Selection of a reference lot of Mueller Hinton agar

0095-1137/86/070001-06$02.00/0

Copyright © 1986, AmericanSocietyforMicrobiology

Selection

of

a

Reference

Lot

of

Mueller-Hinton Agar

HELEN M.

POLLOCK,'*

ARTHUR L. BARRY,' THOMAS L. GAVAN,3 PETER C. FUCHS,4 SHARON HANSEN,5

CLYDE L. THORNSBERRY,6 HARRY FRANKEL,7 AND SARAH B.

FORSYTHE8t

Departmentof Pathology, University of South Alabama, Mobile, Alabama 36617'; The Clinical Microbiology Institute, Inc., Tualatin, Oregon 970622; Departments of Microbiology3 and Biostatistics,8 The Cleveland Clinic Foundation, Cleveland, Ohio 44106; Department of Pathology, St. Vincent's Hospital, Portland, Oregon 972254; Laboratory Service, VeteransAdministration Medical Center, Baltimore, Maryland

212185;

ClinicalBacteriology Section, Centers for Disease

Control, Atlanta, Georgia 303336;andPfizer Pharmaceuticals, Inc., New York,New York 100177 Received 14 January 1986/Accepted 18 March 1986

A collaborative studywas undertakentoevaluate theperformance of currently marketed Mueller-Hinton agars from seven manufacturers by replicate disk diffusion tests with standard quality control strains. Identification of the manufacturerswasconcealed, and theresulting datawereevaluated for the selection ofa physicalreagentstandard against which the performance of future production lots would be tested and made

to conform. A mediumwasselected which wassufficiently close toexisting National Committee for Clinical Laboratory Standards quality control limits that current interpretive criteria would require minimum modification. Two of the seven lots wereeliminated from further consideration because the final pHs were outsideacceptable limits. The remaining four lots had 96% ofmean zonediameters'2mmfromthose of the chosen lot and 65% of the means were <1 mm from those of the chosen lot for all 28 antimicrobial agent-organism combinations. Manufacturers then attempted to produce new lots ofMueller-Hinton agar whichperformed within the prescribed limits of the chosen lot. One lotperformedincloseconformity with the selectedstandard, but the overall performance of the mediawasessentially thesame asthat of therandomly chosenlotsin the initial study. Itwasconcluded thatoneoftheoriginal sevenlots demonstratedproperties which made it a tentative candidate for a physical reagentstandard and that the use ofa physical reagent

standard inevaluating production lots might aid in stabilizing the performance of Mueller-Hintonagar.

The Bauer-Kirby disk diffusion susceptibility test was developed to provide a simple routine procedure which would reliably predict the efficacy of a given antibiotic regimen in the treatmentofaspecific infectious disease (3). Many technical factors affect the size and clarity ofzones around thedisks, including inoculum density, temperature,

depth ofagar, diskpotency, reading ofzones, and medium (1, 2, 4, 5, 7-14, 16-20).

Ericsson and Sherrisreported results fromalarge collab-orative study in Europe, which included a numberof insti-tutions in differentcountries (8). Variations in results were found with all antibiotics, especially tetracycline. Although themethods and media used in these laboratories werenot

always the same, the studypointed to aneed forimproved standardization of the methods used in disk diffusion sus-ceptibility testing.

During the past decade a great deal ofprofessional time has been expended to develop a standard method which would take intoaccountthetechnical factors that contribute

tovariable results (15). Theseefforts included development ofquality control limits for standardized control strains and delineation of interpretive standards. As these standards have been used, it has become apparent that the original quality control limits did not adequately describe the per-formance ofmedia inthe field (12).

Variations in Mueller-Hinton agarbecamemost apparent

with tetracyclines and aminoglycoside antibiotics (5, 9, 11,

16). Cation contentcontributed to manyof these variations (14), andsome workers have suggested that the magnesium andcalciumcontentof Mueller-Hinton agarbe adjustedtoa predetermined levelatthe time that powder lots are

manu-*Corresponding author.

tPresentaddress: 1238ArmacostAve., Los Angeles, CA 90025.

factured (17). Subsequent studies revealed that thiswas not

apractical approach since the cation contribution by theagar isunpredictable (11). In addition, it appeared that only the available soluble cation interfered with antibiotic activity,

notthetotal cationcontent.The addition ofonecationmay unpredictably move more of another cation into solution from the agar matrix (11). Recent attempts to reduce the thymidine content of Mueller-Hinton agar for clearer sulfonamideandtrimethoprimzoneshadaninhibitoryeffect onthegrowth ofsome staphylococci. Preliminary evidence suggested that this phenomenon may also be related to

problems in detecting resistance to penicillinase-resistant penicii4ins in staphylococci.

More subtle differences began to emerge which were reflected in theperformance of different media with specific classesof antibiotics. Thesevariations appeared tobe man-ufacturer related, with each manufacturer producing lots with a particular pattern of performance for each class of antibiotics. Withcephalosporins and penicillins, media from some manufacturers gave results that were consistently at

thelowextremeofpublished quality control limits with the standard strains; others were at the upper extreme of the acceptablerange.Itwasnotunusual foranantibiotictogive a zone 1 to2 mmoutside thequality control range. Studies wereundertakenthrough the National Committee for Clini-cal Laboratory Standards (NCCLS) consensus mechanism

to adjust the quality control ranges to better reflect the

spectrum ofperformance given by lots of Mueller-Hinton agarthat werebeing marketed and developa new perform-ancetestforevaluating newly manufactured lots. The need

for tighter control on medium performance seemed

appar-ent.

The studywas designedto determine theextent of varia-tionintheperformance of Mueller-Hintonagarlots currently 1

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available in the marketplace and the relationship of that performance to published control limits. Since the number of

variablesinherent in performing the test was high, this study

was undertaken to determine whether it was possible to

select an ideal lot from those in production to serve as a

physical reagent standard against which the performance of

future lots of Mueller-Hinton agar could be compared. Subsequently, a trial was undertaken to determine whether

newly manufacturedlots could be adjusted to perform

sim-ilarly to the chosen standard.

Performance criteria for selecting a physical reagent stan-dard were set up along with a test protocol for defining a

reference medium.The results of these studies are reported

here.

MATERIALSAND METHODS

Standardstrains. New vials oflyophilized quality control strains of Staphylococcus aureus ATCC 25923,Escherichia coli ATCC 25922, and Pseudomonas aerguinosa ATCC 27853 were obtained from the American Type Culture Col-lection. There was one set per participating laboratory. A supplementary study was performed by one ofus (A.L.B.)

with Streptococcusfaecalis ATCC 29212 and ATCC 33186

and two methicillin-resistant strains ofStaphylococcus au-reuschosen for their particular sensitivity to the influence of agar media in detecting resistance to the penicillinase-resistant penicillins by disk diffusion.

Media. Powdered Mueller-Hinton agars (20 kg of each) were donated by the seven manufacturers that provide Mueller-Hinton agar for sale in the United States. These included Acumedia, Baltimore, Md.; BBL Microbiology Systems, Cockeysville, Md.; Difco Laboratories, Detroit,

Mich.; GibcoLaboratories, Madison, Wis.; Inolex Spectrum Diagnostics, Glenwood, Ill.; Oxoid, Columbia, Md.; and Scott Laboratories, Fiskeville, R.I. All lots of agar were shipped by each manufacturer to the Centers for Disease

Control (CDC).

Distribution of media. Bottles for transporting media lots

weredonated byDifcoLaboratories and shipped to the CDC Biologic Products Division. Through the courtesy of Morris

Suggs and R. Knox Harrell, this division served as the collection and distribution point for all media lots. Each manufacturer submitted 20 kg of a representative lot for

testing. At CDC these lots were repackaged in the uniform

bottles donated by Difco Laboratories and given a code

number. Each participating laboratory had a different set of

codenumbers to ensure that investigators could not compare results. In addition, the name of the manufacturer ofeach coded lot was not revealed to any of the investigators or committee members. The codedpowder lotswereshippedto eachof the fiveparticipating laboratories from CDC.

Disks. Disks were provided through the courtesy ofBBL Microbiology Systems. All disks were assayed at greater than 100% stated potency, except for cefoperazone and oxacillin, which had 93 and 97% of the stated potency, respectively. Antibiotics were selected to represent the

spectrumofagents known to be affectedby medium

varia-tions (Table 1).

Laboratories. To eliminate as much variation aspossible,

independent laboratories at different institutions with expe-rience in doing studies of this type and with a history of performing the disk test in a reproducible manner were

selected. To firm up the performance of the standardized

disk susceptibility test, technologists from each of the

par-ticipating laboratories went to the CDC Antibiotic

Investi-gational Laboratory forafinal

briefing

on thedetails ofthe

protocol andtheneedtofollow the

protocol

precisely

andto

correct any

misconceptions

about the

study

design.

Antimicrobial susceptibility test. The standardized disk

diffusion test was

performed

with strict adherence to the

procedures and time restrictions listed in NCCLS M2-A2

(15). All

plates

were

poured

within 24 h of use. Two

representative plates foreach lotofagarmediumweretested

at the same time withthe same

adjusted

inoculum for each

quality control strain. Tests with each strain were initiated

and

completed

before thenextstrainwastestedtominimize time

delays.

The diameter of each zone of inhibition was

measured to the nearest 0.1mmafter16to18hof incubation

at 35°C. All zones were measured

independently

by

two

observers from the back of the

plate.

The

plates

were

illuminated with reflected

light

and read

against

a black

background.

Criteria for selecting the primary reference reagent. The

control limits established in NCCLS Standard M2-A2 and

supplements were assumed to be the best estimate of how

the ideal medium should

perform.

The

primary

reference

reagent was selected

by

the

following

criteria:

(i)

closeness

tothe

existing

midpoint

ofcurrentNCCLScontrol

limits;

(ii)

low

variability

in zone

readings

reflecting clarity

ofzones

and distinctness ofzone

edges; (iii)

ability

of mediumto be

produced

in

plated

form without

adjustment

of

pH;

and

(iv)

abilitytodetectmethicillin-resistant

staphylococci

and

mea-sure

trimethoprim

and sulfonamide

susceptibility

of entero-cocci.

Statisticalevaluation. Statistical

analyses

ofall datawere

done

separately

for each of the 28

microorganism-drug

combinations. The basic

study

design

was a

complex

multifactorial

design.

The modelcontains the

following

five

factors:

(i)

institution,

a random factor with five

levels; (ii)

agar lot, a fixed factor with seven

levels; (iii)

reader,

a

randomfactorwithtwo

levels;

(iv) day,

arandomfactorwith

fivelevels; and (v)

plate,

a randomfactor with twolevels.

Thesevenlotsof Mueller-Hintonagarwerefrom different

manufacturers. Five institutions

(investigator

laboratories)

participated:

CDC,

the Cleveland Clinic

Foundation,

the

Clinical

Microbiology

Institute,

Tualatin, Oreg.,

and St.

Vincent's

Hospital,

Portland,

Oreg.,

and the Veterans

Ad-ministration Medical

Center,

Baltimore.Two

plates

for each

lot ofagarwere tested each

day;

each test was

repeated

on

five days; each oftwo readers from each ofthefive

institu-tionsmeasured and recordedzonediametersonall

plates

for

each

day.

A total of100 measurements wastaken for each

microorganism-drug

combination on each lotofagar.

The mathematical model for the

analysis

of variance

(ANOVA)

may be written

explicitly

as

Yijklm = t+ Oti +

P

+ 'Yfi)k + 8Wi +

4(ijI)m

+

(OLI)1i

+

(P-Y)(i3yk

+

(38))(i)jl

+

(Y8)(i)kl

+

(.Yy)(ijl)km

+

(P-Y8)(i)jkl

where ,u, overall mean; ai, institution

effect;

P,

lot

effect;

'Y(i)k,

reader effect;

8(j)y,

day

effect;

(ùjl)m,

plate

effect;

(OP)ij,

interaction effect between institution and

lot;

(p-y)(i2k,

inter-action effect between lot and

reader;

( interaction

effect between lot and

day;

(Y8)(i)kl,

interaction effect

be-tween reader and

day;

(7f)(ffl)km,

interaction effect between reader and plate; and

(I78)()kI,

interaction effect between

lot, reader, and

day.

We assume that

j'1

pj

= 0 and that

(Xi, 'Y(i)kg 8(i)lg4ij/)m,

(afflii, (P5^Y)(i)ji"

S)()l

(-Yb)(i)kl,

(-Y()(ijl)km,

and

(8Y)(i)jkl

are

independent

samples

from Gaussian popu-lations with zero means and variances

o2,

C

Y28,

a2"

2) or(t

(Ir2(1,

(y2(P

(Y2(-Y)2(yg,)

), and

o2(y),

respectively.

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TABLE 1. Mean zone diameters from 100 measurements for each antimicrobialagent-organism combination

Mean zone diam (mm)with NCCLS lot no.: Rangeof

Strain anddrug

1234567means

i 2 3 4 5 6 7men

StaphylococcusaureusATCC 25923

Cephalothin 33.2 32.6 32.5 32.5 33.0 32.6 31.5 1.7

Chloramphenicol 22.3 23.6 22.8 22.8 23.8 22.9 23.4 1.5

Clindamycin 23.8 26.1 24.9 25.8 26.1 25.0 25.7 2.3

Erythromycin 23.7 26.3 25.2 25.8 25.8 24.6 26.1 2.6

Gentamicin 22.8 21.2 24.4 25.1 22.7 22.5 22.3 3.9

Oxacillin 21.1 21.1 21.2 21.2 20.0 19.4 20.2 1.8

Penicillin G 32.8 31.9 32.2 32.4 32.4 31.8 31.5 1.3

Tetracycline 25.4 26.4 25.4 25.8 25.7 25.5 23.5 2.9

Vancomycin 17.6 17.1 16.5 17.2 17.9 17.4 17.2 1.4

Escherichia coli ATCC 25922

Amikacin 21.4 21.4 21.0 23.3 22.7 21.5 21.4 1.9

Ampicillin 18.4 18.8 17.5 18.7 19.1 10.0 18.7 2.5

Carbenicillin 22.6 24.3 23.1 23.8 25.1 25.7 24.5 3.1

Cefotaxime 29.4 30.2 29.4 30.4 30.9 31.3 30.3 1.9

Cefoxitin 23.0 24.5 23.6 24.7 25.4 25.6 24.9 2.6

Cephalothin 16.6 18.9 17.1 19.3 19.2 19.5 19.1 2.9

Chloramphenicol 23.4 23.3 22.5 23.7 23.5 24.2 23.0 1.7

Gentamicin 20.8 20.8 21.3 23.7 22.8 21.5 22.2 2.9

Moxalactam 28.5 29.2 28.6 29.5 30.7 30.2 29.5 2.2

Sulfisoxazole 18.9 20.9 19.2 19.5 19.7 23.3 20.3 4.4

Tetracycline 22.4 21.0 20.8 21.5 21.9 23.0 19.1 3.9

Trimethoprim-sulfamethoxazole 25.4 26.7 25.7 25.4 26.8 27.2 25.7 1.8

Pseudomonas aeruginosa ATCC 27853

Amikacin 21.9 20.5 22.4 21.2 22.0 20.7 20.6 1.9

Carbenicillin 20.6 20.9 20.3 20.3 20.1 20.8 20.8 0.8

Cefoperazone 25.2 25.8 25.1 25.1 25.0 25.1 25.0 0.8

Cefotaxime 20.5 20.2 20.3 20.2 20.1 20.1 20.2 0.4

Gentamicin 18.7 17.4 19.8 19.1 19.1 17.8 17.7 2.4

Moxalactam 20.4 21.7 21.1 20.7 20.3 21.0 20.9 1.4

Piperacillin 28.3 28.9 28.3 28.0 28.3 28.4 28.2 0.9

This model was used to testthe

equality

ofthe mean zone

diameters forthelevels ofeach model

component

aswellas

toobtainanestimate ofthezonediametervariance for each

microorganism-drug

combination. To address the issue of

variability

differences between the

lots,

the ANOVA was

done

separately

foreach lot

(the

components

involving

lot

Pj

wereeliminated fromthemodel

specified above).

All

analy-ses were done with the BMDPstatistical software program

P8V,

which

performs

anANOVA for

general

mixed models.

For each lot of agar, the mean zone

diameter,

95%

confidence

intervals,

and control limitswerecalculated.The

zone diameter variance used in

calculating

the confidence

intervals was estimated

by

the mean square ofthe

interac-tion effect between institution and lot. The control limits

were obtained

by using

the concept of a tolerance limit

(which

estimates a range

containing

a

high proportion

of

individualvalues ina

population).

The

following

formula for

the control limits uses a variance estimate that involves

intralaboratory

variability

and the

appropriate degrees

of

freedom associated with the

study design: yi

+

2.24v'@7,

withyi

equal

tothemeanzone diameter ofthe ithlotand

si2

being

the ith lot zone diameter variance estimated

by

the

sum of the variance

component

estimates for

reader, day,

plate,

reader

by

day,

and reader

by plate.

These control

limits covered 95% ofall zone diameters in the

population

with 95% confidence.

Trial

production

of lots

matching performance

ofproposed

reagentreferencestandard.Eachof thesevenmanufacturers

received

samples

of the

proposed

reagentreferencestandard

whichwasselectedbytheabove criteria.Eachmanufacturer

thenattemptedto adjustthe performanceof anewly

manu-facturedlot of agar to match that of the standard. Evaluation

of thenewly manufactured lotswasaccomplished by testing

eachnewlotin parallelwith the referencereagent with the

same adjusted inoculum and lot of disks. The differences

betweenthe means of30replicates tested on the same day

for each of the 28 microorganism-drug combinations were

statistically

analyzed byapairedt test.The tentative criteria

for accepting a lot of agar as essentially identical to the

reference materialwere that the means ofthe lot forall 28

microorganism-drug

combinations be within 1 mm of those

observed with the

proposed

reference mediumobserved at the same time. Each manufacturer submitted the lot which

they feltmostclosely fitthesecriteriatoCDC forevaluation

by

the sameprotocol. Thirty replicateswere selected, since

it wasdetermined, by using variance estimates fromthefirst

study,

that this would be the number required for a high

probability

ofdetectinga1-mmdifference inmeansbetween

twoagarlotsrunin parallel

(at

= , = 0.01).

RESULTS

pH measurements. Thefive independentlaboratories mea-sured the pH ofeach batch ofagar poured. The

measure-ments wereremarkably similarandrevealed that lots 1 and 7 should beeliminated fromconsiderationbecause they did

not come within the requiredpH range of 7.2 to 7.4 (Table

2).

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TABLE 2. Pooled pH measurements for seven lots of Mueller-Hinton agar at five independent laboratories

pH (n= 25determinations) Lot no.

Mean SD Minimum Maximum

1 7.05 0.05 6.95 7.20

2 7.28 0.05 7.20 7.40

3 7.28 0.04 7.20 7.35

4 7.36 0.05 7.25 7.45

5 7.30 0.06 7.15 7.40

6 7.20 0.06 7.10 7.33

7 7.41 0.06 7.30 7.52

Mean zone diameters. Mean zone diameters for each

microorganism-drugcombinationwerecalculated by pooling

data from allfive institutions, representing 100 readings for eachof the 28 microorganism-drug combinations (five insti-tutions xtworeaders x 5days x 2plates). Themeansof the zonediametersfor each antibiotic andorganism combination are found in Table 1 together with the range of means. Greater differences occurred dueto medium variation with someantibiotics than with others.

With S. aureus ATCC 25923, gentamicin, erythromycin, clindamycin, and tetracycline had the greatest spread of mean zone diameters between agar lots. Variations in the means for E. coli ATCC 25922, attributable in part to

differences in agar lots, were greater with sulfisoxazole, tetracycline, carbenicillin, gentamicin, and cephalothin. Gentamicin and amikacin demonstrated the greatest varia-tion in means associated with differences between lots of agarwith P. aeruginosa ATCC 27853. The spread between

the means onthe sevenlots was neverless than 1mm with

S. aureus ATCC 25923 and E. coli ATCC 25922;the spread ofzonediameters wasasgreatas4.4 mmwithsulfisoxazole and E. coli.

Ranking of lots. Thesevenlotswererankedby thesumof theabsolute differences between themidpoint for each of the 28 drug and organism combinations and the respective NCCLS midpoint. These ranks are presented in Table 3, with arank of 1 indicating the smallest midpoint deviation. Lot 5gavetheclosest fit, while lots 3 and 7were leastclose

to existing midpoints (lots 7 and 1 had pHs outside the

acceptable range).

The sumsof thecalculated control interval widths forthe

28 microorganism-drug combinations were ranked for the

sevenlots, with the lot having the smallestsumgivenarank

of 1 (Table 3). Lot 7 exhibited the smallest amount of

variation, while lot 1 showed the greatest.

The sum of the control interval widths was derived from control limits calculated with a formula for a variance estimateinvolving intralaboratory variability with 95% con-fidencelimits.Thesumofthewidths of the control limits for

all 28 microorganism-drug combinations derived by this

methodaregiven (Table 3).

Comparison of performance of other lots to the one best meeting the criteria for selection. Alllotswere evaluated for similaritiesand differences and for determination of the lot

which best metthe currentNCCLS midpoint standard and hadthe leastvariability(Table 3). Lot 5appearedtofit these

criteria better than any of the other six lots and was tentatively selected asthe newNCCLS reagent standard.

The means for the other six lots were compared to the

meanfor lot 5todeterminetheextentoftheir variation from

this lot (Table 4). Of the 28 means, 13 were significantly

differentfrom those of lot 5 forlots1and2,7 from those of

lot3,and 9fromthoseof lot 4,and11ofthe 28 meansforlots 6and7 were

significantly

different from themeansfor lot5.

Althoughsomeof themeans werestatistically different from

those of lot

5,

92% of allmeanswere within 2mmand65%

werewithin1 mmofthe meansfor lot5. Acomparison ofthe

four lots with thecorrect

pH

revealed that65% of themeans

werewithin <1mmofthe meanof lot5and

96%

werewithin

c2

mm of this lot.

Streptococcus faecalis and

Staphylococcus

aureus testing.

There was wide variation in the

performance

of the seven

lots ofagar with

trimethoprim-sulfamethoxazole

and

Strep-tococcus

faecalis

(ATCC

29212 and ATCC 33186). Lots 3

and 4 gave no zone, while lots

2, 5,

and 7 gave zones

.20

mm and lots 1 and 6gave smaller zones (<20 mm). Other

species produced large

clear zones

of

inhibition when

tri-methoprim-sulfamethoxazole

disks were tested on lot5.

Two strains of methicillin-resistant

Staphylococcus

au-reus were selected

by

one ofus

(C.T.)

for their

particular

sensitivity

to variations in agar media that may prevent

detection of methicillin resistance. Lot 5 demonstrated

def-inite growth

uptothe

edge

of the oxacillin

disk,

whereasthe

otherlots demonstrated variousamountsof

growth

withina

definite zoneofinhibition. Withoneofthe

strains,

methicil-lin disks tested as sensitive on all media. The other strain

wasresistant onlots 1and 5butgaveintermediate (10to 13

mm) zones on the other lots.

Trial production oflots matching performance ofreagent standard. After selection of lot 5 as the

proposed physical

reagent

standard,

eachof thesevenmanufacturerswas

given

some of this lot to

experimentally

manufacture a new

pro-duction lot with

essentially

similar

performance.

The

per-formanceofthefivenewagarlotswas

compared

with that of

lot5 by

performing

30

replicate

tests oneach mediuminone

ofthe reference laboratories

(CDC).

One manufacturer did

not

participate,

and onelot

presented

technical difficulties.

The number ofantimicrobial

agent-organism

combinations

which had greater than 1 mm difference between the mean

zone diameters had not

clearly

decreased with media

sub-mitted by four of the five manufacturers

(Table

5). One

manufacturer, however, improved

its

product sufficiently

to

allow it to be considered a

possible

second reference stan-dard.

DISCUSSION

Variation in the

performance

of lots of Mueller-Hinton

agar led us to

develop

a

tightly

controlled

protocol

which

compared

the

performance

ofagar media

currently

on the market. The

study

was

designed

to determine whether a

TABLE 3. Sums and ranks of the control interval widths and

midpoint deviationsforthe 28microorganism-drugcombinations Widtha Midpointdeviations

Lotno.

Sum Rank Sum Rank

1 153 7 44.5 6.5

2 143 4 33.5 4

3 139 2 44.5 6.5

4 147 6 31.5 3

5 140 3 25.0 1

6 146 5 30.0 2

7 136 1 35.0 5

aWidthof control interval calculatedfromanestimate of variance.Control

interval width is the difference betweenthe lowandhighlimit of thecontrol limits(inmillimeters).

bAbsolutedifferencebetweenmidpointofexistingNCCLS control inter-valsandmidpointof control interval determinedbyusingmethodIII.

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TABLE 4. Absolute value ofdifference of means compared with the proposed reference medium (lot5)

Absolutedifferencesvs lot 5(mm)

Organism Drug

1 2 3 4 6 7

Staphylococcus aureus Cephalothin 0.2 0.4 0.5 0.5 0.4 1.5*

Chloramphenicol 1.5* 0.2 1.0* 1.0* 0.9 0.4

Clindamycin 2.3* 0.0 1.2 0.3 1.1 0.4

Erythromycin 2.1* 0.5 0.6 0.0 1.2* 0.3

Gentamicin 0.1 1.5* 1.7* 2.4* 0.2 0.4

Oxacillin 1.1* 1.1* 1.2* 1.2* 0.6 0.2

Penicillin G 0.4 0.5 0.2 0.0 0.6 0.9

Tetracycline 0.3 0.7 0.3 0.1 0.2 2.2*

Vancomycin 0.3 0.8* 1.4* 0.7* 0.5 0.7*

Escherichia coli Amikacin 1.3* 1.3* 1.7* 0.6 1.2* 1.3*

Ampicillin 0.7 0.3 1.6* 0.4 0.9* 0.4

Carbenicillin 2.5* 0.8* 2.0* 1.3* 0.6 0.6

Cefotaxime 1.5* 0.7 1.5* 0.5 0.4 0.6

Cefoxitin 2.4* 0.9 1.8* 0.7 0.2 0.5

Cephalothin 2.6* 0.3 2.1* 0.1 0.3 0.1

Chloramphenicol 0.1 0.2 1.0* 0.2 0.7* 0.5

Gentamicin 2.0* 2.0* 1.5* 0.9* 1.3* 0.6

Moxalactam 2.2* 1.5* 2.1* 1.2* 0.5 1.2*

Sulfisoxazole 0.8 1.2* 0.5 0.2 3.6* 0.6

Tetracycline 0.5 0.9* 1.1* 0.4 1.1* 3.9*

Trimethoprim- 1.4* 0.1 1.1* 1.4* 0.4 1.1*

sulfamethoxazole

Pseudomonas Amikacin 0.1 1.5* 0.4 0.8* 1.3* 1.4*

aeruginosa Carbenicillin 0.5* 0.8* 0.2 0.2 0.7* 0.7*

Cefoperazone 0.2 0.8 0.1 0.1 0.1 0.0

Cefotaxime 0.4 0.1 0.2 0.1 0.0 0.1

Gentamicin 0.4 1.7* 0.7* 0.0 1.3* 1.4*

Moxalactam 0.1 1.4* 0.8* 0.4 0.7* 0.6*

Piperacillin 0.0 0.6 0.0 0.3 0.1 0.1

a*, Significantatoverall a = 0.05by theBonferroni inequality test for each lot.

physical reagent standard tested in parallel with newly produced lots would enable manufacturers to control their productswith greaterprecision, resulting ingreater uniform-ity in the performance of products from different

manufac-turers. Written control limits which define zone size limits

foreachantimicrobial agent-organismcombination mustbe broad enough to account for a variety of the technical variables that influence measurement of zone diameters as well as inherent differences in performance of

Mueller-Hinton agar from different manufacturers. We propose a performancetestwhichinvolves replicate testing ofa refer-encemedium inparallel witha testlot. Differences between mean zone sizes would beusedtoevaluatemedium variabil-ity and reduce variability resulting from other factors.

The data demonstrated that some changes in quality controlprotocolswerenecessaryatthemanufacturing level. First, the measurements for control strains on currently marketed media were slightly different from existing stan-dards. Secondly, media produced by five manufacturers performed within 2 mm ofthe proposed standard for most

microorganism-drug combinations. Statistical analyses of

the variances indicatedthat amanufacturer would have to

perform 30 replicates on the same day to have 99%

confi-dence ofdetecting1-mmdifferences betweentwolotsofagar runinparallel. This number of replicates exceeded the usual

number oftests performed in manufacturing agarmedia. In addition, current quality control practices compare tested performance against a written rather than a physical stan-dard. The use of a physical standard with an increased

number ofreplicates could theoretically provide

manufac-turerswithabasisforproducingmoreuniform lots of media

and the ability to detect smaller drifts in performance than is currently possible.

Reliable detection of methicillin resistance in

Staphylo-coccusaureuspresentsanumberof technical difficulties. On

currently marketed Mueller-Hinton agar, some of these strains appear to be susceptible with methicillin disks but

resistant (double zones) with oxacillin disks. The character

of the growth within the zones of inhibition appears to be medium dependent with the strains that were tested. This problem requires careful consideration in setting any

stan-TABLE 5. Comparison of means withthose of lot S No.of meansfalling Firstorsecond outsidestandardby:

Manufacturer lot submitteda

>1mm >2 mm

A 1 12 7

2 9 2

B 1 9 0

2 7 3

C 1 16 2

2 10 3

D 1 6 1

2 9 2

E 1 8 2

2 0 0

a Firstlot, Meanzone size of 100readingson theproposedreferencelot comparedwiththat for lot5(5 days x2 readers x 5institutions x 2days); second lot,meanzone size of 30replicatetests on the sameday compared with that for lot5at CDC.

bThe standard was the mean zone of inhibitionobtainedwith lot5of the firstsetof agar lotssubmitted.

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dard for Mueller-Hinton agar. Trimethoprim-sulfamethox-azole susceptibility of Streptococcus faecalis could be

de-termined on only two of the seven lots of Mueller-Hinton

agar.

The major question in developing a physical reagent

standard for Mueller-Hinton agar was whether additional

lotscould be produced which performed essentially the same

as the selected reference lot. In the initial study, overall

performance for the media which had the proper pH was

relatively uniform for production lots, but therewere no data

to demonstrate how closely it was possible to duplicatethe

performance ofa new lot ofagarmedium with areference

reagent available for comparison. Additional studies were

performed to answer that question.

When additional lots ofagar wereproducedand tested in

parallel with the selected physical standard, one

manufac-turersubmitteda mediumwhichwas essentially the same in

performance asthereference lot,thatis,within 1 mmofthe

mean zone size for all microorganism-drug combinations.

Fourmanufacturers submitted lots with performances which

werenotmuch closer to the selected standard than those of

randomlotsthat met thewritten standard.

Changing performance associated with variations in raw

materials over a period of time may be eliminated by maintaining a standard reference medium with which manu-facturers may compare theperformance of future lots. While it appears that different manufacturers may not be able to produce lots of Mueller-Hinton agar with greater similarity inperformance than is currently accomplished, the problem of drift inbehavior of these media may be circumvented by

comparing the performance of new lots with that of a

physicalreagent as wellasagainstwrittencriteria. A

statis-tically valid performancetestbasedonthisprinciple is being

developed.

ACKNOWLEDGMENTS

Wewishtothank LesCunningham, Acumedia; DavidPowerand George Evans,BBLMicrobiologySystems;Aaron L. Lane,Difco;

Mary Nichols, Gibco; VernonL. Olson, Spectrum; Eric Bridson, Oxoid; andTomScott, Scott Laboratories, for their participation in and contributions to this study. We thank the Association of Microbiologic Media Manufacturers for their support and contribu-tions.Weappreciate the help of Morris Suggsand hisgroupatCDC, without whose assistance this entire project would not have been

possible. We also thank George Williams, Cleveland Clinic, for reviewing the manuscript. The administrative assistance of NCCLS wasinvaluable, in particularthatof John Bergen andJohn McCon-nell.

Funds for support of this researchweredonated tothe NCCLS

Microbiology Development Fund.

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