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Exercise No. 7 - Ast

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12 views

Exercise No. 7 - Ast

Uploaded by

jvmartin.sjc
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BACTERIOLOGY LABORATORY

EXERCISE NO. 7-BSMT 3A


ANTIMICROBIAL SUSCEPTIBILITY TESTING

MATERIALS AND INSTRUMENTS


Mueller Hinton agar (MHA) plate 0.5% McFarland Standard
Sterile cotton swab
Antibiotic disks
Forceps
Alcohol lamp
Caliper
Culture
18- to 24-hour culture of Staphylococcus aureus
18- to 24-hour culture of Escherichia coli
.
METHOD: Disk Diffusion (Kirby-Bauer Test)
Principle: A standardized suspension of organism is inoculated onto Mueller-Hinton agar
and paper disks impregnated with specific antibiotic concentrations are placed
afterwards. The diameters of the zones of inhibition are measured using a caliper after
overnight incubation

PROCEDURE
A. Preparation of the Turbidity Standard (0.5 McFarland)
1.Add 0.5 ml of 1.175% BaCl2 to 99.5 ml of 1% H2SO4
2. Using a spectrophotometer, verify the density of the standard. The absorbance of the
3. standard at 625nm should be 0.08 to 0.1 with a 1 cm pathway.

B. Preparation of Inoculum for Susceptibility Testing


1. Pure inoculum is obtained by selecting 4 to 5 colonies of the same morphology from a
nonselective pure culture plate.
2. Using sterile loop, transfer aseptically the 18- to 24-hour old colonies into sterile saline.
3. Compare the turbidity of the inoculum with the Mac Farland standard.
4. If the bacterial suspension initially does not match the standard's turbidity, the
suspension is diluted with sterile saline or supplemented with more organisms.
5. Matching the turbidity is facilitated by holding the bacterial suspension and McFarland
tubes side by side against a Wickerham card.
6. Use the inoculum within 15 minutes.

C. Inoculation of the Test Organism onto Mueller-Hinton Agar (MHA)


1. Aseptically dip the sterile cotton swab into the bacterial suspension.
2. Remove the excess fluid by rotating the swab firmly against the inner wall of the test
tube
3. Inoculate the entire agar surface by overlapping streaking or back and forth motion.
4. Rotate the plate at a 60-degree angle after each streak to ensure even distribution of
the inoculum.
5. Perform the overlapping streaking three times without resuspending the swab into the
inoculum.
6. After streaking, rim the entire sides of the plate to remove excess fluid from streaking.
7. Discard swab into the designated container.
8. Allow inoculum to dry for 3 to 5 minutes with a lid before placing the antibiotic disks.

D. Placement of Disk onto MHA


1.Using sterile forceps or dispenser, place the antimicrobial disks onto the medium.
2. Press gently each disk with the sterile forceps after placement. Do not press all the
disks at the same time to prevent contamination.
3. The disk must be positioned 24 mm from disk center to disk center to avoid overlapping
of zones.
4. Within 15 minutes after applying the disks, incubate the plates at 35°C for 18 to 24
hours.
Note: Do not move the disk once it has contacted the agar since the antibiotic diffuses
immediately. The timing of antibiotic diffusion and incubation are critical for reliable
results.

E. Measurement of the Zone of Inhibition


The end point is defined as that area showing no visible growth when observed with the
unaided eye.
1.To be accepted for plate reading, the growth must be confluent.
2. Using a caliper or ruler, measure the zone of inhibition against a nonreflecting
background.
3. Measure the zone of inhibition from the backside of the plate.
4. Compare the measurement with the interpretive tables of the Clinical and Laboratory
Standards Institute (CLSI).
5. Interpret the results.
6. Record your observations.
Note: If growth between inhibitory zones around each disk is nonconfluent, the test should
not be interpreted and should be repeated. Zone sizes are not included in the reporting
of results.

Quality Control
Antibiotics for Gram-positive bacteria: Staphylococcus aureus ATCC 25923
Antibiotics for Gram-negative bacteria: Escherichia coli ATCC 25922
Monitoring of the Susceptibility Media: Pseudomonas aeruginosa ATCC 27853

Reporting of Results
Susceptible. Presence of a zone of inhibition around the antibiotic disk
Intermediate: Smaller zone of inhibition, range of susceptibility approaches or exceeds
the concentration of the antimicrobial agent
Resistant: No zone of inhibition or the inhibition did not meet the criteria for susceptible
or intermediate
EXERCISE NO. 7- BSMT 3A
ANTIMICROBIAL SUSCEPTIBILITY TESTING

NAME and GROUP NO.: DATE:

POST LAB ACTIVITY

A. DATA AND RESULTS

Name of Organism:
Antimicrobial agent Disk code Zone size S, I, R *
1.
2.
3.
4.
5.
6.
*S= Susceptible; I= Intermediate; R= Resistant

B. QUESTIONS (Maximum of 4 sentences)- 20 pts

1. Which phase of the bacterial growth is best for antimicrobial testing? Why?

2. What is the ideal thickness of the MHA? How does thickness of the medium affects
the result of the susceptibility testing?

3. Enumerate factors affecting zone of inhibition.


CLSI STANDARD CHART FOR ZONE DIAMETER INTERPRETATION
TABLE 1. Zone diameter interpretative standards for Staphylococcus spp.
Staphylococcus species
(Zone diameter, nearest whole mm)

Antibiotic disk Resistant Intermediate Susceptible


Erythromycin (15 ug) </= 13 14-22 >/= 23
Gentamicin (10 ug) </= 12 13-14 >/= 15
Oxacillin (1 ug) </= 10 11-12 >/= 13
Vancomycin (30 ug) -- -- >/= 15
Novobiocin (30 ug) </= 15 -- >15
Kanamycin (30 ug) </= 13 14-17 >/= 18

TABLE 2. Zone diameter interpretative standards for E.coli and other enteric
Gram Negative Rods
E.coli and other enteric Gram Negative Rods
Antibiotic disk Resistant Intermediate Susceptible
Ampicillin (10 ug) </= 13 14-16 >/= 17
Tetracycline (30 ug) </= 14 15-18 >/= 19
Chloramphenicol (30 ug) </= 12 13-17 >/= 18
Gentamicin (10 ug) </= 12 13-14 >/= 15

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