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0095-1137/78/0007-0298$02.00/()

Copyright(C 1978 American Society forMicrobiology

Evaluation of

an

Automated

Agar

Plate Streaker

RICHARD C. TILTON* ANt)RAYMONI) W. RYAN Uniuersityof ConnecticutHealthCenter, Farmington,Connecticut06032

Receivedfor publication25August 1977

Anautomatedagarplate streakerwasevaluated. The Autostreakermechanizes

the agar plate streaking process by providing storage for plates, labeling and

streakingone or more plates for eitherisolationorquantitation, and stackingin

oneofseveral racksfor subsequent incubation. Results showedtheAutostreaker

to produce agarplateswithwell-separated colonies and accurate colonycounts.

A total of 1,930 clinicalspecimens wereprocessed eitherin parallelwithmanual

methods or solely by the Autostreaker. Technologist acceptance of

machine-streaked plateswasoutstanding.

Solidifiedgrowthmedium inrounddishes has

been used forover acenturytoisolate

microor-ganisms. Onlyinthelast decade haveattempts

been made to automate the laborious task of

inoculatingaspecimenformicrobialanalysison

one or moreagarplates.

Methodsforautomatedagarplateinoculation

includethat in which inoculumwasspread only

after itwasmanuallyplacedontheagarsurface

(3) as wellas that of Campbell (1) who added

inoculuminthe form ofanArchimedesspiralto

a rotating plate. Wilkins et al. (4) described a

device for the automatic surface inoculation of

rectangularagartrays.

This report describes the in-use testing ofa

machinethat inoculatesone or moreagarplates

for quantitation or isolation and labels and

stacks them inoneof three racksforincubation. MATERIALS AND METHODS Autostreaker description. The Autostreaker (TomTec, Orange, Conn.) (Fig. 1) isdesigned to

au-tomate theentireprimaryagarplatingprocess. The onlymanualinputisthe conversion of thespecimen toaliquid phase bythetechnologist.Thespecimenis

streakedontherequirednumber ofplates according

toaselected preset program,eitherfor isolation(IM, isolationmode)orforquantitation (CM,countmode). The plates are labeled with an accession number,

sorted, and stacked for transfer to the appropriate incubator.

The Autostreaker selects agar platesfroma large,

self-contained magazine capable ofholdingover 800 plates. lThebottomportion ofthe magazine is refrig-erated toallowstoringa several-daysupply.Thetop

portion,from which the platesarefed,ismaintained at room temperature so that specimens are not streakedoncoldplates.

A pin plug board is used to program the Auto-streaker. Forany1of 10 different kinds ofagarplates, the machine determines(i)whethertheplateistobe streaked for isolationorforcount, (ii) the volume of inoculumtobedepositedperplate (fourpreset

varia-bleamounts), and (iii) oneof three incubatorstacks.

Thestreakingmethod(patentpending)isuniqueto the Autostreaker. The agar plate spins at 300 rpm.

The specimen is drawn into a soft piece of plastic tubing andexpelledontothe agarplatesurface. The inoculating tubingis oscillatedat200strokespermin as it comes in contact with the spinning agar, off-centerofthe plate. The spinning platemoves under

the oscillating tubing so that the specimen being streakedis drawn tothe outeredgeoftheagarplate. After the specimen isprocessed, the tubingis auto-maticallycutoff and discarded. Anewlengthofsterile

tubingisexpelledforthe nextspecimen.

Thecontrolled andadjustablevariablesare as fol-lows:(i)the mode ofdepositofinoculum,inthecenter only(IM)oruniformlyacrossthewhole plate surface

(CM); (ii) the volume of inoculumdeposited; and (iii)

the time allowed forstreakingeachsectionof theagar

platefrom the middletotheouteredge.

In theIM,theentire inoculum volumeisdeposited

inthecentersection. Organismsaretransferredfrom

thecentersectiontothesecondandthird sections by theoverlappingaction oftheoscillatingtube.Aheavy concentration of bacterial colonies will occur in the center section, with isolated colonies in the outside ring. Forthe evaluation of the IM, the centerofthe

agarplatewasstreaked for 8s,the middlesectionwas

streaked for4 s, and the outersection was streaked

for 2s.

In the CM, the inoculum is dispensed from the tubingatauniformrateoverthe entireplatesurface.

For the evaluation, 10[L of inoculumwas deposited

uniformlyovertheagarsurface.

Study protocol. The Autostreaker was used in threehospital laboratories,each foradifferentphase

ofthe evaluation. Laboratories included Waterbury Hospital, Waterbury,Conn.(calibrationofthe instru-ment);St. FrancisHospitaland MedicalCenter (clin-icalspecimensand timestudy);andtheJohnDempsey HospitaloftheUniversityofConnecticut Health Cen-ter(laboratoryevaluation andclinicalspecimens).The manualagarplate streakingusedroutinelyineachof the three hospitalswas considered as the reference method. Ingeneral, thestreakingprocedurefollowed that describedinthe ManualofClinicalMicrobiology 298

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(2). Because of the unique streaking pattern of the Autostreaker, a true blind study was not possible. However, agarplateswereevaluatedindependentlyas

afunction oftheinoculation method and bytwo or moremicrobiologists.

Colonycounts.Colonycounts wereperformedon 1,500preparedliquid specimensovera4-monthperiod toobtainacomparisonofcolonycounts onthe Auto-streaker andbyamanual method.

Controlorganisms with obvious differences in

col-ony morphology (Serratiamarcescens [pigmented],

Escherichia coli, hemolytic Staphylococcus aureus, and Staphylococcus epidermidis) were prepared in suspensions of 1.5 x 107 to 3 x 107 colony-forming units (CFU) per ml. The inocula were individually standardizedbyusingthenephelometerof the Auto-bac (Pfizer Diagnostics, Groton, Conn.). From these individual suspensions, a stock inoculum was made consisting of1 to2partsS.marcescens,2to8partsE. coli,20partsS. aureus, and20partsS. epidermidis. Thestock inoculumwasdilutedto 1 x104, 1 x103,

and 1 x 102 CFU/mland addedtotheAutostreaker

cups. Amounts of1, 5, and10,Awerestreaked in both CM and IM. Eachtestincludedcontrolplatesstreaked manually witha10-,ul calibratedloop.

For a given volume of inoculum dispensed anda given methodofstreaking (IM,CM, and manual), all colony counts performed in either IM or CM were averaged. The mean, standard deviation, and coeffi-cient of variation(CV)weredetermined. The CVwas employed for purposes of comparing the accuracy of the Autostreakerversusthe manualsemiquantitative loop method.

Clinical specimens: (i) urine. A small quantity (1.5to 2ml)orurinewaspoured into the sample cup. The machinewasprogrammed to streak a blood agar plate (BAP, 7.5% sheep blood) with5ytL of urine for quantitation (CM) and a MacConkey agar plate for isolation (IM). A

10-pl

platinum loop was used to streak agarplates manually.

All urinespecimenswererefrigerated from the work of thepreviousdaystotestthedeterioration of urine samples sitting in the machine awaiting processing. Those specimens with either significant growth of bacteria (>1 x 104 CFU/ml) or a mixture of skin contaminants werereprocessed after adelay on the machine carousel of0to 4h.

Afterparalleltesting, the Autostreaker was used to streak 750 clinical urine specimens in place of the manual method. To provide Autostreaker colony counts comparable to the manual method, 10 ul of urinewasdispensedper agarplate.

(ii) Throat cultures. Aprogram wasestablished with Pediatrics Associates of New Haven, Conn. to provideduplicateswabs from 144patients suspected ofstreptococcalpharyngitis.One swabwasinoculated manually, the other wasinoculated by machine. The swab wastwirled briefly in asample cup containing 1.5 ml ofmodifiedStuart transport medium.A

15-pl

amountofsamplewasstreakedonBAPand a

choco-late agarplate.Afterduplicate testing,anadditional

127 specimens were processed solely by the Auto-streaker.

(iii)Sputum cultures.Atotal of163 clinical spu-tum specimens were processed, of which 152 were

performed inparallel with the conventional method. Sputumwasremoved from its container withaswab and added to 1.0 ml of sputolysin (Calbiochem, La Jolla, Calif.) inacapped, sterile test tube (13 by 100 mm). Thecontentsweremixedvigorously and trans-ferredtotheAutostreaker sample cup.BAP, chocolate agarplate, MacConkey agar, and mannitol salt agar wereinoculated.

(iv)Genital specimens. A total of30genital spec-imenswereprocessed, 16 in parallel with the conven-tional method. All specimens were received on swabs, and transferredto modified Stuart medium. A 15-,ul

portion of fluidwasinoculatedonBAP, chocolate agar plate, and Thayer-Martin agar.

(v)Otherswab-type specimens (wounds, ear, eye,etc.). Thesamespecimenpreparation procedure wasused for these swabspecimensasfor throat cul-tures.

(vi) Fecal cultures.Afecalsamplewasremoved from its container withaswab and addedtothesample cupcontaining modified Stuart medium. A total of 52 specimenswereprocessed,8inparallel with the con-ventional method on BAP,MacConkey agar, Hektoen agar, and mannitol salt agar. The contents of the sample cup werepoured intoSelenite Fbroth after processing.

Time factors. Time studies wereperformed ata 750-bed community hospital todetermine potential laborsavings. Theprimary plating processwasdefined asthatpart ofhandlingthe incoming specimenthat the Autostreakermechanizes, i.e.,selectingandsorting agarplates from the refrigerated storage,labelingthe plate,inoculating, streaking, sorting, and stacking for transferto an incubator. That part ofthe specimen-handling process, suchasaccessionlogging, slide prep-aration, andinoculatingtubedmedia,wasconsidered tobecommon toboth manual and automated meth-ods.

Technologists were timed performing various phases ofthe process. Thestreaking exercise depended onseveralfactors suchassize ofloop used and how manytimesitwasflamed and cooled.Separatetests wereconducted onloop cooling times. Various loops wereheatedtoredness, and the time requiredtocool totouchwasrecorded.

Contaminationpotential. The Autostreaker has a plastic hood that covers the workingarea. When thereare nosamplesintheinstrument,twoultraviolet lamps are automatically turned on. They provide a calculated radiation level at 253.7 of 200 ,iW/s per cm2 onthestreakingarea.To testtheeffectiveness of the ultraviolet system, 15BAPswereinoculated with 10 ,ld of S. aureus and E. coli (1 x 107 CFU/ml) and exposed to theultraviolet lamp for 1, 2, 5, 10, and 15

min.

All testingwasconducted withagar plates (Balti-more Biological Laboratory) supplied by BioQuest, Cockeysville, Md.

RESULTS

Figure 1 shows the Autostreaker. Figure 2

showsthosecomponents oftheAutostreaker in

sequence that hold the specimens, label and

streak the agarplates, andsort forincubation.

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300 TILTON AND

Table 1summarizes thedataon 1,500 labora-tory-preparedspecimens containing four differ-ent microorganisms. Based on mean colony

count of all specimens streaked, the

Auto-streakerwasless accuratewhendispensing1

IlI

(CV=50%) thanwhendispensing5 or 10

t,l

(CV

FIG. 1. The Autostreaker, an automated agar plateprocessingmachine.

= 21 to 22%, IM) of inoculum. The IM was

slightlymoreprecise than the CM (CV = 21 to

22%, IM versus 23 to26%,CM). This difference

is not statistically significant. The CV of the

calibrated loop delivery was 37%. On all

ma-chine-streaked plates, the four microorganisms

were present in approximately equal

numrbers

and well-separated so that distinctive colonial morphology waseasilyrecognized.

Table 2 presents the variety of clinical

speci-mensprocessed in this study. Of the818

speci-mens,42% weretested inparallelwith the

con-ventionalmethod.

Of the319 urinespecimens processedin

par-allel, 68 (21%) had a colony count >100,000 CFU/ml; 102 (32%) failed to grow on either MacConkeyagarorBAP; 149 (47%) had colony

counts of either questionable significance (1 x

104 to 1 X 105

CFU/ml)

or representative of contamination (<104CFU per

ml).

Of this group

of 149 specimens, 87 showed microbial growth

onlyon the BAP. Ifthoseagarplateswithless

thanfive colonieswere ignoredbecause of

sta-tistical inaccuracy, then there was

complete

agreementas to

biological

types of

microorga-nisms recovered by both methods. There was

complete

quantitive

agreementwith those spec-imens considered

positive

and those of

question-able significance. In

general,

the machine-streaked

plates

showed more uniform

colony

lvIa

mnCP

\v4'r ( S

iscubator stacks

N.

,tubecsjt;vIs GbL. :....r w

-tubeoscilatI

l-spinning

plaiV,

---- -- .. inoculatinct:g+ 4 P

plateIlah&-rl. r

pro r e ".

re.mov,i.Il.

./

progr;im

cT

P.I'!

FIG. 2. Individual components of the Autostreaker in the sequence of operation.

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distribution withconsistentlybetter isolation of colonies.

The Autostreaker plates were more easily

evaluated on specimens

containing

2105

CFU/ml, even inthe heavy areas ofinitial

in-oculation. The colonies were better separated.

Theseplatesenabled the

microbiologist

tomake

a more accurate judgement as to the type of

organismpresent and the

relationship

of various

microbialtypestothe totalplatecount.

Afterduplicate testing,theAutostreakerwas

used to process 750clinicalurine

specimens

on

aroutine basis inplace of the manual method.

Technologist acceptance of the Autostreaker

plates was excellent. Theircommentsand

sub-jectivejudgements on plate quality confirmed

those heard and observed in the

duplicate

test-ingphaseof theproject.

Of the routine urinespecimensinoculatedby

the Autostreaker, 9.2% had

colony

counts

greater than1 x105

CFU/ml,

42%werebetween

1 X 104 and 1 x 105

CFU/ml,

and 35.7%were

eithersterileorhad<104

CFU/ml.

Results of the specimen deterioration study revealed no change in

colony

count in the 2-h waiting period.

However,

at the end of 4 h, significant overgrowth of thespecimenwas ob-served (from 20 to between 150 and 200

addi-tional coloniesperplatein4h.

TABLE 1. Accuracyof the Autostreaker comparedto aconventionalsemiquantitative streakingmethod

on1,500specirnens

t1IDispensed Mode Mean CV( CFUa V %

1 IM 24 50

CM 22 64

5 IM 54 21

CM 47 23

10 IM 31 22

CM 27 26

1O-fld Calibrated loop 62 37

(manual)

aMean CFUpervolumedispensed.

Atotal of 144throatcultureswereprocessed

induplicate.Of thespecimens,39% hadnormal

throatflora and 59% containedhemolytic

colo-niesofwhich27%werebeta-hemolytic

strepto-cocciresemblinggroupAbyabacitracin

suscep-tibilitytest.

Ingeneral the Autostreaker plateswere more

easily evaluated. The heavily inoculatedareain

the center of theplatewasdiffusedmore than

the solid mass ofgrowth seen when the swab was applied tothe plate manually. This

facili-tated observing the various organisms present

and their relationship to the total microbial

mass.Isolationofcolonies around theperimeter

of theplatewasexcellent.

Atotal of 127 clinical throat specimenswere

then processed routinely by the Autostreaker.

As with the urine specimens, technologist

ac-ceptanceofthe plateswasexcellent. Theyonce

again confirmed the conclusion that theplates providedmoreinformationonthe total ecology

of thespecimen. Of the throatculturesstreaked

bymachine, 10.6%werepositive for

beta-hemo-lyticstreptococcus (group A).

Of the 152sputumspecimens processed, there

was complete microbiological agreement

be-tween the types of colonies recovered by both

methods. Itwasoftenaproblemonthemanually

streaked platestospread out theprimary area

ofinoculation duetotheadhesivenatureofthe

specimen. A typical manually streaked plate

showed aheavy massofcolonies in the initial

quadrant, butrelativelyfewisolatedcolonies in

the other quadrants. This problemwas

accen-tuated when comparing them to the

Auto-streakerplates,whichshowed excellent

separa-tion of colonies even in the heavily inoculated

centerarea.

Results ofotherswab-typespecimens,suchas

wound exudate, genital, eye, nasopharyngeal,

and feces, revealed good correlation between

manualandAutostreakerplatesastoboth the

typesoforganismspresentas well astheir

rela-tive numbers. Consistently, the Autostreaker

TABLE 2. Summary of clinical specimens processedbyconventional methodsand by theAutostreaker Specimentype Inparallel Autostreaker Total % of total

Urine 319 750 1069 55

Throat 144 127 271 14

Group A Streptococcus 25 25 1

screen

Trachealaspirates 15 17 32 2

Genital 16 30 46 2

Wounds 142 27 169 9

Feces 8 44 52 3

Staphylococcusscreen 8 69 77 4

Ear 4 4 8 0.5

Eye 10 8 18 1

Sputum 152 11 163 8

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302

plates were superior to the conventionally

streaked ones.

Figure 3 shows representative agar plates

streaked in both IM and CM. The separation of colonies in the IM is apparent as is the uniform spreading of the organisms over the plate in the CM.

A number of diluents were tested but the

survival rate of microorganisms was higher in

agar-freemodifiedStuart transport medium.

Ex-periments revealed that groupAbeta-hemolytic

streptococciat aninitial inoculum concentration

of either 1 x 103 or 1 x 104 CFU/ml suffered

onlya slight diminution ofcolony countover a 2-h period. SimilartestsusingNeisseria

gonor-rhoeaeplated on chocolate agar indicated a

two-FI(G. 3. (A) BAP streakedforisolation (IM). Beta-hemolytic streptococciwerepredominantin this throat

culture. (B)BAP streakedforisolation (IM). S. aureus waspredominant inthis uwoundculture. (C) Urine

specimenfrom whichtwoagarplateswerestreaked:MacConkeyagarforisolation(IM)and blood agarfor quantitation (CM).ThespecimencontainedE.coli>100,000CFU/ml.

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fold reduction in numbers in 1 h and an addi-tional twofold reduction in 2 h.

Results of the time study indicated that the

meantime foradministrativespecimen handling

exclusive ofstreakingtheplate was 45 s(range,

30 to 110 s depending on specimen type). The streakingtimedependedonseveralfactors,such

as loop size and the number of times it was

flamed and cooled. A 25-gauge nichrome loop

required 15 sto coolto touch, anda 20-gauge,

10-Al

platinum loop required 30 s to cool to

touch. With the nichrome loop, the average

manualstreakingtime perplatewas 30s.

The comparable sample handling time using

theAutostreakerwas21 sforliquid samplesand

32 sforspecimens collectedon aswab, regardless

of the numbers ofplatesstreaked.

Results of thetest todetermine the

effective-ness of the ultraviolet light in the streaking

portion of the machine suggested that the

poten-tial for contaminationwas minimal. Agar plates

containingaheavyinoculum of either E. colior S. aureusexposed forvarying times under the

ultraviolet source showed no growth aftera

1-to2-min exposure.

DISCUSSION

The Autostreaker can automate the entire agarplatestreakingprocess.It isalabor-sparing

machine thatprovidesresults at least equivalent

tomanual methods.Byself-programming,a

lab-oratory can "fine tune" the Autostreaker to

processeachspecimenasthemicrobiologist

de-sires. In addition to providing a refrigerated

storagearea for

plates,

the type of solid media

can be chosen for each specimen and the

ap-pearance of the colonies on the plate can be

controlledby

adjusting

theamountofinoculum dispensed, the timerequiredtostreak theplate,

and the pattern of streaking. Such

flexibility

allowsaspecimen tobeprocessedfor

quantita-tionaswellasfor

analysis

of mixed

colony

types.

Certain specimensreceived in the clinical

mi-crobiologylaboratorymustbeprocessed for col-ony count as well as for isolation. Tradition-ally, quantitativeurinecolonycountshave been

done either

by

the agar pourplatemethodorby

uniformly streakingthesurfaceareaof theplate with a pipette or platinum loop calibrated to

deliver a certainvolume of fluid. Many studies

haveconfirmed thereliabilityof bothmethods.

TheAutostreaker streaksaplate quantitatively

byprecisely deliveringaknownamountoffluid ontheplatesurface andthenuniformly

spread-ingit. Test results indicated thatcolonycounts

onprestandardized inoculawere moreaccurate

by machine than by hand if the volume

dis-pensedbymachinewas 5

ll

orgreater.

Dispens-ing volumes of 1 ul resulted in unacceptable

variation. A reason for this may be that the

delivery mechanism is a positive displacement

system within thetubing. However, due tothe

small volumebeingdispensed,surfacetensionat

the tube outlet hasaneffectonthe actual

deliv-eryvolume. Ifnothingwere totouch the end of

thetubing,theexpelledliquidwouldformadrop

that would accumulate until the drop weight

just exceeded the surface tensiononthetubing

orifice.However,onthe Autostreaker thetubing

is nothanging free, but in contact with the agar

surface. As soon as sufficient liquid is expelled

totouch the agarsurface,thebalance is "wicked

away."Thus,theactualamountofliquid

trans-ferredtothe agarsurface will varyslightlyfrom

thetheoreticaldisplacementvolume. This effect

is difficulttodetect in the IM wherevolumes in

excessof1

,ll

arebeing dispensedatonetime in the centersection of theplate. However, inthe

CM, there is intermittent transfer ofliquid to

the agarsurface. This effect is minimizedby the

longertimesofstreakingused inCM, becauseit

allows the tubing to spread potential colony

clumps overalargerarea.

Therendering of specimensto aliquidphase

beforeloadinginthe Autostreaker isadeparture

fromusualpractice. Althoughtheability ofthe

machine to process plates rapidly with better

colonial distribution thanby handwasshown in

the early phases of the study, the biological similarityof theliquified specimensversus

spec-imensdirectlyinoculatedrequired study.

Initially, phosphate-buffered saline was used as a"transport"medium. However, rapid loss of

groupAbeta-hemolyticstreptococci and N.

gon-orrhoeaewasobserved.Althoughdeath ofthese

two organisms was also seen in the modified

Stuart medium, it wasmarkedly diminished in

the time period (0 to 2 h) that the specimens

might await processing. In those specimens in

which itwassuspectedthatdilution of the swab

contents in the "transport" medium might

re-duce the number of bacteria below thelevels of

detection, the inoculum volume was increased

tocompensate fordilution.

At notimeduring the 6-month period of

ma-chineuse was contamination observed on agar

platesthat could be attributed to the machine.

In addition to streaked plates that were sterile

after incubation, hundreds of Trypticase soy

agarplatesweredryprocessed (no inoculum) to test avarietyof machine parameters. Innocase

weretheseplates cross-contaminated.

Althoughthisstudywasprimarily concerned

with themicrobiological capabilities of the

Au-tostreaker, knowledgeof thepotential

labor-sav-ing benefitsis essential. Thetimestudy revealed

thattheadministrativehandlingofthespecimen

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AND

consumedanaverage of 45s. Thetimerequired

to streakaspecimen manually was a function of

the numberofagar platesused. At a 30-s mean

streaking time per plate, per-specimen times

rangedfrom 36 s for urine to 121 sfor feces.

Exclusive of the 45-s administrative time,

technologistsconsumed from 21 to 32s toload

a specimen onthe Autostreaker. The total

ma-chinetimeperspecimenis of concernonly when

it becomesthelimitingfactorin"through-put."

At the presenttime, the machine processes

ap-proximately85 plates perh (680 plates per 8-h

shift). On subsequent models, the cycle times

can be compressed so that the mean time to

streak a plate is30 sorless (960 plates per 8-h

shift).

In actual laboratory conditions the

Auto-streaker resultedin an average time savings of

53% (range, 42% for urine cultures to 73% for

fecalcultures).

Apotential disadvantage of theAutostreaker

is itslack ofprovisionfor(i)inoculation oftubed

media and (ii) rapid processing of anaerobic

specimens. Laboratory experience dictated that

critical specimens suchascerebrospinal fluid or

oxygen-protected specimens for anaerobic

cul-turebe processedmanually.

ACKNOWLEDGMENTS

We thank thefollowingindividualsfor theirassistance in theconduct ofthisresearch:PaulA.Granato,Veterans Ad-ministration Hospital, Syracuse, N.Y.; SallyJo Rubin, St. FrancisHospital,Hartford, Conn.;and ThomasAstle, Orange, Conn.

LITERATURE CITED

1. Campbell,J. E. 1971. Newsystemfor plating and count-ing is recommended for aerobic bacteria. Clin. Lab. Forum 3:2.

2. Lennette,E.H.,Spaulding,E.H., andJ.C.Truant. 1974.Manual of ClinicalMicrobiology,2nd ed. Ameri-canSocietyforMicrobiology, Washington,D.C. 3.Trotman,R. E. 1971. The automaticspreadingof

bacte-rial cultureoverasolid agarplate.J.Appl.Bacteriol. 34:615-616.

4. Wilkins,J.R., Mills,S.M.,and E. H.Boykin. 1972. Automaticsurface inoculation of agar trays.Appl. Mi-crobiol.24:778-785.

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