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NAMES: Vanessa Olga J.

Dagondon

20135- 52850

DATE
PERFORMED:

09/06/16

DATE
SUBMITTED:

09/13/16

Laboratory Exercise No. 6


DISINFECTANTS
RESULTS AND DISCUSSION
Table 1. Illustrations of E. coli and S. Aureus agar plates after being treated with disinfectants
E. coli

S. aureus

Disinfectant
24h incubation
Betadine

5% NaOCl

0.5% NaOCl

95% Alcohol

75% Alcohol

Merthiolate

48h incubation

24h incubation

48h incubation

1% Lysol

5% Lysol

Formaldehyde

1% Hydrogen
Peroxide

3% Hydrogen
Peroxide

Listerine

Table 2. Summary of Results


Disinfectant

Bacteria

Betadine

E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus

5% NaOCl
0.5% NaOCl
95% Alcohol
75% Alcohol
Merthiolate
1% Lysol
5% Lysol

After 24h incubation


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10 15 C
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After 48h incubation


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10 15 C
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Formaldehyde
1% Hydrogen
Peroxide
3% Hydrogen
Peroxide
Listerine

E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus
E. coli
S. Aureus

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-

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In this experiment, the activity of some disinfectants with respect to the importance of time,
germicidal concentration and microbial species in disinfection was studied. The following
disinfectants were utilized in the experiment: Betadine, Sodium hypochlorite, Alcohol,
Merthiolate, Lysol, Formaldehyde and Listerine. The activity of these disinfectants were used
against the bacteria E. coli and S. aureus. E. coli is a gram-negative, rod-shaped bacterium while
S. aureus is a gram-positive coccal bacterium. Table 1 shows the images of the agar plates
inoculated with the bacteria with its corresponding disinfectant treatment after 24h and 48h of
incubation. Each agar plate is divided into four parts indicating the number of minutes (5, 10, 15)
the disinfectant interacted with the bacteria. Table 2 summarizes the results of the experiment in
which a (-) indicates a no growth and (+) indicates growth.
Betadine is a povidone-iodine and is an example of an iodophor. It is a surface-active
iodophor that improves the wetting action and serves as a reservoir of free iodine (Tortora, 2010).
It is an intermediate level disinfectant and is mainly used for skin disinfection and wound
treatment. Sodium hypochlorite is an intermediate level disinfectant and is usually used as a
household disinfectant. Sodium hypochlorite, at concentrations of 0.5 to 5.25%, is an effective
disinfectant (Zand, et. al, 2011). Alcohol are intermediate level disinfectants and can effectively
kill bacteria and fungi but not endospores and nonenveloped viruses. Alcohol works as a
disinfectant by denaturation of protein. The recommended optimum concentration of alcohol is
between 60% and 90%. Pure alcohol is less effective than aqueous solutions because denaturation
requires water (Tortora, 2010). Merthiolate is a derivative of thiolsalicylate where ethylmercury is
attached though the sulfur or thiol group. It has a very broad spectrum of activity but is primarily
bacteriostatic. Lysol is formulated with O-phenylphenol as its main ingredient. O-phenylphenol is
a cresol, a phenolic derived from coal tar. Phenolics are low/intermediate level disinfectants that
remain active in the presence of organic compounds, are stable, and persist for long periods after
application (Tortora, 2010). It is bacteriostatic at concentrations of 0.1%1% and is
bactericidal/fungicidal at 1%5%. Formaldehyde is a high level disinfectant and sterilant both in
liquid and gaseous states. It is commonly available as formalin, a 37% aqueous solution of
formaldehyde gas. The aqueous solution is bactericidal, tuberculocidal, fungicidal, virucidal and
sporicidal (Gamage, 2003). Hydrogen peroxide is another high level disinfectant which is often
used as antiseptics to clean wounds. Its activity is greatest against anaerobic bacteria. It is useful
for cleaning surgical sites after closure, not before, because at high concentrations, it causes
damage to tissues (Gamage, 2003). Listerine is mainly composed of phenols and antibacterial
essential oils that are proven to penetrate biofilm formation. The mechanism of action of Listerine
involves bacterial cell wall destruction, bacterial enzymatic inhibition, and extraction of bacterial
lipopolysaccharides (Mandel, 1994).
Based on the results, 1% Lysol, 3% Hydrogen peroxide and Listerine were affective against
E. coli since all of these chemicals were able to eradicate the bacteria at all minute intervals for
both 24h and 48h incubation. The least effective are Betadine, 0.5% NaOCl, 5% Lysol,
Formaldehyde, 1% Hydrogen peroxide. For S. aureus, Hydrogen Peroxide and Listerine were
effective disinfectants while betadine, alcohol and others were not. However, errors might cause
this result. These errors include contamination and inaccuracy in plating. Microorganisms differ
in their response to microorganism because each organism has different structure and thus
havedifferent susceptibility in disinfectants. To achieve maximum effectiveness against both
organisms, formaldehyde is the chemical that should be used because it is a high level disinfectant
that would definitely work against the said bacteria. Listerine was the mouthwash tested and it
turned out to be as effective as the other disinfectants and maybe more. This could be attributed
the composition of the mouthwash which is a mixture of phenolics, alcohol and essential oils.

Study Questions:
1. Define disinfection. Compare/contrast it with sterilization, antisepsis and bacteriostasis.
Disinfection is a process that eliminates many or all pathogenic microorganisms, except bacterial
spores, on inanimate objects. In contrast, sterilization is the process that destroys all forms of
microbial life while antisepsis is the application of germicides to living tissue and bacteriostasis
is the inhibition of the growth of bacteria without destruction (Rutala and Weber, 2008).
2. Give the modes of action of the different antiseptics/disinfectants used in the activity.
Betadine (povidone-iodine) penetrates into microorganisms and attacks key groups of proteins
(in particular the free-sulfur amino acids cysteine and methionine), nucleotides, and fatty acids,
which culminates in cell death (McDonnell and Russell, 1999).
NaOCl The mode action of NaOCl has not yet been fully elucidate but it is believed that its
germicidal activity is due to the inhibition of enzyme activity essential for the growth, damage to
the membrane and DNA, and possibly an injury to membrane transport capacity (Fukuzaki,
2006).
Alcohol causes membrane damage and rapid denaturation of proteins, with subsequent
interference with metabolism and cell lysis (McDonnell and Russell, 1999).
Merthiolate When merthiolate breaks down, it releases ethyl-mercury that can penetrate cell
membranes and bind to intracellular enzymes, inhibiting them, and causing cell death (Haley,
2004).
Lysol As a phenolic compound, it acts by disruption of membranes, precipitation of proteins
and inactivation of enzymes (Rao, 2008).
Formaldehyde inactivates microorganisms by alkylating the amino and sulfhydral groups of
proteins and ring nitrogen atoms of purine bases (Rutala and Weber, 2008).
Hydrogen Peroxide works by producing destructive hydroxyl free radicals that can attack
membrane lipids, DNA, and other essential cell components (Rutala and Weber, 2008).
Listerine involves bacterial cell wall destruction, bacterial enzymatic inhibition, and extraction
of bacterial lipopolysaccharides (Mandel, 1994).
3. Which microbial forms are most resistant to disinfectants?
The most resistant microbial forms are prions, followed by bacterial endospores (its spore coat and
cortex act as a barrier), mycobacteria (contains a waxy cell wall that prevents disinfectant entry),
cysts of protozoa, vegetative protozoa, then Gram-negative organisms (possess an outer membrane
that acts as a barrier to the uptake of disinfectants), with cocci generally being the most sensitive
(Tortora, 2010; Rutala and Weber, 2008; Russell, 1999).
4. What is an iodophor? What is its value?
An iodophor is a combination of iodine and a solubilizing agent or carrier which provides a
sustained-release reservoir of iodine and releases small amounts of free iodine in aqueous solution.
Iodophors retain the germicidal efficacy of iodine but unlike iodine generally are nonstaining and
relatively free of toxicity and irritancy (Rutala and Weber, 2008).
References:
Fukuzaki, S. (2006, October 13). Biocontrol Science. Mechanisms of Actions of Sodium
Hypochlorite in Cleaning and Disinfection Processes, 114, 147-157. Retrieved September 12,
2016, from https://www.jstage.jst.go.jp/article/bio1996/11/4/11_4_147/_pdf.
Gamage, B. (2003). Selection and Use of Disinfectants. Retrieved September 13, 2016, from
https://uwaterloo.ca/safety-office/sites/ca.safety-office/files/uploads/files/guide-selection-use-ofdisinfectants.pdf
Haley, B. E. (2004). Biomedical Aspects of Thimerosal Exposure. Retrieved September 13, 2016,
from http://www.hyperactif.net/images/AffadavitThimerosal.pdf
Mandel, I. (1994, August). J Am Dent Assoc. Antimicrobial Mouthrinses: Overview and Update,
125 Suppl, 2:2s-10s. doi:8064061

McDonnell, G., & Russell, A. (1999, January). Clin Microbiol Rev. Antiseptics and Disinfectants:
Activity, Action, and Resistance, 12(1), 147-179. doi:PMC88911
Rao, S., PN. (2008, June). Sterilization and Disinfection. Retrieved September 13, 2016, from
http://www.2ndchance.info/Salmonella-Rao2008.pdf
Russell, A. (1999, December). J Hosp Infect. Bacterial resistance to disinfectants: present
knowledge and future problems, 43 Suppl:S57-68. doi: 10658759
Rutala, W. A., & Weber, D. J. (2008). (USA, Centers for Disease Control and Prevention,
Healthcare Infection Control Practices Advisory Committee). Chapel Hill, North Carolina:
Guideline for Disinfection and Sterilization in Healthcare Facilities.
Tortora, G. J., Funke, B. R., & Case, C. L. (2014). Microbiology: An Introduction (10th ed.). San
Francisco, CA: Pearson Education, Inc.