Vol. 7, No.3JOIRNAI OF ClINICAL MICOBIOLOG;Y, Mar. 1978, p. 298-3040095-1 137/78/0007-0298$02.00/()Copyright (C 1978 American Society for Microbiology

Evaluation of an Automated Agar Plate StreakerRICHARD C. TILTON* ANt) RAYMONI) W. RYAN

Uniuersity of Connecticut Health Center, Farmington, Connecticut 06032

Received for publication 25 August 1977

An automated agar plate streaker was evaluated. The Autostreaker mechanizesthe agar plate streaking process by providing storage for plates, labeling andstreaking one or more plates for either isolation or quantitation, and stacking inone of several racks for subsequent incubation. Results showed the Autostreakerto produce agar plates with well-separated colonies and accurate colony counts.A total of 1,930 clinical specimens were processed either in parallel with manualmethods or solely by the Autostreaker. Technologist acceptance of machine-streaked plates was outstanding.

Solidified growth medium in round dishes hasbeen used for over a century to isolate microor-ganisms. Only in the last decade have attemptsbeen made to automate the laborious task ofinoculating a specimen for microbial analysis on

one or more agar plates.Methods for automated agar plate inoculation

include that in which inoculum was spread onlyafter it was manually placed on the agar surface(3) as well as that of Campbell (1) who addedinoculum in the form of an Archimedes spiral toa rotating plate. Wilkins et al. (4) described a

device for the automatic surface inoculation ofrectangular agar trays.This report describes the in-use testing of a

machine that inoculates one or more agar platesfor quantitation or isolation and labels andstacks them in one of three racks for incubation.

MATERIALS AND METHODSAutostreaker description. The Autostreaker

(TomTec, Orange, Conn.) (Fig. 1) is designed to au-

tomate the entire primary agar plating process. Theonly manual input is the conversion of the specimento a liquid phase by the technologist. The specimen isstreaked on the required number of plates accordingto a selected preset program, either for isolation (IM,isolation mode) or for quantitation (CM, count mode).The plates are labeled with an accession number,sorted, and stacked for transfer to the appropriateincubator.The Autostreaker selects agar plates from a large,

self-contained magazine capable of holding over 800plates. lThe bottom portion of the magazine is refrig-erated to allow storing a several-day supply. The topportion, from which the plates are fed, is maintainedat room temperature so that specimens are notstreaked on cold plates.A pin plug board is used to program the Auto-

streaker. For any 1 of 10 different kinds of agar plates,the machine determines (i) whether the plate is to bestreaked for isolation or for count, (ii) the volume ofinoculum to be deposited per plate (four preset varia-

ble amounts), and (iii) one of three incubator stacks.The streaking method (patent pending) is unique to

the Autostreaker. The agar plate spins at 300 rpm.

The specimen is drawn into a soft piece of plastictubing and expelled onto the agar plate surface. Theinoculating tubing is oscillated at 200 strokes per minas it comes in contact with the spinning agar, off-center of the plate. The spinning plate moves underthe oscillating tubing so that the specimen beingstreaked is drawn to the outer edge of the agar plate.After the specimen is processed, the tubing is auto-matically cut off and discarded. A new length of steriletubing is expelled for the next specimen.The controlled and adjustable variables are as fol-

lows: (i) the mode of deposit of inoculum, in the centeronly (IM) or uniformly across the whole plate surface(CM); (ii) the volume of inoculum deposited; and (iii)the time allowed for streaking each section of the agar

plate from the middle to the outer edge.In the IM, the entire inoculum volume is deposited

in the center section. Organisms are transferred fromthe center section to the second and third sections bythe overlapping action of the oscillating tube. A heavyconcentration of bacterial colonies will occur in thecenter section, with isolated colonies in the outsidering. For the evaluation of the IM, the center of theagar plate was streaked for 8 s, the middle section wasstreaked for 4 s, and the outer section was streakedfor 2 s.

In the CM, the inoculum is dispensed from thetubing at a uniform rate over the entire plate surface.For the evaluation, 10 [L of inoculum was depositeduniformly over the agar surface.

Study protocol. The Autostreaker was used inthree hospital laboratories, each for a different phaseof the evaluation. Laboratories included WaterburyHospital, Waterbury, Conn. (calibration of the instru-ment); St. Francis Hospital and Medical Center (clin-ical specimens and time study); and the John DempseyHospital of the University of Connecticut Health Cen-ter (laboratory evaluation and clinical specimens). Themanual agar plate streaking used routinely in each ofthe three hospitals was considered as the referencemethod. In general, the streaking procedure followedthat described in the Manual of Clinical Microbiology

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AUTOMATED AGAR PLATE STREAKER EVALUATION

(2). Because of the unique streaking pattern of theAutostreaker, a true blind study was not possible.However, agar plates were evaluated independently asa function of the inoculation method and by two ormore microbiologists.Colony counts. Colony counts were performed on

1,500 prepared liquid specimens over a 4-month periodto obtain a comparison of colony counts on the Auto-streaker and by a manual method.

Control organisms with obvious differences in col-ony morphology (Serratia marcescens [pigmented],Escherichia coli, hemolytic Staphylococcus aureus,and Staphylococcus epidermidis) were prepared insuspensions of 1.5 x 107 to 3 x 107 colony-formingunits (CFU) per ml. The inocula were individuallystandardized by using the nephelometer of the Auto-bac (Pfizer Diagnostics, Groton, Conn.). From theseindividual suspensions, a stock inoculum was madeconsisting of 1 to 2 parts S. marcescens, 2 to 8 parts E.coli, 20 parts S. aureus, and 20 parts S. epidermidis.The stock inoculum was diluted to 1 x 104, 1 x 103,

and 1 x 102 CFU/ml and added to the Autostreakercups. Amounts of 1, 5, and 10,A were streaked in bothCM and IM. Each test included control plates streakedmanually with a 10-,ul calibrated loop.

For a given volume of inoculum dispensed and agiven method of streaking (IM, CM, and manual), allcolony counts performed in either IM or CM wereaveraged. The mean, standard deviation, and coeffi-cient of variation (CV) were determined. The CV wasemployed for purposes of comparing the accuracy ofthe Autostreaker versus the manual semiquantitativeloop method.

Clinical specimens: (i) urine. A small quantity(1.5 to 2 ml) or urine was poured into the sample cup.The machine was programmed to streak a blood agarplate (BAP, 7.5% sheep blood) with 5 ytL of urine forquantitation (CM) and a MacConkey agar plate forisolation (IM). A 10-pl platinum loop was used tostreak agar plates manually.

All urine specimens were refrigerated from the workof the previous days to test the deterioration of urinesamples sitting in the machine awaiting processing.Those specimens with either significant growth ofbacteria (>1 x 104 CFU/ml) or a mixture of skincontaminants were reprocessed after a delay on themachine carousel of 0 to 4 h.

After parallel testing, the Autostreaker was used tostreak 750 clinical urine specimens in place of themanual method. To provide Autostreaker colonycounts comparable to the manual method, 10 ul ofurine was dispensed per agar plate.

(ii) Throat cultures. A program was establishedwith Pediatrics Associates of New Haven, Conn. toprovide duplicate swabs from 144 patients suspectedof streptococcal pharyngitis. One swab was inoculatedmanually, the other was inoculated by machine. Theswab was twirled briefly in a sample cup containing1.5 ml of modified Stuart transport medium. A 15-plamount of sample was streaked on BAP and a choco-late agar plate. After duplicate testing, an additional127 specimens were processed solely by the Auto-streaker.

(iii) Sputum cultures. A total of 163 clinical spu-tum specimens were processed, of which 152 were

performed in parallel with the conventional method.Sputum was removed from its container with a swaband added to 1.0 ml of sputolysin (Calbiochem, LaJolla, Calif.) in a capped, sterile test tube (13 by 100mm). The contents were mixed vigorously and trans-ferred to the Autostreaker sample cup. BAP, chocolateagar plate, MacConkey agar, and mannitol salt agarwere inoculated.

(iv) Genital specimens. A total of 30 genital spec-imens were processed, 16 in parallel with the conven-tional method. All specimens were received on swabs,and transferred to modified Stuart medium. A 15-,ulportion of fluid was inoculated on BAP, chocolate agarplate, and Thayer-Martin agar.

(v) Other swab-type specimens (wounds, ear,eye, etc.). The same specimen preparation procedurewas used for these swab specimens as for throat cul-tures.

(vi) Fecal cultures. A fecal sample was removedfrom its container with a swab and added to the samplecup containing modified Stuart medium. A total of 52specimens were processed, 8 in parallel with the con-ventional method on BAP, MacConkey agar, Hektoenagar, and mannitol salt agar. The contents of thesample cup were poured into Selenite F broth afterprocessing.Time factors. Time studies were performed at a

750-bed community hospital to determine potentiallabor savings. The primary plating process was definedas that part of handling the incoming specimen thatthe Autostreaker mechanizes, i.e., selecting and sortingagar plates from the refrigerated storage, labeling theplate, inoculating, streaking, sorting, and stacking fortransfer to an incubator. That part of the specimen-handling process, such as accession logging, slide prep-aration, and inoculating tubed media, was consideredto be common to both manual and automated meth-ods.

Technologists were timed performing variousphases of the process. The streaking exercise dependedon several factors such as size of loop used and howmany times it was flamed and cooled. Separate testswere conducted on loop cooling times. Various loopswere heated to redness, and the time required to coolto touch was recorded.Contamination potential. The Autostreaker has

a plastic hood that covers the working area. Whenthere are no samples in the instrument, two ultravioletlamps are automatically turned on. They provide acalculated radiation level at 253.7 of 200 ,iW/s per cm2on the streaking area. To test the effectiveness of theultraviolet system, 15 BAPs were inoculated with 10,ld of S. aureus and E. coli (1 x 107 CFU/ml) andexposed to the ultraviolet lamp for 1, 2, 5, 10, and 15min.

All testing was conducted with agar plates (Balti-more Biological Laboratory) supplied by BioQuest,Cockeysville, Md.

RESULTSFigure 1 shows the Autostreaker. Figure 2

shows those components of the Autostreaker insequence that hold the specimens, label andstreak the agar plates, and sort for incubation.

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300 TILTON AND RYAN

Table 1 summarizes the data on 1,500 labora-tory-prepared specimens containing four differ-ent microorganisms. Based on mean colonycount of all specimens streaked, the Auto-streaker was less accurate when dispensing 1 IlI(CV = 50%) than when dispensing 5 or 10 t,l (CV

FIG. 1. The Autostreaker, an automated agarplate processing machine.

= 21 to 22%, IM) of inoculum. The IM wasslightly more precise than the CM (CV = 21 to22%, IM versus 23 to 26%, CM). This differenceis not statistically significant. The CV of thecalibrated loop delivery was 37%. On all ma-chine-streaked plates, the four microorganismswere present in approximately equal numrbersand well-separated so that distinctive colonialmorphology was easily recognized.

Table 2 presents the variety of clinical speci-mens processed in this study. Of the 818 speci-mens, 42% were tested in parallel with the con-ventional method.Of the 319 urine specimens processed in par-

allel, 68 (21%) had a colony count >100,000CFU/ml; 102 (32%) failed to grow on eitherMacConkey agar or BAP; 149 (47%) had colonycounts of either questionable significance (1 x104 to 1 X 105 CFU/ml) or representative ofcontamination (<104 CFU per ml). Of this groupof 149 specimens, 87 showed microbial growthonly on the BAP. If those agar plates with lessthan five colonies were ignored because of sta-tistical inaccuracy, then there was completeagreement as to biological types of microorga-nisms recovered by both methods. There wascomplete quantitive agreement with those spec-imens considered positive and those of question-able significance. In general, the machine-streaked plates showed more uniform colony

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

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AUTOMATED AGAR PLATE STREAKER EVALUATION

distribution with consistently better isolation ofcolonies.The Autostreaker plates were more easily

evaluated on specimens containing 2105CFU/ml, even in the heavy areas of initial in-oculation. The colonies were better separated.These plates enabled the microbiologist to makea more accurate judgement as to the type oforganism present and the relationship of variousmicrobial types to the total plate count.

After duplicate testing, the Autostreaker wasused to process 750 clinical urine specimens ona routine basis in place of the manual method.Technologist acceptance of the Autostreakerplates was excellent. Their comments and sub-jective judgements on plate quality confirmedthose heard and observed in the duplicate test-ing phase of the project.Of the routine urine specimens inoculated by

the Autostreaker, 9.2% had colony countsgreater than 1 x 105 CFU/ml, 42% were between1 X 104 and 1 x 105 CFU/ml, and 35.7% wereeither sterile or had <104 CFU/ml.

Results of the specimen deterioration studyrevealed no change in colony count in the 2-hwaiting period. However, at the end of 4 h,significant overgrowth of the specimen was ob-served (from 20 to between 150 and 200 addi-tional colonies per plate in 4 h.

TABLE 1. Accuracy of the Autostreaker compared toa conventional semiquantitative streaking method

on 1,500 specirnens

t1I Dispensed Mode Mean CV (CFUa V %1 IM 24 50

CM 22 645 IM 54 21

CM 47 2310 IM 31 22

CM 27 261O-fld Calibrated loop 62 37(manual)a Mean CFU per volume dispensed.

A total of 144 throat cultures were processedin duplicate. Of the specimens, 39% had normalthroat flora and 59% contained hemolytic colo-nies of which 27% were beta-hemolytic strepto-cocci resembling group A by a bacitracin suscep-tibility test.

In general the Autostreaker plates were moreeasily evaluated. The heavily inoculated area inthe center of the plate was diffused more thanthe solid mass of growth seen when the swabwas applied to the plate manually. This facili-tated observing the various organisms presentand their relationship to the total microbialmass. Isolation of colonies around the perimeterof the plate was excellent.A total of 127 clinical throat specimens were

then processed routinely by the Autostreaker.As with the urine specimens, technologist ac-ceptance of the plates was excellent. They onceagain confirmed the conclusion that the platesprovided more information on the total ecologyof the specimen. Of the throat cultures streakedby machine, 10.6% were positive for beta-hemo-lytic streptococcus (group A).Of the 152 sputum specimens processed, there

was complete microbiological agreement be-tween the types of colonies recovered by bothmethods. It was often a problem on the manuallystreaked plates to spread out the primary areaof inoculation due to the adhesive nature of thespecimen. A typical manually streaked plateshowed a heavy mass of colonies in the initialquadrant, but relatively few isolated colonies inthe other quadrants. This problem was accen-tuated when comparing them to the Auto-streaker plates, which showed excellent separa-tion of colonies even in the heavily inoculatedcenter area.

Results of other swab-type specimens, such aswound exudate, genital, eye, nasopharyngeal,and feces, revealed good correlation betweenmanual and Autostreaker plates as to both thetypes of organisms present as well as their rela-tive numbers. Consistently, the Autostreaker

TABLE 2. Summary of clinical specimens processed by conventional methods and by the AutostreakerSpecimen type In parallel Autostreaker Total % of total

Urine 319 750 1069 55Throat 144 127 271 14Group A Streptococcus 25 25 1

screenTracheal aspirates 15 17 32 2Genital 16 30 46 2Wounds 142 27 169 9Feces 8 44 52 3Staphylococcus screen 8 69 77 4Ear 4 4 8 0.5Eye 10 8 18 1Sputum 152 11 163 8

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302 TILTON AND RYAN

plates were superior to the conventionallystreaked ones.

Figure 3 shows representative agar platesstreaked in both IM and CM. The separation ofcolonies in the IM is apparent as is the uniformspreading of the organisms over the plate in theCM.A number of diluents were tested but the

survival rate of microorganisms was higher inagar-free modified Stuart transport medium. Ex-periments revealed that group A beta-hemolyticstreptococci at an initial inoculum concentrationof either 1 x 103 or 1 x 104 CFU/ml sufferedonly a slight diminution of colony count over a2-h period. Similar tests using Neisseria gonor-rhoeae plated on chocolate agar indicated a two-

FI(G. 3. (A) BAP streaked for isolation (IM). Beta-hemolytic streptococci were predominant in this throatculture. (B) BAP streaked for isolation (IM). S. aureus was predominant in this uwound culture. (C) Urinespecimen from which two agar plates were streaked: MacConkey agar for isolation (IM) and blood agar forquantitation (CM). The specimen contained E. coli >100,000 CFU/ml.

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AUTOMATED AGAR PLATE STREAKER EVALUATION 303

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

Results of the time study indicated that themean time for administrative specimen handlingexclusive of streaking the plate was 45 s (range,30 to 110 s depending on specimen type). Thestreaking time depended on several factors, suchas loop size and the number of times it wasflamed and cooled. A 25-gauge nichrome looprequired 15 s to cool to touch, and a 20-gauge,10-Al platinum loop required 30 s to cool totouch. With the nichrome loop, the averagemanual streaking time per plate was 30 s.The comparable sample handling time using

the Autostreaker was 21 s for liquid samples and32 s for specimens collected on a swab, regardlessof the numbers of plates streaked.

Results of the test to determine the effective-ness of the ultraviolet light in the streakingportion of the machine suggested that the poten-tial for contamination was minimal. Agar platescontaining a heavy inoculum of either E. coli orS. aureus exposed for varying times under theultraviolet source showed no growth after a 1-to 2-min exposure.

DISCUSSION

The Autostreaker can automate the entireagar plate streaking process. It is a labor-sparingmachine that provides results at least equivalentto manual methods. By self-programming, a lab-oratory can "fine tune" the Autostreaker toprocess each specimen as the microbiologist de-sires. In addition to providing a refrigeratedstorage area for plates, the type of solid mediacan be chosen for each specimen and the ap-pearance of the colonies on the plate can becontrolled by adjusting the amount of inoculumdispensed, the time required to streak the plate,and the pattern of streaking. Such flexibilityallows a specimen to be processed for quantita-tion as well as for analysis of mixed colony types.Certain specimens received in the clinical mi-

crobiology laboratory must be processed for col-ony count as well as for isolation. Tradition-ally, quantitative urine colony counts have beendone either by the agar pour plate method or byuniformly streaking the surface area of the platewith a pipette or platinum loop calibrated todeliver a certain volume of fluid. Many studieshave confirmed the reliability of both methods.The Autostreaker streaks a plate quantitativelyby precisely delivering a known amount of fluidon the plate surface and then uniformly spread-ing it. Test results indicated that colony countson prestandardized inocula were more accurateby machine than by hand if the volume dis-pensed by machine was 5 ll or greater. Dispens-

ing volumes of 1 ul resulted in unacceptablevariation. A reason for this may be that thedelivery mechanism is a positive displacementsystem within the tubing. However, due to thesmall volume being dispensed, surface tension atthe tube outlet has an effect on the actual deliv-ery volume. If nothing were to touch the end ofthe tubing, the expelled liquid would form a dropthat would accumulate until the drop weightjust exceeded the surface tension on the tubingorifice. However, on the Autostreaker the tubingis not hanging free, but in contact with the agarsurface. As soon as sufficient liquid is expelledto touch the agar surface, the balance is "wickedaway." Thus, the actual amount of liquid trans-ferred to the agar surface will vary slightly fromthe theoretical displacement volume. This effectis difficult to detect in the IM where volumes inexcess of 1 ,ll are being dispensed at one time inthe center section of the plate. However, in theCM, there is intermittent transfer of liquid tothe agar surface. This effect is minimized by thelonger times of streaking used in CM, because itallows the tubing to spread potential colonyclumps over a larger area.The rendering of specimens to a liquid phase

before loading in the Autostreaker is a departurefrom usual practice. Although the ability of themachine to process plates rapidly with bettercolonial distribution than by hand was shown inthe early phases of the study, the biologicalsimilarity of the liquified specimens versus spec-imens directly inoculated required study.

Initially, phosphate-buffered saline was usedas a "transport" medium. However, rapid loss ofgroup A beta-hemolytic streptococci and N. gon-orrhoeae was observed. Although death of thesetwo organisms was also seen in the modifiedStuart medium, it was markedly diminished inthe time period (0 to 2 h) that the specimensmight await processing. In those specimens inwhich it was suspected that dilution of the swabcontents in the "transport" medium might re-duce the number of bacteria below the levels ofdetection, the inoculum volume was increasedto compensate for dilution.At no time during the 6-month period of ma-

chine use was contamination observed on agarplates that could be attributed to the machine.In addition to streaked plates that were sterileafter incubation, hundreds of Trypticase soyagar plates were dry processed (no inoculum) totest a variety of machine parameters. In no casewere these plates cross-contaminated.Although this study was primarily concerned

with the microbiological capabilities of the Au-tostreaker, knowledge of the potential labor-sav-ing benefits is essential. The time study revealedthat the administrative handling of the specimen

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304 TILTON AND RYAN

consumed an average of 45 s. The time requiredto streak a specimen manually was a function ofthe number of agar plates used. At a 30-s meanstreaking time per plate, per-specimen timesranged from 36 s for urine to 121 s for feces.

Exclusive of the 45-s administrative time,technologists consumed from 21 to 32 s to loada specimen on the Autostreaker. The total ma-chine time per specimen is of concern only whenit becomes the limiting factor in "through-put."At the present time, the machine processes ap-proximately 85 plates per h (680 plates per 8-hshift). On subsequent models, the cycle timescan be compressed so that the mean time tostreak a plate is 30 s or less (960 plates per 8-hshift).

In actual laboratory conditions the Auto-streaker resulted in an average time savings of53% (range, 42% for urine cultures to 73% forfecal cultures).A potential disadvantage of the Autostreaker

is its lack of provision for (i) inoculation of tubed

J. CLIN. MICROBIOL.

media and (ii) rapid processing of anaerobicspecimens. Laboratory experience dictated thatcritical specimens such as cerebrospinal fluid oroxygen-protected specimens for anaerobic cul-ture be processed manually.

ACKNOWLEDGMENTS

We thank the following individuals for their assistance inthe conduct of this research: Paul A. Granato, Veterans Ad-ministration Hospital, Syracuse, N.Y.; Sally Jo Rubin, St.Francis Hospital, Hartford, Conn.; and Thomas Astle, Orange,Conn.

LITERATURE CITED1. Campbell, J. E. 1971. New system for plating and count-

ing is recommended for aerobic bacteria. Clin. Lab.Forum 3:2.

2. Lennette, E. H., Spaulding, E. H., and J. C. Truant.1974. Manual of Clinical Microbiology, 2nd ed. Ameri-can Society for Microbiology, Washington, D.C.

3. Trotman, R. E. 1971. The automatic spreading of bacte-rial culture over a solid agar plate. J. Appl. Bacteriol.34:615-616.

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

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