Research II
Quarter 2 - Module 1 Screening for Antimicrobial
Activity
Arrange the following pictures depicting the sequence of this activity by writing the
number on the space provided before each letter.
The ASEPTIC TECHNIQUE which ensures that no
contamination of culture media occurs during handling
and inoculation is done during the screening of plant
extracts for antimicrobial activity. It also ensures that the
microorganisms do not contaminate the environment and
the handler.
Screening for Antimicrobial Activity
Antimicrobial agents or antibiotics treat infectious diseases caused by
bacteria and fungi. They either kill or slow down the growth of these
microorganisms. However, in recent years, commercial antimicrobial agents are
growing ineffective in controlling diseases caused by microorganisms. Thus, there
is a continuing search for effective antimicrobial agents.
Screening of antimicrobial agents involves searching for antimicrobial
activity. The antimicrobial activity of extracts or compounds either kills or inhibits
the growth of microorganisms. Testing for antimicrobial activity also determines
whether the inhibitory spectra of the compounds/extracts are broad or narrow. It
also recommends the use of several test organisms representing major groups of
microorganisms in screening for microbiological activity (Guevarra, 2001) such as:
 Staphylococcus aureus- gram-positive bacteria
 Escherichia coli- gram-negative bacteria
 Saccharomyces cerevisiae- unicellular fungus (yeast)
 Aspergillus niger - filamentous fungus (black mold)
 Candida albicans and Trichophyton mentagrophytes, which are pathogenic fungi,
are also used for testing.
 disinfectant (5% aqueous phenolic solution or 1% aqueous sodium
hypochlorite (Chlorox) solution or 70% isopropyl alcohol) and swabbing it
before and after working.

2. Wash hands thoroughly with soap and water before and after working.
3. Work in an area beside a lighted alcohol lamp or Bunsen burner inside a
Laminar
flow hood or an improvised chamber free from air draft.
4. Inoculating loops and needles are sterilized by inserting in a Bunsen burner
until red-hot and allowed to cool dipping into the inoculum.
5. The cap of a tube is flamed before inserting a cooled loop or needle or sterilized
Testing uses reference strains of these microorganisms obtained as culture cotton swab. The cap is kept in your hand and not on the lab table. After
collections (i.e., American Type Culture Collection (ATCC), UP-NSRI Culture inoculation, the tube mouth is flamed again and recapped.
Collection (UPCC)) from members of the Philippine Network of Microbial Culture
6. Agar plates are left closed. When swabbing, the plate cover is raised slightly
above the plate to protect the agar surface from contamination.

7. Label the bottom of the plates and incubate plates upside down.
8. Used agar cultures and plates are autoclaved before final disposal.
9. Autoclave all used loops and glass wares before cleaning.
10. Used disposable items such as cotton swabs, tissue paper, and cotton should
be
discarded in containers to be sterilized in an autoclave at 121oC under 15
psi for least 30 minutes.

Collection (PNMCC). It uses methods based on standard assay procedures such as

the disc or agar cup/well diffusion method. Both diffusion methods recommend
the use of specific age and concentration of inoculum, incubation times and
temperatures, agar thickness, and other parameters. Changes to these parameters
affect the results. Aseptic Techniques Observed During Screening for Antimicrobial
Activity

1. Disinfect the working area by wetting a piece of tissue paper or cotton with a
Evaluation of Antimicrobial Activity of Plant Extracts
 There are several procedures involved in the screening of plant extracts for
antimicrobial activity such as preparation of the plant extract, preparation of the
test microorganism or inoculum, screening methods for antimicrobial activity,
documenting the results, and aseptic techniques observed during screening for
antimicrobial activity.
Preparation of the Plant Extract
Extract the authenticated
plant sample using the method
and solvent of choice. The resulting
extract should be dry, syrupy, and
free from extracting solvent and
dissolved in normal saline solution
for water-soluble extracts, and
saline solution with 0.2% Tween 80
for resinous extracts.
Screening Methods for Antimicrobial Activity
Two methods are used to evaluate the antimicrobial activity of compounds
or extracts. Procedures for these methods are usually done in a biosafety level 2
(BSL-2) laboratory. In these methods, paper discs or agar wells/cups containing
the compound or extract are placed in agar plates inoculated with a standardized
inoculum of the test microorganism. The plates are incubated under suitable
conditions and the diameters of the inhibition growth zones are measured to
determine whether the microorganism is susceptible or not. The zone of inhibition
is the region where no bacterial colonies grow and is seen as clear areas
surrounding the discs. Its size is a measure of the compound's (or extract’s)
effectiveness: the larger the clear area around the disc, the more effective the
compound (or extract).
Paper discs or wells containing a standard antibiotic to which the test
microorganism is susceptible are used as standard antimicrobials for comparison.
The negative control is usually the solvent used for extraction. Results are
expressed as mean ± standard deviation and analysis is done by comparing the
mean diameters of the zones of inhibition to a standard antibiotic.

Preparation of the Test Microorganism 1. Agar Disc Diffusion Method

or Inoculum The agar disc diffusion method, a modification of the Kirby Bauer test,
Transfer a loopful of bacteria from operates on the principle that an antimicrobial agent impregnated disc will diffuse
a plate, slant, or a broth and transfer to 50 into an agar medium containing a test organism. As the antimicrobial agent
ml of fresh nutrient or Mueller Hinton diffuses into the agar culture, it inhibits the growth of a susceptible
broths and incubate at 35oC ± 2oC for 18- microorganism. The inhibition of growth is observed as a clear zone surrounding
24 hours. Prepare the inoculum by the disc.
suspending bacteria from the overnight
cultures in sterile saline solution. Mix the
suspended bacterial inoculum and compare
its turbidity to a 0.5 McFarland standard.
Adjust the turbidity by adding either sterile
saline solution or culture broth and
compare it with again with the standard. A
0.5 McFarland standard provides 1.5 x 108
CFU/ml of the microorganism.

For filamentous fungi, transfer a loopful of conidia to a slant or plate of

potato dextrose agar or Sabouraud dextrose agar and incubate at room
temperature until conidia have luxuriantly developed. To prepare the spore
suspension, scrape 5 loopfuls of conidia and transfer it to saline-Tween 80 in a
screw-capped tube. Agitate the contents by shaking or placing it in a vortex
mechanical mixer for 1 minute. Compare the turbidity of the spore suspension to a
0.5 McFarland standard. Adjust the turbidity by adding either saline-Tween 80
solution or spore scrapings and compare it with again with the standard. To
prepare the fungal inoculum to be used in swabbing, transfer 0.5 ml of the
adjusted spore suspension to 20 ml of saline-Tween 80.
For yeasts such as Saccharomyces cerevisiae or Candida albicans, transfer
several loopful of organism from slants to 50 ml of Sabouraud glucose broth and
incubateat room temperature for 18 hours. To prepare the yeast cell suspension,
follow the procedure for filamentous fungi, but use sterile isotonic saline instead of
saline-Tween 80 as a suspending medium.
2.Agar Cup/Well Diffusion Method
The Agar cup/well diffusion method is like the
disc diffusion method in that the antimicrobial agent
placed in the cup/well diffuses in the agar medium to
inhibit the growth of the microorganism tested. Instead
of paper discs, however, a hole (6-8 mm in diameter) is
made in the agar plate using a sterile cork-borer, and
20-100 μl of the extract or antimicrobial agent at the
desired concentration is placed in each well. Zones of
inhibition are measured after incubation under the
conditions specified for the microorganism used.

Documenting the Results

Turn the plates upside down and place it above a black background
illuminated with reflected light then measure the zone of inhibition. Alternatively,
you may remove the cover of the plate and measure the zone of inhibition
illuminated with reflected light.
Write the results as diameter of the zone of inhibition in millimeters.
Record the size of the paper disc (or the agar well) used in the test in millimeters,
and the test organism. Any changes done to the standard method should also be
recorded.
 II. The following illustration shows an agar plate with zones of inhibition.
DR. FELIPE DE JESUS NATIONAL HIGH SCHOOL Identify the areas pointed to by the arrows from the following choices.
Agnaya, Plaridel, Bulacan
1. Disc containing the extract
SECOND QUARTER
ACTIVITY 3 IN RESEARCH II 2. Zone of inhibition
Name: ____________________________________ Score: ______________ 3. Ineffective extract
Section: ___________________ Date: _______________ 4. Bacterial growth
5. Effective extract
I. The diagram below shows a plate after incubation. Measure the zones of
inhibition and record your answers in Table 2. Then answer the questions
that follow.

III. Fill in the blanks by choosing the correct word/words from the word

box.

Antimicrobial screening of plant extracts is done in a (1)

____________ area beside a lighted (2)____________inside a (3)____________. It
is done using microorganisms representing major groups of microbial flora
such as: (4) ____________, a gram positive bacteria, (5) ____________, a gram
negative bacteria, and a yeast like Candida albicans. The media used for
testing is sterilized by (6) ____________ at 15 psi for 30 minutes. Swabbing
of (7) ____________ plates is done by using a/an (8) ____________ that is
standardized using a 0.5 McFarland standard. Plates are incubated (9)
____________, and the zones of inhibition are measured using a ruler or
caliper. Spent media, used loops and glass wares, and other used
disposable items are (10) ____________ after the testing is done.