JOURNAL OF CLINICAL MICROBIOLOGY, July 1983, p. 84-91 Vol. 18, No. 10095-1137/83/070084-08$02.00/0Copyright C 1983, American Society for Microbiology

Evaluation of the Prompt Inoculation System for Preparationof Standardized Bacterial InoculaMARLYS E. LUND'* AND RONALD W. HAWKINSON2

Medical Products Division1 and Riker Laboratories, Inc.,2 3M, St. Paul, Minnesota 55144

Received 7 January 1983/Accepted 4 April 1983

Standardized inocula prepared with the 3M Prompt inoculation system wereevaluated by (i) performing colony counts on the inocula and (ii) employing theseinocula in disk diffusion or broth microdilution susceptibility tests. The inocula-tion wand delivered 1.85 X 108 ± 0.6 log10 CFU to the various diluents tested.Inocula prepared in 1 ml of saline and used to inoculate disk diffusion testsresulted in 96.5% agreement between Prompt and standard tests when Promptinocula were used within 15 min of preparation and 95.6% agreement betweenpaired tests when Prompt inocula were allowed to sit at room temperature for 2 hbefore use. With 15-min Prompt inocula, only one (0.04%) major discrepancybetween paired tests was observed. The 2-h Prompt inocula gave three (0.13%)major or very major discrepancies. The 15-min Prompt inocula prepared in 30 mlof diluent resulted in 98.2% of Prompt minimal inhibitory concentrations within+1 log2 dilution step of the standard test result, whereas the 2-h inocula resulted in97.4% agreement between paired minimal inhibitory concentrations at this level.Thus, the Prompt inocula were found to give results equivalent to those obtainedfrom inocula prepared by conventional procedures.

Recently D'Amato and Hochstein (3) andBaker and Thornsberry (1) have reported thesuccessful application of a direct suspensionmethod for the preparation of standardized in-ocula for antimicrobic susceptibility tests(ASTs). Direct suspension of bacteria from anagar plate in a suitable diluent until the turbiditymatches that of a 0.5 McFarland standard addsconsiderable flexibility to the setup procedurefor ASTs by eliminating the conventionally re-quired 2- to 8-h incubation period for prepara-tion of a log- or stationary-phase broth culture.The 3M Prompt inoculation system (hereinshortened tO Prompt) offers this flexibility and inaddition elimninates the time-consuming and te-dious process of turbidity adjustment. The sys-tem includes a special inoculation wand (10) anda selection of prepackaged diluents. The wandpicks up atnd delivers a constant number ofbacteria. The diluents are packaged to provide(i) an inoculum equivalent to the 0.5 McFarlandturbidity standard or (ii) an inoculum suitable forbroth microdilution tests that involve a 1:20dilution of the inoculum in the final inoculationstep (e.g., 5 ,ul of inoculum into 0.1 ml ofantimicrobic solution). Furthermore, the dilu-ents have been selected so that the inocula arestable when held at room temperature, obviatingthe need to use the standardized inocula within15 min of preparation.

This study was undertaken to determine thereproducibility of the inoculation wand's pickupand delivery and to evaluate the performance ofinocula prepared with Prompt in antimicrobicsusceptibility tests set up (i) within 15 min ofinoculum preparation and (ii) 2 h after inoculumpreparation.

MATERIALS AND METHODSMicroorganisms. The 100 clinical isolates tested in

this study were kindly supplied by C. Thomsberry andC. Baker, Centers for Disease Control, Atlanta, Ga.The organisms were selected to represent a widevariety of susceptibility profiles. Only amikacin andvancomycin were not tested over the entire range ofpossible response because resistant strains of orga-nisms were not available. Table 1 gives the distributionof the species and also the number of strains subjectedto replicate testing. Organisms were maintained at-20°C, frozen in sterile, defibrinated sheep's blood.Before testing, a small portion of a frozen, bacterialsuspension was thawed and plated to a sheep bloodagar plate. This initial subculture was incubated over-night at 35 ± 2°C. Study subculture plates (SSPs) wereprepared by suspending one colony from the initialsubculture plate in 5 ml of sterile 0.85% saline; thissaline suspension served as the source of inoculaforthe requisite number of either blood agar plates forgram-positive bacteria or MacConkey agar plates forgram-negative bacteria. These SSPs were incubatedfor 18 to 22 h at 35 + 2°C.AST. Standard ASTs were performed by the meth-

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PROMPT INOCULATION SYSTEM 85

TABLE 1. Organisms testedTotal no. No. of strains

Organism of subjected tostrains replicate testing

Escherichia coli 20 5Klebsiella pneumoniae 10 5Providencia rettgeri 3 2Morganella morganii 4 2Proteus vulgaris 3 2Proteus mirabilis 12 5Providencia stuartii 5 2Serratia marcescens 5 2Enterobacter cloacae 3 2Enterobacter aerogenes 3 2Enterobacter agglomerans 2 1Pseudomonas aeruginosa 5 5Staphylococcus aureus 12 4Streptococcus faecalis 4 3Streptococcus faecium 2 2Streptococcus faecium var. 2 2durans

Streptococcus bovis 3 3Staphylococcus epidermidis 2 1

ods recommended by the National Committee forClinical Laboratory Standards (NCCLS) for disk diffu-sion (6) or broth microdilution (7) ASTs with theexception that the final diluents for the microdilutiontests were those recommended by Micro-Media Sys-tems, Inc.: 0.02% polysorbate 80 (P80) and 0.02%polysorbate 80 supplemented with the chloride salts ofcalcium and magnesium (CSP80). The Prompt testsdiffered from standard tests only in the preparationand handling of the inocula.Inoculum preparation and handling. A single SSP

served as the source of inocula for all parallel tests.Bacteria taken from three to five colonies were inocu-lated into 5 ml of soybean-casein digest broth for thestandard tests. The broth was incubated for 2 to 4 h at35 ± 2°C and then was diluted in sterile saline to matchthe recommended barium sulfate standard. The stan-dardized bacterial suspension was used to inoculate adisk diffusion AST; 0.5-ml portions of the same sus-pension were diluted 1:30 in P80 and in CSP80. Thesedilutions were used to inoculate the standard microdi-lution tests. All standard ASTs were set up within 15min of inoculum preparation.Prompt inocula were prepared using the Prompt

inoculation wand and one of four prepackaged sus-pending solutions or diluents. The diluent for the diskdiffusion ASTs was 1 ml of sterile 0.85% saline. Threediluents-P80, CSP80, and 0.85% saline-in 30-mlportions were tested in broth microdilution ASTs. Thewand was used to pick five colonies >1 mm indiameter or 10 colonies 0.5 to 1 mm in diameter froman SSP. The 2-mm tip of the wand served as a guide tocolony size. The wand, filled with bacteria, was placedinto a diluent vial, and the bacteria were released fromthe wand by vortexing (disk diffusion test inocula) or

shaking vigorously (microdilution test inocula). EachPrompt inoculum was used within 15 min of prepara-tion to set up the time zero (To) test. The wand-stopperwas used to recap the vial with its remaining inoculum,and the vial was held at room temperature for 2 h (T2).

The aged bacterial suspension was then mixed againand used to inoculate the T2 tests.

Colony counts. To determine the number of bacteriadelivered by the wand to the diluent, Prompt Toinocula were diluted, and the dilutions were used toprepare soybean-casein digest pour plates. The 1-mlsaline suspensions were diluted 10-'; triplicate 0.1-mlportions of the final dilution were used to inoculateeach of three pour plates. The 30-ml suspensions werediluted lo-4 and plated similarly. After overnightincubation of the pour plates, colonies were countedwith a Quebec or 3M colony counter. To assess thestability of the bacterial suspensions, the count proce-dure was repeated on the aged Prompt inocula (T2counts). T2 counts were not determined for the 30-mlsaline diluent.

Reproducibility study. One-half of the bacterial iso-lates-35 gram-negative strains and 15 gram-positivestrains-were subjected to replicate testing (Table 1).Both ASTs and counts were replicated. For any givenorganism replicate tests were set up on the same dayon the same lots of materials. Triplicate tests wereperformed for the 1-ml saline and 30-ml P80 and CSP80diluents, and duplicate tests were performed for the30-ml saline diluent. Parallel standard tests were alsoreplicated.

RESULTSThe overall mean, based upon 742 counts, for

the wand's pickup and delivery was 1.85 x 108CFU. The 95% confidence interval for an indi-vidual pick was 4.66 x 107 to 7.36 x 108 CFU, arange of 1.2 log1o. Data from five counts derivedfrom colonies of either Staphylococcus epider-midis or Klebsiella pneumoniae which had notreached the requisite minimum size (0.5 mm) atthe time of inoculum preparation have beenexcluded. Counts for the excluded data rangedfrom 1.0 x 106 to 1.4 x 107 CFU. Figure 1 showsthe mean wand delivery and 95% confidenceinterval for the various genera and related bacte-rial groups tested.The mean To and T2 inoculum densities for the

1-ml saline and 30-ml P80 and CSP80 diluentsappear in Fig. 2. In the saline diluent, countsremained essentially stable. In P80, there was asignificant tendency (paired Student's t test, P <0.001) for decreased counts at T2 with gram-negative organisms. The most extreme decreasein counts was seen with strains of Pseudomonasaeruginosa. T2 counts for these organismsdropped, on the average, to 47.3% of the startingvalue. The Proteus and Enterobacter-Serratiagroup counts dropped to approximately 65% ofthe starting value, and those for the Morganella-Providencia group dropped to approximately73%. Escherichia coli, Klebsiella pneumoniae,and the gram-positive organisms remained rela-tively unaffected by the P80 diluent. In thecation-supplemented system, the opposite effectwas noted. That is, counts increased significant-ly (P < 0.001). All organism groups except P.

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86 LUND AND HAWKINSON

1O10-

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(742) (4J hoW nd 0 (52) (114)

ELoN Ent_ Po-StsPotus Stsphyloccw(115) (101 (107) (9)

FIG. 1. Mean number and 95% confidence intervalfor viable microorganisms picked up by the Promptinoculation wand.

aeruginosa showed a significant increase in theT2 counts. The magnitude of the increase was,however, much smaller than that of the decreaseseen in the unsupplemented system. The aver-age increase in counts was only 12.7%.

Disk diffusion susceptibility results were cate-gorized as susceptible, intermediate, or resistantaccording to the published zone diameter breakpoints (8). Disagreements between paired stan-dard and Prompt results were scored in thetraditional manner into three categories of dis-crepancy: (i) minor, intermediate in one systemand either susceptible or resistant in the other;(ii) major, resistant by the Prompt system andsusceptible by the standard method; and (iii)very major, susceptible by the Prompt systemand resistant by the standard method. The datafor the To comparison are summarized in Table2, and those for the T2 comparison are summa-

rized in Table 3. The results were in total accordfor 96.54% of To paired tests and 95.57% of T2paired tests. At To only one major and no verymajor errors were observed in 2,396 paired tests(agreement plus minor discrepancies or essentialaccord, 99.96%). At T2 there were one verymajor and two major errors in 2,395 paired tests(essential accord, 99.87%). Only the combina-tion of penicillin and streptococci at To andpenicillin and streptococci and kanamycin andstreptococci at T2 showed significantly less than90% agreement. For those antimicrobic agent-organism combinations essential accordequalled 100%; i.e., all discrepancies betweenPrompt and standard tests were minor ones.

Figures 3 and 4 show, for the combined dilu-ent systems, the percentages of paired minimum

TABLE 2. Interpretive agreement between standard method and Prompt To disk diffusion susceptibility tests

Gram-negative species Staphylococcus spp. Streptococcus spp.Antimicrobial % % Majorb % % Major % % Major

agent Agree- Minor or very nd Agree- Minor or very n Agree- Minor or very nment majorc ment major ment major

Amikacin 99.3 0.7 0.0 145Ampicillin 97.2 2.8 0.0 145 100.0 0.0 0.0 24 100.0 0.0 0.0 31Carbenicillin 96.5 3.5 0.0 144Cefamandole 96.6 3.4 0.0 145Cefoxitin 98.6 1.4 0.0 145Cephalothin 97.9 2.1 0.0 145 100.0 0.0 0.0 24 96.8 3.2 0.0 31Chloramphenicol 94.4 4.9 0.7 144 100.0 0.0 0.0 24 100.0 0.0 0.0 31Clindamycin 100.0 0.0 0.0 24 100.0 0.0 0.0 31Erythromycin 95.8 4.2 0.0 24 93.5 6.5 0.0 31Gentamicin 100.0 0.0 0.0 145 100.0 0.0 0.0 24 100.0 0.0 0.0 31Kanamycin 91.7 8.3 0.0 24 83.9 16.1 0.0 31Nitrofurantoin 93.1 6.9 0.0 145Oxacillin 87.5 12.5 0.0 24 100.0 0.0 0.0 31Penicillin 100.0 0.0 0.0 24 77.4e 22.6 0.0 31Tetracycline 90.3 9.7 0.0 145 100.0 0.0 0.0 23 100.0 0.0 0.0 31TMSf 97.2 2.8 0.0 144 100.0 0.0 0.0 24 96.7 3.3 0.0 31Tobramycin 96.6 3.4 0.0 145Vancomycin 100.0 0.0 0.0 24 100.0 0.0 0.0 31

a A minor error is classified as intermediate by one system and either susceptible or resistant by the other.b A major error is classified as resistant by Prompt and susceptible by the standard method.c A very major error is classified as susceptible by Prompt and resistant by the standard method.d n, Number of paired tests.' Significantly less than 90%o agreement.f TMS, Trimethoprim-sulfamethoxazole.

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PROMPT INOCULATION SYSTEM 87

TABLE 3. Interpretive agreement between the standard method and Prompt T2 disk diffusion susceptibilitytestsa

Gram-negative species Staphylococcus spp. Streptococcus spp.Antimicrobial % % Major % % Major % % Majoragent Agree- Minor or very n Agree- Mm or very n Agree- M or very n

ment major ment or major ment Minor majorAmikacin 99.3 0.7 0.0 145Ampicillin 97.2 2.1 0.7 145 100.0 0.0 0.0 24 100.0 0.0 0.0 31Carbenicillin %.6 3.4 0.0 145Cefamandole 96.6 3.4 0.0 145Cefoxitin 97.2 2.8 0.0 145Cephalothin 99.3 0.7 0.0 144 100.0 0.0 0.0 24 80.8 19.4 0.0 31Chloramphenicol 89.6 9.7 0.7 144 100.0 0.0 0.0 24 100.0 0.0 0.0 31Clindamycin 95.8 0.0 4.2 24 100.0 0.0 0.0 31Erythromycin 95.8 4.2 0.0 24 100.0 0.0 0.0 30Gentamicin 97.9 2.1 0.0 145 100.0 0.0 0.0 24 100.0 0.0 0.0 31Kanamycin 91.7 8.3 0.0 24 74.2b 25.8 0.0 31Nitrofurantoin 91.7 8.3 0.0 145Oxacillin 91.7 8.3 0.0 24 100.0 0.0 0.0 31Penicillin 100.0 0.0 0.0 24 67.7b 32.3 0.0 31Tetracycline 91.0 9.0 0.0 145 100.0 0.0 0.0 23 96.8 3.2 0.0 31TMSC 97.9 2.1 0.0 144 100.0 0.0 0.0 24 80.0 20.0 0.0 31Tobramycin 97.9 2.1 0.0 145Vancomycin 100.0 0.0 0.0 24 100.0 0.0 0.0 31

a See footnotes a through d of Table 2 for an explanation of the headings.b Significantly less than 90%o agreement.c TMS, Trimethoprim-sulfamethoxazole.

inhibitory concentrations (MICs) which agreed was 98.2% at the one-dilution step level andwithin ±1 and ±2102 dilution steps. The overall 99.8% at the two-step level. The respectiveagreement between 7,074 pairs of MICs at To values for the agreement between the 7,046 pairs

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88 LUND AND HAWKINSON

Percent Agreementoi111co1 I It IoiiD i 1

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at T2 were 97.4 and 99.6%. These data includeagreement between systems for off-scale as wellas on-scale endpoints. At To there were 2,194MIC pairs with on-scale endpoints. Of thesepairs, 97.0% agreed within one dilution step and99.5% agreed within two dilution steps. At T2there were 2,126 on-scale paired MICs with94.4% (not significantly <95%) agreement at onedilution step and 99.2% agreement at two dilu-tion steps.MIC pairs showing significant disagreement-

i.e., paired results greater than 2 log2 dilutionsteps apart-were tested for significant skew-ness. Because so few tests (<1%) showed signif-icant disagreement, the data were pooled overtime (i) by Gram stain reaction of the organismswithin each Prompt diluent system and (ii) byeach of the organism groups over all diluentsystems. For the diluent systems, the T2 Promptresults for the P80 system showed significantbias toward low MICs with gram-negative orga-nisms. On the other hand, the staphylococci

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Carbenicillin

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Cephalothin

Chloramphenicol

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Nitrofurantoin

Tetracycline

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VOL. 18, 1983

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PROMPT INOCULATION SYSTEM 89

Percent Agreement

t1 t 1i SSD 5 80 aa @ sco co a -A C C." co cOn_ M11 X X

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showed a significant tendency for MICs higherthan the standard test in both Prompt To and T2tests.The data from the intrasystem reproducibility

studies appear in Tables 4 and 5. For the diskdiffusion susceptibility test (Table 4), each sys-tem showed, overall, >97% of replicate tests intotal accord and >99.7% in essential accord. Nosignificant difference in reproducibility was de-tected between systems. Similarly, no differencein reproducibility was detected between systemsin the broth microdilution test (Table 5) at the 1and 2 log2 dilution step levels. The Prompt Totest, however, gave the same MIC significantlymore times than did the standard test (P < 0.05).Nevertheless, all test systems showed excellentintrasystem reproducibility, with >98% of repli-cate tests falling within one dilution step of eachother.

DISCUSSIONAs Barry points out (2), the density of the

inoculum in any AST is "extremely critical" and"must be controlled in order to obtain reliableresults." The importance of the organism'sphase of growth has been less certain. Thestandard test procedures of the NCCLS give asthe growth phase of choice the log phase. Sta-tionary-phase cultures of rapidly growing bacte-ria and direct suspension offastidious organismstaken from an overnight agar culture are recog-nized as acceptable alternative sources for stan-dardized inocula in the agar disk diffusion test(6). Recently, D'Amato and Hochstein (3) re-ported the equivalence of the direct suspensionmethod to the traditional preparation of a stan-dardized suspension of organisms in the logphase of growth in disk diffusion ASTs. Thework of Baker and Thornsberry (1) confirms this

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90 LUND AND HAWKINSON

TABLE 4. Intrasystem reproducibility for diskdiffusion susceptibility testsa

Total No. (%)Organism Test system no. of No. (%) major orgroup

T compari- minor verysons majorb

Gram NCCLS 1,258 26 (2.07) 1 (0.08)negative

Prompt ToC 1,260 30 (2.38) 0 (0)Prompt T2d 1,260 24 (1.91) 2 (0.16)

Gram NCCLS 538 18 (3.35) 0 (0)positive

Prompt To 540 12 (2.22) 0 (0)Prompt T2 538 22 (4.09) 2 (0.37)e

Totals NCCLS 1,796 44 (2.45) 1 (0.06)Prompt To 1,800 42 (2.33) 0 (0)Prompt T2 1,798 46 (2.56) 4 (0.22)

a See footnotes a through c of Table 2 for anexplanation of the headings.

b The usual distinction between major and verymajor errors cannot be made in an intrasystem com-parison.

c Prompt tests set up within 15 min of inoculumpreparation.

d Prompt tests set up with an inoculum aged for 2 hat room temperature.

e Errors are traceable to the appearance of resistantvariants in one in a set of triplicate tests for S. aureusand clindamycin.

observation and extends the successful applica-tion of direct suspension to the microdilutionprocedure.The current study reconfirms the efficacy of

direct suspension of organisms from an agarplate culture as a method of preparing standard-ized inocula for either disk diffusion or microdi-lution susceptibility tests. Furthermore, the T2data indicate that if such a suspension is pre-pared in a suitable nonnutritive diluent, one is no

longer constrained to use the standardized in-oculum within 15 min of preparation. That is notto say that the bacteria may be allowed toremain in the starvation environment indefinite-ly. How long the inoculum will remain suitablefor susceptibility testing is both organism anddiluent dependent. For example, dilute, aqueousP80 is obviously a bad environment for bacteria.Although the T2 Prompt microdilution tests em-ploying this diluent still give satisfactory results,the inimical effect of the diluent is apparent inthe trends toward decreased counts and signifi-cantly lower MICs for the gram-negative orga-nisms. The effect of this nonionic surfactant,particularly upon gram-negative organisms, isnot surprising; one would expect the hydropho-bic portion of the polysorbate molecule to inter-act with and disrupt the lipopolysaccharides andlipoproteins of the gram-negative cell wall. Itwould appear that 2 h represents the outer limitof exposure to the dilute P80 environment forsusceptibility test inocula, and shorter exposuretimes are undoubtedly preferable. For suscepti-bility test systems requiring the absence of cat-ions in the diluent, saline proved to be superiorto P80, especially over prolonged periods oftime. No trend in either change of inoculumdensity or skewness in MIC results developedfor inocula held for 2 h in saline. Thus, the outerlimit of exposure time to the saline diluent hasnot been defined, nor has this limit been definedfor the cation-supplemented P80 system. Thetrend toward increased counts over time in theCSP80 illustrates the sparing effect of divalentcations, especially Mg2+, on bacteria in a starva-tion environment (4). How long the bacteria canmaintain the vigor exhibited in the supplementeddiluent is uncertain.The trend for significantly higher MICs with

the Prompt and staphylococci is attributable tothe inoculum effect rather than to the phase ofgrowth or the diluent. The significant disagree-

TABLE 5. Intrasystem reproducibility for broth microdilution susceptibility testsCumulative % agreement between tests at the

Organism group Test comparisons following log2 dilution steps:0 1 2 >2

Gram negative Standard 2,555 91.04 98.83 99.92 100Prompt To 2,555 92.72 99.14 99.92 100Prompt T2 2,541 91.66 98.86 99.80 100

Gram positive Standard 693 90.04 98.27 99.57 100Prompt To 693 91.34 98.70 100Prompt T2 691 92.62 99.13 99.57 100

Overall totals Standard 3,248 90.83 98.71 99.85 100Prompt To 3,248 92.43 99.05 99.94 100Prompt T2 3,232 91.86 98.92 99.75 100

a See footnotes c and d of Table 4.

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PROMPT INOCULATION SYSTEM 91

ments between standard and Prompt tests oc-curred with penicillin or ampicillin and beta-lactamase-producing strains of S. aureus. Tiltonet al. (9) note that these particular antimicrobicagent-organism combinations are especially sub-ject to inoculum effect, but that reasonable re-producibility of MICs may be expected if theinoculum density is maintained between 105 to106 CFU/ml in the susceptibility test itself.When the contents of the growth control well inthe microdilution tray were quantitatively cul-tured, the inoculum density in the standard testswas found to range from 2.4 x 104 to 6.7 x 104CFU/ml for the staphylococci. Correspondingdensities for the Prompt tests were 2.8 x 105 to6.1 x 105 CFU/ml. The tendency for conven-tionally standardized suspensions of staphylo-cocci to err on the low side of the ideal densitymay be noted in other studies (3, 5).The excellent agreement (>95%) between

Prompt and standard tests reflects the perform-ance of the inoculation wand. The pickup anddelivery of the wand were quite consistent. Oneoften hears that a bacterial suspension matchinga 0.5 McFarland turbidity standard will containbetween 5 x 107 to S x 108 (1.6 x 1080 - 0.5log10) CFU/ml. Although this may represent theideal situation, little if any data have been pub-lished which support this claim. If, for example,one determines the 95% confidence interval forthe 24 counts reported by Matsen et al. (5), onefinds the span of the interval to be 1.4 log10 whena variety of organisms are tested. In this light,the 1.2 log10 span for the wand's delivery with alarge variety of organisms seems very respect-able.To have the wand perform properly, it is

important to follow the simple strictures in thedirections for use. Data in this study, the exclud-ed counts, indicate that when colonies are lessthan 0.5 mm in diameter, the process of touching10 colonies will not fill the wand. Baker andThornsberry (1) found it necessary to touch upto 30 colonies to fill the wand with some of thefastidious organisms they tested. The report ofWicks et al. (10) indicates that touching fivecolonies is sufficient to fill the wand when colo-nies are larger than 1 mm in diameter. It is alsoimportant that organisms be taken from fresh(<24-h) plate cultures to assure that viable orga-nisms are selected and to prevent the occurrenceof an unduly long lag phase in the susceptibilitytest. The wand may be depressed to the surfaceof the agar, but it must not penetrate the surface.The mixing procedure must be vigorous becausegentle inversion of the vial will not release theorganisms from the wand's tip. The foaming thatoccurs upon shaking the surfactant-containingdiluents will not interfere with susceptibilitytests because the bubbles formed tend to be

trapped in the bottle when the inoculum isdispensed.

If one follows the directions outlined above,the wand will pick up and deliver a consistentnumber of bacteria, 1.85 X 108°0 - 0.6 loglo CFU.The desired density of the inoculum to be pre-pared is controlled by the amount of diluent intowhich the wand is placed. Within practical lim-its, it is possible to package diluents to providestandardized suspensions of bacteria over arange of concentrations while saving time andeliminating the tedium of manual turbidity ad-justment. The nature of the diluent may bedictated by the test procedure in which theinoculum is to be used. The 1-ml system de-scribed in this study provides inocula functional-ly equivalent to those matching a 0.5 McFarlandturbidity standard and gives reliable results(>99%o essential accord with standard tests) inagar disk diffusion susceptibility tests. The 30-mlsystems described here, with >95% pairedMICs within ±1 log2 dilution of the standardtest, provide inocula appropriate for those brothmicrodilution systems with inoculator pegs de-livering approximately 5 ,ul per well.

ACKNOWLEDGMENTSWe are especially grateful to J. M. Kelly and J. H. Wicks

for their technical assistance.LITERATURE CITED

1. Baker, C. N., and C. Thornsberry. 1983. Inoculum stan-dardization in antimicrobial susceptibility testing: evalua-tion of overnight agar cultures and the rapid inoculumstandardization system. J. Clin. Microbiol. 17:450-457.

2. Barry, A. L. 1976. The antimicrobic susceptibility test:principles and practice. Lea & Febiger, Philadelphia, Pa.

3. D'Amato, R. F., and L. HochsteLn. 1982. Evaluation of arapid inoculum preparation method for agar disk diffusionsusceptibility testing. J. Clin. Microbiol. 15:282-285.

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VOL. 18, 1983

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