0095-1137/78/0007-0298$02.00/()
Copyright(C 1978 American Society forMicrobiology
Evaluation of
anAutomated
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 achoco-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 10t,l
(CVFIG. 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 perml).
Of this groupof 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 ofmicroorga-nisms recovered by both methods. There was
complete
quantitive
agreementwith those spec-imens consideredpositive
and those of question-able significance. Ingeneral,
the machine-streakedplates
showed more uniformcolony
lvIa
mnCP
\v4'r ( Siscubator 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
2105CFU/ml, even inthe heavy areas ofinitial
in-oculation. The colonies were better separated.
Theseplatesenabled the
microbiologist
tomakea more accurate judgement as to the type of
organismpresent and the
relationship
of variousmicrobialtypestothe totalplatecount.
Afterduplicate testing,theAutostreakerwas
used to process 750clinicalurine
specimens
onaroutine 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
countsgreater than1 x105
CFU/ml,
42%werebetween1 X 104 and 1 x 105
CFU/ml,
and 35.7%wereeithersterileorhad<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 200addi-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 totouch. 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 mediacan 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 mixedcolony
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 pourplatemethodorbyuniformly 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, intheCM, 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.