0095-1137/78/0008-0288$02.00/0
Copyright©) 1978 AmericanSocietyforMicrobiology Printed in U. S.A.
Evaluation of
Twenty-Three Blood Culture Media
JEGDISH P. BABU, RONALDF. SCHELL,* ANDJACK L. LE FROCK
DepartmentofMedicine(Division ofInfectious Diseases)andDepartment of MicrobiologyandImmunology, Albany Medical College,andClinicalMicrobiology Laboratory, AlbanyMedical CenterHospital,*Albany,
New York12208
Received for publication20June1978
Severalinvestigators have evaluated clinicallyavariety ofcommercially avail-able blood culture media. Noagreementhas been reachedastowhich of these media isoptimal for detection of bacteremia. Thepurpose ofthisstudywasto determine the rate of recovery ofmicroorganisms from various blood culture
media. A totalof 23 bloodculturemediawereinoculated with7 to 15 microor-ganismsper bottle inthepresenceorabsence ofanerythrocyte-serum mixture. The results demonstrated that blood culture media differed in their ability to support thegrowthofmicroorganisms. At 4 days after inoculation, only 10 of the 23 blood culture media supported the growth of 91% (10 of the 11)or moreof the test microorganisms. The recovery rate ofmicroorganisms depended not only
uponthe typeof medium but also uponthe manufacturer ofthe type ofblood culture medium. The addition of an erythrocyte-serum mixture to the blood culture media didnotinfluencethedifference in therecoveryrateof microorga-nismsamongmedia and thesametypeof médium prepared by different manu-facturers. Themajority (15 ofthe23) ofthe bloodculture mediasupplemented with theerythrocyte-serum mixture failedtosupportthegrowth of 91%or more of the test microorganisms at 4 days after inoculation. These results have demonstrated that blood culture media need to be improved. Better quality control measures should also be implemented to evaluate commercial blood culture media.
Thepromptandaccurateisolation ofthe
eti-ological
agentof bacteremia isoneofthemostimportant
functionsperformed by
clinicalmicro-biology
laboratories. It isimportant
that theinfecting organism be detected and identified
rapidly
toguide
propertherapy.
One of themajor variables
affecting
the isolation of bacter-emicagentsis the blood culturemedium. Several investigators(1-3, 6, 7, 11, 12, 16, 17, 19, 20) have evaluatedclinically
the blood culture mediatodetermine the
optimal
medium forisolation ofmicroorganisms. Blood culture media were in-oculated in
parallel
with the blood ofpatients
andtherecoveryratesofthebacteremicagents were compared. Unfortunately, no agreement has been reached as to which ofthecommer-cially available blood culture media is
optimal
forthe isolationofawidevarietyof microorga-nisms (5-7,10, 13-15, 17, 21,22).
Itis estimated that thereareover 200separate
blood culturemedia available
today.
The paral-lel clinical evaluation of these multiple blood culture media wouldrequire alarge
volumeofbloodfromabacteremic individual and
possibly
lead to medical complications. To circumventanyclinical
complication,
wechose toevaluatemultiple blood culture media
by
using simulated clinicalspecimens.
A total of 23 blood culturemedia were inoculated with asuspension of
in-dividual microorganisms with or without an
erythrocyte-serummixture (RBC-SM). The re-covery rates ofmicroorganismsfromindividual
blood culture mediawerecompared.
MATERIALS AND METHODS
Blood culture media. A total of23blood culture mediawerepurchased fromsevencommercial manu-facturers. Blood culture bottles contained approxi-mately 50ml of medium supplemented with either sodiumpolyanetholesulfonateorsodium amylosulfate.
Thetypesandcommercialsourcesofthe blood culture mediaarelisted in Table1.
Microorganisms. The microorganisms prepared foruse asstockcultures in this investigation are listed
in Table 2. These microorganisms were chosen to represent thosefrequently isolated from bacteremic individualsorhaving fastidious growth requirements.
Seedcultures wereprepared by inoculating brain heart infusion (BHI) broth with a single colony obtained froma recentclinical isolate.Separate 100-ml samples of BHI broth were inoculated with individual seed cultures.Acoenzyme-vitamin-amino acid enrichment (Grand Island Biological Co. [Gibco]) wasadded to
BHIbrothtofacilitatethegrowth ofmicroorganisms.
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TABLE 1. Commercial blood culture media inoculated withaerobic and facultative anaerobic
microorganisms
Typeofblood culture medium'
BHIbroth
Columbiabroth Trypticasesoybroth
Columbia broth
Supplementedpeptonebroth Trypticasesoybroth
BHIbroth Columbiabroth
Thiol broth
TSB BHIbroth
Columbiabroth
Columbia broth+10%sucrose
Columbia broth+20%sucrose
Dextrosephosphatebroth
TSB
Lederlebloodculturemedia BHIbroth
Brucellabroth
Brucellabroth+10%sucrose
Columbiabroth TSB
Columbia broth
aMedia contained sodium polyanetholesulfonate except forColumbia and Trypticase soy brothsfrom BD, which contained sodium amylosulfate. The con-centrations ofthesecompounds rangedfrom 0.025 to 0.05%.
TABLE 2. Aerobic andfacultativeanaerobic microorganismsusedforinoculationofblood
culture media
Inoculum(no. Microorganism of viable
orga-nissmaper
me-dium)' Acinetobacter calcoaceticussubsp.lwoffi 13
Candida albicans 7
Escherichia coli ... ... 15
Haemophilus influenzatypeb .. 12
Klebsiellapneumoniae 14
Neisseria meningitidis .... 14
Pseudomonasaeruginosa 12
Staphylococcus aureus 8
Streptococcus faecalis 10
Streptococcus pneumoniae 14
Streptococcuspyogenes 10
aThe standarderrorassociatedwith eachinoculum
was+1.3organisms.
Afterincubationat 35°C for18h, the cultureswere
centrifugedat10,000rpmfor 20 min. Thepelletswere
suspended and washed three times with fresh BHI broth.Afterthe finalcentrifugation, thepelletswere
suspendedin 20mlof BHIbroth,and 2-mlsamplesof eachmicroorganism wereplacedinvials, sealed,and storedinliquid nitrogen.
Determination of the number of microorga-nismsused forinoculation of blood culture me-dia. Frozen vials containing themicroorganisms were thawed, and the bacterial suspensionswere serially dilutedtoyieldafinal concentrationof 7 to 15 micro-organisms per ml(Table 2). This inoculumwaschosen
tosimulatealowgradebacteremia(4, 8, 9). To deter-minethe actual number ofmicroorganisms introduced into eachblood culture medium, a 0.5-ml sample of the inoculum was plated on chocolate agar plates. Colonieswerecounted aftera24-hincubationat35°C, and the number of microorganisms per milliliter of
inoculum was determined.
Preparation of human serum and erythro-cytes. Asingle lot ofpooled humanserum wasused in thisinvestigation. Healthy volunteers who were not
on antibiotic therapy during the preceding 2 weeks servedasdonors of blood. Theserumwas separated bycentrifugationat2,000 rpmfor 15min, sterilized by filtration (0.22-,um pore size; Millipore Corp.), and storedat-70°C untilusetoprevent theinactivation ofcomplement. Group Oerythrocyteswereobtained from thehospital blood bank and washed three times with saline. Theserum anderythrocytesweretested forsterility.
Inoculation and evaluation of blood culture media. The rubber stopper of each blood culture bottlewascleansed with 70%alcohol,disinfected with BetadineSurgicalScrub/Skin Cleanser for1min, and washed with 70% alcohol. A 1-ml quantity of BHI broth containingtest microorganisms (Table 2) was injected into duplicate blood culture bottles with or withoutRBC-SM. The RBC-SM contained2.75ml of erythrocytes and2.25 ml ofserum.The bottles were agitated, incubated unventedat35°C, and subcultured
at1, 4, 7, and14daysafterinoculation.Subculturing wasperformed by inoculating chocolate agar plates with 0.1to 0.2 ml of blood culture medium. Recovery of 91% (10 of 11) ormoreof thetestmicroorganisms from the blood culture media within 4 days after inoculation was consideredtobe a satisfactory recov-eryrate.
RESULTS
Evaluation of blood culture media. The
purposeof thisexperimentwas todetermine the ability of 23 blood culture media (Table 1) as obtained fromthe manufacturerstosupport the
growthof 11 testmicroorganisms (Table2).
Du-plicate blood culture mediawereinoculated with individualsuspensions ofmicroorganisms,
incu-bated,andsubcultured. Thepercentages of mi-croorganismsrecovered from the bloodculture
mediaareshowninTable3.Peptonebroth from
Becton, Dickinson & Co. (BD) supported the
growth of 100% of the test microorganisms
within 24 h after inoculation. At the same time
interval, an additional six blood culture media
supported thegrowthof 91% ofthe test
micro-organisms.These results demonstrated that only 7 of the 23 blood culture mediasupported the growth of 91% or more of the test microorga-Commercial
source
BBL BBL BBL BD BD BD Difco
Difco
Difco Difco Gibco Gibco Gibco Gibco Gibco Gibco Lederle Pfizer Pfizer Pfizer Pfizer Pfîzer Scott
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290 SCHELL, AND LE FROCK
TABLE 3. Cumulative percentages ofmicroorganismsrecoveredfromindividual blood culturemedium Cumulative% ofmicroorganismsrecovered after: Blood culture medium (source)
1day 4days 7days 14days
1. BHIbroth(BBL) 91 100 100 100
2. BHIbroth(Difco) 91 100 100 100
3. BHIbroth (Gibco) 91 100 100 100
4. BHIbroth(Pfizer) 82 82 91 91
5. Brucella broth(Pfizer) 73 73 82 82
6. Brucellabroth + 10% sucrose (Pfizer) 36 64 91 91
7. Columbia broth(BBL) 91 100 100 100
8. Columbia broth (BD) 45 91 100 100
9. Columbiabroth(Difco) 45 45 73 73
10. Columbia broth (Gibco) 91 91 100 100
11. Columbia broth + 10% sucrose (Gibco) 73 82 100 100
12. Columbiabroth + 20% sucrose (Gibco) 45 64 91 91
13. Columbia broth (Pfizer) 45 45 45 73
14. Columbia broth(Scott) 36 91 91 91
15. Dextrosephosphate broth (Gibco) 73 82 82 91
16. Lederleblood culture medium(Lederle) 82 91 91 91
17. Supplementedpeptonebroth (BD) 100 100 100 100
18. Thiolbroth(Difco) 27 27 55 55
19. TSB (BBL) 45 45 82 82
20. TSB (BD) 45 82 82 100
21. TSB (Difco) 45 73 91 91
22. TSB (Gibco) 91 100 100 100
23. TSB(Pfizer) 82 82 82 82
nisms within 24 h after inoculation. At 4 days
after inoculation, BHI broths from Baltimore Biological Laboratory (BBL), Difco Laborato-ries, and Gibco, Columbia broth from BBL, and tryptic soy broth (TSB) from Gibco
sup-ported the growth of 100% of the test
microor-ganisms.Inaddition,Columbiabroths from BD
and Gibco,ScottLaboratories, Inc., and Lederle
bloodculture medium from American Cyanamid
Co., Lederle Laboratories Div. supported the
growthof 91% of the testmicroorganisms.
Haemophilus influenza,Neisseria
meningi-tidis, and Streptococcuspneumoniae were the most frequent microorganisms that the blood
culturemedia failedtosupport.The most fastid-iousmicroorganism,H. influenza,wasisolated
in24h fromonly1of the23bloodculture media. At4daysafterinoculation,fouradditionalblood
culture mediasupportedthegrowthof H.
influ-enzae. The less fastidious microorganisms N.
meningitidisand S.pneumoniaewererecovered from six and nine of the blood culture media at 1and4days after inoculation,respectively. Non-fastidiousmicroorganisms, suchasEscherichia
coli,Klebsiella
pneumoniae,
andStreptococcus
faecalis,wererecoveredfrom100% of theblood culture media within 4 days after inoculation.Evaluation of blood culture media
sup-plementedwithRBC-SM.A total of23 blood culture media(Table1)weresupplementedwith RBC-SM, inoculated with individual
suspen-sions of test microorganisms, incubated, and
subcultured.The results of thisstudyareshown inTable4.BHI broth fromGibcosupportedthe growth of100%of thetestmicroorganismsat 24 h after inoculation. At thesame time interval,
BHIbroths from Difco and Pfizer Inc.,Columbia broth from Gibco, and TSB from Difco
sup-portedthegrowthof 91% of thetest
microorga-nisms. At4daysafterinoculation,100% recovery ofthemicroorganisms wasobtainedfromeight additional blood culture media
(Table
4).Supplementation ofthe blood culture media withRBC-SM (Table4) improvedtherecovery of H.
influenza
whencomparedwith medianotsupplemented with RBC-SM
(Table
3).Im-proved isolation of H.
influenza
wasdetected in BHIbrothsfromGibcoandPfizer, Columbiabroths from Gibco and Scott, Dextrose phos-phate brothfrom Gibco, and TSB from Difco. TheRBC-SM alsoimprovedthe recoveryofN.
meningitidisandS.
pneumoniae,
butnostatis-ticallysignificant improvementwasdetected in theisolation rates ofless fastidious
microorga-nisms, such as E. coli, K. pneumoniae, and S.
faecalis.Theseresults
suggested
thatthe RBC-SMplayedanimportantrole in the recovery of fastidiousmicroorganisms
fromsomeblood cul-ture media. The majority ofthe blood culture media(15 of the23)showed eithernochangeor a decrease in the recovery ofmicroorganisms
uponaddition ofRBC-SM.
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TABLE 4. Cumulative percentagesofmicroorganismsrecoveredfrom individualblood culture media supplemented with humanserumanderythrocytes
Cumulative % ofmicroorganisms recovered after: Blood culture medium (source)
1day 4days 7days 14days
1. BHI broth (BBL) 73 82 82 100
2. BHI broth(Difco) 91 100 100 100
3. BHI broth(Gibco) 100 100 100 100
4. BHI broth(Pfizer) 91 100 100 100
5. Brucella broth(Pfizer) 36 45 45 45
6. Brucella broth+ 10%sucrose(Pfizer) 0 27 45 55
7. Columbia broth (BBL) 82 82 82 82
8. Columbia broth(BD) 73 82 100 100
9. Columbia broth(Difco) 73 82 100 100
10. Columbia broth(Gibco) 91 100 100 100
11. Columbia broth+10% sucrose(Gibco) 45 64 64 64
12. Columbia broth+20% sucrose(Gibco) 55 64 73 73
13. Columbia broth(Pfîzer) 55 55 73 82
14. Columbia broth(Scott) 82 100 100 100
15. Dextrosephosphate broth(Gibco) 64 100 100 100
16. Lederle blood culture medium(Lederle) 64 64 73 73
17. Supplementedpeptonebroth(BD) 73 73 73 73
18. Thiol broth(Difco) 55 64 64 73
19. TSB(BBL) 55 73 73 73
20. TSB(BD) 64 73 82 82
21. TSB(Difco) 91 100 100 100
22. TSB (Gibco) 73 100 100 100
23. TSB(Pfizer) 64 73 82 82
DISCUSSION
Selection ofanoptimalblood culture medium
fortherapiddetection of the
etiological
agent of bacteremia is one of the most importantdeci-sions made
by
clinicalmicrobiologists.
Fre-quently,
aclinicalmicrobiologist
selectsabloodculture medium baseduponclinical studies that haveevaluated
multiple
blood culture media in parallel (3, 5, 6, 14, 16-18,20)
oruponinforma-tion
supplied
by
themanufacturers of the bloodculture media. Areview of theliterature (4, 7,
10, 15, 17, 19,22) has shown thatno consensus
existsas towhichofthe available blood culture media is the
optimal
system for recovery ofawide spectrum ofaerobic and
facultatively
an-aerobic
microorganisms.
Anumber ofvariables,
such as
variety
of blood culturemedia,
density
of bacteria in the blood ofa
patient,
time of bloodcollection,
and immune response of the host have contributed to the confusion as towhich of the available commercial (6, 16, 17, 20) and "homemade"(12) blood culture mediaisthe optimal one forrapid detection ofbacteremia. We have demonstrated that the type of
me-dium,the
manufacturer,
and theRBC-SM haveaneffect on theabilityof blood culture mediato
supportthegrowthof
microorganisms.
Evalua-tion of23bloodculture media withor withoutRBC-SM demonstratedthat themajorityof the mediafailedtosupport thegrowthof 91%(10 of
11) or more of the testmicroorganismswithin 4
days after inoculation. Table 3 shows that the
blood culture media differed in their ability to support the growth of fastidious aerobic and
facultatively anaerobic microorganisms.In
gen-eral, BHI broth showed enhanced isolation of
fastidious
and nonfastidious microorganismswhencompared with TSB, Columbia broth, and
the other blood culture media. This suggested
that BHI broth contained nutritional factors
that enhanced therecovery ofmicroorganisms
orcontainedfewermicrobial inhibitors than did
the othermedia.
The recovery ofmicroorganisms from blood culture medianot
supplemented
withRBC-SMwas also
dependent
upon the manufacturer ofthe type of medium (Table 3).
Although
BHI broth showed an enhanced recovery ofmicro-organismswhencompared with the othertypes
of
media,
aslight
difference in the percentageand time ofrecovery wasdetected based upon
the manufacturer of thistype ofmedium(Table
3). BHI broths from BBL and Difcosupported the growthof 100% of thetest microorganisms
within 4daysafterinoculation, whereas91% or
less recovery of microorganisms was obtained from BHI broths from Gibco and Pfizer within 4 daysafter inoculation. The differencesin re-covery rates ofmicroorganisms weremore pro-nounced when the othertypes ofcommercially
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prepared media were compared (Table 3).
Co-lumbia broths from Gibco and BBL supported the growth of 91% of the test microorganisms, whereas Columbia broths prepared by Difco, Pfizer, BD, andScott recovered less than 50% of thetestmicroorganisms in24h.Similarly, TSB from Gibco and Pfizer supported the growth of 81%or moreof thetestmicroorganisms, whereas TSBprepared byDifco, BBL, and BD recovered only 45% of the microorganisms in 24 h.These resultsemphasized that blood culturemedia
pre-pared by different manufacturers influenced the
recovery rateofmicroorganisms.
Inoculation of blood culture media with RBC-SM failedtoalleviate thedifferenceinrecovery ratesofmicroorganismsamongthetypesof
me-diaand the same typeof mediumprepared by
different
manufacturers(Table
4).Infact,
incor-poration of RBC-SMintothe blood culture
me-dia failedto improve therecovery rates of
mi-croorganisms from themajority (15 of the 23) of the blood culture media when compared with blood culture media not supplemented with RBC-SM(Table 3).
Rapid detection of the
etiological
agent of bacteremia isextremely important.
Evaluation of the23blood culture mediasupplemented
with RBC-SM showed that BHIbroths fromDifco,
Gibco, andPfizer,
Columbia broths from Gibcoand
Scott,
TSB from Difco andGibco,
andDex-trose
phosphate
broth fromGibcosupported
the growth of 100% of the testmicroorganisms
within 4days
afterinoculation(Table
4). BHIbroth from Gibco was the
only
blood culture medium thatsupported
thegrowth
of 100% ofthetest
microorganisms
within24hafterinoc-ulation. Cautionmustbe exercised in
interpret-ing these results as recommendations for the selection of anoptimal
blood culture medium until additional clinical studies have beenper-formed.
Theresults of this
investigation
clearly
dem-onstrate that blood culture media need to be improved. Tosupport the
growth
of fastidiousmicroorganisms,
the nutritionalcapacity
ofcer-tainblood culture media should be
augmented,
ortheinhibitorysubstancesshouldberemoved.
It is apparent that better
quality
control meas-ures areneededtoevaluate thegrowth-promot-ingabilityof commercialblood culturemediaby clinical
microbiologists
and the manufacturers.Quality
control of the blood culture media isabsolutely
necessary, because there is adiffer-encein recovery rates ofmicroorganisms from the samemediumprepared by different manu-facturers. Inoculation of blood culture media
with
microorganisms
should beanintegralpartof the
quality
controlprogramtoevaluatecom-mercialand homemade blood culture media.
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