Activity 9: Antimicrobial Disk Susceptibility testing

I. INTRODUCTION
The antimicrobial disk susceptibility is used often due to its efficiency and cost.
Usually use Mueller-Hinton agar. MH agar is considered the best medium to use for
testing nonfastidous bacteria for the following reasons
1. It shows acceptable batch-to-batch reproducibility for susceptibility testing
2. It is low in sulfonamide, trimethoprim, and tetracycline inhibitors
3. It supports satisfactory growth of most nonfastidious pathogens

This activity tries to test the effectivity of an antibiotic against bacteria and is
compared to the effect of the choice of local herbal. Upon performing this activity, as
positive result, the dish will show decreasing concentrations of bacteria further away
from the disk. This means that there is no growth of bacteria happened around the
disk.
Further the zone around the antibiotic disk that has no growth is referred to as the
zone of inhibition since this approximates the minimum antibiotic concentration
sufficient to prevent growth of the test isolate. (*)

II. OBJECTIVES
The General objective of this activity is to test possible resistance of local herbal
plants to pathogens provided in the laboratory and compare it to the effect of an
established drug in the pharmacy.
The specific objectives of this activity are;
a. Demonstrate the antimicrobial disk diffusion,
b. Analyze the effectiveness of the choice herbal plant against bacteria, and
c. Communicate oral and written results.

III. METHODS
MATERIALS USED
Test tubes Incubator
Cotton swab
Micropipette Plant extract
Petri dish Beaker
Dropper Refrigerator

PROCEDURE
1. Sterilizing of the glass wares to be used.
2. Prepare agar plates.
3. Obtain plant extract.
4. Streak bacteria on the plate.
5. Label the 4 parts of the dish.
 6. Place one disk each part.
7. Add 20µl of amoxicillin on one part another 20µl of the herbal on the other 3
disks.
8. Cover the dish and wrap with paper. Observe after 18-24 hours.

IV. RESULTS AND DISCUSSIONS

The dish was observed after more than 24 hours due to part negligence, also the
suspensions of class. The Microbiology laboratory is located at the top floor of the
building and is very tedious to visit whenever we’re not so obliged to be there (well
I’m not blaming it totally t the location and the negligence).
The observers prepared the petri dishes for these activity and placed it in the
incubator. After 2 weeks (give or take, I can’t remember), the dish was contaminated
due to the possibility of having bacteria other than the bacteria placed by the
performers of this activity. So the dish was to be repeated.
The students had to repeat it until the third try to keep up with the other groups but
failed. The failure was due to the inability of the students during the 18-24 hours that
the bacteria are to be observed and inspected. Other members of the group are
nowhere during that time also, the room with the refrigerator and the incubator is
locked. Thus, the students cannot get inside the room.

The contamination of the petridish: the acquisition of contaminants in the petridish.

There is a possibility that there are moulds in the dish. Also, the bacteria and yeast
could be a problem. The slimy pasty bacterial and yeast colonies grow over the top of
young cultures and smother them. (**). Next, is the airborne contaminants due to lack
of disinfection of the work area. The number of airborne spores can be reduced by
using bleach and disinfectant sprays, and that the windows and doors should have
been closed while working. Further, while transferring the bacteria into the plates the
lids should be opened in a short period of time. Spores could also stick in the opening
of tubes and the lids of the petri dishes.
According to references, the incorrect concentration of divalent cations (calcium and
magnesium) will affect the results of aminoglycoside and tetracycline tests against P.
aeruginosa.  Excess cation concentration will result in reduced zone sizes and low
concentration will increase zone sizes.  (***)

V. CONCLUSION
In conclusion, the disk susceptibility test was not successful. The result was due to
negligence, unavailability and the lack of unity among members.
VI. REFERENCES

(*)http://amrls.cvm.msu.edu/microbiology/detecting-antimicrobial-resistance/test-
methods/examples-of-antibiotic-sensitivity-tesing-methods
(**)http://website.nbm-mnb.ca/mycologywebpages/Moulds/Contamination.html
(***)http://www.microbelibrary.org/component/resource/laboratory-test/3189-kirby-
bauer-disk-diffusion-susceptibility-test-protocol