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Agar Plates Inoculating and Spreading Samples Onto Evaluation of an Automated Instrument for Inoculating and Spreading Samples onto Agar Plates J. H. Glasson, L. H. Guthrie, D. J. Nielsen and F. A. Bethell J. Clin. Microbiol. 2008, 46(4):1281. DOI: 10.1128/JCM.01687-07. Published Ahead of Print 13 February 2008. Updated information and services can be found at: http://jcm.asm.org/content/46/4/1281 Downloaded from These include: REFERENCES This article cites 7 articles, 3 of which can be accessed free at: http://jcm.asm.org/content/46/4/1281#ref-list-1 http://jcm.asm.org/ CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on April 2, 2013 by guest Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2008, p. 1281–1284 Vol. 46, No. 4 0095-1137/08/$08.00ϩ0 doi:10.1128/JCM.01687-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved. Evaluation of an Automated Instrument for Inoculating and Spreading Samples onto Agar Platesᰔ J. H. Glasson,1* L. H. Guthrie,2 D. J. Nielsen,1 and F. A. Bethell1 Institute of Medical and Veterinary Science, Adelaide, South Australia,1 and LabTech Systems Ltd., Kent Town, South Australia2 Received 24 August 2007/Returned for modification 4 October 2007/Accepted 6 February 2008 The findings from a preliminary assessment of a new instrument designed for the inoculation and spreading of specimens for microbiological analysis onto agar plates are described. The study found that the instrument was able to select full or biplates from a number of input cassettes, each containing different agar types. Samples were then inoculated by the instrument onto the agar surfaces and spread by a novel plastic applicator. Following this, the instrument labeled the plates and sorted them into a number of specified output stations. It was found that the instrument was able to inoculate and spread samples over a greater proportion Downloaded from of the agar plate surface than the manual loop-to-plate method. As a consequence, up to 44% more usable colonies were produced per plate from clinical specimens and standard cultures. Viable counts showed that the instrument was able to detect as few as 102 CFU/ml in fluids and also facilitated the enumeration of organisms, particularly in specimens such as urine. The processing of specimens in many routine diagnostic MATERIALS AND METHODS laboratories is a manual and time-consuming activity. At- MicroStreak. MicroStreak (LabTech Systems, Kent Town, South Australia) is http://jcm.asm.org/ tempts to gain more efficiency in this area have evolved slowly an automated instrument that is able to select standard 90- to 100-mm-diameter since 1881, when Robert Koch first published a technique for full and biplates from a number of input cassettes. The instrument incorporates a precision pipettor using a separate, plugged, disposable pipette tip for each the isolation and examination of bacterial colonies using pools patient, to inoculate defined volumes of liquid sample across the surfaces of of gelatin-based media on glass slides (4). At that time, Koch selected plates, and another robotic arm applies a single-use, multitined plastic described the inoculation and spreading of samples using pins disposable applicator (international patent application PCT/AU2005/000079) to or vaccination needles sterilized by heat. Koch’s technique was the inoculum. While the applicator is in contact with the inoculum on the agar surface, the instrument rotates the plate at a defined speed. On completion of revolutionized in 1887, when his assistant, Richard Petri, de- this spreading cycle, the plates are sorted and delivered to specified output scribed a method using two round glass dishes, one for the stations for removal by the operator. on April 2, 2013 by guest culture medium and the other, a little larger, to act as a lid (6). Study design. A prototype MicroStreak instrument was tested at the Institute Since that time, apart from the development of molded of Medical and Veterinary Science (Adelaide, South Australia) over a period of plastic petri dishes and plastic disposable loops, the main tools 10 weeks. Different types of prepared plate media from a number of manufac- turers from Australia, the United States, and France were used, and standard used by microbiologists for the inoculation of samples onto bacterial strains and human clinical specimens were assessed using both the agar surfaces have remained relatively unchanged. The devel- instrument and the traditional manual loop-to-plate inoculation method (7). opment of inoculating and spreading devices, such as spiral Dilution studies. Standard cultures of Staphylococcus epidermidis ATCC 14990 platers and wire loop-based robotic systems, have made con- and Escherichia coli ATCC 25922 were grown overnight in brain heart infusion broth (Oxoid, Australia), and 10-fold dilutions of each culture were prepared in tributions to automating the process but have not found their sterile saline to a dilution of 10Ϫ7. Ten-microliter samples of each dilution were place in many routine diagnostic laboratories. then dispensed by the instrument onto five Columbia agar plates with 5% horse This report describes the preliminary assessment of an au- blood (HBA) (Oxoid, Australia), and each plate was rotated at speeds ranging tomated specimen and agar plate management instrument that from 10 to 100 rpm. After overnight aerobic incubation at 35°C, the growth on each plate was described and discrete colonies were counted. Bacterial counts utilizes a novel plastic applicator to spread inocula. The instru- were determined from these dilutions. A similar methodology was followed for ment is able to select from a variety of cassettes containing biplates containing HBA and cystine-lactose-electrolyte-deficient agar (CLED) different agar types in standard 90- to 100-mm petri dishes, (Oxoid, Australia). inoculate liquid samples onto the media, spread the samples, Assessment of clinical samples. (i) Enrichment broths. One hundred sixty selenite broths (Oxoid, Australia) were inoculated with 1 ml of fecal-saline label the inoculated plates, and distribute them to a number of suspensions prepared from samples submitted for routine analysis and incubated specific output cassettes for incubation. aerobically for 18 h at 35°C. Following incubation, 10 ␮l of each selenite broth The objectives of the study were to assess the ability of the was dispensed by the instrument onto xylose-lactose-desoxycholate agar (XLD) instrument to inoculate commonly encountered clinical speci- (Oxoid, Australia). Fifty of the inoculated plates were rotated at 40 rpm and 110 plates at 50 rpm before being incubated aerobically for 18 h at 35°C. Manually mens and to compare the microbiological results with the man- streaked control plates using sterile 10-␮l loops were also prepared from each ual loop-to-plate method. selenite broth (5). Following incubation, 54 blood samples previously inoculated into Bactec blood culture bottles (Becton Dickinson, County Clare, Ireland) were processed by the instrument and manually. Samples were removed from the Bactec bottles manually using sterile syringes and inoculated onto agar plates for manual * Corresponding author. Mailing address: Institute of Medical and Vet- streaking. An aliquot was also transferred aseptically to sterile tubes, and 10 ␮l erinary Science, Frome Road, Adelaide, South Australia 5000. Phone: 61 was inoculated from these tubes by the instrument onto selected plates, which 8 8222 3423. Fax: 61 8 8222 3538. E-mail: [email protected]. were rotated at 50 rpm. The plates inoculated by both methods were chocolate ᰔ Published ahead of print on 13 February 2008. agar (Oxoid, Australia), HBA, CLED, and HBA/CLED biplates. The CLED 1281 1282 GLASSON ET AL. J. CLIN.MICROBIOL. FIG. 2. S. aureus from a wound swab preparation plated on Columbia agar with 5% horse blood. The plate on the left was inoc- ulated by the manual loop-to-plate method and the plate on the right by MicroStreak. Downloaded from FIG. 1. Influence of plate rotation speed on the formation of iso- lated colonies from broths and saline dilutions of S. epidermidis ATCC epidermidis and 109 CFU/ml for E. coli. The lowest concentra- 14990 and E. coli ATCC 25922. tion of bacteria detected in any of the diluted broths was 102 CFU/ml. With a total of 600 plates from the S. epidermidis and E. coli plates were incubated in air, the chocolate agar and HBA/CED plates in 5% broths and dilutions counted, the number of isolated colonies CO2, and the HBA anaerobically before being examined. (ii) Swabs. Thirty-eight swabs submitted for routine microbiological assess- produced, in addition to areas of confluence, was found to be ment from wounds, ears, genital tracts, and eyes were processed by both the dependent on the rotation speed of the plates (Fig. 1). At http://jcm.asm.org/ manual method and MicroStreak. All swabs tested had been placed in Amies bacterial concentrations in the range from 106 to 108 CFU/ml, (gel) transport medium (Eurotube; Deltalab, Rubi, Spain). an average of more than 50 isolated colonies were produced Following the preparation of slides for routine stains and microscopy, manual control plates were prepared by rolling each swab onto the surfaces of selected per plate at speeds above 50 rpm. It was also noted that when plates and streaked using the manual loop-to-plate method (5). The remaining bacteria were present in smaller numbers, particularly in the contents of these swabs were then expressed into 1.0 ml of saline within 1.5-ml samples with bacterial concentrations in the range from 102 to microtubes, and 30 ␮l of these preparations was inoculated by the instrument 103 CFU/ml, optimal results were obtained at rotation speeds onto selected plates and rotated at a speed of 40 rpm.
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