Name of Laboratory : Chitungwiza Central Hospital Revision Number : 0

Department : Quality Effective Date : 26/09/2012

Document Number : Page number
Title : ANTIMICROBIAL SUSCEPTIBILITY TEST Authorized By :



1.PURPOSE/Test Summary : The purpose of this procedure is to describe the in-vitro
susceptibility testing of bacteria to antimicrobial agents for selection to be used for effective
treatment as described by CLSI.

2.RESPONSIBILITY : Medical Laboratory Scientist/Medical Laboratory Technician.

3.REAGENTS,EQUIPMENT,MATERIALS :
3.1 Reagents
0.5 Mcfarland standard
Saline
3.2Materials
Candle jar (CO2)
Candles
Antimicrobial disks
Screw cap bottle
Stainless steel forceps
Burner
Sterile cotton swabs
Control strains
Calibrated loop
3.3Media
Sterile saline
Peptone water/Mueller Hinton broth
Sheep or Horse blood
Mueller-Hinton agar medium


4.PRINCIPLE
Disc diffusion antimicrobial sensitivity testing is an in-vitro antimicrobial sensitivity testing
based on the inhibition of bacterial growth around the disc of blotting paper. Impregnated with a
known volume and appropriate concentration of an antimicrobial place on a plate of sensitivity
testing agar inoculated with the test organism. The antimicrobial diffuses from the disk into the
medium.
Following overnight incubation, the culture is examined for areas of no growth around the disk
(zones of inhibition). Bacterial strains sensitive to the antimicrobial are inhibited at a distance
from the disk whereas resistant strains grow up to the edge of the disk.



5.METHOD
5.1Inoculation preparation : Growth method
1.Select 4 to 4 well isolated colonies from a fresh culture of the organism on a non inhibitory
agar medium (BA, HB1, 75a and MH).
2.Transfer into a tube/bottle containing 4 5m,l of suitable broth medium (TSB HIB MH).
3.Mix and incubate broth culture at 35
0
C until it achieves or exceeds the 0.5 McFarland standard
or its optical equivalent (2 6 hours).
4.Compare turbidity using a photometric device (spectrophotometer) or visually against a card.

5.2Direct Colony Suspension Method : This is the recommended method.
1.Make a direct saline suspension (4 5 colonies) from a fresh culture.
2.Adjust suspension to match the 05 McFarland turbidity standard as above.

N.B. : Never use undiluted overnight broth culture or other un-standardized inoculums.

5.3Innoculation
1.Inoculate Mueller Hinton agar plate using sterile cotton wool swab within 5 minutes after
adjusting turbidity of inocula.
(N.B. Use HTM for Haemophilus , MH +5% sheep blood for neisseria and streptococcus
pneumonia as media for sensitivity).
2.Allow for excess surface moisture to be absorbed (3 5 minutes but not more than 15
minutes).

5.4Application of Discs
1.Place the discs individually with sterile forceps or mechanical dispensing apparatus.
2.Place no more than 4 discs on a 90mm plate in general.
Do not relocate a disk once placed onto the media.
3.Incubate plates for 16 18 hours at 35
0
C within 15 minutes of applying discs.


QUALITY CONTROL
1.Include appropriate control organism with each test to monitor overall performance of the test
system. Test control strains in the same way using the same condition and methods that are used
to test clinical isolates.
2.Repeat test if controls are out of range if controls continue to be out of range troubleshoot.
NB: When testing is performed daily for each antimicrobial agent/organism combination, 1 out
of every 20 consecutive results may be out of the acceptable range (95% confidence limits). Any
more than 1 out of 20 control result requires corrective action.








6.RESULTS:
Measure and record in millimeters zone diameter of disc for control organisms and compare with
acceptable zone diameter control limits.

Annex
1.Take zone margin as area showing no obvious visible bacterial growth that can be detected by
the unaided eye.
2.Repeat test if controls are out of range or troubleshoot if this continues.
3.Measure and record in millimeters zone diameter including diameter of discs of clinical
isolates.
4.Compare zone sizes with the zone size interpretive criteria.
5.Report results as susceptible (S), intermediate (I) or resistant on agent tested.
NB : Disk diffusion zone diameters correlates well with minimum inhibitory concentrations
(MICS) from standard dilution tests.
































Appendices
Acceptable zone diameter (mm) quality strains limits.

Disc content E. coli
ATCC25922
S.aureaus
ATCC25923
P.aeruginosa
ATCC27853
E. coli
ATCC35218
Amikacin 30g 19 - 26 20 - 26 18 - 26 -
Amoxillin 20/10g 19 25 28 36 - 18 22
Ampicillin 10g 16 22 27 35 - -
Carbenicillin 100g 23 29 - 18 24 -
Ceftizoxime 30g 30 36 27 35 12 17 -
Chloramphenicol 30g 21 27 19 26 - -
Ciprofloxacin 5g 30 40 22 30 25 33 -
Clindamycin 2g - 24 30 - -
Doxycyline 30g 18 24 23 29 - -
Erythromycin 15g - 22 30 - -
Gentamicin 10g 19 26 19 27 16 21 -
Kanamycin 30g 17 25 19 26 - -
Methicillim 5g - 17 22 - -
Nalidixic acid 30g 22 28 - - -
Nitrofurantoin 30g 20 25 18 22 - -
Norfloxacin 300g 28 35 17 28 22 29 -
Penicillin 10g - 26 37 - -
Streptomycin 10g 12 20 14 22 - -
Tetracycline 10g 18 - 25 24 30 - -
Trimethoprim 5g 21 28 19 26 - -
Vancomycin 30g - 17 - 21 - -
























Antimicrobial
agent
Disc content H. influenzae
ATCC49247
H. influenzae
ATCC49766
N. gonorrhoea
ATCC49226
S. pneumonia
ATCC49619
Amoxillin 20/10g 15 23 - - -
Ampicillin 10g 13 21 - - 30 36
Ceftizoxime 30g 29 39 - 42 51 28 34
Chloramphenicol 30g 31 40 - - 23 27
Ciprofloxacin 5g 34 42 - 48 58 -
Clindamycin 2g - - - 19 25
Erythromycin 15g - - - 25 30
Nitrofurantoin 300g - - - 23 29
Norfloxacin 10g - - - 15 21
Penicillin 10units - - 26 34 24 30
Tetracycline 30g 14 22 - 30 42 27 31
Trimethoprim/
sulfamethoxazole
(Cotrimoxazole)
1.25/23/75 g 24 32 - - 20 28
Vancomycin 30g - - - 20 - 27

NB : Ampicillin disc content for urines is 25g; Gentamicin is 30g; Tetracycline is 100g;
Nicene is 30g
NB : Limits have not incorporated all new changes that are in Volume 26.

























Zone diameter (nearest whole mm) interpretive standards for
routine testing of different organisms.

a) Enterobacteriaceae


Test/Report
Group
Antimicrobial
agent
Disc content R I S
A Ampicillin 10g <13 14 16 >17
U Carbenicillin 100g <19 20 22 >23
B Amoxicillin 20/10g <13 14 17 >18
B Ceftizoxime 30g <14 15 19 >20
A Gentamicin 10g <12 13 14 >15
B Amilkacin 30g <14 15 16 >17
C Kanamycin 30g <13 14 17 >18
O Streptomycin 10g <11 12 14 >15
C Tetracycline 30g <14 15 18 >19
O Doxycycline 30g <12 13 15 >16
B Ciprofloxacin 5g <15 16 20 >21
U Norfloxacin 10g <12 13 16 >17
O Nalidixic acid 30g <13 14 18 >19
B Cotrimoxazole 1.25/23.75g <10 11 15 >16
U Trimthoprim 5g <10 11 15 >16
C Chloramphenicol 30g <12 13 17 >18
U Nitrofurantoin 300g <14 15 16 >17












b) Pseudomonas aeruginosa

Test/Report
Group
Antimicrobial
agent
Disc content R I S
U Carbenicillin 100g <13 14 16 >17
U Ceftizoxime 30g <14 15 19 >20
A Gentamicin 10g <12 13 14 >15
B Amilkacin 30g <14 15 16 >17
U Tetracycline 30g <14 15 18 >19
O Doxycycline 30g <12 13 15 >16
B Ciprofloxacin 5g <15 16 20 >21
U Norfloxacin 10g <12 13 16 >17
C Chloramphenicol 30g <12 13 - 17 >18
C Cotrimoxazole 1.25/23.75g <10 11 - 15 >16


c) Enterococcus species

Test/Report
Group
Antimicrobial
agent
Disc
content
R I S
A Penicillin 10 units <14 - >15
A Ampicillin 10g <16 - >17
B Vancomycin 30g <14 15 - 16 >17
C Erythromycin 15g <13 14 - 22 >23
C Tetracycline 30g <14 15 - 18 >19
C Doxycline 30g <12 13 - 15 >16
U Ciprofloxacin 5g <15 16 - 20 >21
U Norfloxacin 10g <12 13 - 16 >17
U Nitrofurantoin 300g <14 15 - 16 >17
C Chloramphenicol 30g <12 13 - 17 >18
C Gentamicin 120g <6 7 9 >10
C Streptomycin 300g <6 7 - 9 >10



d) Haemophilus species

Test/Report
Group
Antimicrobial
agent
Disc content R I S
A Ampicillin 10g <18 19 21 >22
O Amoxicillin 20/10g <19 - >20
B Ceftizoxime 30g - - >20
C Tetracycline 30g <25 26 28 >29
C Ciprofloxacin 5g - - >21
A Cotrimoxazole 1.25/23.75g <10 11 15 >16
B Chloramphenicol 30g <25 26 - 28 >29


e) Neisseria gonorrhea


Test/Report
Group
Antimicrobial
agent
Disc
content
R I S
C Penicilline 10units <26 27 46 >47
C Ceftizoxime 30g - - >38
C Tetracycline 30g <30 31 37 >38
C Ciprofloxacine 5g <27 28 - 40 >41














f) Streptococcus pneumoniae


Test/Report
Group
Antimicrobial
agent
Disc content R I S
A Penicillin 1g <19 20 27 >28
B Vancomycin 30/10g <18 19 25 >26
A Erythromycin 15g - - >17
B Tetracycline 30g <18 19 22 >23
A Cotrimoxazole 1.25/23.75g <15 16 18 >19
C Chloramphenicol 30g <20 - >21
C Clindamycin 2g <15 16 - 18 >19



















g) Streptococcus species other than S. pneumonia


Test/Report
Group
Antimicrobial
agent
Disc
content
R I S
A Penicillin 10units <19 20 27 >28
A Ampicillin 10g <18 19 25 >26
B Vancomycin 30g - - >17
A Erythromycin 15g <15 16 20 >21
O Tetracycline 30g <18 19 22 >23
B Chloramphenicol 30g <17 18 20 >21
B Clindamycin 2g <15 16 - 18 >19

NB : Zone diameter interpretive criteria are subject to change and users
are advised to look for latest editions of the CLSI (Clinical and
Laboratory Standards Institute) formerly CLSI.

Key :

Group A - Primary test and report
Group B - Primary test and report selectively
Group C - Supplementary report selectively
Group U - Supplementary for urine only
Group O - Other