Internal Quality Control (IQC)

Antimicrobial Susceptibility Tests Using

Disk Diffusion
National AMR Surveillance Network, NCDC

National Programme on Containment of Antimicrobial Resistance

National Centre for Disease Control, New Delhi
April 2019
CONTENTS
I. Scope .......................................................................................................................................................... 2

II. Selection of Strains for Quality Control ..................................................................................................... 2

III. Maintenance and Testing of QC Strains..................................................................................................... 3

Figure 1: Flow Chart: Maintenance of QC strains in a bacteriology lab......................................................... 4

IV. Quality Control (QC) Results—Documentation - Zone Diameter ............................................................. 5

V. QC Conversion Plan ................................................................................................................................... 5

1. The 20- or 30-Day Plan.......................................................................................................................... 5

2. The 15-Replicate (3× 5 Day) Plan ......................................................................................................... 5

3. Implementing Weekly Quality Control Testing ..................................................................................... 6

4. Out-of-Range Results with Quality Control Strains and Corrective Action .......................................... 6
Figure 2: Flow Chart: Quality Control Plan in bacteriology lab ...................................................................... 7
5. Weekly Quality Control Testing – Out-of-Range Result Due to Identifiable Error .............................. 8
6. Weekly Quality Control Testing – Out-of-Range Result Not Due to Identifiable Error ....................... 9

7. Additional Corrective Action- Checklist ............................................................................................. 11

8. Reporting Patient Results When Out-of-Range Quality Control Results Are Observed ..................... 12

9. Accuracy Checklist before Reporting the Patient AST Results. .......................................................... 12

10. End-Point Interpretation Control ......................................................................................................... 13
VI. Appendix .................................................................................................................................................. 14

Table 1: Disk Diffusion QC Ranges for QC Strains .................................................................................... 14

Table 2: Disk Diffusion QC Ranges for Non-fastidious QC Strains and β-Lactam Combination Agents. . 15

Table 3: Characteristics of Routine QC Strains for Antimicrobial Susceptibility Tests .............................. 16

Table 4: Conditions for Disk diffusion Antimicrobial Susceptibility Test for Non-fastidious Organisms . 17

Table 5: Plan 15-Replicate (3×5 Day): Acceptance Criteria and Recommended Action* .......................... 18

Table 6: Internal Quality Control for monitoring AST by disk diffusion log sheet..................................... 19

VII. Annexures ................................................................................................................................................ 20

a. Mueller Hinton Agar (MHA) ............................................................................................................... 20

a.1 Media QC recording format ................................................................................................................... 20

b. Antimicrobial Disks & Quality Specifications .................................................................................... 21

c. Storage of Antimicrobial Disks............................................................................................................ 21

 d. Batch or Lot Quality Control ............................................................................................................... 22

e. Inoculum Preparation for Disk Diffusion Tests ................................................................................... 22

VIII. References ................................................................................................................................................ 23

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 Internal Quality Control (IQC) for Antimicrobial Susceptibility Tests
Using Disk Diffusion

Quality control (QC) are procedures used to assure that a test run is valid and results are accurate and
reliable.All participating laboratories in the NCDC AMR surveillance network should have internal
quality control procedures in place. This SOP is intended to provide guidance on performing internal
QC for disk diffusion antimicrobial susceptibility tests (AST) in the department of
Microbiology/Clinical Microbiology laboratory of a hospital.

I. Scope
This SOP gives an overview of the purpose of IQC for Disc Diffusion Antimicrobial Susceptibility
Tests:
i. To ensure all testing materials including media and antibiotic disks are maintained properly by
the laboratory.
ii. Description of the selection, maintenance and testing of QC strains
iii. The recommended frequency for performing QC testing including the amount of testing
required to reduce QC frequency from daily to weekly.
iv. Suggestions for troubleshooting out-of-range results with QC strains
v. Factors to consider before reporting patient results when out-of-range QC results are observed
vi. Guidance for confirming noteworthy or uncommon results encountered when testing patient
isolates

II. Selection of Strains for Quality Control

i. Procure strains from a recognized source (e.g., ATCC, NCTC) for QC of antimicrobial
susceptibility tests (disk diffusion) to ensure the test system is working properly
ii. Select and include routine QC strains as recommended by CLSI document1, 2,
iii. CLSI-recommended QC strains should produce results within the expected range listed in
table 1 to 2 of Appendix.
iv. The characteristics of QC strains provided under the national AMR surveillance programme
are described in table 3 Appendix (routine QC strains).
v. Use the recommended testing conditions for the respective organism group as in table 4
Appendix.
vi. Select QC strains that most closely resemble the organism isolated from the clinical specimen.
vii. For commercial test systems, follow manufacturer’s recommendations for all QC procedures
including the QC strains recommended by the manufacturer

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III. Maintenance and Testing of QC Strains
i. For long-term storage, maintain stock cultures at −20°C or below in 10% to 15% glycerol in
Tryptic soy broth (TSB), alternatively strains can be lyophilized. Temperature lower than
−60°C or liquid nitrogen is preferred for long term storage as some QC strains, particularly
those with plasmid-mediated resistance (e.g., E. coli ATCC 35218), have been shown to lose
the plasmid when stored at temperatures higher than −60°C.
ii. Revive the original frozen or freeze-dried ATCC strain as per instructions in the kit insert,
culture on Tryptic soy agar plate and incubate. Label this as F1 culture plate. FollOw steps as
mentioned in Figure 1
iii. From the F1 culture plate, make at least 12/52 F1 (either 12 monthly or 52 weekly) Tryptic soy
broth (TSB) glycerol stock vials which will last for one year for each QC strain. Label these as
F1/month or F1/week (e.g. F1/Jan; F1/Feb…..F1/Dec; F1/week1;
F1/week2……….F1/week52) QC strain glycerol stock and store them at −20°C or below.
iv. For each month/week use particular F1-month/week stock. For eg. for the month of January
use F1/Jan and for February use F1/Feb and so on; in case weekly stock vials use F1/week1 for
week1 and F1/week52 for last week of the calendar year.
v. Subsequently from one F1/month or F1/week QC strain glycerol stock subculture on TSA
plate/slant to get F2 weekly working QC strain for 1 week. This step is to be repeated every
week. After one month next F1/ month vial or after a week next F1/week vial is to be revived.
vi. Store F2 weekly working QC strain plate/slant at 2 to 8°C or as appropriate for the organism
type
vii. F2 weekly working QC strain will be sub cultured for the preparation of F3 QC strain to obtain
isolated colonies. Always use fresh subcultures (e.g., overnight incubation) for inoculum
suspension preparation for AST.
viii. Discard F2 weekly working QC strain after one week. Repeat step (e), starting with F1/month
or F1/week QC strain glycerol stock for getting next F2 weekly working QC strain for week 2,
and so on.
ix. After a month, discard F1/month or after a week, discard F1/week QC strain glycerol stock.

Note:
 If QC test appears contaminated or if QC results are questionable, it may be necessary
to revive a new F1 QC strain glycerol stock.
 Test QC strains using the same materials and methods that are used to test clinical
isolates.

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 Figure 1: Flow Chart: Maintenance of QC strains in a bacteriology lab

Original ATCC QC Strain

Revive on Tryptic Soy Agar (TSA) plate

Prepare at least 12/ 52 TSB glycerol vials to get F1 12 monthly/ 52 weekly

QC strain glycerol stock (Store at−20°C or below)

Revive and Inoculate F1 for the month/week on TSA

plate/slant for every week

After 1 month/1week end,

Week 1 Week 2 Week 3 Week 4 discard F1 and pull another
F1 Glycerol stock from
freezer

F2 Working Weekly QC Strain

F3 Day 1 F3 Day 2 F3 Day 3 F3 Day 4 F3 Day 5 F3 Day 6

2 2 2
F2 Working weekly Strain will be discarded after 1 week and fresh plate will be taken for next week

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IV. Quality Control (QC) Results—Documentation - Zone
Diameter
Document results from all disk diffusion QC test on a QC log sheet (Sample of QC log sheet at
Annexure Table 6.).
Acceptable zone diameter QC ranges for a single QC test (single-drug/single-organism
combination) for select antibiotics are listed in table 1 and 2 in Appendix.
Results for QC strains must fall within CLSI specified ranges for testing to be acceptable.

Note: Refer CLSI M100-29th Ed, tables 4 A1- A 2 Page 150-157 for other antibiotics and β-lactam
combinations.

Results for QC strains must fall within CLSI specified ranges for testing to be acceptable.

Frequency of Quality Control Testing

Two plans are available to demonstrate satisfactory performance with daily QC testing before
going to weekly QC testing.
1. 20- or 30-day plan
2. 15-replicate (3× 5 day) plan.
Either plan allows a laboratory to perform weekly QC testing once satisfactory performance with daily testing
of QC strains is documented.

V. QC Conversion Plan
(Performance Criteria for Reducing Quality Control Frequency to Weekly)

1. The 20- or 30-Day Plan

i. Test all applicable QC strains for 20 or 30 consecutive test days, and document
results.
ii. When no more than 1 out of 20 or 3 out of 30 consecutive tests turn out to be
outside acceptable limit, performance will be considered satisfactory to proceed to
weekly testing. If not, take corrective action.

2. The 15-Replicate (3× 5 Day) Plan

iii. Test three replicates of each applicable QC strain using individual inoculum
preparations for five consecutive test days and document results.
iv. Upon successful completion of the 15-replicate (3 × 5 day) plan, lab can proceed to
go to weekly QC testing.
v. If completion of the 15-replicate (3 × 5 day) plan is unsuccessful, take corrective
action as appropriate, and continue daily QC testing.(Refer Table 5 Appendix)
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Note: testing on “consecutive test days” means testing of QC strains each day tests are performed on
isolates. It does not refer to calendar day.

3. Implementing Weekly Quality Control Testing

i. Implement Weekly QC testing once satisfactory performance with daily QC testing
has been documented.
ii. Along with weekly testing, QC will be performed whenever reagent component of
the test is changed.
iii. If any weekly test result is out of range, take corrective action.

4. Out-of-Range Results with Quality Control Strains and Corrective Action

i. Out-of-range QC results can be categorized into 1) random, 2) identifiable, or 3)
system related.
ii. Out-of-range results with QC strains due to random or identifiable errors can
usually be resolved by a single repeat of the QC test.
iii. Out-of-range QC tests are often the result of contamination or the use of an
incorrect QC strain; corrective action should first include repeating the test with a
pure culture of a freshly sub cultured QC strain.
iv. Out-of-range QC results that are due to a problem with the test system usually do
not correct when the QC test is repeated and may indicate a serious problem that
can adversely affect patient results. Every out-of-range QC result must be
investigated.

Note: See disk diffusion Troubleshooting Guide in M100, 29th edition

Table 4D for trouble shooting and corrective action for out-of –range results
with QC strains.

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 Figure 2: Flow Chart: Quality Control Plan in bacteriology lab

Quality Control using ATCC

Reconstitute the ATCC Strain

Make 52 aliquots and freeze

Weekly use one aliquot to prepare

working culture

Use 30days/15 days conversion plan

every time new discs/media
obtained
Trouble shoot Use fresh culture
in case of every time while
errors putting QC
Switch to weekly plan procedure
Once conversion achieved

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 5. Weekly Quality Control Testing – Out-of-Range Result Due to Identifiable
Error
i. Identify the reason for an out-of-range result, correct the problem, document the
reason, and retest the QC strain on the day the error is observed. If the repeated
result is within range, no further corrective action is required.
ii. Identifiable reasons for the out-of-control results may include, but are not limited
to:
a. QC strain
i. Use of the wrong QC strain
ii. Improper storage
iii. Inadequate maintenance (e.g., use of the same F2 subculture for > 1 month)
Contamination
iv. Non-viability
v. Changes in the organism (eg, mutation, loss of plasmid)
b. Testing supplies
i. Improper storage or shipping conditions
ii. Contamination
iii. Use of a defective agar plate (eg, too thick or too thin)
iv. Use of damaged (e.g., cracked) plates / expired materials (disks)
c. Testing process
i. Inoculum suspensions incorrectly prepared or adjusted
ii. Inoculum prepared from a plate incubated for the incorrect length of time
Inoculum prepared from differential or selective media containing antimicrobial
agents or other growth-inhibiting compounds
iii. Use of the wrong incubation temperature or conditions
iv. Use of wrong disk, ancillary supplies
v. Improper disk placement (e.g., inadequate contact with the agar) or disk falls off
agar
vi. Incorrect reading or interpretation of test results
vii. Transcription error
d. Equipment
o Not functioning properly or out of calibration (e.g., pipettes)

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 6. Weekly Quality Control Testing – Out-of-Range Result Not Due to
Identifiable Error
If the reason for out-of-range result with the QC strain cannot be identified, perform
corrective action, as follows, to determine if the error is random. Test out-of-range
antimicrobial agent/organism combination on the day error is observed or as soon as F2 or F3
subculture of the QC strain is available.
If the repeat results are in range, evaluate all QC results available for the antimicrobial
agent/organism combination when using the same lot numbers of materials that were used
when the out-of-range QC result was observed. If five acceptable QC results are available, no
additional days of QC testing are needed.
Different scenarios of inconsistent QC results and action to be taken is given below for better
guidance

a. Scenario #1:
Ampicillin E. coli ATCC® Day Lot Lot Result Action
25922; acceptable range: Number Number
15 to 22 mm Week (Disks)
(MHA)

1 1 3564 16481 18

2 1 3564 16481 19

3 1 3564 16481 18

4 1 3564 16481 19

5 1 3564 16481 14 Out of range. Repeat QC same day.

5 2 3564 16481 17 In range. Five acceptable in-range QC

tests for E. coli ATCC® 25922 with
ampicillin disks lot 3564 and MHA lot
16481. Resume weekly QC testing.
For more trouble shooting / suggestive action refer to CLSI guideline M100-29th Ed. Table 4D, Pg.164-166.

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b. Scenario #2:
Ampicillin E. coli Day Lot Lot Result Action
ATCC® 25922; Number Number
acceptable range: 15 (Disks)
(MHA)
to 22 mm Week

1 1 9661 16785 18

2 1 9661 16785 19

3 1 9661 16785 14 Out of range. Repeat QC same day.

3 2 9661 16785 18 In range. Three acceptable in-range QC tests for

E. coli ATCC® 25922 with ampicillin disks lot
9661 and MHA lot 16785. Repeat QC 2 more
consecutive days.

3 3 9661 16785 18 In range.

3 4 9661 16785 17 In range. Five acceptable in-range QC tests for E.

coli ATCC® 25922 with ampicillin disks lot 9661
and MHA lot 16785. Resume weekly QC testing.

c. Scenario#3:
Ampicillin E. coli Day Lot Lot Result Action
ATCC® 25922; Number Number
acceptable range: 15 (Disks)
(MHA)
to 22 mm Week

1 1 8541 16922 17

2 1 8541 16922 21

3 1 8541 16922 28 Out of range. Repeat QC same day.

3 2 8541 16922 21 In range. Three acceptable in-range QC tests for

E. coli ATCC® 25922 with ampicillin disks lot
8541 and MHA lot 16922. Repeat QC 2 more
consecutive days
3 3 8541 16922 30 Out of range. Repeat QC same day.

3 4 8541 16922 28 Out of range. Perform corrective action if results

for a QC strain/antimicrobial agent combination
are out of range and the error is not identifiable on
two consecutive days of testing/if > 3 results for a
QC strain/antimicrobial agent combination are out
of range during 30 consecutive days of testing.
Daily QC to be continued until final resolution is
achieved. See section 4.11

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 7. Additional Corrective Action- Checklist
Additional corrective action is required if repeat results with QC strains are still out of range. It is
possible that the problem is due to a system error rather than a random error. If repeat QC errors are
observed then
iii. Continue Daily QC tests until final resolution of the problem is achieved.
iv. If necessary, obtain a new QC strain (either from stock cultures or a reliable source) and
new lots of materials (including new turbidity standards), possibly from different
manufacturers.
v. If the problem appears to be related to a manufacturer, contact and provide the
manufacturer with the test results and lot numbers of materials used.
vi. It may be helpful to exchange QC strains and materials with another laboratory (may be
Reference Lab) in order to determine the root cause of out-of-range QC results where the
reason is not identifiable

As part of the corrective actions, make certain that you check all of the following:

1. Zone diameters were measured correctly. ☐ Yes ☐ No

2. Turbidity standard is homogeneous and free of clumps. ☐ Yes ☐ No

3. Inoculum suspension was prepared properly. ☐ Yes ☐ No

4. Test materials were stored properly. ☐ Yes ☐ No

5. Test materials have not reached or passed their expiration dates ☐ Yes ☐ No
6. Incubator temperature and atmosphere are within the specified range and ☐ Yes ☐ No
other equipment is properly maintained.
7. QC strain used for testing is acceptable. ☐ Yes ☐ No
8. QC strain maintained as per recommendations ☐ Yes ☐ No
9. The person performing testing has been certified as competent to perform ☐ Yes ☐ No
the test
10. The pH of the medium is in the established range ☐ Yes ☐ No
11. The depth of the agar in the plates is 4 mm. ☐ Yes ☐ No

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 8. Reporting Patient Results When Out-of-Range Quality Control Results Are
Observed

When an out-of-range result occurs when testing QC strains or when corrective action is
necessary, each patient test result must be carefully examined to determine if it can be reliably
reported. Factors to consider may include, but are not limited to:
 Size and direction of QC strain error (e.g., slightly increased OR significantly increased
OR decreased zone size)
 Actual patient result and its proximity to the interpretive breakpoint
 Results with other QC organisms
 Results with other antimicrobial agents
 Usefulness of the particular QC strain/antimicrobial agent as an indicator for a
procedural or storage issue (e.g., inoculum dependent, heat labile)

Options to consider for patient results include:

 Suppress the results for an individual antimicrobial agent
 Review individual patient or cumulative data for unusual patterns
 Use an alternative test method or a reference laboratory until the problem is resolved

9. Accuracy Checklist before Reporting the Patient AST Results.

To ensure that antimicrobial susceptibility test results on patients’ isolates are accurate
review all results, make certain:
1. Results with QC strains are within the acceptable range. ☐ Yes ☐ No
2. Growth is satisfactory. ☐ Yes ☐ No

3. Test is not contaminated (mixed) ☐ Yes ☐ No

4. The overall susceptibility profile or antibiograms are consistent with expected

results for agents tested and the identification of the isolate; check results for ☐ Yes ☐ No
all antimicrobial agents tested and not just those that will be reported.
5. The results from individual antimicrobial agents within a specific drug class
follow the established hierarchy of activity rules (eg, third-generation
cephalosporins are more active than first- or second-generation cephalosporins ☐ Yes ☐ No
against Enterobacteriaceae).

6. The isolate is susceptible to those antimicrobial agents for which resistance has
not been documented (e.g. vancomycin and Streptococcus spp.) and for which ☐ Yes ☐ No
only “susceptible” interpretive criteria exist in CLSI M100 S 29, 2019
7. Atypical resistance, if present, is confirmed. ☐ Yes ☐ No

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 While Testing Patient Isolates confirm the above checklist before Reporting Results

NB: Multiple test parameters are monitored by following the QC recommendations.

However, acceptable results derived from testing QC strains do not guarantee
accurate results when testing patient isolates.

Further confirm unusual or inconsistent results by checking for

 Previous results on the patient (e.g. did the patient previously have the same isolate
with an unusual antibiogram?)
 Previous QC performance (e.g., is there a similar trend or observation with recent QC
testing?)
 Problems with the testing supplies, process, or equipment
 If a reason for the unusual or inconsistent result for the patient’s isolate cannot be
ascertained, a repeat of the susceptibility test or the identification, or both, may be
needed.
 Use an alternative test method or a reference laboratory until the problem is resolved

Note: Refer CLSI M100 29th edition page 212-217, Appendix A, page 204. Suggestions for
Confirming Resistant, Intermediate, or Non-susceptible Antimicrobial Susceptibility Test Results
and Organism Identification

Each laboratory must develop its own policy for confirmation of unusual or
inconsistent AST results. This policy should emphasize those results that may
significantly impact patient care.

10.End-Point Interpretation Control

Monitor end-point interpretation periodically to minimize variation in the interpretation of
zone sizes among observers.
 All laboratory personnel who perform these tests should independently read a selected
set of tests.
 Record the results and compare to the results obtained by an experienced reader; or,
when using QC strains, compare to the expected results from M-100 Tables 4 A1-2.
 Generally, zone measurement readings from several individuals should not vary more
than ± 2 mm.

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VI. Appendix
Table 1: Disk Diffusion QC Ranges for QC Strains
Disk Diffusion QC Ranges ( mm)

Antimicrobial Disk Content E. coli Staphylococcus aureus ATCC Pseudomonas aeruginosa ATCC
Agent ATCC 25922 25923 27853
Amikacin 30 µg 19–26 20–26 18–26
Ampicillin 10 µg 15–22 27–35 –
Azithromycin 15 µg - 21-26 -
Aztreonam 30 µg 28-36 - 23-29
Cefazolin 30 µg 21-27 29-35 -
Cefepime 30 µg 31-37 23-29 25-31
Cefotaxime 30 µg 29-35 25-31 18-22
Cefoxitin 30 µg 23-29 23-29 -
Ceftaroline 30 µg 26-34 26-35 -
Ceftazidime 30 µg 25-32 16-20 22-29
Ceftriaxone 30 µg 29-35 22-28 17-23
Cefuroxime 30 µg 20-26 27-35 -
Chloramphenicol 30 µg 21-27 19-26 -
Ciprofloxacin 5 µg 29-37 22-30 25-33
Clindamycin 2µg - 24-30 -
Doxycycline 30 µg 18-24 23-29 -
Ertapenem 10 µg 29-36 24-31 13-21
Erythromycin 15 µg - 22-30 -
b 200 µg
Fosfomycin 22-30 25-33 -
c 10 µg
Gentamicin 19-26 19-27 17-23
Imipenem 10 µg 26-32 - 20-28
Linezolid 30 µg - 25-32 -
Meropenem 10 µg 28-35 29-37 27-33
Minocycline 30 µg 19-25 25-30 -
Nalidixic acid 30 µg 22-28 - -
Nitrofurantoin 300 µg 20-25 18-22 -
Penicillin 10 units - 26-37 -
Teicoplanin 30 µg - 15-21 -
Tetracycline 30 µg 18-25 24-30 -
Tobramycin 10 µg 18-26 19-29 20-26
TMP/SXT 1.25/23.75 µg 23-29 24-32 -
Vancomycin 30 µg - 17-21 -
a. Refer M100-S29 Tables 4A1-2 Pgs 150-157 for complete list.
When a Commercial test system is used for AST refer to manufacturer’s instructions for QC test recommendations and QC ranges
b. The 200-µg fosfomycin disk should contain 50 µg of glucose-6-phosphate.
c. For control ranges of gentamicin 120-µg and streptomycin 300-µg disk use E.faecalis ATCC 29212(gentamicin:16–23 mm; streptomycin: 14–20 mm).

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Table 2: Disk Diffusion QC Ranges for Non-fastidious QC Strains and β-Lactam
Combination Agents.
(Un supplemented Mueller-Hinton medium)

E. coli P.
ATCC a eruginosa S. aureus E. coli ATCC
25922 ATCC ATCC25923 35218 a,b
Antimicrobial Agent 27853
Disk β- β-lactamase
Content lactamase Inducible
negative, mecA TEM-1
negative AmpC
negative
MIC QC ranges, mm
Amoxicillin-
20/10 µg 18–24 – 28–36 17–22
clavulanate (2:1)
Piperacillin 100 µg 24–30 25–33 – 12–18
Piperacillin- 100/10
24–30 25–33 27–36 24–30
tazobactam µg

QC strain is recommended for routine QC of these combination antibiotic discs

a. Careful attention to organism maintenance (e.g. minimal subcultures) and storage (e.g. −60°C or
below) is especially important for this QC strain because spontaneous loss of the plasmid encoding
the β‐lactamase has been documented. If stored at temperatures above −60°C or if repeatedly sub
cultured, the strain may lose its resistance characteristics and QC results may be outside the
acceptable ranges.

b. To confirm the integrity of the QC strain (E.coli ATCC 35218), test the β‐lactam agent highlighted
in orange by disk diffusion when the strain is first sub cultured from a frozen stock culture. In-range
results for the single agent indicate the QC strain is reliable for QC of β‐lactam combination agents.
It is not necessary to check the QC strain again with a single agent until a new frozen stock culture
is put into use, providing recommendations for handling QC strain are followed.

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 Table 3: Characteristics of Routine QC Strains for Antimicrobial Susceptibility
Tests
Organism Disk Diffusion
QC Strains MIC Tests Other Tests Comments
Characteristics Tests

E. faecalis Nonfastidious • Vancomycin Assess suitability of

ATCC 29212 gram- positive agar medium for
bacteria sulfonamide or
• HLAR tests
trimethoprim disk
• High-level diffusion tests a
mupirocin
resistance MIC
test
Escherichia coli  β-lactamase  Nonfastidious  Nonfastidious
ATCC 25922 negative gram-negative gram- negative
bacteria bacteria

E. coli  TEM-1  β -lactam  β -lactam

ATCC 35218 combination combination
agents agents
Pseudomonas  Inducible AmpC  Nonfastidious  Nonfastidious  Assess suitability
aeruginosa β-lactamase gram-negative gram- negative of cation content
ATCC 27853b bacteria bacteria in each batch/lot
of CAMHB.

Staphylococcus  β-lactamase  Nonfastidious  High-level  Little value in

aureus negative gram-positive mupirocin MIC testing due
ATCC 25923  mecA negative bacteria resistance disk to its extreme
 mupA negative diffusion test susceptibility to
 Inducible most drugs
clindamycin
resistance disk
diffusion test (D-
zone test)
S. aureus  Weak β -  Nonfastidious  Oxacillin salt  Assess suitability
ATCC 29213 lactamase– gram- positive agar of cation content
producing strain bacteria  High-level in each batch/lot
 mecA negative mupirocin of MHB for
 mupA negative resistance MIC Daptomycin broth
test microdilution
a.
 Inducible
Disk diffusion and MIC end points should be easy to read as 80% or greater reduction in growth if the medium has acceptable levels of thymidine.
b. May develop resistance to β -lactam antimicrobial agents after repeated subcultures. clindamycin
c. Minimize this risk by sub culturing from a frozen stock culture at least monthly or whenever the strainresistance MIC
demonstrates results outside the
acceptable range. test
 Penicillin zone-
edge test

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 Table 4: Conditions for Disk diffusion Antimicrobial Susceptibility Test for Non-
fastidious Organisms
(Including routine QC Strains)

Organism/ CLSI Medi 0.5 McFarland Incubation Incubation Minimal QC Required

Organism Group M100 um Inoculum Time
Table
Direct colony Escherichia coli ATCC
suspension in 25922
broth or saline,
or growth P. aeruginosa ATCC
Enterobacteriacea method 35°C± 2°C; ambient 27853
2A MHA 16–18 hours
e air for carbapenems

E. coli ATCC 35218 (for

β-lactam/β-lactamase
inhibitor combinations)
Direct colony P. aeruginosa ATCC
suspension in 27853
broth or saline,
Pseudomonas
2B-1 MHA or growth 35°C± 2°C; ambient
16–18 hours E. coli ATCC 35218 (for β
aeruginosa method air -lactam/ β -lactamase
inhibitor combinations)

Direct colony P. aeruginosa ATCC

suspension in 27853
broth or saline,
or growth E. coli ATCC 25922 for
Acinetobacter method 35°C± 2°C; ambient tetracyclines and
2B-2 MHA 20–24 hours
spp. air trimethoprim-
sulfamethoxazole
E. coli ATCC® 35218 (for
β -lactam/ β -lactamase
inhibitor combinations)
Direct colony 35°C ± 2°C; ambient
16–18 hours
*Staphylococcus suspension in air (Testing at Staphylococcus aureus
24 hours for
spp. 2C MHA broth or saline temperatures above ATCC 25923
cefoxitin with
35°C may not detect
CoNS
MRS)
Direct colony Staphylococcus aureus
suspension in 16–18 hours ATCC 25923
**Enterococcus 35°C± 2°C; ambient
2D MHA broth or saline, 24 hours for
spp air
or growth vancomycin
method
* Examine linezolid zones carefully with transmitted light; Vancomycin disk diffusion test is not recommended for S.aureus and CoNS

** Examine vancomycin zones carefully with transmitted light for small colonies or haze inside the zones of inhibition; any growth =resistance

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Table 5: Plan 15-Replicate (3×5 Day): Acceptance Criteria and Recommended
Action*
Number Out of Range Conclusion From Initial Number Out of Range Conclusion After Repeat
With Initial Testing Testing (Based on 15 After Repeat Testing Testing
(Based on 15 Replicates) Replicates) (Based on All 30
Replicates)
0–1 Plan is successful. N/A N/A
Convert to weekly
QC testing
2–3 Test another 3 2–3 Plan is successful.
replicates for 5 days Convert to weekly QC
testing
≥4 Plan fails. Investigate ≥4 Plan fails. Investigate and
and take corrective take corrective action as
action as appropriate. appropriate. Continue QC
Continue QC each each test day
test day
*Assess each QC strain/antimicrobial agent combination separately.

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 Table 6: Internal Quality Control for monitoring AST by disk diffusion log sheet
Disk diffusion QC ranges (in mm)
Antimicrobial Disk
Lot no. E. coli Staph aureus Pseud E. coli
agent content ATCC 25922 ATCC 25923 aeruginosa ATCC 35218
ATCC 27853

Amikacin 30 µg 19–26 20–26 18–26

Ampicillin 10 µg 15–22 27–35

Azithromycin 15 µg 21-26
Aztreonam 30 µg 28-36 23-29
Cefazolin 30 µg 21-27 29-35
Cefepime 30 µg 31-37 23-29 25-31
Cefotaxime 30 µg 29-35 25-31 18-22
Cefoxitin 30 µg 23-29 23-29
Ceftazidime 30 µg 25-32 16-20 22-29
Ceftriaxone 30 µg 29-35 22-28 17-23
Cefuroxime 30 µg 20-26 27-35
Chloramphenicol 30 µg 21-27 19-26
Ciprofloxacin 5 µg 29-37 22-30 25-33
Clindamycin 2 µg 24-30
Coamoxyclav 20/10 µg 18–24 28–36 17–22
Cotrimoxazole 1.25/23.75 µg 23-29 24-32
Doxycycline 30 µg 18-24 23-29
Ertapenem 10 µg 29-36 24-31 13-21
Erythromycin 15 µg 22-30
1 200 µg 22-30 25-33
Fosfomycin
2 10 µg 19-26 19-27 17-23
Gentamicin
Imipenem 10 µg 26-32 20-28
Linezolid 30 µg 25-32
Meropenem 10 µg 28-35 29-37 27-33
Nitrofurantoin 300 µg 20-25 18-22
Norfloxacin 10 µg 28-35 17-28 22-29
Penicillin 10 units 26-37
Piperacillin 100 µg 24–30 25–33 12–18
Piperacillin- 100/10 µg 24–30 25–33 27–36 24–30
Tazobactam
Teicoplanin 30 µg 15-21
Tetracycline 30 µg 18-25 24-30
Tobramycin 10 µg 18-26 19-29 20-26
Vancomycin 30 µg 17-21

1 Fosfomycin 200-µg disk should contain 50 µg of glucose-6-phosphate

2 For Gentamicin HLAR use 120-µg disk (& to monitor MH agar quality) use E. faecalis ATCC 29212; QC range: 16–23 mm

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VII. Annexures

Guidance on MHA and Disks

a. Mueller Hinton Agar (MHA)
a. Prepare MHA from a commercially available dehydrated base according to the
manufacturer’s instructions.
b. Immediately after autoclaving, allow the agar to cool in a 45 to 50°C water bath.
c. Pour freshly prepared and cooled medium into glass or plastic flat-bottomed Petri on a
level, horizontal surface to give a uniform depth of approximately 4 mm (60 to 70 mL of
medium for plates with a diameter of 150 mm and 25 to 30 mL for plates with a diameter
of 100 mm).
d. Fresh plates should be used the same day or stored in a refrigerator (2- 8ºC).
e. Examine a representative sample of each batch of plates for sterility by incubating at
35°C ± 2°C for 24 hours or longer
f. Plates are stable for seven days, but could have a longer shelf life if precautions are
taken to prevent drying (wrapped in plastic to minimize evaporation) and QC is in range
at the time of testing.
g. Just before use, if excess moisture is visible on the surface, plates should be placed in an
incubator (35ºC) or, with lids ajar, in a laminar-flow hood at room temperature until the
moisture evaporates (usually 10 to 30 minutes).
h. Check the pH of each batch of MHA as reference in the a.1 QC reporting format below.
i. The pH of MHA should be between 7.2 and 7.4 at room temperature (refer to CLSI
guidelines M02 and manufacturer instructions for further details).
j. If the pH is less than 7.2 certain drugs will appear to lose potency (eg, aminoglycosides,
macrolides), whereas other antimicrobial agents may appear to have excessive activity
(e.g., tetracycline). If the pH is greater than 7.4, the opposite effects can be expected.

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 a.1 Media QC recording format

Media QC record
Name of the medium
ATCC strains used Expected reactions
Strain 1
Strain 2
Strain 3
Acceptable Expected reactions obtained
Unacceptable Expected reactions not obtained

Date of preparation Lot number Expiry Autoclave PH Physical Sterility Growth Reviewed
Date examination check check with by
ATCC
strains
Hold time Temperature

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For each batch of media prepared, in addition to fertility check, perform following quality control

Positive controls QC Strain Tested for Expected results

Escherichia coli ATCC®

Nonfastidious gram-negative bacteria Good growth
25922
Nonfastidious gram-negative bacteria
Pseudomonas
Assess suitability of cation content in Good growth
aeruginosa ATCC® 27853
each batch/lot of CAMHB
Staphylococcus
Nonfastidious gram-positive bacteria Good growth
aureus ATCC® 25923
E. faecalis ATCC 29212 Assess suitability of medium for
sulfonamide or trimethoprim disk
diffusion tests
Negative control Un-Inoculated medium No growth

b. Antimicrobial Disks & Quality Specifications

Purchase Disks from a reliable commercial vendor.
a) Check for following at the time of receiving:
 The disks should be accompanied, at minimum, by a certificate of analysis stating the
content of the disks.
 Lot number, expiration date, and assurance that they were tested and performed according
to established QC specifications.

c. Storage of Antimicrobial Disks

a. Refrigerate the cartridges at 8°C or below or freeze at −14°C or below in a non-frost-free
freezer until needed.
b. Sealed packages of disks that contain drugs from the ß-lactam class should be stored
frozen (-20ºC), except for a small working supply, which may be refrigerated for at most
one week. Some labile agents (e.g, imipenem, cefaclor and clavulanic acid combinations)
may retain greater stability if stored frozen until the day of use.
c. Remove the disk cartridges from the refrigerator or freezer 1–2 hours before use to
equilibrate to room temperature before use.
d. Once a cartridge of disks has been removed from its sealed package, place it in a tightly
sealed, desiccated container for storage.
e. Refrigerate the container with the disk –dispensing apparatus when not in use.
f. Use only those disks that have not reached the expiry date stated on the label. Discard
disks when they reach the expiry date.

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d. Batch or Lot Quality Control
a. Check each batch or new lot of MHA /antibiotic discs with the appropriate QC strains
before or concurrent with their first use for testing patient isolates. Performance tests with
each lot of MHA must conform to the control limits of disk QC ranges for organisms and
antibiotics.
b. If zone diameter measurements obtained do not fall within acceptable range (Table 2-3
Appendix B) proceed with corrective action.
c. From each batch of prepared media, keep MHA plates and other agar media not
containing antimicrobials overnight at 37ºC for sterility checking prior to inoculation.
d. Maintain records to include, at a minimum, the lot numbers, expiry dates, and date of use
of all materials and reagents used in performing susceptibility tests.

e. Inoculum Preparation for Disk Diffusion Tests

i. Turbidity Standard for Inoculum Preparation

a. Use a BaSO4 turbidity standard equivalent to a 0.5 McFarland standard to standardize the
inoculum for susceptibility testing.
b. Verify the correct density (0.08 to 0.13) of the standard using a spectrophotometer at
absorbance 625nm.
c. The density of BaSO4 standard must be verified monthly, otherwise should be made
fresh. For further details see Annexure

ii. Direct colony suspension method for Inoculum Preparation

Prepare a saline suspension of the isolate from an overnight incubated agar plate (use a
nonselective medium, such as blood agar) to obtain 0.5 McFarland turbidity (1.5 x 108
cfu/ ml of E. coli ATCC®25922).
iii. Growth method for Inoculum Preparation
a. With a sterile straight wire touch the top of each of four to five colonies of the same
morphological type, and inoculate tryptic soya or any suitable broth.
b. Incubate tube at 35ºC till turbidity of 0.5 McFarland tube or more is achieved (usually 2
to 6 hrs). Then with sterile normal saline adjust turbidity to exactly 0.5 McFarland.
c. Use of the spectrophotometric method is preferable over visual determination.
d. For visual comparison with the 0.5 McFarland standard, look through the broths in
transmitted light against a white background with contrasting black lines.

iv. Inoculation of Test Plates

a. Inoculate MHA plate within 15 minutes of adjusting the inoculum turbidity to 0.5
McFarland standards.
b. Dip a sterile cotton swab into the suspension, rotate several times, and gently press onto
the inside wall of the tube above the fluid level to remove excess inoculum from the
swab.

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 c. Inoculate the dried surface of an MHA plate by streaking three times over the entire
surface of the agar, with the plate rotated approximately 60o each time to ensure even
distribution of the inoculum.
d. Make a final sweep of the swab around the agar rim. The lid may be left ajar for 3 to 5
minutes but no longer than 15 minutes to allow any excess surface moisture to be
absorbed before the drug-impregnated discs are applied.

v. Application of Disks to Inoculated Agar Plates

e. Selected antimicrobial discs should be dispensed evenly onto the agar plate individually
or with dispending apparatus.
f. They discs must be distributed evenly so they are no closer than 24mm from centre to
centre.
g. No more than 12 discs are applied on a 150 mm plate or 6 discs on a 100 mm plate or 5
discs on a 90mm plate
h. Each disc must be pressed down to ensure complete contact with the agar surface.
i. Dispensing too near to the edge of the plate should be avoided.
j. Because some of the drugs diffuse instantaneously, a disc should not be relocated once it
has come in contact with the agar surface.
k. Incubation– Invert the plates and place in an incubator set at 35º ± 2ºC within 15
minutes after discs are applied.

VIII. References
1. CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—
Twelfth Edition. CLSI document M02-A12. Wayne, PA: Clinical and Laboratory Standards
Institute; 2015.
2. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-eighth edition.
CLSI document M100-A28 edition. Wayne, PA: Clinical and Laboratory Standards Institute;
2018.

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