JOURNAL OF CLINICAL MICROBIOIOGY. Aug. 1989. p. 1814-18250095-1137/89/081814-12$02.00/0Copyright © 1989, American Society for Microbiology

New Datum Handling Methods for the Quality Control of AntibioticSolutions and Plates Used in the Antimicrobial Susceptibility Test

S. N. McDERMOTT"' AND T. F. HARTLEY2t

Division of Clinli(c(al Microbiologvt aid Division of Clinii(c(al Che/wmistîv, 2 IîstithtUe of Medic al and Veterinary Science,A delaide, Sout/h A IIstra/lil 5000, AiustraliaReceived 27 JuIy 1988/Accepted 17 March 1989

Quality control programs are described for monitoring both the antibiotic solutions and the plates used in thedilution antimicrobial susceptibility test. For the quality control of antibiotic solutions, a simple comparativedisk diffusion assay is used in which the data are expressed as zone ratios calculated from the following formula:zone ratios = mean zones of inhibition for disks impregnated with antibiotic under test/mean zones of inhibitionof previously prepared quality control disks impregnated with the same antibiotic. The rejection limits set forthis method are twice as stringent as for MIC estimation, and its precision is four times greater. For qualitycontrol of the antibiotic agar plates, fluid was absorbed directly into filter paper disks placed on the surface ofthe medium. These disks were then transferred to a seeded plate to produce zones of inhibition. These zones

of inhibition were then converted to zone ratios by dividing the zone of inhibition for the individual antibioticsby the mean of the total zones of inhibition for all the antibiotics tested on the same plate. We furthernormalized these data by expressing each entry as a standard normal deviate calculated from the mean andstandard deviation of the cumulative data for each individual antibiotic. The use of zone ratios reducedbatch-to-batch variation, and standard normal deviate values gave a uniform result for convenience. Themethod has a sensitivity equal to that of an MIC estimation if it were possible to estimate the MICs of antibioticsat these low levels.

Antibiotic solutions. Laboratories which perform the agar

dilution antimicrobial susceptibility test require and muststore al the standard antibiotic powders used in these tests.Most of these powders are unstable and have stated expiry

dates based on a 10% loss of potency under ideal storageconditions. Because it is impractical for laboratories per-forming routine testing to accurately assay these powders,they are obliged to rely on the information on stated potencyand expiry date provided by the manufacturers. Stock solu-tions are prepared from these powders and may be stored bythe laboratory. We believe that it is essential to performquality control (QC) testing of these solutions and havedeveloped a method for monitoring the concentration of theantibiotic in these solutions to ensure that reliable andconsistent results are obtained when pouring antibiotic-containing plates from these solutions or using them in MICestimations.

In one of the methods currently available to perform QCtesting of these solutions, the broth dilution method recom-

mended by the National Committee for Clinical LaboratoryStandards (9), stock solutions which may have an antibioticcontent of between 50 and 200% of the desired target are

regarded as acceptable. A further disadvantage is that thetest does not provide quantitative data on antibiotic concen-

trations, since the results are expressed relative to an

arbitrarily selected dilution series. Antibiotic assays in

which diffusion methods are used are complex and time-consuming to perform, because fresh standards must beprepared to obtain calibration line data needed to calculatethe concentration of the antibiotics. This method, however.has the advantage of compensating for interassay fluctua-tions.

* Corresponding author.t Present address: Clinical Chemistry Department. Royal Hobart

Hospital, Hobart. Tasmania 7000. Australia.

As an alternative, Cooper and Linton (2, 3) have shownthat a single disk containing a known concentration ofantibiotic can be used to calculate the critical concentrationof the antibiotic required to inhibit the growth of an organismunder controlled conditions. We have extended this conceptand developed a method to perform QC testing of antibioticsolutions by using single-strength assay standard disks thatwe refer to as quality control disks (QCD). These diskscontain a lower antibiotic concentration than commerciallyavailable disks, which produce too large a zone of inhibition(DZI) and consequently lack sensitivity. The results of testsare compared with those of the QCDs and expressed as azone ratio (ZR) to compensate for interassay fluctuations.

TABLE 1. Data used to illustrate the ZR approach to the QCof antibiotic susceptibility test solutions

DZI l'ordiskDZfoPlate no. impregnated with QCD (mm) ZR

test solution(mmQ)

1 14.6 12.6 1.162) 14.' 12.0 1.183 14.2 12.1 1.174 14.2 13.9 1.025 14.8 12.7 1.176 15.0 13.0 1.157 14.1 12.0 1.188 14.8 12.8 1.169 14.8 12.7 1.1710 16.2 14.4 1.131i 15.3 13.4 1.1412 14.6 12.6 1.1613 15.1 13.1 1.1514 16.0 14.1 1.13

Mean 14.9 12.9 1.148StD 0.644 0.758 0.0402CVY 4.3 5.9 3.5

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QUALITY CONTROL IN ANTIMICROBIAL SUSCEPTIBILITY TEST 1815

TABLE 2. Summary of the data-processing procedure illustrated with reference to a typical data set

Day DZI (mm) for following Man DZI ZR for following antibiotic: SND for following antibiotic:(mm)A B C D A B C D A B C D

Mon 18 23 31 35 26.8 0.67 0.86 1.16 1.31 0.77 -1.20 1.61 1.00Tues 16 22 27 33 24.5 0.65 0.9( 1.10 1.35 -0.77 0.40 -0.32 0.33Wed 15 20 25 30 22.5 0.67 0.89 1.11 1.33 0.77 O O -0.33Thurs 13 18 22 28 20.3 0.64 0.89 1.08 1.38 -1.54 O -0.97 1.33Fri 12 17 20 24 18.3 0.66 0.93 1.09 1.31 0 1.60 -0.65 -1.00

Mean 0.66 0.89 1.11 1.34SD 0.013 0.025 0.031 0.030

Antibiotic plates. The second procedure in the agar dilu-tion antimicrobial susceptibility test which requires QCtesting is the preparation of the antibiotic plates containing a

wide range of antibiotics at a variety of concentrations. Mostlaboratories rely on the use of QC organisms for which theantibiotic MICs are known (5, 9), but these will detect onlygross errors of preparation. Franklin (6) proposed that QCtesting of antibiotic plates be carried out by producing DZIsfrom cores of agar cut out of the plates; acceptance andrejection limits were set at +1 and ±+ mm of the target DZI,respectively. Unfortunately this method mutilates the plates,and no data were given to support its ability to detect errors.We have developed a method in which filter paper disks

were used to absorb antibiotic solution from the platesurface, thus avoiding mutilation of the plate. The panel ofantibiotic disks containing antibiotic solutions from all thebilayered plates used in the agar dilution antimicrobialsusceptibility test were then tested on a single plate toproduce DZIs. The individual DZIs were then divided by themean DZI of all the DZIs on the bilayered plate to convertthem to ZRs, which compensate for interassay fluctuations.The single-layered plates were treated in a similar manner,but the data were processed separately. From these data.95% confidence limits were established for the test devia-tions, and any fluctuations greater than these were consid-ered abnormal and were a warning to repeat the test. If a

particular antibiotic plate failed the test twice, that batch ofplates was discarded. To simplify the interpretation of theresult from an individual plate, we converted all the resultsto standard normal deviate (SND) values. We have demon-

TABLE 3. Antibiotics used to establish the analyticalsensitivity of the plate method

Use of antibiotic at following concn (,ug/ml)":Antibiotic

0.5 1 4 8 16 32 64 128 256 512

Amoxicillin x x x x x x

Ampicillin x x x x x x

Imipenem x x x x x x

Cephalothin x x x x x x

Azlocillin x x x x x

Cefoxitin (x) (x) x x x

Gentamicin x x x

Tobramycin (x) (X) x X X

Tetracycline x x x x x

Nalidixic acid X X X

Norfloxacin X x xSulfadiazine (X) x x

Trimethoprim (x) x x

U x. The antibiotic was used at this concentration. (x), These platesproduced no DZIs or inconsistent smail DZIs.

strated that this method can clearly differentiate populationswhich have a one-dilution difference in concentration.

MATERIALS AND METHODS

Theoretical considerations and experimental design. (i) An-tibiotic stock solution testing. Our approach is based on thetheory that when a test concentration equals the QCDconcentration, the ZR must be 1.0. In practice, the ratiofluctuates in the vicinity of 1.0. We proposed that byexpressing the results for our test solutions as a normalizedZR, we would reduce the effects of interassay fluctuationsand make it possible to perform a quantitative assay by usingsingle-strength assay standards. The process of the calcula-tions is illustrated in Table 1 for a representative data set,and the reduction in the coefficient of variation (CV%) forthe data expressed as the ZR can be appreciated.

(ài) Antibiotic plate testing. The design of the part of thestudy devoted to antibiotic plate testing was based on thehypothesis that interassay fluctuations in the DZIs are bothrandom within a test and proportional to each other, despitebeing produced by different antibiotics. We proposed that byexpressing individual DZIs as a ZR to the mean DZI for allthe antibiotics tested on a single plate on the same day, wewould reduce the obscuring effect of random intratest fluc-tuation on trends in the cumulative records of data for testson different batches.

Representation of data was further simplified by express-ing al the ZRs in the tables of cumulative results for the

TABLE 4. Concentrations of antibiotics used in routine tests

Use of antibiotic at followingType of plate Antibiotic concn (Lg/ml):

1 2 4 16 32 64 256

Double-layered Amoxicillin x xAmoxicillin- x x

clavulanic acidCephalothin x xImipenem X XNorfloxacin XGentamicin xTobramycin xAziocillin x XImipenem X xNorfloxacin x

Single-layered Tetracycline x xCefoxitin x xTrimethoprim xSulfadiazine xNalidixic acid x

x . The antibiotics \as tised at this concentration.

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1816 McDERMOTT AND HARTLEY

TABLE 5. Antibiotic concentrations in the stock solutionsused to impregnate the QCD

Stock solutionAntibiotic

Concn (mg/ml) p.g/disk

Amoxicillin 1iCephalothin' 1 0.2Tetracycline 1Gentamicin iTobramycin 1Nalidixic acid 3 6Sulfadiazine 16 32Trimethoprim 0.4 0.8Cefoxitin 2 4Norfloxacin 1 2Azlocillin 8 16Imipenem" 1 0.2

' These stock solutions were diluted 1/10 before 2 (1. was pipetted onto eachdisk.

antibiotics as SND units (1). This process of data manipula-tion is illustrated in Table 2 for a hypothetical panel of fourtypical antibiotics, A to D, studied daily for 5 days.The first data set in Table 2 shows the cumulative DZI data

in millimeters. Visual inspection and assessment of thispanel can only be subjective, and this assessment is compli-cated by the wide range in DZIs (12 to 35 mm) encounteredin this selection of four antibiotics. The general observationthat all DZIs appear to be decreasing in size is probably theonly comment that can be made. The ZR data are probablyeasier for most observers to interpret, since the target ZRs(as shown by the column means) are 0.66, 0.89, 1.11, and1.34. However, even the data in this panel cover a widedynamic range (0.64 to 1.38), and this is the principaljustification for calculating the SNDs. All the SND data,regardless of their column, can be subjected to the same

assessment based upon the common statistical acceptancecriterion of mean + 1.97 standard deviations (P < 0.05). Thetarget mean for an SND is zero by definition. From this setof SNDs, it is obvious that the readings for antibiotic C on

Monday and antibiotic B on Friday were the most discrep-ant. In retrospect, it would have been difficult to support thisconclusion from the DZI data alone. An additional advan-tage of the SND data is that they have a statistical meaning,

and a table of "Z values" (1) can be consulted to assignactual statistical probabilities to all the results, e.g., SND =

1.60, P = 0.890. In practice, a method which rejects resultsoutside the 95% confidence limits will reject 1 in 20 sets of

co

E4-.

cn

a4._E

c

oN

40

20

O-

20-

40i

20-

O-

Cephalothin

y=32.6+ 4.84 log x

r = 0.998

I i I 10 1.6 3.2

Sulphadiazifie

y = 7.03 + 3.95 log x

Ir = 0.997

O

Tetracycline

128 256

y = 21.5 + 4.89 log x

r = 0.989

O 10 20

Amount of Antibiotic ug/discFIG. 1. Wide-range calibration curves for cephalothin, sulfadia-

zine. and tetracycline in the disk diffusion method.

plates even though the antibiotic concentration may beacceptable. To overcome this problem, we reject onlybatches of plates which are outside the 95% confidence limitswhen retested.A weakness in this approach is that it requires a number of

plate batches to be tested before the statistics of calculating

TABLE 6. Evaluation of the variability of the DZI measurement versus the ZR measurement for Il antibiotic stock solutions

QCD DZI Stock solution DZI ZR CV% fromAntibiotic No. of calculation

tests Mean CV(Z( Mean CV7 Mean CV%; Cof ZR only

(mm) (mm)

Amoxicillin 29 23.480 7.971 23.598 8.166 1.003 1.30(2 0.195Cephalothin 27 25.434 7.238 25.248 7.077 0.993 2.043 0.161Tetracycline 30 23.805 6.416 23.152 7.268 0.971 2.840 0.852Gentamicin 45 20.392 6.919 20).569 6.675 1.007 1.939 0.244Tobramycin 22 20.741 4.987 20.863 5.052 1.(11 1.749 0.065Nalidixic acid 20) 17.709 8.787 18.108 7.562 1.024 2.423 1.225Sulfiadiazine 25 18.938 16.519 17.674 18.138 0.930 4.673 1.619Trimethoprim 24 21.607 10.316 2'0.834 10.196 0.965 2.318 0.12Cefoxitin 26 24.084 8.241 24.344 7.629 1.()10 2.193 0.612Norfloxacin 24 24.649 3.371 24.732 3.704 1.005 2.352 0.333Azlocillin 20 20.0)83 8.796 20).012 9.478 0.998 1.573 0.682

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QUALITY CONTROL IN ANTIMICROBIAL SUSCEPTIBILITY TEST

TABLE 7. Calibration curves for the 11 antibiotic stock solutions expressed as diameters of the DZI and as ZRs versus concentrations

QCD concn Data S ZR for following concn:

Antibiotic (gdk) sSope interceptA 31 1411

Amoxicillin 2 A 3.212 23.44 0.9781B 2.833C 0.1219

21.730.9113

0.96180.9696 0.911 0.961 1.023 1.045

A 4.665B 4.097C 0.1735

A 3.826B 4.391C 0.1749

A 2.825B 2.675C 0.1'99

A 3.019B 3.157C 0.1558

A 3.984B 4.443C 0.2248

A 7.533B 4.289C 4.652D 3.693E 0.2614

A 4.487B 4.714C 0.2286

A 5.124B 5.159C 0.2048

A 5.218B 5.279C 0.2070

A 3.088B 3.029C 0.1487

33.2331.521.282

21.4920.020.8824

19.8319.000.9148

16.3119.330.8951

12.1810.050.5895

-11.326.8777.240

11.180.0852

21.0421.691.060

18.3417.470.7151

21.7721.460.8546

17.1717.190.8343

0.97890.97360.9748

0.97150.98970.9763

0.98210.95770.9675

0.97510.97640.9736

(.95590.97950.9651

0.97410.97300.98190.96620.8508

0.97950.94560.9598

0.98550.98730.9817

0.99670.9887(.9891

0.96040.96210.9381

(.883 0.953 1.042 1.073

0.882 0.953 1.042 1.075

0.914 0.967 1.034 1.057

0.895 0.958 1.038 1.066

0.851 0.942 1.057

0.810 0.916 1.049

0.851 0.944 1.060

0.8X57 0.940 1.045

0.854 0.938 1.044

0.904 0.964 1.040

1.097

1.097

1.102

1.082

1.082

1.067

' r. Correlation coefficient for the logarithmic equation v = a + blog, V. For the number of datum points, sec the text.

the column mean and column standard deviations of the ZRsbecomes reliable; 10 batches is the minimum number we

were prepared to consider. Unfortunately, it is impractical inroutine work to wait for many months to accumulate thesebase-line data, and we compromised by setting up 10 base-line plates over a 2-week period from the same productionbatch of plates. This compromise provided us with data setswhich actually underestimate the true batch-to-batch varia-

tion. However, experience with this QC system has provedthat this is an advantage, because it raises the sensitivity ofthe approach, particularly when there are gradual drifts in

QC results. In this situation, no action is usually taken. andthe ZRs and SNDs slowly adjust to new plateau values. If.however, the base-line data are always included. the QCprocedure becomes less prone to this problem. We haveincorporated the following rules into our computer program

so that the base-line data are always included: (i) if the batchof 10 plates for base-line data plus the total number of plates.

NP, assayed to date is less than 20, all the data i.e., 10 + NP,are used; (ii) if NP is greater than 20, the base-line data plusdata from the last 10 plates assayed are used. In this way, we

ensure that the latest data are always compared with thebase-line data set. Having defined the statistical basis of themethod, we proceeded to design an experiment to determineits analytical sensitivity. We needed to determine what thestatistically significant deviations, if any, would mean in

terms of antibiotic concentrations in those plates.Our work on the antibiotic solutions reported in this paper

demonstrated that large DZIs were usually associated withpoor analytical sensitivity, and we assumed that the same

restriction would apply to this part of the study. Single platesof each antibiotic and concentration were prepared (Table3). The range of antibiotic concentrations was selected toenable us to compare results for one dilution above andbelow the routinely used concentrations. Since the antibioticconcentrations on numerous plates were tested, it was

Cephalothin

Tetracycline

Gentamicin

Tobramycin

Nalidixic acid

Sulfadiazine

Trimethoprim

Cefoxitin

Norfloxacin

Azlocillin

0.2

6.4

32

0.8

4

3.2

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1818 McDERMOTT AND HARTLEY

Tetracycline

30-

20-

1

1.1

0.9-

0.7-.

Sulphadiazine

(B)

30

I t.-ttt)

1 t

3 435 45

0.5 -

3 15 25

Amount of Antibiotic, ug/discFIG. 2. Narrow-range calibration curves for tetracycline and sulfadiazine expressed as both diameters of the DZI of two separate test runs

(A and B) and ZR (C and E). The equations for the lines of best fit are given in Table 6.

necessary to use three seeded test plates for the completerange of tests. The same plate containing each concentrationof antibiotic was tested 10 times over a 2-week period. TheDZIs of the routinely used antibiotic concentrations, to-gether with the one dilution above and below were dividedby the mean of the routinely used concentration DZIs toproduce ZRs and standard deviations. A 2-standard-devi-ation bar chart was constructed from these results andexamined for possible overlap of the mean + 2 standarddeviations for the routine concentration with those for onedilution above and below this concentration. Overlappingwould have been interpreted as evidence of poor analyticalsensitivity.The base-line data for the routine prospective quality

control of agar plates were established as follows. Bottlescontaining 200 ml of agar were prepared to include thecomplete range of antibiotics and concentrations shown inTable 4. Ten 9-cm agar plates were prepared from each ofthese bottles. The plates were stored at 4°C in plastic

screw-top containers. Each of these plates was tested priorto being used for routine antibiotic susceptibility testing.The QC of routinely used antibiotic plates was performed

by testing one plate from each batch of 10 plates containingthe antibiotics and concentrations shown in Table 4. Thedata obtained from both the routine QC and the base-linedata testing were processed as outlined in Table 2.

Materials and organisms used. Antibiotic assay filter disks(diameter, 6 mm) AA grade filter paper; Whatman, Maid-stone, England) were chosen for the QC method after studiesshowed these disks to have a high uptake of fluid whenplaced on the surface of agar plates. The mean weight of thefluid absorbed was 24.14 p.g, with a 95% confidence limit of1.5756 p.g; subsequent QC testing of new batches of diskshas given good reproducibility of these data. The balanceused to obtain these results was an H1OW (Mettler, Zurich,Switzerland), which gave 95% confidence limits of 0.00092 gwith a QC weight of 0.5586 g.

Ultra-micro Samplers (Oxford Instruments, St. Louis,

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QUALITY CONTROL IN ANTIMICROBIAL SUSCEPTIBILITY TEST 1819

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10 A A

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1S S A16 S A17 S A18 S A19 S A

20 C A21 C A22 C A23 C A

24 C A25 C A26 C A21 C A28 C A29 C A30 C A

31 C A32 C A33 C A34 C A35 C A36 C A

37 C A

38 C A39 C A

40 C A320c A

41 C S42 C S

FIG. 3. QC chart for gentamicin QCD ratio measurements.Batch dates: batch 1, 3 February 1987; batch 42, 13 October 1987.Stock solution dates: solution A, 28 August 1986; solution B. 21January 1987; solution C, 10 April 1987. QCD dates: QCD A. 20January 1987; QCD B, 7 August 1987.

Mo.) delivering 2 ,ul with a specified accuracy of 3% and a

reproducibility of 0.04 p.l were chosen to load the QCDs withantibiotic solution. This volume was selected to avoid dilut-ing stock solutions. Davis et al. (4) considered this the lowestpractical volume usable for antibiotic disk assays.The indicator organism chosen for the assays was a

Bacilluis species (IMVS FD0101) which has been used inantibiotic assays for many years at the Institute of Medicaland Veterinary Science. This organism was used as a sporesuspension in distilled water with a viable count adjusted to108 CFU by the plate count method of Miles and Misra (8)and is stable for many years at 4°C. This organism does notform P-lactamase and gives DZIs which lie in the area ofoptimal sensitivity of the calibration curves used in thispaper.

Large-diameter polystyrene petri dishes (15 cm) and Iso-

e e> mn

* o o °o ~o y-P..................................

j SIl................... j.....................................

'o

C)OoeO wN0.980 1.995 2.911 3.964 4.964 5.897 6.972 7.979 8.957 9.968 10.974 11.991 12.953 13.958 14.960 15.989 16.956 17.956 18.935 19.981 20.925 21.980 22.984 231.008 241.010 251.004 26.983 27.966 281.005 291.004 30

ee

oda

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A) A

At aB A

*0

B 0A AA AA AB A8 A8 AC AS AC AS AS AC AC AC AC AC AC AC AC AC AC AC AC AC A0 AD AO AO Ao AO A

FIG. 4. QC chart for tetracycline QCD ratio measurements.Batch dates: batch 1. 9 December 1986: batch 30. 13 October 1987.Stock solution dates: solution A. 4 November 1986: solution B. 27January 1987; solution C. 26 February 1987; solution D. 18 August1987. QCD A date, 10 January 1985.

Sensitest agar (CM 471; Oxoid, Basingstoke, England)stored in 20-ml aliquots was used throughout this trial,unless otherwise stipulated.

Preparation of bilayered assay plates. The 15-cm bilayeredassay plates were used to assay both antibiotic solutions andantibiotic dilution plates. The bottoms of all the plates usedwere checked for flatness with a straight edge, and pouringwas done on a preleveled surface. A base layer of 20 ml ofIso-Sensitest agar was poured and allowed to set before theseeded layer was poured onto this surface. The seeded layercontained 20 ml of Iso-Sensitest agar which had been cooledto 50°C before addition of 0.1 ml of the Bacillus sporesuspension. After pouring, the plates were left at roomtemperature with their lids off so that the surface dried, andthey were used within 1 h of being prepared to avoidpremature growth of the indicator organism in relation to thediffusion of antibiotic from the disks tested.

Preparation of QCDs. Fifty filter disks were placed acrossthe bottom of a sterile disposable petri dish (diameter, 9 cm),and 2 ,ul of the appropriate antibiotic was pipetted into thecenter of each disk. They were dried for 1 h at 37°C beforebeing placed in plastic containers holding a layer of silica geldesiccant. The lids of the containers were sealed withimbedding wax before they were placed in long-term storageat -20°C. The concentrations of the stock solutions and the

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1820 McDERMOTT AND HARTLEY

eow

*0O O0Co~~~~~~~~N lo O

0 ~~~~~~~~~~

~O O ~ N 000....................................... ...................I S I .941 1 A A1 0 I 1.021 2 A AI S I .955 3 A AI Il .983 4 B A

I S I .945 5 AAI S .958 6 B AI I I .961 7 8 AI O .964 e 8 AI I I .941 9 C AI .967 10 C A

I I I .890 il C Ao .917 12 C A

* I I .803 13 C Ai .923 14 C A

10 .923 1S C AS I I .892 16 C A

I I I .878 17 C ASI I .910 18 C A

S I I .892 19 C A* .915 20 C AI S I .950 21 C AS i .915 22 C Ag i I .940 23 C A

Il .904 24 C AS g .916 25 C A

....................................... ...................

FIG. 5. QC chart for sulfadiazine QCD ratio measurements.Batch dates: batch 1, 10 December 1986; batch 25. 13 October 1987.Stock solution dates: solution A. 10 April 1986; solution B. 11December 1986; solution C. 23 March 1987. QCD A date, 10October 1985.

amounts of antibiotic used on each QCD are shown in Table5.

Routine testing method for antibiotic solutions. Two antibi-otic solutions were tested on each 15-cm bilayered assayplate. Each antibiotic and its QCDs were tested in quadru-plicate. Test disks were placed adjacent to the appropriateQCDs, with no attempt being made to achieve randomdistribution. Templates were used to facilitate the work sothat the disks could be rapidly positioned no closer than 3 cmto each other and to reduce the prediffusion time betweendisks. Our standard procedure was to place the four QCDsfor the first antibiotic on the plate, position the four testdisks, and then pipette 2 p.l of the appropriate antibiotic ontothe center of each of the test disks. This procedure was thenrepeated for the second antibiotic solution. The petri disheswere incubated at 37°C for 18 h with a spacer between thedish and the lid to allow sufficient ventilation to encourage aluxuriant growth of the indicator organism. For reading, thepetri dishes were placed bottom uppermost over an incidentlight source. DZIs were measured with vernier calipers andrecorded to the nearest 0.1 mm. ZRs for antibiotic stocksolution testing were calculated as follows: ZR = mean DZIfor four test disks/mean DZI for four QCDs. Data are validonly when the tests and QCDs are performed on the sameplate.

Routine testing of plates. A filter disk (diameter, 6 mm) wasplaced close to the edge of the agar on each plate to be testedand left to absorb fluid for a minimum of 30 min before beingtransferred aseptically to an appropriately seeded plate.

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FIG. 6. QC chart for triniethoprim QCD ratio rneasurerments.Batch dates: batch 1. 1 December 1986; batch 32. 9 November 1987.Stock solution dates: solution A. 25 November 1986; solution B, 23March 1987.

These seeded plates were incubated and read as above.Disks from up to 20 different antibiotic plates can be sub-jected to QC testing on a single 15-cm plate. The range testedwill be dictated by the normal range used and reported by thelaboratory, but a minimum of five antibiotic plates should beused. The full range of antibiotics in use must be tested oneach run for the results to be valid. If the laboratory changesa single antibiotic in its range, a new data base must becompiled. The ranges used in our laboratory are shown inTable 5. ZRs for antibiotic plate testing were calculated asfollows: ZR = DZI of test disk/mean of all DZIs on the testplate. Data are valid only if all measurements are performedon a single plate containing a full range and all dilutions ofantibiotics. SNDs for antibiotic plate testing were calculatedas follows: SND = (ZR for an antibiotic - mean ZR for thisantibiotic observed to date)/standard deviation of the ZRsfor this antibiotic observed to date. The derivation of thismean ZR is discussed below.

Full concentration range calibration curves to establish theoptimal working range of the disk method. Three antibioticswere chosen in the calibration curve study: cephalothin,sulfadiazine, and tetracycline. Disks were prepared to con-tain nine different concentrations of each antibiotic. Theranges were 0.02 to 4 Fig per disk for cephalothin, 0.1 to 20p.g per disk for tetracycline, and 0.8 to 256 1tg per disk for

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sulfadiazine. A full 15-cm plate was used to test each range,and each range was tested 10 times in a single run. Regres-sion lines were constructed, and the standard deviation wascalculated from the data. All concentrations of these antibi-otics tested, with the exception of the 0.8- and 1.6->tgsulfadiazine disks, produced DZIs.

Narrow-range calibration curves used to establish the ana-lytical sensitivity of the QCD method. New stock solutionswere made up at a 50% higher concentration than the normalstock solution concentration used (Table 5). These stocksolutions were diluted to prepare a series of antibioticconcentrations in solution. The following scheme of dilu-tions was used in all cases: for 150% concentration, 5 ml ofsolution and no distilled water; for 125% concentration, 5 mlof solution and 1 ml of distilled water; for 100% concentra-tion, 2 ml of solution and 1 ml of distilled water; for 75%concentration, 1 ml of solution and 1 ml of distilled water;and for 50% concentration, 1 ml of solution and 2 ml ofdistilled water. Each concentration level of the antibioticwas tested in triplicate on a single 15-cm plate on twoseparate days. All testing was carried out under the sameconditions, and all the DZIs were measured by the sameoperator. The calibration curves obtained from this studywere used to calculate the acceptance and rejection limits ofthe test.

Testing procedure to establish the analytical sensitivity ofthe antibiotic plate method. Plates were prepared to containthe range of antibiotics shown in Table 4. One dilution aboveand below the target range was included for each antibiotic.Because the antibiotic concentrations of numerous plateswere tested, it was necessary to use three seeded test platesfor the complete range of tests. A plate containing the sameset of concentrations of antibiotic was tested 10 times over a2-week period. The DZIs of the routinely used antibioticconcentrations and of the concentrations above and belowthis were divided by the mean of the routinely used concen-tration DZIs to produce ZRs. The standard deviations of theZRs were also calculated. A mean + 2 standard deviationsbar chart for the three concentrations was constructed fromthese results and examined for overlap of results (see Fig. 7).

Establishing base-line data for the plate testing method.Bottles containing 200 ml of agar were prepared to includethe complete range of antibiotics and concentrations uscd inour laboratory. Ten agar plates (diameter, 9 cm) were pouredfrom each of these bottles. The plates were stored at 4°C inplastic screw-top containers. Each of these plates was testedbefore being used for routine antibiotic susceptibility testing.

RESULTS AND DISCUSSION

Antibiotic solutions. In Fig. 1 we present calibration curvedata for three antibiotics, cephalothin, sulfadiazine, andtetracycline, over very wide concentration ranges. It can beseen that the slopes of the curves became very flat atconcentrations equal to and greater than 0.8 ,ug of cephalo-thin per disk, 64 ,ug of sulfadiazine per disk, and 4 ,ug oftetracycline per disk. The error bars shown on these graphsare equal to +2 standard deviations, and each point repre-sents the mean of 10 zone sizes. In our test system thecommercial susceptibility disks produced by Oxoid (cepha-lothin, 30 Ag per disk; sulfadiazine, 256 pg per disk; andtetracycline, 10 ,ug per disk) would theoretically give DZIs of49, 29, and 33 mm, respectively, and a single experimentconfirmed that these antibiotics gave DZIs of 52, 25, and 32mm, respectively, for these antibiotics. Diameters of thissize fall on the portion of the curve where the error bars

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QUALITY CONTROL IN ANTIMICROBIAL SUSCEPTIBILITY TEST 1823

TABLE 10. DZI, ZR, and SND data for five antibiotics showing increase in CV%(- from DZI to ZR for cefoxitin'

Day DZI MatrixA B C

1 14.6 17.1 14.52 13.3 15.6 13.43 12.3 16.2 12.64 14.2 17.2 15.65 13.6 16.9 14.76 13.1 16.6 13.67 13.3 16.9 14.28 12.1 16.4 15.19 13.7 18.0 15.710 14.4 17.6 14.6A 14.5 16.0 16.5B 14.5 16.0 17.0C 13.5 14.8 15.9D 14.1 15.2 15.9E 14.3 15.5 16.1F 13.5 15.0 16.8G 14.5 16.3 17.2H 12.8 15.9 14.9I 13.5 17.5 16.5J 12.0 15.8 15.8

MeansofRows

D14.211.811.717.214.912.914.714.713.414.919.518.316.918.017.517.618.013.715.014.5

E12.511.110.811.711.711.511.811.612.412.512.013.512.312.612.412.013.112.012.011.5

14.58013.04012.72015.18014.36013.54014.18013.98014.64014.80015.70015.86014.68015.16015.16014.98015.82013.86014.90013.920

Zone Ratio MatrixF G1.001 1.1731.020 1.1960.967 1.2740.935 1.1330.947 1.1770.968 1.2260.938 1.1920.866 1.1730.936 1.2300.973 1.1890.924 1.0190.914 1.0090.920 1.0080.930 1.0030.943 1.0220.901 1.0010.917 1.0300.924 1.1470.906 1.1740.862 1.135

H0.9951.0280.9911.0281.0241.0041.0011.0801.0720.9861.0511.0721.0831.0491.0621.1211.0871.0751.1071.135

I0.9740.9050.9201.1331.0380.9531.0371.0520.9151.0071.2421.1541.1511.1871.1541.1751.1380.9881.0071.042

J0.8570.8510.8490.7710.8150.8490.8320.8300.8470.8450.7640.8510.8380.8310.8180.8010.8280.8660.8050.826

Standard Normal Deviate TableA B C1.72 0.52 -1.312.19 0.78 -0.560.83 1.64 -1.400.02 0.08 -0.560.32 0.57 -0.650.85 1.11 -1.090.09 0.73 -1.15

-1.77 0.53 0.620.03 1.15 0.450.99 0.70 -1.49

-0.28 -1.18 -0.04-0.52 -1.29 0.43-0.38 -1.30 0.69-0.11 -1.36 -0.090.22 -1.14 0.21

-0.86 -1.37 1.55-0.46 -1.05 0.78-0.28 0.24 0.51-0.73 0.54 1.23-1.86 0.10 1.86

" Means of columns A to E (DZI): 13.59. 16.32. 15.33. 15.47. and 12.05. SDs of columns A to E (DZI): 0.82. 0.89. 1.26. 2.26. and 0.64. CVk of columns A toE (DZI): 6.0. 5.4. 8.2. 14.6. and 5.3. Means of the columns F to J: 0.935. 1.126. 1.053. 1.059. and 0.829. SDs of columns F to J: 0.039. 0.090. 0.044. 0.101. and0.027. CVÇi of columns F to J: 4.2. 8.0. 4.2. 9.5. and 3.3. Column headings: A and F. tetracycline (4 ,g/ml ) B and G. cefo`xitin (16 ,ug/ml() C and H. trimethoprim(4 p.g/ml); D and 1. suifadiazine (256 p.g/ml) E and J. nalidixic acid (32 ,ug/ml). Underlined SND values exceed the +1.97 rejection limits.

indicate considerable overlap. Hence, if these disks wereused, it would be difficult to discriminate either the nextlowest or the next highest concentration from the targetconcentration. We concluded that these commercial disks,although readily available, could not be used as a convenientsource of QCDs.

In Table 6 we present data which illustrate that the CVcfor DZIs (column 6) fell from a median of 7.6%c (range, 3.7 to18.1%) to a median of 2.2% (range, 1.3 to 4.7%) when thedata were expressed as ZRs (column 8). This improvementwas not due to the combination of statistical errors in thecalculation of the ZRs. This calculation is shown in column9 of Table 6. The calculation of the ZRs does not appear tobe a statistical artifact (7). We concluded that the use of theratio was a very effective means of minimizing those com-ponents of batch-to-batch variation which were not due tolaboratory errors.Having minimized the batch-to-batch variation by this

technique, we evaluated the ZR measurement as a potentialQC parameter. Our first task was to determine action limits

TABLE 11. Improved CVc with problematic antibiotics using B.suhlfiis NCTC 8236 (ATCC 11774)

Antibiotic and DZI (CVc() ZR (CV%concn (pg/lmI)

Tetracycline1 9.7 9.34 11.4 9.5

Cefoxitin4 6.8 7.016 10.8 8.6

Sulfadiazine (25) 10.5 7.8Trimethoprim (4) 9.6 9.1

for each antibiotic. We expected that these limits would beasymmetric, because the response curve of antibiotic con-

centrations (in micrograms per disk) on the xv axis versus

either the DZI or the zone ratio on the axis is logarithmicand is of the form v = a + blog, x. Calibration curves were

constructed from triplicate readings on two separate occa-

sions at five concentration levels. namely, 50, 75, 100, 125,and 150%c of the concentration normally used to impregnatethe QCDs (Table 5) for each of the Il antibiotics. Forsulfadiazine these experiments were performed on fourseparate occasions. because this antibiotic gives highlyvariable DZIs. The data obtained for the 11 antibiotics are

summarized in Table 7. Least-squares regression analyses ofthe data given for DZI versus log. .v were used to obtain theequations for the lines of best fit for the data expressed as

concentration versus DZI and as the concentration of anti-biotic in each QCD versus ZR.

Excellent fits were obtained. as indicated by the correla-tion coefficient, r. which at the P = 0.001 level of significancefor 15 datum points is 0.7419. It was possible to pool the datafrom data sets A and B (Table 7) after they had beenexpressed as ZRs. This gave 30 datum points per calibrationcurve (60 datum points for sulfadiazine), and again thecorrelation coefficients confirmed an excellent agreementwith the logarithmic model. For 30 datum points, r = 0.5620is significant at the P = 0.001 level of significance. Figure 2

illustrates the graphs obtained for tetracycline (data sets A.B, and C) and sulfadiazine (data sets A, B, and E).The equations for these calibration curves permitted us to

calculate some practical QC action limits in ZR units. whichcould be used prospectively in our QC scheme. We arbitrar-ily elected to use warning limits of 75 and 125%r and rejectionlimits of 50 and 150% of the target concentration of theantibiotic on the QCD. These limits are considerably more

D-0.83-1.51-1.370.73

-0.21-1.04-0.22-0.07-1.41-0.511.810.940.911.270.941.150.78

-0.69-0.51-0.17

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-2.15-0.520.760.130.040.680.59

-2.380.830.340.09-0.40-1.02-0.031.37

-0.87-0.10

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1824 McDERMOTT AND HARTLEY

Zone Ratios

Amoxycillin 1 2 48 19f 32

0.7 0.8 0.

j'

.9 1.0 1.1 1I

Il i-4I-I -4 I |

Ampicillin 1 2 4 - -I I8 16 32 - - -I

Imlpenem 1 2 4 I - I8 16 32 -I-.i

CephalothIn 1 2 48 16323l

Azlocillin 8 JQ 32 I- I- I FI-32 64 128 1- 1-1 41_

CefoxîtIn 8 16 32 - - -

48L48 Il IlIGentemicin 2 4 8 - h h-I

Tobramycin 2 4 8 f-1

Tetracycline 2 4 8 - I-t

122 H-ISulphadiazine 266 612

Trimethoprin 4 8 F

FIG. 7. Analytical sensitivity 2-standard-deviation bar chart for antibiotic plate testing. See text for calculation of ZR data. The numbersafter the antibiotic names are the concentrations of the antibiotics (in micrograms per milliliter) incorporated in the plates. The underlinedvalues are the concentrations incorporated into routinely produced plates.

stringent than those in the conventional MIC approach,which has acceptance limits of 50 and 200% and rejectionlimits of 25% below and 400% above the target MIC. Theactual values of the ZR limits for each antibiotic were

calculated by substituting values of 50, 75, 125, and 150% ofthe QCD concentrations (Table 7, column 2) into the regres-sion equations. The results of these calculations are shownin Table 7, columns 7 to 10.We have used these limits prospectively to evaluate the

QC data accumulated for the Il antibiotics over an 8-monthperiod. Figure 3 illustrates the data we have collected forgentamicin; they are typical of the data we obtained forantibiotics giving the most highly reproducible results, i.e.,amoxicillin, cephalothin, gentamicin, tobramycin, nalidixicacid, cefoxitin, norfloxacin, and azlocillin. The data fortetracycline, sulfadiazine, and trimethoprim are shown inFig. 4, 5 and 6, respectively. The ZRs for these threeantibiotics tended to be low, with means of 0.970, 0.928, and0.966, respectively, probably because the solutions used toprepare the QCDs of batch A had 17 to 21% higher concen-trations than anticipated when the stock solutions were

produced. Some further support for this hypothesis wasdiscernible in the data for trimethoprim (Fig. 6), whichshowed some improvement when QCDs of batch B wereused. The four datum points for batch B had a mean of 0.994.Comparison of ZRs and SNDs with the diameters of DZIs in

antibiotic plates. Table 8 is a summary of a typical data set

for gentamicin, tobramycin, azlocillin, imipenem, and nor-floxacin at the various concentrations given in Table 3. Thedata for 10 base-line plates plus data from 10 routine assaysare shown. Means, standard deviations, and CV% are givenin the footnote. The CVYc ranged from 5.3 to 10.6% for theDZI results. The corresponding ZR data are given in thesecond panel, and the individual statistics for ZR are alsogiven in the footnote. A column-by-column comparison ofthe CVs clearly demonstrated that the theoretical advan-tage of calculating ZRs was again realized in practice. TheseCVs ranged from 4.1 to 9.9%. The panel of SND datacould therefore be confidently scanned for items whichexceeded the ±1.97 rejection limits. Eight items highlightedin this third panel exceeded these limits, indicating that theplates tested on days A and B were unacceptable. Thisconclusion was reached easily from the SND data but was

harder to detect from the ZR data matrix and even lessdiscernible from the DZI data matrix.Table 9 presents the DZI, ZR, and SND data for amoxi-

cillin. amoxicillin-clavulanic acid (Augmentin), gentamicin,cephalothin, imipenem, and norfloxacin at the concentra-tions given in Table 3. Again, a column-by-column compar-ison of the CVs substantiated the theoretical advantage ofcalculating the ZRs. For this set of six antibiotics, 9 of the 10columns of data showed improvements in the CV%s ofbetween 0.3 and 4.2%. However, no apparent improvementwas shown for norfloxacin, and this may be attributed to the

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QUALITY CONTROL IN ANTIMICROBIAL SUSCEPTIBILITY TEST 1825

exceptionally good DZI CV% result obtained for this antibi-otic. The SND data showed several instances when the valuewas equal to or greater than the + 1.97 warning limits. Threeof these occurred singly on days 9, A, and F. Days H and Jboth had two values outside these warning limits. Thisappeared to be a relatively high warning rate and was one ofthe reasons we chose to retain seemingly unacceptable data,such as those for imipenem at 16 pg/liter on day 9, within the10 base-line data sets. Their intentional inclusion was de-signed to reduce the sensitivity of this QC test to realisticand practical levels. When batches of plates which hadexceeded the warning limit were retested, the results almostinvariably fell within the acceptance limits, confirming thisobservation of oversensitivity. This is illustrated by the factthat the probability of a deviation of +1.97 is P = 0.025 andthe probability of two consecutive deviations of ± 1.97 is P =(0.025)2 = 0.0006. An example of a test failure is shown inTable 8, rows A and B, for azlocillin plates with concentra-tions of 16 and 64 mg/liter.The data in Table 10 illustrate a further feature of this QC

approach. In Tables 8 and 9 there was an improvement in theCV%s of the ZR compared with the DZI data, but the data inTable 10 demonstrate that this is not always the case. TheCV%s for the cefoxitin data increased from 5.4% for the DZIdata to 8.0% for the ZR data. This was unexpected, and afurther examination revealed that this phenomenon was dueto the highly variable data for trimethoprim and sulfadiazine(CV% = 8.2 and 14.6% respectively), which distorted theresults for cefoxitin. If the columns of data for trimethoprimand sulfadiazine were omitted and the data for tetracycline,cefoxitin, and nalidixic acid were retained and processedagain, the following satisfactory results for the CV% of theZR data were obtained: tetracycline, 3.5%; trimethoprim,4.4%; nalidixic acid, 3.6%. Hence, in all cases, the CVs ofthe ZR data were reduced, as was previously anticipated.

Further work has revealed that the indicator organismused in this study may not be optimal for testing fortrimethoprim and sulfadiazine. Use of B. siibtilis NCTC 8236(ATCC 11774) for a similar panel of antibiotics which in-cluded the problematic sulfadiazine and trimethoprim pro-vided more satisfactory results (Table 11).

Analytical sensitivity. Figure 7 presents a graphical sum-mary of the data collected for the 13 antibiotics in the platesexamined in this final part of the study. When using the ZRmethod, it was quite clear that it was possible to distinguishbetween the target concentration of any of these antibioticplates and one dilution above or below this target concen-tration. The statistical confidence with which we could makethis distinction was better than P = 0.05 for the fourantibiotics sulfadiazine, trimethoprim, cefoxitin, and tetra-cycline; i.e., we were 95% confident that we would be able toidentify differences. It was possible to detect errors in onedirection only, i.e., one dilution below their target concen-trations.

Conclusions. QC should be easy to perform, inexpensive,and reproducible. These were our priorities in designing themethods; however, it would appear that in the case of theQCDs, we have achieved improvements in analytical preci-sion. The methods are simple, do not involve complicated

procedures, and can be easily incorporated into the routinework of the laboratory.The criteria used for assessment of antibiotic solution QC

are quantitative and are suitable for cumulative assessmentin graphical form. We have developed a computer programto assist with the filing and display of the results to encour-age the ready acceptance of this method in the busy routineof the laboratory. Should the method gain wide acceptance,a commercial source of QCDs would further simplify theadoption of this approach in smaller laboratories and facili-tate-both interlaboratory QC surveys and method develop-ment.The criteria used for the assessment of antibiotic plates are

qualitative and do not assay the antibiotic concentrationcontained in the agar; however, they do detect abnormalfluctuations within the normal deviations of the method. Thismethod of QC is a further extension of the ZR techniqueapplied to the QC of antibiotic susceptibility testing plates. Itis simple to apply and provides a uniform criterion, the SND,on which to make QC decisions which have statisticalrespectability and confidence. It can be easily incorporatedinto a computerized QC data recording system and should beapplicable to the particular antibiotic susceptibility testingpanels in any laboratory, provided that care is taken torecognize that inclusion of antibiotics which are known tohave highly variable performance characteristics may de-grade the overall performance and potential of this ap-proach.

ACKNOWLEDGMENT

We are grateful to Margaret Priede for her patience and willing-ness to help us at all times in the preparation of this paper.

LITERATURE CITED1. Adler, H. L., and E. B. Roesslèr. Introduction to probability and

statistics, 6th ed., p. 116 and 361-363. W. H. Freeman and Co.,San Francisco.

2. Cooper, K. E. 1964. The theory of antibiotic inhibition zone, p.1-86. In F. Kavanagh (ed.), Analytical microbiology. AcademicPress, Inc., New York.

3. Cooper, K. E., and A. H. Linton. 1952. The importance of thetemperature during the early hours of incubation of agar plates inassays and note on the theory of inhibition zones. J. Gen.Microbiol. 7:8-17.

4. Davis, W. W., and T. R. Stout. 1971. Disc plate method ofmicrobiological antibiotic assay. I. Factors influencing variabilityand error. Apple. Microbiol. 22:659-665.

5. Erisson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivitytesting. Report of an international collaborative study. ActaPathol. Microbiol. Scand. Sect. B Suppl. 217:1-90.

6. Franklin, J. C. 1980. Quality control in agar dilution sensitivitytesting by direct assay of the antibiotic in the solid medium. J.Clin. Pathol. 33:93-95.

7. Kirshbaum, A., J. Kramer, and B. Arret. 1960. The assay andcontrol of antibiotic discs. Antibiot. Chemother. 10:249-259.

8. Miles, A. A., and S. S. Misra. 1938. The estimation of thebactericidal power of the blood. J. Hyg. 38:732-748.

9. National Commiittee for Clinical Laboratory Standards. 1985.Methods for dilution antimicrobial susceptibility tests for bacte-ria that grow aerobically, p. 591-594. National Committee forClinical Laboratory Standards, Villanova, Pa.

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