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