Culture Media used for Antimicrobial Testing according to the standard guideline CLSI.

1. MEDIA USED FOR AST
TESTING
Tuba Bashir

2. •Disk diffusion method
•Agar dilution method
•Broth microdilution
Methods of Performing AST

3. Media Used for DST testing
CLSI
Mueller-Hinton agar (MHA)
Mueller-Hinton agar (MHA) with 5% sheep blood
Haemophilus Test Medium (HTM)
EUCAST
Mueller-Hinton agar (MHA)
Mueller-Hinton + 5% mechanically defibrinated horse blood + 20 mg/L
β-NAD
BSAC 2015
Iso-Sensitest agar
Iso-Sensitest agar supplemented with 5% defibrinated horse blood
(ISA + %5 horse blood + 20mg/L NAD may also be used)

4. Requirements of Isolated Organisms
Organism Medium Inoculum Incubation
time
Incubation
Condition
Reference
Enterobacteriaceae Mueller-Hinton
agar (MHA)
Growth Method or Direct
colony suspension,
equivalent to a 0.5
McFarland standard
16 -18 hours 35 °C ± 2;
ambient air
CLSI 2016
Pseudomonas aeruginosa Mueller-Hinton
agar (MHA)
Growth Method or Direct
colony suspension,
equivalent to a 0.5
McFarland standard
16 -18 hours 35 °C ± 2;
ambient air
CLSI 2016
Stenotrophomonas
maltophilia
Mueller-Hinton
agar (MHA)
Growth Method or Direct
colony suspension,
equivalent to a 0.5
McFarland standard
20-24
hours
35 °C ± 2;
ambient air
CLSI 2016
Acinetobacter spp. Mueller-Hinton
agar (MHA)
Growth Method or Direct
colony suspension,
equivalent to a 0.5
McFarland standard
20-24
hours
35 °C ± 2;
ambient air
CLSI 2016
Burkholderia cepacia
complex
Mueller-Hinton
agar (MHA)
Growth Method or Direct
colony suspension,
equivalent to a 0.5
McFarland standard
20-24
hours
35 °C ± 2;
ambient air
CLSI 2016
Staphylococcus spp. Mueller-Hinton
agar (MHA)
Direct colony suspension,
equivalent to a 0.5
McFarland standard
16 -18 hours;
24 hours CoNS
and Cefoxitin
35 °C ± 2;
ambient air
CLSI 2016

5. Enterococcus spp. Mueller-Hinton
agar (MHA)
Growth Method or Direct
colony suspension,
equivalent to a 0.5
McFarland standard
20-24hours 35 °C ± 2;
ambient air
CLSI 2016
Streptococcus spp. β –
Hemolytic Group
Mueller-Hinton
agar (MHA) with
5% sheep blood
Direct colony suspension,
equivalent to a 0.5
McFarland standard
20-24
hours
35 °C ± 2;
5% CO²
CLSI 2016
Streptococcus pneumoniae Mueller-Hinton
agar (MHA) with
5% sheep blood
Direct colony suspension
(18-20 hr. blood agar plate),
equivalent to a 0.5
McFarland standard
24hours 35 °C ± 2;
5% CO²
CLSI 2016
Streptococcus spp.
Viridans Group
Mueller-Hinton
agar (MHA) with
5% sheep blood
Direct colony suspension
(18-20 hr. blood agar plate),
equivalent to a 0.5
McFarland standard
24hours 35 °C ± 2;
5% CO²
CLSI 2016
Neisseria meningitidis Mueller-Hinton
agar (MHA) with
5% sheep blood
Direct colony suspension
(18-20 hr. Chocolate agar
plate preferably, blood agar
may be used),equivalent to
a 0.5 McFarland standard
24hours 35 °C ± 2;
5% CO²
CLSI 2016
Haemophilus influenzae
and Haemophilus
parainfluenzae
Haemophilus
Test Medium
(HTM)
Direct colony suspension,
equivalent to a 0.5
McFarland standard
16 -18 hours 35 °C ± 2;
5% CO²
CLSI 2016
The suspension should optimally be used within 15 min and always within 60 min of preparation.
Note: Chocolate Agar is used in replace of Haemophilus Test Medium (HTM)
Human Blood (expired blood bag-from blood bank) in replace of sheep blood

6. Mueller-Hinton agar
it is a non-selective, non-differential
medium
it contains starch. Starch is known to absorb
toxins released from bacteria, so that they
cannot interfere with the antibiotics
it is a loose agar. This allows for better
diffusion of the antibiotics than most other
plates. A better diffusion leads to a true
zone of inhibition.

7. Media Preparation
Materials
•Mueller Hinton dehydrated powder
•Weighing balance, weighing boats, spatula, flasks,
thermometer, sterile petri dishes, autoclave, autoclave tape,
pH meter, water bath
•Distilled water

8. Procedure
Weigh Mueller Hinton agar according to the instructions given on the label of the
media powder. Suspend the powder in distilled water. completely dissolve the
powder.
Autoclave at 121oC for 15-20 minutes.
Immediately after autoclaving, allow the agar to cool to 50oC waterbath.
Pour the freshly prepared and cooled medium into sterile petri dishes on a level
horizontal surface to give a uniform depth of approximately 4 mm.
This corresponds to 60 ml to 70 ml of medium for plates with a diameter of 150
mm
25 - 30 ml for plates with a diameter of 100 mm
& 20 ml for 90mm plate.
Allow the agar plates to cool further to room temperature.
Note: Media Bottles labelled with media prepare (MHA), on autoclave tape.
Media dispensing is best done inside the BSC to minimize contamination.

9. TO PREPARE MUELLER HINTON AGAR WITH SHEEP BLOOD (MHB): Cool
medium to 50oC and aseptically add 5% sterile defibrinated sheep blood.
Mix well.
•Check the pH of each batch of MHA and MHB when the medium is
prepared. The agar medium should have a pH of 7.3 ± 0.1 at room
temperature.
•If the pH is outside the range, the batch of the Mueller Hinton plates
should be discarded and a new batch of plates prepared.
•If the pH for every batch is too high or low, the entire lot of
dehydrated medium may have to be returned to the manufacturer as
unsatisfactory.
•Allow the medium to cool further to room temperature to remove
excess moisture.
Do not leave lids ajar because this medium is easily
contaminated.
a.Label plates with media name and preparation date.
b.Wrap plates in plastic bags, 10 plates per bag. Leave appropriate
number of plates outside for quality control (QC).
c.Label media bags with media name, preparation date, expiry date,
and storage temperature.
d.Record media preparation on appropriate form.
e.Perform media QC.

10. Storage
Store wrapped plates at 2o to 8oC for up to 8 weeks.
Prior to inoculation with patient specimens, prepared media that have been
refrigerated should be removed from refrigeration and equilibrated to room
temperature. This is to allow water of condensation to evaporate or dissipate and to
avoid temperature shock to the inoculum.
Just before use, if excess moisture is on the surface, MHA plates can be placed in
the incubator (35o to 37oC) until the moisture evaporates (usually 10–30 minutes).
•Quality Control
For MHA, the media preparation record is merged with the media QC form.
•Incubate at least one un inoculated agar plate from each batch or lot
overnight or longer to verify sterility of the medium.
•Test each new batch or lot of agar plates or disks with the appropriate QC
strains (see antimicrobial susceptibility testing AST QC forms) to determine if
zone sizes obtained with the batch or lot fall within the expected range (see
M100 table).
•Do not use media with unacceptable QC results. Document QC failures and
corrective action taken. Inform tech-in-charge of all QC failures.
Inoculation Procedure
Refer to SOP for Antimicrobial Susceptibility Testing AST
Interpretation of Results:
Refer to SOP for Antimicrobial Susceptibility Testing

11. Procedural Notes
•When dissolving media base, add the exact weight to approximately one
half of the volume of purified water. After thorough mixing, add the
remainder of the water with care being taken to wash down the sides of the
container.
•Dissolution of the media base is enhanced by allowing agar preparations to
stand for 5 minutes with occasional agitation Agar plates stored in
refrigerator for several days should be wrapped in plastic bags prior to
refrigeration to prevent moisture loss.
•Mueller Hinton agar deeper than 4 mm may cause false resistant results,
and agar less than 4 mm deep may be associated with a false susceptibility
report.
•A pH outside the range of 7.3 ± 0.1 at 25oC may adversely affect
susceptibility test results. If the pH is less than 7.2, certain drugs will
appear to lose potency (aminoglycoside and macrolides); while other agents
may appear to have excessive activity (e.g., tetracyclines). If the pH is greater
that 7.4, the opposite effects can be expected.

12. Preparation
date
Media Powder AST QC
Result
(Passed/Faile
d)
Sterility test
(Growth/No
growth)
Quantit
y
prepare
d
pH
Prepared by:
Tech
Initial/date
Supervis
or
initial/dat
e
Manufactur
er
# Expiry date
MICROBIOLOGY LABORATORY
Media Preparation and Quality Control Record: Mueller Hinton Agar
Test for sterility: Incubate uninoculated media at 35oC for 48 hours. Acceptable result - No growth
QC Organisms and Expected Results: See weekly AST QC form
QC Procedure: See AST procedure
QC Frequency: Perform QC on each batch of prepared media.
If QC failed, do not use batch of media. Inform supervisor immediately. Document all QC failures and corrective action taken.
QC failure/Corrective Action:
_____________________________________________________________________________________________________
__________________________________________________________________________________________________________
4.2 Media QC Form: Mueller Hinton Agar

13. Automated Media Preparation
Mediclave 9
APS 320

14. Quality tests
chemical and biological parameter
checked to ensure end products
meet product quality specification,
packaging,labelling and storage are
important

15. Organism Strain Characteristics
Enterobacteriaceae Escherichia coli ATCC 25922
Pseudomonas aeruginosa 27853
Escherichia coli ATCC 35218
Susceptible, wild-type
(for Carbapenems)
(for β-lactam/ β –lactamase inhibitor
combinations)
Pseudomonas aeruginosa Pseudomonas aeruginosa 27853
Escherichia coli ATCC 35218
(for Carbapenems)
(for β-lactam/ β –lactamase inhibitor
combinations)
Acinetobacter specie Pseudomonas aeruginosa 27853
Escherichia coli ATCC 35218
Escherichia coli ATCC 25922
Susceptible, wild-type
(for β-lactam/ β –lactamase inhibitor
combinations)
(for tetracyclines and trimethoprim-
sulfamethoxazole)
Burkholderia cepacia complex Pseudomonas aeruginosa 27853
Escherichia coli ATCC 35218
Escherichia coli ATCC 25922
Susceptible, wild-type
(for β-lactam/ β –lactamase inhibitor
combinations)
(for Chloramphenicol, minocycline, and
trimethoprim-sulfamethoxazole)
Stenotrophomonas maltophilia Pseudomonas aeruginosa 27853
Escherichia coli ATCC 35218
Escherichia coli ATCC 25922
Susceptible, wild-type
(for β-lactam/ β –lactamase inhibitor
combinations)
(for Chloramphenicol, minocycline, and
trimethoprim-sulfamethoxazole)
Staphylococcus aureus Staph. aureus ATCC 25923
Enterococcus spp. Staph. aureus ATCC 25923
Streptococcus pneumoniae Strept. pneumoniae ATCC 49619
Streptococcus spp. β-hemolytic group Strept. pneumoniae ATCC 49619
Streptococcus spp. Viridans hemolytic
group
Strept. pneumoniae ATCC 49619
Neisseria meningitidis Strept. pneumoniae ATCC 49619
See Table 4A & 4B of CLSI (M100-S26) or QC file for QC ranges
Use specified control strains to monitor the performance of the test.