 4 and 7 days incubation the pH values of the inocul ated culture mediawere measured with a Digital pH Meter.
Evaluatio n of Growth
Each stage of dilution was evaluated as follows: very heavy grow th = 5 ; heavy grow th= 4; kissing colonies = 3 ; > 50- 100 colonies = 2; > 20-50 colonies = 1.5 ; > 10-20colonies = 1; < 10 colonies = 0.5.
The average values calculated for each of the 3 corresponding stages of diluti on wereadded toge ther. Accord ingly, the growth intensity of the individua l test strai ns dependingon incubat ion time was ascerta ined.
Results
Conventional blood culture media were totally unsuccessful in cultivating H. in{luenzae. With N. gonorrhoeae, C. neojormans and N. asteroides neither of the mediacould provide acceptable results (Table 2).
The abovementioned conclusions led us to develop new culture media for thedetection of microorganisms in CSF and other blood-free body fluids (2). MOPSElectrolyte Broth A is one example (Table 1).
The SPS- and blood-free MOPS Electrolyte Broth A as well as the MOPS Electrolyte Broth A with 0.025% SPS and 10% fresh human blood proved to be very suitableand equally effective, as shown by Tables 3 and 4. The addition of 0.025% SPS tothe blood-free MOPS Electrolyte Broth A did not influence the growth of C. albicans,H. iniluenzae, S. aureus, S. epidermidis and C. fetus. Howev er, it did not providegood results with most of the fastidious and/or SPS-sensitive species (Table 3). Theaddition of 10% human blood to the SPS-free MOPS Electrolyte Broth A completlyinhibited the growth of N. asteroides, H. iniluenzae and C. fetus, but did not seriously influence the growth of N. meningitidis and S. aureus as shown in Table 4.
112 M.A.-F.Abdou and H. St6ckel
Table 1. Composition of two well known blood culture media and the recentl y-developedMOPS electrolyte broth A for recovery of microorganisms in CSF and other body fluids
Ingredients
Dipeptone HFQuadtonePentatoneProteose PeptoneCalf 's brain infusionBovine heart infusionGelat inDextroseSodium chlorideCalcium chlorideMa gnesium sulfatep-Aminobenzoic acidThyminePenicillinaseSodium polyanethole sulfonate!L-Cystein . HClSodium dithioniteGlutamic acidL-GlutamineProlineAdenineGuanineCocarb oxylaseNico tinamide Adenine Dinucleotid eVitamin B 12ThiamineMenadioneHemindi-Sodium hydrogen ph osphateMorpholino propane sulfonic acid'Sodium carbonateAqua dest.Final pH value
1 SPS, an ant icoagulant.• MOPS, a biological buffer.
Discussion
Fluid Culture MediaBrain Heart Supplemented MOPS Electro-Dipeptone Peptone lyre Broth A
++
++++
+ ++ + ++ + +
++ ++
++
+ ++ +
++
+ + +++ ++ ++ ++ ++ +
+++ +
+ ++
+ + ++ + +7.3 ± 0.1 7.2 ± 0.1 7.0 ± 0.1
Due to a lack of special culture media, clinicians and microbiologists use convention al blood culture media for the detection of microorganisms in CSF and otherbody fluids.
This method is unsuitable for at least 3 reasons:
1. These culture media contain no blood. Thus all the growth-promoting substancesof blood which are essential for fastidious microorganisms are absent (2).
NT
able
2.U
nsu
itab
ilit
yof
conv
enti
onal
bloo
dcu
ltur
em
edia
1co
ntai
ning
SPS
wit
ho
ut
the
addi
tion
ofbl
ood
for
the
dete
ctio
nof
fast
idio
usae
robi
c?= '"
and
facu
ltat
ive
anae
robi
cm
icro
orga
nism
sin
CSF
and
othe
rbo
dyfl
uid
s" ?f :r:
Tes
tS
trai
ns2
Gro
wth
inte
nsit
yex
pre
ssed
infi
gure
s(c
umul
ativ
e)in
-e TB
rain
Hea
rtD
ipep
tone
Bro
thw
ith
0.02
5%SP
SS
uppl
emen
ted
Pep
ton
eB
roth
wit
h0.
03%
SPS
~In
cuba
tion
tim
eIn
cub
atio
nti
me
>8
h1
d2
d3
d4
d7
d8
hId
2d
3d
4d
7d
~ 0 ::1.
C.
neof
orm
ans
00
0.5
5.0
9.7
514
.75
00
00
02.
25'!" >
DS
M70
219
N '"N
.as
tero
ides
00
0.5
2.75
6.75
13.2
50
00
00
0....
AT
CC
3311
8N
.go
norr
hoea
e0
00
00
00
00
00
0A
TC
C19
424
N.g
onor
rho
eae
00
00
00
00
0.5
3.0
7.75
13.7
5M
.U.
Wie
sbad
en3
N.
men
ingi
tid
is0
1.0
8.25
15.2
523
.25
31.2
50
2.25
7.75
14.2
522
.026
.75
M.U
.W
iesb
aden
H.i
nflu
enza
e0
00
00
00
00
00
0A
TC
C19
418
H.
intl
uen
zae
00
00
00
00
00
00
M.
U.
Wie
sbad
ent;l
j
S.pn
eum
on
iae
0.5
9.0
16.2
522
.528
.032
.25
0.5
8.75
10.5
10.5
410
.510
.50- 0
AT
CC
E63
030
- oS.
pneu
mon
iae
09.
015
.75
24.5
31.
537
.25
0.5
9.5
12.5
12.7
512
.75
512
.75
E..A
TC
C9
163
2 .., (1)
1V
enti
lati
on
ofcu
ltur
ebo
ttle
imm
edia
tely
afte
rin
ocul
atio
n.~ (1
)
•In
ocul
ump
ercu
ltur
ebo
ttle
=25
-150
CFU
(ave
rage
valu
e=
77C
FU).
0-
3M
ediz
inal
Unt
ersu
chun
gsst
elle
Wie
sbad
en(D
irec
tor:
Pro
f.D
r.m
ed.
H.-
H.S
chas
san)
.~
.
•F
rom
this
poin
tth
esu
bcul
ture
was
neg
ativ
ean
dth
epH
-val
uesh
ifte
dto
4.79
.•
Fro
mth
isp
oin
tth
esu
bcul
ture
was
nega
tive
and
the
pH
-val
uesh
ifte
dto
4.93
.~ ~ w
..... ..... -l>-
Tab
le3.
Inhi
biti
onof
gro
wth
ofae
rob
ic,
facu
ltat
ive
anae
robi
can
dm
icro
aero
phil
icor
gani
sms
inb
loo
d-fr
eeb
roth
1th
rou
gh
the
addi
tion
of0.
025
%SP
S~ ?>
Tes
tS
trai
ns'
Gro
wth
inte
nsity
expr
esse
din
figu
res
(cum
ulat
ive)
inbl
ood-
free
MO
PS
Ele
ctro
lyte
Bro
thA
.~
wit
ho
ut
SPS
wit
h0.
025%
SPS
>In
cuba
tion
time
Incu
bat
ion
tim
esr 0
-
8h
1d
2d
3d
4d
8h
1d
2d
3d
4d.
0 l: I'l ::l
C.
neof
orm
ans
00.
54.
7510
.516
.50
0.5
3.25
7.5
9.25
0-
DS
M70
219
;r: Vl
N.a
ster
oide
s0
00
3.5
9.5
00
00
3.5
0:A
TC
C33
118
()
~
N.g
onor
rhoe
ae0
8.25
16.7
523
.75
32.2
50
5.5
13.2
520
.528
.75
!!..
AT
CC
1942
4N
.gon
orrh
oeae
0.5
6.75
15.2
524
.031
.75
0.25
5.75
10.5
17.2
522
.5M
.U.
Wie
sbad
en3
N.
men
ingi
tidi
s1.
09.
016
.75
24.0
31.7
50.
58.
516
.25
23.5
31.5
M.U
.W
iesb
aden
H.i
nflu
enza
e0
8.5
17.5
25.0
31.2
50
8.75
17.5
25.2
531
.25
AT
CC
1941
8S.
pneu
mon
iae
0.2
59.
2515
.75
21.5
27.5
08.
7516
.022
.028
.0A
TC
CE
6303
S.pn
eum
onia
e0
05.
7512
.75
19.7
50
00
0.25
5.5
AT
CC
9163
1V
enti
lati
onof
cult
ure
bott
leim
med
iate
lyaf
ter
inoc
ulat
ion
.•
Inoc
ulum
per
cult
ure
bott
le=
25-1
50C
FU(a
vera
geva
lue
80C
FU)
3M
ediz
inal
Unt
ersu
chun
gsst
eIIe
Wie
sbad
en(D
irec
tor:
Prof
.D
r.m
ed.
H.-
H.S
chas
san)
Tab
le4.
Inhi
biti
onof
gro
wth
ofae
robi
c,fa
cult
ativ
ean
aero
bic
and
mic
roae
roph
ilic
orga
nism
sin
SPS-
free
bro
th1
thro
ugh
the
addi
tion
of10
%h
um
anb
loo
d
Tes
tS
trai
ns2
Gro
wth
inte
nsity
expr
esse
din
figu
res
(cum
ulat
ive)
inM
OP
SE
lect
roly
teB
roth
A+
10%
hu
man
bloo
dw
ith
ou
tSP
Sw
ith
0.02
5%
SPS
Incu
bati
onti
me
Incu
bati
onti
me
8h
1d
2d
3d
4d
8h
Id2
d3
d
C.a
lbic
ans
00
1.0
5.7
513
.00
1.75
8.0
14.5
DS
M70
014
C.
neof
orm
ans
00
2.0
8.0
14.2
50
0.5
4.25
10.5
DS
M70
219
N.
gono
rrho
eae
01.
06.
2513
.75
21.0
04.
011
.019
.25
AT
CC
1942
4N
.gon
orrh
oeae
00
00
00
1.25
7.7
516
.75
M.U
.W
iesb
aden
3
S.ep
ider
mid
is0
09.
018
.027
.00
.25
5.0
14.0
23.0
AT
CC
1499
0S.
pneu
mon
iae
04.
513
.019
.526
.25
1.0
6.0
14.7
521
.25
AT
CC
E63
03S.
pneu
mon
iae
08.
517
.25
24.7
531
.75
1.75
10.7
519
.25
27.0
AT
CC
9163
1V
enti
lati
onof
cult
ure
bott
leim
med
iate
lyaf
ter
inoc
ulat
ion
.2
Ino
culu
mp
ercu
ltur
ebo
ttle
=25
-150
CFU
(ave
rage
valu
e80
CFU
).3
Med
izin
alU
nter
such
ungs
stel
leW
iesb
aden
(Dir
ecto
r:Pr
of.
Dr.
rned
.H
.-H
.Sch
assa
n).
4d
--
22.5
16.5
25.7
5
24.2
5
32.0
27.7
5t:O 0'
34.7
50 0
-(J c 2' .., " ~ "0- ~. .....
......
. ""
116 M.A.-F.Abdou and HStockel
2. Conventional blood culture media contain anorganic phosphate buffer systems .These have a weak buffer capacity. In high carbohydrate culture media with aweak buffer system the pH value can shift below 5.0. S. pneumoniae and otheracid-sensitive microorganisms die out after only 3 days incubation (2). In addition,phosphates cause precipitation of the bivalent and trivalent cations if these arepresent in the culture medium. The use of these cations and their inactivatingeffect on residues of chemotherapeutica in samples from patients on antibiotictherapy has therefore reluctantly been abandoned.
3. Conventional blood culture media contain an anticoagulant. In most cases, thisis sodium polyanetholesulfonate (SPS), and in a very few cases sodium arnylosulfate (SAS). It has been established in a series of studies that a SPS content ofmort' than 0.03% in blood culture media can, to a greater or lesser extent, inhibitthe growth of N. gonorrhoeae, N. meningitidis, H. influenzae, Streptobacillusmoniliiormis, Streptococcus uiridans, Peptostreptococcus anaerobius and somespecies of Bacteroides (5, 6, 7, 10, 11). Because of this the SPS concentration inblood culture media was reduced from 0.05% to 0.025%-0.03% (13). However,the sensitivity of the abovementioned microorganisms to SPS apparently dependson several factors, e.g, composition of culture media, size of inoculum and percentual proportion of blood (9).Blood culture media containing 1.2% gelatine and 0.025%-0.03% SPS werereported to have no inhibitory effect on N. gonorrboeae, N. meningitidis andP. anaerobius (4, 11, 12, 15). However, this seems not apply to conventional bloodculture media to which no blood has been added, as shown in Table 2.
For the detection of SPS-sensitive and /or fastidious microorganisms, in particularfrom samples with too few CFU or from patients undergoing antibiotic treatment,it is necessary to use culture media which have been supplemented with growthpromoting substances and other compounds which inactivate inhibitory substances.Moreover, such culture media should have a stable buffer system.
If CSF and other blood-free body fluids are being examined the culture mediaused must contain no SPS or SAS, as anticoagulants are in such cases not only superfluous but also partly inhibit growth, as shown in Table 3.
MOPS electrolyte broth A has been developed especially for the cultivation offastidious aerobic and facultative anaerobic microorganisms in CSF and other bloodfree body fluids (2). However, the authors do not advise the use of this medium fordetecting microorganisms in blood, as it is free of anticoagulants. Blood would therefore inhibit the growth of some clinically important microorganisms.
References
1. Abdou, M.A.-F.: Vor- und Nachteile kultureller und nichtkultureller Verfahren zumErregernachweis in Aspiraten und Punktaten. I. Mikroskopische, biochemische undkulturelle Verfahren. Med. Lab. 35 (1982) 123-132
2. Abdou, M.A. -F. und HiStockel : Vergleichsuntersuchungen mit 2 neuen und 8 bekannten Nahrrnedien zur Anziichtunganspruchsvollerund anspruchsloserErreger aus Liquorund anderen Korperfliissigkeiten. Zbl. Bakt. Hyg. , I.Abt. Orig. A 252 (1982) 116-128
3. Edberg, S. C., C. j. Bottenbley, and j. M. Singer: The mechanismof inhibition of aminoglycoside and polymyxin class antibiotics by polyanionic detergents. Proc. Soc. expoBioI. (N.Y.) 153 (1976) 49-51
Blood Culture Media 117
4. Eng,]. and E. Holten: Gelatin neutralization of the inhibitory effect of sodium polyanetholesulfonate on Neisseria meningitidis in blood culture media. J. Clin. Microbiol.6 (1977) 1-3
5. Eng,]. and H.lveland: Inhibitory effect in vitro of sodium polyanetholesulfonate onthe growth of Neisseria meningitidis. J. Clin . Microbiol. 1 (1975) 444-447
6. Graves, M. H., [ ,A. Morello, and F. E.Kocka: Sodium polyanetholesulfonate sensitivityof anaerobic cocci. Appl. Microbiol. 27 (1974) 1131-1133
7. Kocka, F.E., E.].Arthur, and R.1.Searcy: Comparative effects of two sulfated polyanions used in blood culture on anaerobic cocci. Amer. J. Clin. Path . 61 (1974) 25-27
8. Lawrance, B.1. and W. H. Traub: Inactivation of the bactericidal activity of humanserum by Liquoid (sodium polyanetholsulfonate). Appl. Microbiol. 17 (1969) 839-842
9. Rintala, 1. and H. M. Pollock: Effects of two blood culture anticoagulants on growthof Neisseria meningitidis. J. Clin. Microbiol. 7 (1978) 332-336
10. Rosner, R.: Evaluation of four blood culture systems using parallel culture methods.Appl. Microbiol. 28 (1974) 245-247
11. Staneck,].1. and S. Vincent: Inhibition of Neisseria gonorrhoeae by sodium polyanetholsulfonate. J. Clin . Microbiol. 13 (1981) 463-467
12. Traub, W. H. and I.Kleber : Inactivation of classical and alternative pathway-activatedbacterial activity of human serum by sodium polyanetholsulfonate. J. Clin. Microbiol. 5(1977) 278-284
13. Ullmann, U., M .A.-F.Abdou, R.Dahn, Ri l.iaticken und E.Herrero: Nachweis vonBakterien und Pilzen aus Blutproben. DGHM-Verfahrensrichtlinien (Hrsg. F.Burkhardt). Zbl. Bakt. Hyg ., I. Abt, Orig . A 252 (1982) 1-8
14. Von Haebler, T. and A.A. Miles: The action of polyanetholesulfonate (Liquoid) onblood cultures. J. Path. Bact. 46 (1938) 245-252
15. Wilkins, T.D. and S.E .H.West: Medium dependent inhibition of Peptostreptococcusanaerobius by sodium polyanetholesulfonate in blood culture media. J. Clin. Microbiol.3 (1976) 393-396
Dr . M.A.-F.Abdou, Department of Microbiology, Biotest-Serum-Institur GmbH, Postfach 730260, D-6000 Frankfurt /Main 73