EXERCISE 7

In Vitro Effects of Antimicrobial on Microorganisms

Objectives:

Upon completion of the laboratory activity, the students should be able to:

1. Determine the effectiveness of selected antimicrobials based on their relative

sensitivity and resistance on microorganisms.

2. Determine the degree of potency of selected antimicrobials by comparing the

diameter of the zones of inhibition.

Introduction:

The incidence of infectious diseases is rising that necessitates effective and potent
antimicrobials to reduce their alarming numbers of cases. It is important, therefore, that such
chemotherapeutic agents should be used rationally, as indicated in one (1) of the thrusts of
the Philippine National Drug Formulary. Rational use of drugs means that chemotherapeutic
agent should be appropriate for each disease. As applied in infectious diseases,
antimicrobials should target the specific offending organism.

The evaluation of the sensitivities of an antimicrobial agent is employed in this activity

through indirect antibiotic sensitivity test using selected antimicrobials which are impregnated
in small sterile paper disc and later spread on lawn of known microorganisms on plated
media. This type is called disk method. The drug diffuses into the medium during its
incubation producing concentration gradient as microbes proliferate. The width of the
halo/plaque or technically termed ‘zone of inhibition’ (ZOH) is measured; thus, making it a
qualitative determination.

Materials:

sterile cotton swabs with equally sized tips/calibrated loops

sterile thumb forceps
commercially prepared antibiotics disks with equal concentrations
calibrated ruler (in millimeters)
sterile agar plates of Mueller-Hilton
autoclave

24-hour culture of Staphylococcus aureus in tubed broth

24-hour culture of Escherichia coli in tubed broth
Procedures:

1. Obtain sterile plated media prepared by the laboratory personnel. Label them accordingly
with the name of microorganism to be inoculated on. Using a marker, make an equal
four-quadrant division on their undersides while keeping them closed with their
appropriate lid covers.

2. Perform inoculation techniques (either by using sterile cotton swabs with equally sized
tips or calibrated loops) separately by using the above-named bacterial cultures on
Mueller-Hinton media while observing aseptic techniques. Set aside the inoculated
media to permit absorption of the cells for 10 minutes.

3. Lay the one (1) antibiotics disk centrally on each quadrant of the medium by using sterile
thumb forceps to allow contact of the disks and the inoculated media.

(Note: Use overlapping technique of streaking to ensure an even distribution of the

bacterial culture. This is recalled by swabbing the plate horizontally first, then
vertically, and finally at 45-degree angle.)

4. Incubate the plated media at 37 degrees Celsius for 24-48 hours.

5. After incubation, measure the ZOH of each antibiotics disk and record it in millimeters.
Compare actual findings to a standard.

Safety first

The water, which is used in washing glassware and other materials not
contaminated with chemicals, should be disposed of into the sink.

Used antibiotics should be disposed into the designed bin.

Disposable gloves must be collected in a container with appropriate lid

cover. The same is done with used disposable masks.
 SUGGESTED ANTIMICROBIAL DISCS FOR ROUTINE USE WITH INTERPRETATION OF
ZONE OF INHIBITION
CATEGORIES OF GENERIC NAME OF CODE POTENCY RESISTANT INTERMEDIATE SUSCEPTIBLE
BACTERIA ANTIMICROBIAL (in (in millimeters) (in
millimeters) millimeters)
Amikacin An 30 14 15-16 17
Ampicillin- Sam 11 12-14 15
Sulbactam
Aztreonam Atm 30 15 16-21 22
*Co-trimoxazole Sxt 1.25/23.75 10 11-15 16
Ceftriaxone Cro 30 13 14-20 21
Imipinem Ipm 10 13 14-15 16
FOR Chloramphenicol C30 30 12 13-17 18
ENTEROBACTERIACEAE
Ciprofloxacin Cip 5 15 16-20 21
Gentamycin Gm 10 12 13-14 15
Netilmicin Net 30 12 13-14 15
Ofloxacin Ofx 5 12 13-15 16
Piperacillin Pip 100 17 18-20 21
Co-amoxiclav Aug 30 14 15-17 18
**(Augmentin)
Azithromycin Azm 13 14-17 18
ADDITIONAL Nalidixic An 14 15-18 19
ANTIMICROBIALS FOR
URINE ISOLATES Nitrofurantoin Fd 14 15-16 17
Oxacillin/Methicillin Ox 1 Staph 10: 11-12 Staph 13:
CNS 17 CNS 18
Erythromycin E 10 13 14-22 23
Clindamycin Cc 15 14 15-20 21
Tetracycline Te 30 14 15-18 19
Chloramphenicol C30 30 12 13-17 18
FOR STAPHYLOCOCCUS
(Staph) AND Co-trimoxazole Sxt 1.25/23.75 10 11-15 16
COAGULASE-NEGATIVE Gentamycin Gm 10 12 13-14 15
STAPHYLOCOCCI (CNS)
Netilmycin Net 30 12 13-14 15
Amikacin An 30 14 15-16 17
Ciprofloxacin Cip 5 15 16-20 21
Imipinem Ipm 10 13 14-15 16
Ceftazidime Caz 14 16-17 18
 CATEGORIES OF BACTERIA GENERIC NAME CODE POTENCY RESISTANT INTERMEDIATE SUSCEPTIBLE
OF (in (in millimeters) (in
ANTIMICROBIAL millimeters) millimeters)
Ceftazidime Caz 30 14 16-17 18

Piperacillin Pip 100 17 18-20 21

Gentamycin Gm 10 12 13-14 15

Netilmycin Net 30 12 13-14 15

FOR Amikacin An 30 14 15-16 17

Pseudomonas
Ciprofloxacin Cip 5 15 16-20 21
aeruginosa
Aztreonam Atm 30 15 16-21 22

Ceftazidime Cro 30 13 14-20 21

Ofloxacin Ofx 5 12 13-15 16

Imipenem Ipm 13 14-15 16

NON- Ceftazidime Caz 30 14 15-17 18

FERMENTATIVE
GRAM- Piperacillin Pip 100 17 18-20 21
NEGATIVE
Gentamycin Gm 10 12 13-14 15
BACTERIA
Netilmicin Net 30 12 13-14 15
FOR
ACINOBACTER Tobramycin Nn 10 12 13-14 15
spp.
Amikacin An 30 14 15-16 17

Ciprofloxacin Cip 5 15 16-20 17

Co-trimoxazole Sxt 10 11-15 16

Ampicillin- Sam 10 11 12-14 15

Sulbactam
Aztreonam Azm 15 16-21 22

Cefepime Fep 30 14 15-17 18

Imipenem Ipm 15 13 14-15 16

Chloramphenicol C30 30 12 13-17 18

Co-trimoxazole Sxt 10 11-15 16

Nalidixic Acid Na 30 14 15-18 19

FOR
ENTERIC/STOOL Ciprofloxacin Cip 5 15 16-20 21
ISOLATES
Tetracycline Te 30 14 15-18 19

Ampicillin Am

Nitrofurantoin Fd 14 15-16 17

*Co-trimoxazole= combination of Trimethropim and Sulfamethoxazole

**Co-amoxiclav= combination of Amoxicillin and Clavulanic Acid
(These tables, modified by the authors, are courtesy of the Bacteriology section of Cagayan Valley Medical Center)
 Diagram Of Inoculated Agar Plates With Equidistant Placement Of Antimicrobial Discs

(Based on source:https://www.nuffieldfoundation.org/sites/default/files/PB_investigating-antimicrobial-
action-sterile-paper-discs.jpg)
 Report Sheet

Name:___________________________________ Date submitted:_______________

Course/Year/Section/Set:______________________________ Score:____________

Documentation of Results:

Zones of Inhibition of Different Antimicrobials (in millimeters)

Bacterial Species Antimicrobials Antimicrobials Antimicrobials Antimicrobials

A B C D
Staphylococcus aureus
Escherichia coli

Study Questions for Exercise 7:

1. Which of the antimicrobials used is/are effective in controlling microbial growth?

2. What is the significance of using calibrated loops in making a lawn of microorganisms on

prepared agar plates?

3. Differentiate potency and effectiveness?

4. What are the factors considered in the standardization of the antibiotic’s sensitivity test?

5. Predict the effect of the following antimicrobial agents when used on bacterial cultures such
as Staphylococcus aureus and Escherichia coli in antibiotics sensitivity test. Briefly support
your final answers.

a. Metronidazole

b. Acyclovir

c. Ketoconazole