Name of Laboratory : Chitungwiza Central Hospital

Department : Quality
Document Number :
Title : ANTIMICROBIAL SUSCEPTIBILITY TEST

Revision Number : 0
Effective Date : 26/09/2012
Page number
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 350C 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 350C 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
Amikacin
Amoxillin
Ampicillin
Carbenicillin
Ceftizoxime
Chloramphenicol
Ciprofloxacin
Clindamycin
Doxycyline
Erythromycin
Gentamicin
Kanamycin
Methicillim
Nalidixic acid
Nitrofurantoin
Norfloxacin
Penicillin
Streptomycin
Tetracycline
Trimethoprim
Vancomycin

30g
20/10g
10g
100g
30g
30g
5g
2g
30g
15g
10g
30g
5g
30g
30g
300g
10g
10g
10g
5g
30g

E. coli
ATCC25922
19 - 26
19 25
16 22
23 29
30 36
21 27
30 40
18 24
19 26
17 25
22 28
20 25
28 35
12 20
18 - 25
21 28
-

S.aureaus
ATCC25923
20 - 26
28 36
27 35
27 35
19 26
22 30
24 30
23 29
22 30
19 27
19 26
17 22
18 22
17 28
26 37
14 22
24 30
19 26
17 - 21

P.aeruginosa
ATCC27853
18 - 26
18 24
12 17
25 33
16 21
22 29
-

E. coli
ATCC35218
18 22
-

Antimicrobial
agent
Amoxillin
Ampicillin
Ceftizoxime
Chloramphenicol
Ciprofloxacin
Clindamycin
Erythromycin
Nitrofurantoin
Norfloxacin
Penicillin
Tetracycline
Trimethoprim/
sulfamethoxazole
(Cotrimoxazole)
Vancomycin

Disc content
20/10g
10g
30g
30g
5g
2g
15g
300g
10g
10units
30g
1.25/23/75 g

H. influenzae
ATCC49247
15 23
13 21
29 39
31 40
34 42
14 22
24 32

H. influenzae
ATCC49766
-

N. gonorrhoea
ATCC49226
42 51
48 58
26 34
30 42
-

S. pneumonia
ATCC49619
30 36
28 34
23 27
19 25
25 30
23 29
15 21
24 30
27 31
20 28

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
A
U
B
B
A
B
C
O
C
O
B
U
O
B
U
C
U

Antimicrobial
agent
Ampicillin
Carbenicillin
Amoxicillin
Ceftizoxime
Gentamicin
Amilkacin
Kanamycin
Streptomycin
Tetracycline
Doxycycline
Ciprofloxacin
Norfloxacin
Nalidixic acid
Cotrimoxazole
Trimthoprim
Chloramphenicol
Nitrofurantoin

Disc content R

10g
100g
20/10g
30g
10g
30g
30g
10g
30g
30g
5g
10g
30g
1.25/23.75g
5g
30g
300g

14 16
20 22
14 17
15 19
13 14
15 16
14 17
12 14
15 18
13 15
16 20
13 16
14 18
11 15
11 15
13 17
15 16

>17
>23
>18
>20
>15
>17
>18
>15
>19
>16
>21
>17
>19
>16
>16
>18
>17

<13
<19
<13
<14
<12
<14
<13
<11
<14
<12
<15
<12
<13
<10
<10
<12
<14

b) Pseudomonas aeruginosa
Test/Report
Group
U
U
A
B
U
O
B
U
C
C

Antimicrobial
agent
Carbenicillin
Ceftizoxime
Gentamicin
Amilkacin
Tetracycline
Doxycycline
Ciprofloxacin
Norfloxacin
Chloramphenicol
Cotrimoxazole

Disc content R

100g
30g
10g
30g
30g
30g
5g
10g
30g
1.25/23.75g

14 16
15 19
13 14
15 16
15 18
13 15
16 20
13 16
13 - 17
11 - 15

>17
>20
>15
>17
>19
>16
>21
>17
>18
>16

<13
<14
<12
<14
<14
<12
<15
<12
<12
<10

c) Enterococcus species
Test/Report
Group
A
A
B
C
C
C
U
U
U
C
C
C

Antimicrobial
agent
Penicillin
Ampicillin
Vancomycin
Erythromycin
Tetracycline
Doxycline
Ciprofloxacin
Norfloxacin
Nitrofurantoin
Chloramphenicol
Gentamicin
Streptomycin

Disc
content
10 units
10g
30g
15g
30g
30g
5g
10g
300g
30g
120g
300g

<14
<16
<14
<13
<14
<12
<15
<12
<14
<12
<6
<6

15 - 16
14 - 22
15 - 18
13 - 15
16 - 20
13 - 16
15 - 16
13 - 17
79
7-9

>15
>17
>17
>23
>19
>16
>21
>17
>17
>18
>10
>10

d) Haemophilus species
Test/Report
Group
A
O
B
C
C
A
B

Antimicrobial
agent
Ampicillin
Amoxicillin
Ceftizoxime
Tetracycline
Ciprofloxacin
Cotrimoxazole
Chloramphenicol

Disc content

10g
20/10g
30g
30g
5g
1.25/23.75g
30g

<18
<19
<25
<10
<25

19 21
26 28
11 15
26 - 28

>22
>20
>20
>29
>21
>16
>29

e) Neisseria gonorrhea

Test/Report
Group
C
C
C
C

Antimicrobial
agent
Penicilline
Ceftizoxime
Tetracycline
Ciprofloxacine

Disc
content
10units
30g
30g
5g

<26
<30
<27

27 46
31 37
28 - 40

>47
>38
>38
>41

f) Streptococcus pneumoniae

Test/Report
Group
A
B
A
B
A
C
C

Antimicrobial
agent
Penicillin
Vancomycin
Erythromycin
Tetracycline
Cotrimoxazole
Chloramphenicol
Clindamycin

Disc content R

1g
30/10g
15g
30g
1.25/23.75g
30g
2g

20 27
19 25
19 22
16 18
16 - 18

>28
>26
>17
>23
>19
>21
>19

<19
<18
<18
<15
<20
<15

g) Streptococcus species other than S. pneumonia

Test/Report
Group
A
A
B
A
O
B
B

Antimicrobial
agent
Penicillin
Ampicillin
Vancomycin
Erythromycin
Tetracycline
Chloramphenicol
Clindamycin

Disc
content
10units
10g
30g
15g
30g
30g
2g

<19
<18
<15
<18
<17
<15

20 27
19 25
16 20
19 22
18 20
16 - 18

>28
>26
>17
>21
>23
>21
>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