1.0 INTRODUCTION:
The diffusion method is widely used to evaluate the
antimicrobial activity of plants or microbial extracts. Similar to the
procedure used in the disk-diffusion method, the agar plate surface is inoculated
by spreading a volume of the microbial inoculum over the entire agar surface.
Then, a hole with a diameter of 6 to 8 mm is punched aseptically with a
sterile cork borer or a tip, and a volume (20–100 µL) of the antimicrobial
agent or extract solution at desired concentration is introduced into the well.
The antimicrobial agent diffuses in the agar medium and inhibits the growth of
the microbial strain tested.
2.0 METHODOLOGY:
·
Media Preparation
·
Mueller-Hinton Agar (MHA) will
be prepared from the dehydrated medium according to the manufacturer’s
instructions. Media will be prepared using distilled water or deionized water.
·
Media will be completely dissolved
by heating with frequent agitation followed by sterilization by autoclaving at
121°C for 15 min.
·
pH will be measured of each
preparation after sterilization using pH meter (pH shall be in between 7.2 and
7.4 at room temperature).
· Agar will be poured into a
sterile glass or plastic petri dish on a flat surface to a uniform depth of 4
mm and allow for solidifying.
·
Agar well diffusion
· The agar plate surface will be
inoculated by spreading a volume of the microbial inoculum over the entire agar
surface.
·
Then, a hole with a diameter
of 6 to 8 mm will be punched aseptically with a sterile cork borer or a
tip
· A volume (20–100 µL) of
the antimicrobial agent or extract solution at desired concentration will be
introduced into the well.
· Agar plates will be incubated
under suitable conditions depending upon the test microorganism.
·
The antimicrobial agent
diffuses in the agar medium and inhibits the growth of the microbial strain
tested.
3.0 ENDPOINT PARAMETER(S):
·
Zone of inhibition
·
Minimum Inhibitory Concentration
(MIC)
4.0 REFERENCE(S):
4.1 Mounyr
Balouiri, Moulay
Sadiki, and Saad
Koraichi Ibnsouda.
Methods for in vitro evaluating antimicrobial activity: A review. J
Pharm Anal. 2016
Apr; 6(2): 71–79
4.2 Romika Dhiman, Neeraj Aggarwal, Kamal Rai
Aneja, and Manpreet Kaur. In Vitro Antimicrobial Activity of Spices and
Medicinal Herbs against Selected Microbes Associated with Juices. International
Journal of Microbiology Volume 2016, Article ID 9015802, 9 pages http://dx.doi.org/10.1155/2016/9015802.
4.3 Demeke Debalke, Mastewal Birhan, Amebaye
Kinubeh, Muluken Yayeh, "Assessments of Antibacterial
Effects of Aqueous-Ethanolic Extracts of Sida rhombifolia’s Aerial Part", The Scientific World
Journal, vol. 2018, Article
ID 8429809, 8 pages, 2018. https://doi.org/10.1155/2018/8429809.
END OF DOCUMENTS
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