Statistical evaluation of a quality control method for isolation of pathogenic Vibrio species on selected thiosulfate citrate bile salts sucrose agars

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0095-1137/82/121110-07$02.00/0

Statistical

Evaluation of

a

Quality

Control Method for

Isolation of

Pathogenic

Vibrio

Species

on

Selected

Thiosulfate-Citrate-Bile

Salts-Sucrose Agars

PAUL A.WEST,' ESTELLE RUSSEK,2PHYLLISR. BRAYTON,' ANDRITA R. COLWELL'*

DepartmentofMicrobiology'andDepartment of DairySciences,2 University ofMaryland, CollegePark, Maryland 20742

Received7July 1982/Accepted 23 September1982

The recovery of Vibriocholerae,

Vibriofluvialis,

Vibrioparahaemolyticus, and Vibrio vulnificus, employing eight strains of each species, was studiedby using four brands ofthiosulfate-citrate-bile salts-sucrose (TCBS)agarprepared accord-ing to manufacturers' instructions and following a standardized procedure. A standardized broth inoculum of each strainwasplacedonduplicateplates of each brand of TCBSagarandalsoontryptic soy agar(DifcoLaboratories) containing 1% (wt/vol) NaCl, the latter serving asthe control. Plates were inoculated in a sequence designed to compensate for bias associated with multiplication of the bacteria during the inoculation procedure. Colony counts and quality of growth were recorded afterincubation for 18 hat 35°C. The comparison procedure was repeated four timesat weeklyintervals. Datawereanalyzed by usingananalysis of variance model. The recovery and quality ofgrowth of each species varied

significantly onthedifferent brands ofTCBSagar. Significant variabilitywasalso

identifiedforsomecomponentsof theinoculation procedure. Modificationsofthe inoculationprocedurearesuggested tominimizesourcesof variance. Asimplified

statisticalprocedure, basedonthettest, is described for mediaqualitycontrolfor

laboratories routinely isolating pathogenic Vibrio spp.

Thiosulfate-citrate-bile salts-sucrose (TCBS)

agarwasoriginally developedfor the isolation of Vibrio spp. pathogenic for humans (11). The medium is recommended by the World Health

Organization fordiagnosis of cholera when iso-lation ofV. cholerae from fecal material is the clinical procedure employed (25). TCBS agar

wasusedin thestudyof the outbreak of cholera

which occurred in Louisiana during 1978 (2). TCBS agar has been widely recommended for

isolation of V. cholerae, both 01 and non-01 serovars, V.parahaemolyticus, V.fluvialis, and V. vulnificus from clinical

specimens (6,

24). Interestingly,evidence has accumulated indicat-ing that the pathogenic Vibriospecies, including

V. cholerae, are naturally occurring organisms in the aquatic environment (3, 5, 9, 10, 12, 13, 23). Furthermore, theprimary isolation medium

mostcommonlyusedinthe ecological studies of

theseorganisms is TCBS agar.

Major commercial sources of TCBS agarare

located in Japan (Eiken Chemical Co., Tokyo),

the United States (BBL

Microbiology Systems,

Cockeysville,

Md., andDifco

Laboratories,

De-troit, Mich.), and England

(Oxoid Ltd.,

Basing-stoke). Severalindependent studies have

report-ed brand-to-brand variations in the efficacy of

TCBSagartogrowpathogenic Vibrio spp. (14-17, 22). However, allof the earlierstudies used differentplatingtechniques, andnoneevaluated

thereproducibilityand sourcesof error

associat-ed with a particular procedure employed to

inoculate the TCBS agar plates. Accordingly,

recovery rates for pathogenic Vibrio spp. on

TCBS agar reported by various investigators show considerable variation,asituationthathas

prompted the World Health Organization to

recommend establishment of minimum accept-able guidelines for recovery of Vibrio spp. on

TCBS agar (26).

Because ofthe significant use of TCBS agar

for isolation of pathogenic Vibrio spp. from clinical and environmental samples, we have evaluated thereproducibility and major sources

of error associated with the quality control

method described by Nicholls et al. (17) for TCBS agar to improve the procedures so that

sucherrors areminimized. Inthestudy reported

here, we also consider recovery rates and growth of selected pathogenic Vibrio spp. on

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STATISTICAL EVALUATION OF TCBS AGARS 1111

T S AS

6ZL

TCBS agar brand

88L Eiken Difco Oxoid

Ii@

P

Plate

1

7

m

1®

Plate 2

FIG. 1. Inoculation ofdivided, duplicate plates of TCBS agar and the reference agar (TSAS). Numbers

indicate sequenceof inoculation.

TCBS agar and suggest guidelines for quality control ofthe medium.

MATERIALS AND METHODS

The studywasconducted over a period of4weeks

and involved twoinvestigators, each being assigned

specific tasks inthestudy. Astandardized

experimen-tal procedure was employed to minimize error arising from differences in expertise between the investiga-tors.

Bacterial strains. Eight strains of each of four

spe-cies were included, the majority of which had been

isolated from the aquatic environment. The strains, to

the best of ourknowledge, had been originally isolated

on TCBS agar. All strains had been identified by

recommended methods and schemes (24). Two 01

serovarsofV.cholerae, including strain ATCC 14035,

isolated from cholera stools, were studied together

with six non-O1 serovars isolated from Chesapeake

Bayand Louisiana coastal water or sediment samples

within the last 3 years. All but one of the strains

(ATCC 17802) ofV. parahaemolyticus were isolated

from different sites in Chesapeake Bay since 1980.

Strains of V. fluvialis represented isolates from a

diverse range ofhabitats, viz., waters in England and

Chesapeake Bay andstoolsamples collected in

Ban-gladesh and Indonesia. All strains of V. vulnificus

were isolated from Chesapeake Bay. Each of the

Vibrio strains were stored in liquid nitrogen after

suspension in a cryoprotectant of tryptic soy broth

(Difco) plus 8% (vol/vol) glycerol. During the study,

benchcultures were keptat ambient temperature on

tryptic soy agar(Difco) containing1% (wt/vol) NaCl

andweresubculturedat5-dayintervals. Strains

repre-sentingisolates fromfeces of normalindividuals, i.e.,

without clinical disease, were received asfresh, pure

cultures onblood agarplates fromWalterReedArmy

MedicalCenter, Washington, D.C., and used

immedi-ately fortheinhibitiontestsdescribed below.

Platingmedia.The brands and batchesof TCBS agar

used were: BBL, C4DLJ1; Difco, 684845; Eiken,

15NOBA/E-MDO1;andOxoid, 188/28193. All brands

were prepared for use by the sameinvestigator,

ac-cordingtothemanufacturers' instructions.After

boil-ing,allmediawereplacedat55±2°C for5±0.1 h and

then were dispensed in 20- + 0.2-ml aliquots into

triple-vented petridishes (90mmdiameter). Platesof

themedium were allowed to dry at ambient

tempera-ture(25+ 3°C) for 72 ± 3h.The non-inhibitory plating

medium used as control was tryptic soy agar (Difco)

containing1%(wt/vol) NaCl (TSAS). The incubation

temperature used throughout the study was 35 +

0.50C.

Inoculationofplating media.Each strain was grown

overnight in5 mlof tryptic soy broth (Difco) with 1%

(wt/vol) NaCl (TSBS). Approximately 0.1 ml of each

culture was inoculated into 5 ml of fresh TSBS and

incubated for4 ± 0.1 h, after which the number of

bacteria was estimated from the absorbance at 560 nm,

measured in aSpectronic 20 (Bausch & Lomb, Inc.,

Rochester, N.Y.) spectrometer. Ten-fold dilutions in TSBSwere made toprepare astandardized inoculum

intherangeof 30 to 150 colony-forming units per 0.1

ml.Each of two divided sections, in duplicate plates of

each brand of TCBS agar and of the reference TSAS

medium, were spot inoculated with 100,l- amounts,

using the bias compensation technique described by

Nicholls et al. (17) and illustrated in Fig. 1. The

inoculum was not spread over the plate surface. Five

drops were usually required to deliver each 100,l-d

amount, and these drops were placed away from each

otheron the plate surface. After inoculation, plates

wereincubated at ambient temperature for 1±0.1 h to

provide for absorption of the inoculum into the

medi-um,after which the plates were incubated aerobically

for18 + 2 hbefore colonies on each divided section

were counted manually.

RankingofTCBS agars. After colonieswere

count-ed, eachbrand ofTCBS agarwasarbitrarily ranked

from 1 (worst) to 4 (best) for each strain, using as

criteria abundance ofgrowthaswell asthose

charac-teristics which aid inrecognizing colonies, i.e.,

colo-nial appearance andcolonycolor.Apreliminaryblind

trial of the ranking method demonstrated that the

media could be ranked consistently by one of the

investigators.

Reproducibilityof theplating regime.The

reproduc-ibility ofthecomparisonmethodforTCBS, described

by Nichollsetal. (17),wasevaluatedby repeating the

procedure entirely four timesatweekly intervals.

InhibitoryactivityofTCBSagars.Theabilityof each

brandof TCBS agar to inhibitgrowth of non-Vibrio

strains, viz., four strains of Escherichia coli, two

Proteusspp.,twostrains of Pseudomonas aeruginosa,

and twostrains ofStreptococcusfaecalis, wastested VOL. 16,1982

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TABLE 1. Unbiased estimates of the variance components associated with the ANOVA evaluation of the

qualitycontrol methodology

Effecta Brand of

Species_Speie

_|agar

TCBS Reproduc- Reproduc- Rsda

ibility

RerStrain

ibility x Plate

Resridoual

Recovery

agaribility

~~~~~~strain

err rate

Vibriocholerae BBL 0.0018b(3) 0.0295c (7) 0.0369f(21) -0.0012b (32) 0.0112 (64) 58.64

Difco 0.0021b 0.0112d 0.0093f 0.0087 0.0057 75.88

Eiken -0.0040b 0.0448e 0.0967f 0.0025e 0.0053 65.30

Oxoid -0.0002b 0.0044d 0.002le 0.0020e 0.0047 86.14

Vibriofluvialis BBL 0.1376f(3) 0.1661f(7) 0.0834f(21) 0.0217f(32) 0.0156 (64) 17.18

Difco 0.0206e 0.0073b 0.0578f -0.0008b 0.0116 66.45

Eiken 0.0516d 0.0181b 0.0652f 0.0033e 0.0088 54.03

Oxoid 0.0090e 0.0094e 0.0175f 0-.0011b 0.0126 65.75

Vibriovulnificusg BBL -0.0087b(3) -0.0053b(5) 0.1027f(15) -0.0015b(24) 0.0090 (48) 55.01

Difco -0.0031b 0.0259b 0.0677f -0.0010 0.0118 54.08

Eiken -0.0050b -0.0073b 0.0394f 0.0036e 0.0096 66.53

Oxoid -0.0012 00.ob

0.0095f

-0.0029b 0.0123 64.61

Vibrio BBL -0.0010b(3) 0.0078d(7) 0.0064d(21) 0.0032e (32) 0.0077 (64) 62.34

parahaemolyticus Difco 0.0007b 0.0059d 0.0056c 0.0004b 0.0101 62.49

Eiken 0.0069b 0.0186b 0.0594f 0.0057d 0.0081 54.19

Oxoid 0.0204b 0.0266c 0.0080c -0.0001b 0.0127 47.61

a Numbers inparentheses indicate degrees of freedom foreacheffect.Fordescriptions of theeffects, see the

text.

bNotsignificant.

cP<0.001.

dP<0.01. 'eP<0.05.

fp<0.0001.

8Only six strains of this specieswereexamined.

by streaking 100 ,u ofanovernight TSBS culture of

these strainsontoseparate agarplates and examining

growth under low magnification(x5)after incubation

for 18 t 2 hat35°C.

Analysisof data.Ratiosof the total bacterialcounts

for each plate of each TCBS agar brand and the

corresponding non-inhibitory reference agar (TSAS)

platewerecompiledtorepresent the recoveryratefor

eachstrainand eachbrand of TCBS agar. Theratios

werenormalized byaloglotransformationtoimprove

homogeneityof variances.Thetransformed datawere

analyzed byusingarandomeffects(model II)analysis

of variance (ANOVA) technique designed to assess

thesignificanceofvariancesassociated with technical

components within the quality control method. The

followingcomponentswereexamined:week-by-week

overall recovery ratefor each species

(reproducibili-ty);individual strainperformanceaveragedoverthe4

weeks (strain); week-by-week variation in individual

strain performance (reproducibility x strain), and

within-week variability between counts from each

plate foreachstrain ofaspecies,averagedoverthe 4

weeks(plate).Theunbiased variance component

esti-mates were computed by deriving expected mean

squarevalues(7, 18).Theaveragerankingofeachtest

mediumwascomputedover a3-weekperiodforeach

species. Rankswere compared by usingaFriedman

rank-summultiplecomparison procedure (8)to

deter-minethestatisticallydifferentvalues.

Instudies oferrorassociated withthe plating

com-ponentwithin thequality control method, the ANOVA

tablewasrecomputed, usinglog1o-transformed counts

from eachportion of each TCBSandTSAS agar plate.

Ratioswere notused since recovery, per se,was not

considered in this aspect of the study. The best

estimatefor the variance ofa strain in agiven week

was taken as twice the mean square of the plate

component and was computed, as previously

de-scribed, over the 4-week period. The formula

de-scribed by Cochran (4) was used to determine the

relative number of TCBS agar and TSAS agar plates

requiredtominimize thevariance of the log of the ratio

of counts on eachmedium.

RESULTSANDDISCUSSION

The unbiased estimates of variance for the components ofthe quality control method are givenin Table1,togetherwith the meanpercent recovery rateforeachspecies duringthetesting

period. Where significant strain variance was observed, itwas interpreted as indicating that, forthespeciesunder study,the isolatesdidnot yield approximately the same recovery rate

within a week. In some cases, strain variation was notsignificantwithinaweek but was highly

significant when considered on a week-to-week

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TABLE 2. Averageranking of test media

Rank(from1 to4)forthefollowingspecies':

TCBSagar Vibrio

brand Vibriocholerae Vibriofluviali.s Vibriolvulnificus

parahaemolvtic

s

BBL 1.5b 1.3" 2 Ob 2.6"

Difco 3.1' 3.5' 3.4'

2.3b

Eiken 1.8b 2.4"1 1.5b 2.lb

Oxoid 3.5c 2.8" 3.0c 3 b

aNumbersandcriteria usedforrankingareexplainedin the text.

b-dWithin each column,entries with the same superscript are not significantlydifferent (P <0.05), usinga

multiplecomparison procedure basedon Friedman rank sumstatistic.

basis(reproducibility x straineffect), suggesting

that theisolaterecoveryrate wasnotstablefrom week to week and thus diminishing the useful-ness of the isolates for quality control.

Signifi-cant differences between counts on each plate

(plateeffect)canbe attributed tovariation in the

quality control method and in the inherent

re-coverability ofthe strains. Potential sources of errorincludedpipetting, distribution of the

bac-teria in broth serving asthe inoculum, and lack of homogeneity in distribution of the selective agents in the unreconstituted TCBS agar batch

as well as in the medium after dispensing into

petri dishes. There are significant differences among each of the pathogenic Vibrio spp. with respect tosuitability for qualitycontrol ofTCBS

agar (Table 1). All were associatedwith

signifi-cant variance in at least one component of the

procedure.

Two strains of V.

vulnificus

failed to

grow on the TSAS agar on two occasions and were,thereafter,eliminatedfromthefinal

analy-ses.

Examination of rankings for the medium re-vealed significant differences in the ability of

each brand to recover each of the pathogenic

Vibrio spp. included in the study, as well as in the production ofcolonial morphology charac-teristic of each species (Table 2). Characteristic

yellow, i.e., sucrose-fermenting, colonies were observed on all brands of TCBS agar when growth was luxuriant. The rendition of green,

i.e., non-sucrose-fermenting, colonies was supe-rior on the Oxoid brand. Media performance rankings did not always correlate with the per-centage recovery rateforeachspecies. Percent-age recovery rates only indicated the ability of strains to grow on TCBSagar, whereas empha-sis was placed in the arbitrary ranking scheme

on the quality, rather than the abundance, of growth. Low mediarankingbuthighpercentage recovery rateimplies that anorganism grew on TCBS agar with a poorly defined colonial ap-pearance.

The plate component of the quality control method was studied further to determine the

sample size required to minimize differences in

variability, or range, of counts on the test (TCBS) and control (TSAS) agars. Approxi-mately equal count variances should improve

the precision (measurement of the closeness of repeatedtestings) of the quality control method. The mean squares were computed for

logl0-transformedplate counts from over the4weeks oftesting and pooled to give an indication of the inherent variability, i.e., variance, of strain counts each week(Table 3).

For all species, the variance of counts was greater on test agar, i.e., TCBS, than on the corresponding control agar. The square roots of the values werecalculated toprovidean appro-priate standard deviation (s) and were used in the ratio ofsample sizeformula (4) to calculate the number (n2) of control agar plates and the number(n1) of test agar plates needed to mini-mize differences invariance, wheren.= s,(n1 +

n-,)/(1

+

s5).

Forthis

calculation,

variance and standarddeviationcould have been computed in moretraditionalways rather than by determina-tion of the mean squares within an ANOVA. By

using square root values for V. cholerae from

Oxoid brand

(sl)

and thecorresponding control agar (S2), the formula predicts that a ratio of

approximately two TCBS agar plates to one TSAS plate will minimize differences in count variability (Table 3). The ratio is, however, greater for other combinations of TCBS agar brand and pathogenic Vibrio spp., due to the greatervariabilityof counts on the test medium. Results obtained from the growth studies of non-Vibrio teststrains plated onto each brand of

TCBS agar indicated that some brands were moreinhibitorythanothers. None of the strains grew on BBL orEiken brand, whereas S.faeca-lis and the Proteus spp. grew weakly, but

visi-bly, onOxoid and Difco brands, respectively. Theimportanceof thepresent study is empha-sizedby the re-emergence of cholera as a health problem throughout the world. Outbreaks of the disease are no longer confined to cholera-en-demic areas, asevidenced by outbreaks in Italy (1) and the United States (2). In addition, the incidence of human disease associated with

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TABLE 3. Strain variance of plate counts derived from expected mean squares after a

log,(

transtormation

of the plate counts averaged over the 4-week study period

PlatecouLntvariance for the following species: Platingmedium

Vibriocholerae Vibriofluvialis Vibrio .//1ifica. _{parahacllnolV-icus.}i

TSAS 0).00175 0.00361 0.0030)2 t).00413

TCBS

BBL 0.00572 0.05672 0.00554 0.00766

Difco 0.00524 0.00778 0.00722 0.00544

Eiken 0.00614 0.01275 0.02005 0.01414

Oxoid 0.00530 0.00672 0).00781 0.00665

er pathogenic Vibrio spp. is also increasing, as

the epidemiology of the causative agents

be-comesbetter understood (3). Besides V.

(holer-(ae, related pathogenic Vibrio species, viz., V.

f)arUaIemolyllti(cus, V. flivialis, and V.

*,iifi-c.s, are increasingly being isolated from the

natural aquatic environment in the absence of

diseaseinthe surroundinghumanpopulation (3,

5, 9. 13). A comparison ofbrain heart infusion

agar(BHI) and TCBS forrecovery of V. parza-lI(ileiolvyticIIs had previously showed areduced

recoveryofV.parahuamolyticusonTCBS

com-pared with BHI (19).Thus,it isimperativethata

reliable, reproducible method be available for

the isolation ofthese organisms.

TCBS agar is the most commonly used

pri-mary selective medium for the isolation of

hu-man pathogenic Vibr-io spp. from clinical and environmentalsamples. The medium isdesigned

toinhibitthegrowth of normalgutbacterial flora

but allow growth ofthe Viibr-iospp.Although the

compositionof each brandof TCBSagarshould beidentical, several reportshave indicated vari-ation in the ability ofdifferent brandsto inhibit

gut flora withoutcompromisingrecoveryof

Vib-1-io species(14. 16, 17. 22).

It was not ourprimary intention, inthe

pres-ent study, to evaluate the efficacy of specific

brands ofTCBSagarforrecovery ofpathogenic

Vibrio

spp. sincesuch studies havealreadybeen reported. However, wehaveconfirmed the

pre-vious studies in demonstrating that there are

considerable differences in recovery between

brands of this medium. All brands performed

well in inhibiting representatives of normal gut

flora, but some brands were apparently too

inhibitory for certain

Vibri.i

spp. as well. We

report here for the first time the usefulness of

TCBS agarforrecovery ofthe newly described

pathogens, V. fluvialis and V.

vulnificius.

Several technical problems arose withthe use

of TCBS agar during collaborative studies,

promptingustoevaluate statistically a

previous-ly described quality control method for TCBS

agar. The significant differences reported in the

literature forrecovery ofpathogenic

Vibr-io

spp.

on TCBS agar meant that guidelines for

accep-tance or rejection of a batch of medium could

notbemadewithconfidence. Inaddition,

Vibri-i

spp. often occur in small numbers in environ-mental samples. It is likelythat clinical

labora-tories also encounter small numbers of

orga-nisms, either in poorly collected samples or in

fecal samples from "shedders", i.e.,individuals

whoexcrete small numbersofthe organism. No

data were available to indicate which brand of TCBS agar would be sensitive enough in such

situations to recover

Vibri-i

spp. most effective-ly.

An overriding consideration in enumeration

was the need to standardize the quality and

abundance of growth, as well as the colony

appearance, of different pathogenic

Vibrio

spp.

onTCBS agar. Unfortunately, almost total

reli-anceis placedoncolonial morphologyonTCBS

agar for primary identification of these

orga-nisms. In our preliminary studies, we had

no-ticed that colonies of cholera toxin-producing V.

(clolerae serovar 01 grew so poorly on certain

brands of TCBS agar that we suspected that

many, if not most, laboratories would not

per-form furthertests toidentify the organisms, but

would discard the samplesas negative.

The quality control method described by

Ni-cholls et al. (17) and statistically evaluated in this study appears to be useful for determining acceptable recovery rates of TCBS agar before the TCBS agar is used in the laboratory for isolation of pathogenic vibrios.Significant

varia-tion wasnoted, however, in certain components

ofthe method, although these are amenable to

improvement. For example, strain variation within aweekoftestingandfrom weektoweek oftesting maybe the result ofstorage ofstrains

between testing on plates in the

laboratory.

Variation could be reduced if the test strains

were freeze-dried or stored in liquid nitrogen

after preparation of the storage stock from a

commonbroth source. Thestrainscould thus be

reconstituted whenever needed.

Variation in plate counts can be reduced ifa

predictive formula to determine the optimum

numberofcontrol and testplates is used. If the

colony count variance between test agar and

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control agar plate counts is equal, then equal numbers of plates canbe used for each medium.

The variances, however, werenot equal (Table 3). Thus,alargenumberof platesisrequired for amediumgivingthegreatervariance,if

approxi-mately equal variances of counts on test agar

and control agar areto be achieved.

Statistical-ly, it is preferable to use as many plates as

possible, but this is not economically feasible.

Accordingly, individual laboratories must

de-cide the sizeof thesampletobe taken from each

batch of medium tobe used forqualitycontrol.

Although recovery rates approaching 100%

forV. choleraeonTCBS agarhave been

report-ed (14), more realistic rates appearto be 60 to

70%(15, 17).Inthepresentstudy,we investigat-ed recovery rates which could be expected for purecultures ofV. cholerae underrepeatedand

controlled testing. Our data confirm that a rate

of 60to70%, compared with therate for TSAS

agar, is readily achievable. However, different

laboratories will undoubtedlyusedifferent

crite-ria, different reference media, andguidelines for

acceptanceorrejection of TCBS agar, basedon

quality of growth and recovery rates.

The problem remainsasto howtodecide the

point atwhich onerejects abatchof medium, if

the recovery rate is less than the chosen

guide-line. A t test can be constructed to determine

whether aTCBS agarcount is significantly less than the predetermined guideline (20). In the

example which follows, the guideline is set at 70% orgreaterasacceptable recovery. Howev-er, any recovery rate can be employed in the

formula, in accordance with the preference of

the individual laboratory. For the following, let

log10X be the average log count from nI test

medium plates and

log1(Y

be the average log

count fromn. plates of the corresponding

refer-ence medium. A one-tailed t test, as follows,

examines whether the average log ratio of

countsissignificantly less than the log (0.7). Let

(logo0X

- log(Y)

-logl0

(0.7)

t =

wheresj and s,are thevariances of thetest and

reference media, using

loglo-transformed

counts.

The t value has the following degrees of

freedom(df):

ni n,) df sr] |s] -2

n+

n1+1 n,-41

orifsj isapproximately equal tos., df = (n +

n,) - 2. Ifthe calculated t valueisless thanthe

negative of the tabulated t-table value

(one-tailed test) at the designated degreesoffreedom

(e.g., df = 6; table value = 1.943) and 0.05

probability level, then the observed counts are

significantly less than the70% guideline for the

acceptance of the medium (21).

Recovery rates are, in general, based on the

best recoveryperformancesof the strains. Some

workers, however, advocate using the most

poorly recovered strains for quality control, suggesting that those strains will detect poorly prepared or inhibitory batches of TCBS agar with greatersensitivity (22). A suitable compro-mise may well betoemploytwo or more strains

which together provide reproducible recovery rates, as well as alikelihood of nonrecovery of

strains very sensitive to the inhibitory

com-poundswithin the medium.

In conclusion, the present study provides the

most extensive set of datato date on recovery

rates of pathogenic Vib1io spp., using different

brands of TCBS agar. Methods for ensuring adequate quality control of this mediumarealso

provided. Nevertheless, the choice of brand and guidelinesfor recoveryrateultimatelyrestswith

the individual laboratory orinvestigator.

ACKNOWLEDGMENTS

This work was supported in part byfunds from National Science Foundation grant no. DEB77-14646,National Ocean-ic andAtmospheric Administration/Sea Grant no.

NA81AA-D00040,andOffice of Naval Research Contract N00014-81-K-0638. All computeranalyseswereperformed byusingtheSAS analysis of variance and generallinear models proceduresat the U.S.DepartmentofAgriculture. Washington,D.C.

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Annu.Rev.Microbiol.34:341-367.

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