disc plate method of microbiological antibiotic assay

APPLIED MICROBIOLOGY, Oct. 1971, p. 659-665Copyright © 1971 American Society for Microbiology

Vol. 22, No. 4Printed ilt U.S.A.

Disc Plate Method of Microbiological Antibiotic AssayI. Factors Influencing Variability and Error1

W. W. DAVIS AND T. R. STOUT

The Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Inidiana 46206

Received for publication 7 June 1971

Several factors are investigated that normally cause variation in zone diametersin conventional disc plate diffusion assay procedures. Of these factors the mostserious is the unequal exposure of the individual plates at top or bottom of stacksto temperatures above and below room temperature. This unequal temperature ex-posure is avoided by novel handling and incubation procedures. A major variable,but one which can be controlled, is the varying time interval between pouringseeded agar and the time of applying the pads with antibiotic to the plates. Thisinfluence of time of setting and the effects of several other sequential operationsare combined into a composite variable. This variable is then accounted for andnormalized by interposing "external" reference plates set with a reference solutionin the sequence of approximately 100 plates. No "internal" reference zones are em-ployed. Such factors as volume of agar poured, wedge shape of agar in a dish,volumetric errors in dilutions, and timing considerations are studied and discussed.The results of this study form the basis for a test protocol which is presented in afollowing paper.

The diffusion assay of antibiotics employing adisc plate method has great virtue of convenience,simplicity, sensitivity, efficiency, and dependa-bility (5, 10). The relation of diameter of inhibi-tory zones to concentration of antibiotic in asolution applied in cups has been consideredtheoretically (2, 7). In practice, the diameter isdirectly responsive to antibiotic concentration,and the limitations on accuracy are the recognitionand control of the many subtle variables. Theapplicability of the system is general, andmaterials and equipment for the conduct of theprocedure are widely available. Paper discs andblow-molded plastic petri dishes are available. Aninstrument for accurate reading of zone diameterswas described by Davis et al. (3) and is manu-factured and sold by Fisher Scientific Co., Pitts-burgh, Pa.When studies conducted with antibiotics ap-

pear to the experimenter to demand high accuracyof assay, the alternatives of microbiological orphysiochemical assay present themselves. Somecomparisons between several microbiologicalprocedures are presented by Gavin (5, 6).A chemical or physicochemical test, if validated

as completely specific for the active component,may be more accurate than results generally

I Presented at Analytical Microbiology Section, A and IMDivision of the American Society for Microbiology, Detroit,Mich., 6 May 1968.

achieved with microbiological procedures (1, 6).A chemical or physical test is often more amenableto automation than microbiological procedures(9). For instance, automation has recently mademore efficient and accurate the use ofturbidimetricand colorimetric assays (8). A chemical orphysical test is likely to be very narrow in itsapplication and much more difficult to establishthan a microbiological test.

Microbiological assay procedures provide avalid measure of antibiotic activity with littledanger, generally, of interference from biologic-ally inactive components or degradation products,and the disc plate method persists in wide use(10).In our own experience, surprisingly large in-

accuracies have been encountered in the results ofsuch testing. Accordingly, we reexamined someof the factors in the disc plate diffusion methodand attempted to improve the accuracy of themethod without sacrificing its great generality,sensitivity, and convenience.

MATERIALS AND METHODSCertain a priori commitments to equipment and

procedures were made in initiating the present study.Disposable 3.75 inch (ca. 9.53 cm) blow-moldedplastic petri dishes and 0.25 inch (6.3 mm) diameterantibiotic discs were used; application of all solutionswere from the same automatic self-filling and -deliver-ing capillary pipette. Six discs were placed on each

659

DAVIS AND STOUT

petri dish, all filled with the same solution, consistingof either a known or an unknown. This is in contrastto the more common practice of using "internalstandards," i.e., two or three discs with referencesolution on each petri dish (10).The automatic filling and emptying pipette em-

ployed here is similar in construction to that de-scribed by Davis and McGuire (4) except that it ismade with a smaller capacity (17 jAliters) to deliveran appropriate amount of fluid into the 0.25 inch(ca. 0.64 cm) pads employed. The total capillary ca-pacity of the pads placed on a dry surface is about 27uliters. When a pad is placed on an agar surface, someof this capacity is filled by absorbing fluid from theagar within the few seconds before applying the testsolution from the pipette. Enough capillary capacitymust remain in the pad when the pipette is applied topull all of the fluid out of the pipette.A single antibiotic solution [2 ,ug of Keflin per ml

in pH 6.9 (0.2 molar) phosphate buffer] was employedthroughout, the same amount of which was appliedto each pad of every plate. By this procedure, variablessuch as the order of pouring the plates, the time andorder of "setting" the plates (i.e., placing pads andsolution), and placing the plates in stacks for incuba-tion, etc. revealed their separate influences. Even verysmall variation of zone diameters could be tied tothese controlled variables. Zone diameters weremeasured reproducibly to plus or minus 0.05 mm em-ploying a Fisher-Lilly Zone Reader (3). The use ofKeflin solution with Bacillius sutbtilis as the test or-ganism resulted in sharp zone edges.

Usually a total of 102 plates, each bearing six pads,Nvere set and handled in a standard way each day by asingle operator. All variations of zone diameterswithin plates, between plates in each stack, and be-tween stacks were examined for systematic and ran-dom relationships, and the variability was interpretedas to cause and as to the possibility of control.

Throughout this study B. suibtilis was employed asthe test organism. A suspension of spores held at 5 Cin distilled water was added to hot nutrient agar. Thehot seeded agar was generally poured at 50 C. Certainprocedures or materials were changed in the course ofthe study, with only minor accounting for their effectupon accuracy. For instance, we determined thatsome disposable petri dishes were superior to others,and, after an examination of significant structural

features of the dishes, the practice of using only onespecific product was adopted. This was DiamondPlastics petri dishes from mold 3.The most feasible operation with stacks of plates

appeared from the beginning to be to make a unit ofhandling of about 30 plates with blanks at top andbottom of stacks. The quantity of seeded agar pouredper plate was varied, being either 6, 8, or 10 ml, butthe 8-ml pour was adopted and considered standard.The composition of nutrient agar was arbitrarilychosen for well-defined zones. [The composition ofnutrient agar employed with B. subtilis was for liter:1.5 g of beef extract (Difco), 6.0 g of peptone (Difco),3.0 g of yeast extract (Difco), and 20.0 g of agar(BBL). This was adjusted to 6.0 with dilute HCl orNaOH and was unbuffered.]An early decision was made to employ only volu-

metric displacement burettes in making up the sam-ples of standard or unknown solutions. In this way,inaccuracies due to drainage errors were avoided. Alsoanticipating the necessity to employ the same diluentfor standard solutions as for unknowns (rat plasmadog plasma, tissue juices, etc.), procedures werechosen so as to be amenable to the employment ofmicroquantities of fluid. Accordingly, standard dis-placement microburette procedures were establishedto make all dilutions and to provide accurate and con-venient volumetric deliveries in the microliter range.The displacement microburettes were of either 0.2 mlor 2.0 ml total capacity.

RESULTS

Application of test solution to pad. The quantityof solution applied to a pad has a large propor-tional influence upon the size of resulting zonediameters. A comparison was made of the vari-ability of volumes of solutions applied to pads bya commonly used procedure and the procedureadopted here. Weighed pads were filled with buffersolution by one of the two procedures and thenreweighed. The first procedure was that of touch-ing the pad to a supply of the solution in as uni-form a manner as feasible saturating the capillarycapacity of the pad. The second procedure in-volved applying solution with the self-filling and

TABLE 1. Statisticail comparisont of the dippinig anid pipette methodls of delivering test solhtioiionto assay discs

Volume delivered (X)

Liquid delivered Disc dipping method Capillary pipetting method

Na X Sz > S (%) N XS2 IS(%)S2 S S (%C)

10%,.NaCi 10 27.38 1.008 1.004 3.65 10 17.99 0.125 0.353 2.00Water 10 27.21 2.096 1.447 5.35 10 16.86 0.020 0.143 0.83Water 10 27.09 1.401 1.183 4.36 10 16.88 0.050 0.223 1.32Average 4.45 I 1.31

N is the number of pads in each group, X is the mean for the group, S2 is the variance, S is the standarddeviation, and S(%O) is the coefficient of variation.

660 APPL. MICROBIOL.

MICROBIOLOGICAL ANTIBIOTIC ASSAY. I

AGARTHICKNESS

(mm)15.0-

12.5-

10.0-

7.5-

5.0-

2.5-

010

z

POURVOLUME

(ml.)

12 14 16 1870NE DIAMETER - mm

FIG. 1. Depenzdence of zonte diameter uponi pourvolume anid agar thickness. Zone diameters are averagesofsix zonzes formed in each of two dishes represenzted bytwo data points at each poutr volume.

-delivering pipette as described above. Table 1 setsforth the resulting weights of solution and thevariability. The variability of weight or volumeapplied is at least three times as great from thefilling of pads by touching solution as from theuse of a delivery pipette. The pipette may be ex-

pected also to circumvent errors in other pro-cedures due to varying surface tension andviscosity of the sample solution.

Preparation of test solution. Large errors gener-ally occur in volumetric dilutions employingdelivery pipettes for small volumes, especiallywhere viscous solutions with implicit largedrainage errors must be employed. To circumventthese errors, the use of volumetric displacementdevices was adopted instead of volumetric de-livery devices. Significant errors of delivery fromthese displacement microburettes occur whendelivery of less than 1 ,-liter is attempted. Theprotocol presented in the following paper antici-pates the delivery of never less than 2.0 uliters inthe preparation of solutions.Agar thickness. The thickness of the test agar

layer greatly affects the resulting zone diameter.Accordingly, the relation of zone diameter versusthickness of test agar layer was examined. Theresults in Fig. 1 show that the thickness or thepour volume in a petri dish is very critical at pourvolumes usually employed. An error in thickness

is a direct and proportional consequence of anyerror in pour volume. A pouring device allowingdelivery of constant volume is therefore essential.

In addition to such error in agar thicknessfrom plate to plate resulting from pour volumeerror, there is also the possibility of variation inagar thickness within a plate. Two sources of suchthickness errors exist in disposable petri dishes.The first is the formation of a wedge-shapedlayer of agar as a result of tilt of the plate bottomwhile the melted agar is cooling and setting up.This can be due either to an imperfect plate orto slope of the surface on which the plates areplaced for pouring.The extent of such wedge shape of the agar has

been measured by employing a depth gauge tomeasure the inside contour of the plate bottom.We found that the mold in which the plasticdishes are made generally imposes a sizeable tiltto the dish characteristic of the mold. The amountof tilt characteristic of four mold numbers ofDiamond Plastics plates was 0.025, 0.125, 0.15,and 0.175 mm across the 2.5 inch (ca. 6.4 cm)diameter of the circle of zone centers. Theproducts of several other manufacturers weresimilarly examined and the range of tilt en-countered was generally greater than 0.125 mm.The average thickness of agar in an 8-ml pour ina petri dish is approximately 1.25 mm. It is there-fore obvious that a few tenths millimeter ofwedge shape is a large percentage of variation inthickness. Figure 2 shows for one experiment theinterrelationship of tilt (from two sources), wedgeshape of agar, position of zones, and zone diam-eters.The wedge effect leaves the average thickness

of agar in a plate unaffected. Whereas the wedgeeffect causes major variation of zone diameterswithin a plate, there is of course a tendency of thelarger zones resulting on the thin side to compen-sate for and average out the smaller zone diam-eters which result from thicker agar on theopposite side. Although this cancelling may notbe complete, no regular effect could be observedon the average of the six zone diameters of plateswhen tilt of 0.00, 0.45, 0.72, 0.90, and even 1.20mm were deliberately imposed. When averagingof all zones in a plate is depended upon toeliminate this source of error, as is done here,the opposite zone should be eliminated also ifany zone in a plate is imperfect or unreadable.Such averaging out of the wedge effect would notoccur in unsymmetrical arrangement of like zones,as where three unknowns and three internalstandard zones are employed (10).A second systematic variation in the shape of

plates which leads to variation of the thickness ofagar from zone to zone within a plate is a cup-

VOL. 22, 1971 661

DAVIS AND STOUT

2 3

1 ~~4

6 5

"ll< ~~~~~~~DISIFOmH TILT 0.10 mmIPOSITION 1 TO 4.-BENCH TILT0.20mm PER 2'2"-LEVEL

0.3mm COMBINED TILT

14

mm

ZONE DIAMETERSl1mm(AVG. 18 DISHES)

FIG. 2. Relation of zone diameter to pad positionand agar thickness in a plate with wedge-shaped agar.The tilt characteristic of the dish is uniformly related toposition of the sprue visible on the blow-moulded dish.These plates were oriented and placed on a tilted sur-face and poured.

shaped contour of the bottom of the plate. Theinverse deformity, a center bulge, is also promi-nent in the products of some manufacturers. Thisdeformity is minimal in the Diamond Plasticsplates.The effect of this second shape factor is not

cancelled by averaging. The placement of pads atunequal distances from the center when a cupshape or center bulge exists would cause variationin the zone diameters due to the variation of agarthickness along the line from center to edge. Theuse of a visual guide for placing the pads is essen-tial. The location of pads, as regards distancefrom the center, is more critical the greater suchdeformity of the bottom of the plate and thesmaller the amount of agar poured. We havemade no measurements on tilt or other deformitiesin the bottom of glass petri dishes, but they areso conspicuously misshapen that they should notbe considered for use.

Inoculum concentration. An influence of inoc-ulum concentration on resulting zone size iswidely recognized (11). Experiments were per-formed to determine how critical the density ofspore inoculum might be in the present systemwhen other factors are constant. Although widevariation in spore density did produce zonediameter differences (smaller zones for higherinoculum), the most serious consequence was inthe quality of resulting zone edges, which were

granular and poorly defined when a too diluteinoculum was used. An appropriate inoculumconcentration was found after preparation ofeach new batch of spores and kept constant byemploying a uniform dilutuion into heated agar.

Temperature-time exposure. Major attentionwas given to several details of the operation whichmight impose an inequality of temperature ex-posure of the individual plates handled in a singleday. Attention was given to every step of thehandling procedure between pouring of platesand grow-out of the test organisms.

In a preliminary experiment, the temperatureof seeded agar was measured as it emerged froma pouring tube from the automatic pipettingmachine. This was done by having a copper-constantan thermocouple in the outlet tube. Thethermocouple EMF was directly recorded on astrip chart recorder throughout this experimentalpouring. The seeded agar was held in a constanttemperature water bath at 50 C during the pour-ing. The passage of hot agar through the pipetterand tube gradually heated the pipetter severaldegrees. Inversely, the agar was initially cooledin passing through the pipetter and the first agarto emerge was not up to the steady state pouringtemperature. Accordingly, when the pouring is tobe done at 50 C, water at 50 C is pumped throughthe pipetter until the temperature at the pouringtip is stabilized before pumping the heated seededagar.A record was made of the temperature of

emerging seeded agar at 50 C after brief (3 min)flushing with water at 50 C and then rejectingfive pours (40 ml) of seeded agar at 50 C. Thetemperature was constant throughout a pour of100 plates to better than 0.5 C.An experiment was performed to determine the

rate at which 8 ml of hot agar cooled in petridishes after pouring. A thermocouple was placedin such plates and the temperature was followedwith a strip chart recorder. The course of tem-perature decline to room temperature was asymp-totic and rapid. In general, the temperature hadfallen to within 1 C of room temperature within15 min after pouring.The placing of poured plates in stacks before

they reach room temperature slows and prolongsthe cooling time. The major consequence of thisslower cooling in a stack is that a plate removedfrom the bench top and placed in a stack at ahigher temperature (shorter time after pouring)experiences a much longer exposure to elevatedtemperature than one placed in the stack afterlonger cooling on the bench top.

If the relatively slow operation of pouring (10mi), was followed quickly by the relatively fastoperation of stacking (2 min), the first plates



TABLE 2. Dependentce of zone diameteron incubation temperature

Determination Stack 37 C 30 C 26 C

Zone diameter 1 12.76 13.49 14.88(mm)a 2 12.40 12.42 14.40

Concn with sim- 1 1.70 2.00 2.77ulated standard 2 1.77 2.00 2.80curve at 30 C

a Average of 12 plates.

poured would have cooled much further whenstacked than the last plates poured. Experimentsin which plates were stacked immediately afterpouring has confirmed this expectation, producingsignificantly smaller zones. Accordingly, a regularpractice was adopted of letting the plates cool onthe bench top for approximately 20 min aftercompleting the pouring before stacking.To judge how critical is temperature exposure

during holding or incubation on the resulting zonediameter and on the test results, incubation wascarried out at several temperatures, and the resultsare presented as zone diameter versus temperaturein Table 2. All the zone diameters for each stackwere averaged. Data for only two stacks are pre-sented. A typical conversion of zone diameter to"concentration" shows the high sensitivity of testresults, expressed as concentration, which wouldresult from unaccounted variation of incubationtemperature.Upon cooling in a refrigerator or warming in

an incubator, heat flows into or out of the stackthrough some thermal contact of the stack with aheat source or heat sink. For instance, the bottomplates of the stack would deviate prominently inthermal exposure if the stack were placed on acold refrigerator shelf or on a warm incubatorshelf. In all experiments in which both refrigera-tion and incubation at 37 C were employed, suchend-effects were seen. The consequence of refriger-ation was always to imprint a depressed tempera-ture exposure (and consequent high zonediameter) upon the plates that had been in closestcontact with the refrigerator shelf. Even wrappingthe stack and use of an insulating pad did noteliminate the top and bottom effect.When stacks were placed in an incubator at

37 C for grow-out, similar but more elusive topand bottom anomalies of zone diameters wereobserved within stacks. Wrapping the stacks inaluminum foil and suspending them by means ofmasking tape to eliminate solid contact with theincubator failed to eliminate completely the topand bottom anomalies of resulting zone diam-eters. In persistent effort to eliminate the top and

bottom anomalies in stacks caused by incubationat 37 C, changes were tried to restrict, redirect,or eliminate air currents in the incubator. Suchefforts resulted in alteration but not removal oftop and bottom anomalies. Since the anomalieswere generally most exaggerated in the topmostand bottommost plates, but still detectable severalplates from top or bottom, this source of errorcould be minimized by leaving unset the lastfour plates on the top and on the bottom. Eventhis practice did not completely eliminate theanomalies.

Evaporation of even a very small amount ofwater from a poured plate would cause a largelowering of temperature. Evaporation wouldoccur if the plates in stacks were allowed to bein contact with an unsaturated atmosphere. If thethermal flow into or out of the plate is slowcompared to the rate of evaporation, one couldanticipate the lowering of the temperature of 8ml of agar by 5.0 C by evaporation of only 80 mgof water (1 %7, of the 8-ml pour volume). Accord-ingly, enclosure of stacks was provided to secureand maintain a saturated atmosphere about theplates to avoid the cooling effect of such evapora-tion.

All efforts to attain uniform or linearly dimin-ishing zone diameters within stacks ofplates failedwhen refrigeration was employed between pour-ing and setting, and when incubation at 37 C wasused. Therefore, a room temperature procedurewas devised for storing and incubation to circum-vent large temperature swings. There are inherentadvantages in room temperature (26 C) handlingover conventional procedures that employ re-frigeration and incubation at 37 C. These are theabsence of temperature swings which cannot bemade exactly equivalent for all plates in one ormore stacks. Subsequently, work in these labora-tories (J. F. Quay, unpublished data) has shownthat some test organisms produce better zoneswhen the stacks, enclosed in close-fitting steelcylinders, are incubated at 30 C, or even at 37 C.Relatively little end anomaly results from suchincubation if the steel cylinders are employed asdescribed before without intervening refrigeratorstorage.Other factors affecting germination rate. In

addition to temperature, other factors that in-fluence rate of germination include the chemicalcomposition of the medium (which, of course,can be fixed) and pH, which can be adjustedinitially and stabilized by inclusion of buffers.Without buffer, pH of agar seeded with B.subtilis spores generally drifts upward duringgrow-out. Such pH changes during grow-outhave been observed, but pH variability from plateto plate is not great, and growth is often more

VOL. 22, 1971 663

DAVIS AND STOUT

favorable in a very weakly buffered or un-

buffered solution.Timing considerations. To find and control the

sources of variability in diffusion microbiologicaltesting, attention has been centered upon pa-

rameters of the diffusion process and upon pa-

rameters of the process of growing out of the testmicroorganisms. The result of a diffusion-inhibition test is the result of a race betweenspreading of antibiotic by diffusion and the grow-out of the test organisms. The conditions thatinfluence diffusion rate are largely controllable,and small temperature variation has a relativelysmall influence upon diffusion.The moment at which the germinating spores

reach some critical stage-perhaps where the re-

sulting vegetative organism first doubles-is the"timing" mechanism for determining the positionof the zone edge. Inside this circle the germinatingorganisms are prevented from passing the criticalstage (doubling), whereas outside this circle theconcentration of antibiotic is too small to preventthe event. It is generally understood that whenthe concentration of inoculum increases (heredoubles) more antibiotic is required to preventfurther growth. Thus, the line within whichdoubling is prevented by just sufficient antibioticconcentration becomes a fixed zone edge whichbecomes sharper but does not move significantlywith the further divisions and doubling of num-bers of test organisms. Outside this circle, a con-tinuing increase of density occurs as the doublingand redoubling of organisms continues.The initiation of germination for all plates is

perhaps that moment when the spore suspensionis added to hot agar. Accordingly, the last platepoured should come to the critical timing eventreferred to above at about the same time, becauseall plates are poured within a 10-min period. Thecooling to room temperature of the first and lastplates poured is separated only by this shortinterval. The cooling is equally rapid for all platessince they all cool on the bench top.The initiation of diffusion, however, is at the

moment when the antibiotic solution is appliedto the pad. From the first plate set to the lastplate set in a series of 102 plates may be 1 hr.Thus, when "time" is called by the germinationevent, the last plates set will have had 1 hr lessto diffuse, and the zones determined at thatmoment must be smaller for a given concentrationof test solution.

Ifthe setting ofthe test pads is done on a regulartime schedule, this factor can be accounted for inthe subsequent treatment of data. The function ofa constant standard solution (2 ug/ml) set atregular intervals throughout the schedule is to de-fine the influence of the order and time sequence

of this and all other operations carried out in thesame numerical sequence. The influence of orderof handling within the experiment of 1 day canthen be accounted for in the data handling.Representative data show that the diameters de-crease approximately linearly with the settingorder, or with the time of setting, if a regularschedule of setting is followed. This slope must begreater in proportion to the time required forsetting a given number of plates.An experiment was performed to determine the

effect of the time of holding the poured andstacked plates at room temperature before settingthem. The time between setting and grow-out(the time allowed for diffusion) must be reducedby longer holding between pouring and setting. Aregular decline occurred in the zone diametersproduced by 2.0 ,ug of antibiotic per ml appliedto plates held for intervals up to about 5 hr afterpouring (see Table 3). When the plates were setat later times, small, unusable, poorly definedzones were formed. From this experiment, weconcluded that resulting zones are sharp andusable from plates that have been held up to asmuch as 3 hr after pouring. This limitation mayof course vary greatly with the test organism.The following paper presents a convenient

method of accounting for all the factors of timing;

TABLE 3. Influence of room temperature holdingtime before setting plates

Holding time Zone diameters Observations(hr) (mm)'

Expt 11 15.18 Normal zones2 14.43 Normal zones3 13.57 Normal zones4 12.71 Normal zones5 11.55 Normal zones6 10.74 Diffuse zone edges

7 9.8 Diffuse zone edges8 9.9 Diffuse zone edges10 10.16 Diffuse zone edges12 10.20 Diffuse zone edges

Expt 21 15.04 Normal zones2 13.96 Normal zones4 11.83 Normal zones5 10.75 Normal zones6 9.71 Normal zones7 8.60 Normal zones8 8.10 Diffuse zone edges

9 8.15 Diffuse zone edges10 8.25 Diffuse zone edges

a Average of 24 zones.



namely, the order of pouring, stacking, andsetting. Fortunately, although the effects of thesevariables cannot be removed, they can be regular-ized, combined, and accounted for in a manner

that removes them as a source of variability or

error in assay results.

DISCUSSION

Of the factors that influence variability anderror, some can be controlled by choice of condi-tions and some by handling procedures. Otherfactors cannot feasibly be controlled, but, byestablishing an order and timing of the opera-

tions, the consequence of the combination offactors is a regular decline in zone diameter for a

given concentration as the plate sequence numberincreases. Reference plates inserted at regularintervals permit normalizing this combinedvariable.Among the controllable factors, we have shown

that special care must be taken to apply a constantvolume of solution to the pads. The superiority ofusing an automatic filling and emptying capillarypipette for applying antibiotic solution over an

alternative procedure is demonstrated. The use ofthe pipette is rapid and feasible. Setting plateswas possible at a regular rate of two plates or 12pads per minute.

Accuracy of diluting test solutions is poor byconventional volumetric delivery glassware if verysmall volumes are employed. Such difficulties canbe circumvented by employing displacementmicroburettes, which not only provide accuratereading of volumes but also eliminate the hazardof drainage error.Agar thickness is a critical factor in determining

the zone diameter. The factors that contribute tovariation in agar thickness are the amount of agarpoured (causing a directly proportional error inthickness) and wedge shape. The latter resultsfrom sloping petri dishes or from pouring on asurface with tilt.Another fault of petri dishes which causes un-

controlled variation in thickness of agar is thepresence of a nonplanar bottom. Whereas wedgeshape produces very large zone diameter varia-tions within a plate, averaging aU zone diametersin a plate removes most of the error from thissource. In test protocols where "internal" refer-ence zones are employed, both reference and un-

known zones should be set only as opposed pairs,and, if one zone of agar is unusable, the oppositezone should be dropped.

Different temperature exposure of plates in thedifferent positions within stacks (viz., top andbottom) is unavoidable but can be minimized byenclosure of stacks in close-fitting steel cylindersand avoidance of large temperature shifts, partic-ularly between room and refrigerator tempera-ture. Refrigeration should not be used, andincubation should be at room temperature if thesatisfactory growth of test organisms allows.Several factors associated with pouring and stack-ing have been found that cause inequalities oftemperature exposure. Corrective measures werefound.

Characteristic growth of test organism and thezone size and sharpness respond to changes in pHand buffering. No attempt was made here tooptimize these factors for the test. This should bedone for each test organism and for each anti-biotic, if repetitive testing is to be done.

ACKNOWLEDGMMENTS

We acknowledge helpful discussions with Jack Westhead andgenerous technical assistance by members of his laboratory staff.

LITERATURE CITED

l. Alicino, J. F. 1946. lodometric method for the assay ofpenicillin preparations. Ind. Eng. Chem. Anal. 18:619-620.

2. Cooper, K. E., and A. H. Linton. 1952. Importance of tem-perature during the early hours of incubation of agar platesin assays. J. Gen. Microbiol. 7:8-17.

3. Davis, W. W., J. M. McGuire, and T. V. Parke. 1949. Somenew procedures and instruments useful for microbiologicalantibiotic testing by diffusion methods. I. A new zone

reader. J. Amer. Pharm. Soc. Sci. Ed. 38:459-462.4. Davis, W. W., and J. M. McGuire. 1949. Some new pro-

cedures and instrument useful for microbiological anti-biotic testing by diffusion methods. II. An automaticdelivery pipette for use with porous discs. J. Amer. Pharm.Ass. Sci. Ed. 38:462-463.

5. Gavin, J. 1956. Microbiological process report. Analyticalmicrobiology. II. The diffusion methods. AppI. Micro-biol. 5:25-33.

6. Gavin, J. 1956. Microbiological process report. Analyticalmicrobiology. III. Turbidimetric methods. Appi. Micro-biol. 5:235-243.

7. Humphrey, J. H., and J. W. Lightbown. 1952. A general theoryfor plate assay of antibiotics with some practical applica-tions. J. Gen. Microbiol. 7:120-143.

8. Kuzel, N. R., and F. W. Kavanaugh. 1971. Automated sys-tem for microbiology. It. Construction of system andevaluation of antibiotics and vitamins. J. Pharm. Sci.60:767-773.

9. Niedermayer, A. O., F. M. Russo-Alesi, C. A. Lendzian,and J. M. Kelly. 1960. Automated system for the continu-ous determination of penicillin in fermentation mediausing hydroxylamine reagent. Anal. Chem. 32:664-666.

10. Simon, J. S., and E. J. Yin. 1970. Microbioassay of anti-microbial agents. Appl. Microbiol. 19:573-579.

11. Thompson, F. V. 1964. The relation between inoculum andzone size in sensitivity tests by the agar diffusion method.

Acta Pathol. Microbiol. Scand. 61:303-316.

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