Republic of the Philippines

CATANDUANES STATE UNIVERSITY

Virac, Catanduanes

Catanduanes State University

College of Health and Sciences

Microbiology and Parasitology Laboratory

Lab Activity #5

STERILIZATION AND DISINFECTION

Submitted by:

MONICA JOYCE B. TITO

BSN-1C

Submitted to:

MARIA XUYEN B. MAGTANGOB

Instructor
 Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

LABORATORY ACTIVITY NO. 5

Sterilization and Disinfection

Introduction

There are several methods that can be employed to kill organisms or inhibit their
growth. These can be classified into physical and chemical methods. Of the physical
methods, heating is the most reliable and whenever possible, it should be the method of
choice. It is also the method that is readily accessible and universally accepted.
Chemical agents can also be used to achieve sterilization and disinfection.
Disinfection is important in infection control, not only in hospitals but also at home. A
wide variety of chemical agents belonging to several groups can be used. Different
modes of action have been ascribed to them. These chemical agents may interfere with
the functions of the cell membrane, denature proteins, or destroy or modify the
functional groups of proteins.

Materials and Equipment

Physical Methods of Inactivation of Organisms

Figure 1.

4 broth cultures of Bacillus subtilis

4 broth cultures of Escherichia coli

8 tubes of nutrient broth

Procedure
 Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

Figure 2. Figure 3.
First tube - autoclave at 15 pounds per Second tube - stand in a pan of boiling
square inch (psi) for 15 minutes water for 30 minutes

Figure 4. Figure 5.
Third tube - stand in a pan of water at Fourth tube - control tube
60 degrees Celsius for one hour

Materials and Equipment

Chemical Method of Inactivation of Organisms

 Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

Figure 6.

 One broth culture with light suspension of Staphylococcus aureus

 Four 1ml water blanks
 Nutrient agar plate

Figure 8.
Figure 7. Transfer a loopful of the S. aureus suspension to each of the
Divide the nutrient agar into four water blanks.
quadrants
Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

Figure 8.
Transfer a loopful of the suspension of
the microorganism from each tube to
another tube of nutrient broth and
incubate for 35 degrees Celsius for 24
hours

Figure 9.
Record the growth with sub-cultured
tubes (no growth, minimal growth,
moderate growth, or heavy growth)
 Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

What did you learn in the activity?

In this activity, the Power Rangers group have learned the differences of the 2
methods of sterilization (physical and chemical) when it comes to the growth of
microorganisms. In the physical method, the growth in sub-cultured tubes varies. For
Bacillus subtilis, minimal growth was observed in autoclave and heating tubes while
moderate growth was experienced in boiling water tube. For Escherichia coli, moderate,
heavy and minimal growths were observed in autoclave, boiling water and heating tubes
respectively. In this method, we have noticed that the heating technique (60 degrees
Celsius for one hour) only exhibited one type of microorganism growth regardless of
which kind of bacteria is being employed: minimal growth.

Meanwhile, it’s the opposite in the chemical method. All quadrants of the nutrient
agar plate with chemical agents (control, 1:1000 Zephiran, 70% alcohol and Povidone
iodine) have observed positive growth of microorganisms regardless of which type is
being used. Therefore, sterilization and disinfection are so important to reduce the
number of microorganisms on all things and decrease the risk of infection transmission.
Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

Catanduanes State University

College of Health and Sciences

Microbiology and Parasitology Laboratory

Lab Activity #7

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Submitted by:

MONICA JOYCE B. TITO

BSN-1C

Submitted to:

MARIA XUYEN B. MAGTANGOB

Instructor
 Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

LABORATORY ACTIVITY NO. 7

Antimicrobial Susceptibility Testing

Introduction

Antibiotics are given to treat infectious diseases. The physician faces the
problem of deciding which antibiotic to use for a given infectious disease. To make sure
that the antibiotic to be given is suited for a specific organism, an antimicrobial
susceptibility test must be requested. This test will tell the physician if the organism
involved in the disease process is susceptible to or resistant to a particular antibiotic,
thereby saving the patient from spending money on a drug that will not work on the
particular organism involved in the first place. Susceptibility testing is most often
indicated when the etiologic agent involved is known to be capable of developing
resistance to commonly used antimicrobial agents. It is rarely done if the organism is
not known to develop resistance against a given antibiotic.

Materials and Equipment

Figure 1.

 Prepared cultures of Staphylococcus aureus and E.

Coli

 Commercially prepared antibiotic discs (Amoxicillin,

Chloramphenicol, Tetracycline, Clindamycin,
Erythromycin).

Procedure
 Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

Figure 3.
Figure 2. Incubate at 35 degrees Celsius for 16-18
On the culture plates, place equidistant from hours.
each other and in a circular fashion, one disc
each of the commercially prepared antibiotic
disc.

Figure 4. Figure 5.

After incubation, measure with a ruler the widest The results.

diameter of the zones of inhibition of each
antibiotic (expressed in millimeters)
 Republic of the Philippines
CATANDUANES STATE UNIVERSITY
Virac, Catanduanes

What did you learn in the activity?

In this activity, the Power Rangers group have learned that most antibiotics that
have been tested for antimicrobial susceptibility through disc diffusion method are
resistant to microorganisms. Tetracycline, Erythromycin and Chloramphenicol have all
been recorded with a 0.7 cm zone size and are resistant. Meanwhile, Amoxicillin has a
zone size of 1.5 cm and is susceptible to microorganisms while Clindamycin has 2.4 cm
of zone size and is intermediate susceptible. The importance of antimicrobial
susceptibility testing is to tell the physician if the organism involved in the disease
process is susceptible (can be treated with a drug), intermediate susceptible (may be
treatable with the drug but may require adjusted dosage) or resistant (cannot be treated
with drug) to a particular antibiotic. That way, it can help the patient from saving a lot of
money to a drug that doesn’t work on a particular microorganism. Therefore, this test is
essential and crucial not only for the doctor’s right prescription of the medicine but also
for the patient’s health betterment.