Exercise 4

DISINFECTANTS

GEROLAGA, WINSTON JAKE C.

BS in Chemistry IV
wcgerolaga@up.edu.ph

BIO 120.1
Section 3

12th September 2018

Introduction

Practicing disinfection is an essential part in microbiological laboratories (Sandle, 2015).

The act of disinfecting refers to the elimination of such pathogenic microbes from inanimate
objects, while antiseptic refers to pathogen removal from body tissues or skin (Reichert & Young,
1997).
Microbes are minute organisms that grow, multiply, and flourish in the human system and
other life, which sometimes give complications and various diseases. These microorganisms can
be found everywhere, and they easily gain entry to the human body, since the human body is a
very suitable place for their growth and replication. This is when diseases manifest and can
sometimes be spread by communication and exposure to other potential hosts (Tortora et al.,
2013). Hence there is a need to eliminate such microorganisms.
A lot of disinfectants of different types and varieties have been used over the years.
Through years of advancement, disinfectants have developed to exterminate a wider range of
pathogens and therefore claiming to be more effective than the others. Disinfectants work in
various ways such as asphyxiation of microbes by the oxygen that they contain, prevention of
oxygen access, or acts as direct poisons. Some remove or change the chemical properties of the
substance the microbes are dependent to, making the microbes unable to survive in such
conditions and therefore leading to elimination. (Kingzett, 2012).
In this exercise, the disinfectant in focus is 1% hydrogen peroxide (for the group).
Hydrogen peroxide exhibits microbicidal effects against viruses, bacteria, and a variety of fungi.
It is also sporicidal and is used as a liquid sterilant, which makes it corrosive to copper, brass,
and zinc (Dreyer, 2007).
This exercise aims to study the activity of disinfectants and learn the importance of time,
germicidal concentration, and microbial species in disinfection.

1
Materials and Methods

A. Materials

Material/Equipment/Specimen/
Description/Function
Sample/Organism

A general purpose medium supporting

Nutrient agar
growth of microorganisms

A laboratory equipment that uses denatured

Alcohol lamp alcohol to produce and open flame; used for
inoculation and sterilization purposes

E. coli A short, Gram-negative bacillus

S. aureus A Gram-positive coccus

Sodium hypochlorite (bleach) A sterilizing chemical

Absolute alcohol A disinfectant

Lysol A brand of cleaning/disinfecting products

An oxidizer, bleaching agent, and an

Hydrogen peroxide (H2O2)
antiseptic

Mouthwash (Listerine) A liquid disinfectant for oral purposes

2
B. Methods

The groups in the class were assigned with a particular disinfectant to evaluate.
In this exercise, the assigned disinfectant was 1% hydrogen peroxide.

1. Disinfection and Sanitation of Workplace and Exposed Hands

A generous amount of 5% Lysol solution was sprayed to the worktable. A clean

piece of cloth was used to spread the solution. Then, 70% isopropyl alcohol was then
sprayed onto the worktable, then letting it stand to dry.
To the hands, a generous amount of 70% isopropyl alcohol was poured. A
clean pair of sterile gloves were put on, and another round of 70% isopropyl alcohol
was poured into the gloved hands.

2. Evaluation of Other Disinfectants

A volume of 5.0 mL of the assigned

control 5 min
disinfectant (1% H2O2) was transferred into a
sterile test tube. Then, 0.5 mL of the bacterial
culture (E. coli and S. aureus) were transferred
into the sterile test tubes with the disinfectants. E. coli in 1% H2O2
Incubation time: 15:39
The tubes were shaken gently to distribute the
organisms uniformly.
A prepared nutrient agar (NA) plate was
obtained. The plate was divided into four sections 15 min 10 min

(quadrants) with a marking pen. The correct

labelling of the plate is shown in Figure 1. Figure 1. A correctly-labelled plate for
analysis. E. coli should be
At 5, 10, and 15-minute intervals, one changed with whatever name
depending on the organism
loopful of the disinfectant-culture mixture was inoculated. The time of
transferred to a section of the NA plate. Figure 2 incubation should also be noted.

shows the correct way of streaking.

3
 control 5 min
For the control, the inoculum was taken directly
from the culture.
All the plates were sealed and incubated at 35°C
E. coli in 1% H2O2 for 24 and 48 hours. The plate sections were all observed
Incubation time: 15:39
for growth after 24 and 48 hours.

3. Effects of Handwashing: The Fingerprint Technique

15 min 10 min

Before performing this exercise, the hands of the

Figure 2. The proper way of streaking the
disinfectant-culture mixture. The
streak (black thick line) should be
a straight line as depicted in the
figure.
4 3
SURNAME
Incubation time: 17:05
1 2
performer of the experiment was not disinfected.
A NA plate was divided into 4 quadrants and
labelled properly as shown in Figure 3. The nutrient agar
in sections 1 and 3 were touched with the thumb and the
forefinger of the right hand, respectively. Then, the right
hand was disinfected with 70% isopropyl alcohol, and then
touched sections 2 and 4 with the thumb and forefinger,
respectively.
The plate was sealed and incubated at 35°C for 24
and 48 hours. The plate sections were observed for growth
after the mentioned time. Finally, the plate was stored in a
refrigerator at around 4°C after analysis.

4. Cleaning and Disinfection of Workplace

Figure 3. A correctly-labelled plate for
analysis. E. coli should be
changed with whatever name
depending on the organism After all the laboratory work, the worktable was
inoculated. The time of
incubation should also be noted. disinfected with 5% Lysol, followed by 70% isopropyl
alcohol.

4
Results and Discussion

After 24 and 48 hours of incubation, the plates were all observed for bacterial growth.
Table 1 shows the legend for bacterial growth description.

Table 1. Growth description legend.

Growth Symbol

Maximum growth 4+
Moderate growth 3+
Some growth 2+
A little growth 1+
No growth 0

A. Effects of Handwashing: The Fingerprint Technique

After 24 hours, there had been growth in all quadrants of the plate, even those with the
disinfected fingers. This implies that even after disinfection, there may be microbes that are still
present in the fingers of a person and these might not be easily affected or killed by disinfectants.
The growth after 48 hours was even more intense relative to that of the growth after 24 hours.
The results are summarized in Table 2. This implies that time is a factor in bacterial growth. Figure
4 shows the appearance of the NA plates after 24 and 48 hours.

Table 2. Summary of the results for bacterial growth in the fingerprint technique.

Section of plate After 24 hours After 48 hours

1 (thumb without disinfection) 2+ 4+

2 (disinfected thumb) 1+ 2+
3 (forefinger without disinfection) 3+ 4+
4 (disinfected forefinger) 1+ 2+

5
 4 3 4 3

GEROLAGA GEROLAGA
Incubation time: 17:09 Incubation time: 17:09

1 2 1 2

A B

Figure 4. Nutrient agar plates from the fingerprint technique. Observation after 24 hours is seen in plate A; observation
after 48 hours is seen in plate B.

B. Evaluation of Other Disinfectants

In this part, E. coli and S. aureus were observed for their behavior after exposure to 1%
hydrogen peroxide solution. Figure 5 shows the appearance of the NA plates with E. coli-1%
hydrogen peroxide mixture after 24 and 48 hours. Figure 6 shows that of S. aureus-1% hydrogen
peroxide solution.

control 5 min control 5 min

E. coli in 1% H2O2 E. coli in 1% H2O2

Incubation time: 15:39 Incubation time: 15:39

15 min 10 min 15 min 10 min

A B

Figure 5. Nutrient agar plates with E. coli-1% H2O2 mixture. Observation after 24 hours is seen in plate A; observation
after 48 hours is seen in plate B.

6
 control 5 min control 5 min

S. aureus in 1% H2O2 S. aureus in 1% H2O2

Incubation time: 16:20 Incubation time: 16:20

15 min 10 min 15 min 10 min

A B

Figure 6. Nutrient agar plates with S. aureus-1% H2O2 mixture. Observation after 24 hours is seen in plate A;
observation after 48 hours is seen in plate B.

In both NA plates with the E. coli-1% H2O2 mixture after 24 and 48 hours, the bacteria with
the disinfectant did not show any signs of growth. This only shows that the disinfectant used is an
effective one and has eliminated E. coli. In the control, growth became intense after 48 hours.
On the other hand, the plates with S. aureus as the sample, there was a diagonal-
mannered growth across the plate. This may be caused by improper inoculation of the
microorganism. Supposed to be, there should be little to no growth in the quadrants with the
disinfectant-culture mixture. However, because of some errors in the inoculation, excess sample
from the control seeped its way across the plate, causing the error to propagate.
Table 3 summarizes the results after 24 and 48 hours of incubation for both organisms.

Table 3. Summary of the results for the growth of E. coli and S. aureus with 1% H2O2.

E. coli S. aureus
Growth
Control 5 min 10 min 15 min Control 5 min 10 min 15 min

After 24 hours 3+ 0 0 0 3+ 0 1+ 1+
After 48 hours 4+ 0 0 0 4+ 0 1+ 2+

7
 Other disinfectants were also observed for its activity on E. coli and S. aureus. The
following disinfectants were used: 1% Lysol, 5% Lysol, 3% H2O2, 0.5% bleach, 5% bleach,
Betadine, and mouthwash (Listerine). Summarized in Table 4 are the results.

Table 4. Summary of the results for the growth of E. coli and S. aureus with various disinfectants. *

E. coli S. aureus
Growth
C 5 min 10 min 15 min C 5 min 10 min 15 min

After 24 hours 4+ 2+ 1+ 0 4+ 0 0 0
1% Lysol
After 48 hours 4+ 2+ 1+ 0 4+ 0 0 0
After 24 hours 4+ 1+ 1+ 1+ 4+ 2+ 2+ 3+
5% Lysol
After 48 hours 4+ 1+ 0 0 4+ 2+ 2+ 3+
After 24 hours 4+ 1+ 0 0 4+ 1+ 0 1+
3% H2O2
After 48 hours 4+ 1+ 0 0 4+ 1+ 0 1+
After 24 hours 1+ 0 0 0 1+ 0 0 0
0.5% bleach
After 48 hours 2+ 1+ 1+ 1+ 1+ 0 0 0
After 24 hours 1+ 0 0 0 1+ 0 0 0
5% bleach
After 48 hours 2+ 1+ 1+ 1+ 1+ 0 0 0
After 24 hours 4+ 2+ 1+ 1+ 4+ 1+ 1+ 0
Listerine
After 48 hours 4+ 2+ 1+ 1+ 4+ 2+ 1+ 0
After 24 hours 4+ 0 0 1+ 4+ 0 0 1+
Betadine
After 48 hours 4+ 0 0 1+ 4+ 1+ 1+ 2+
* C: control. Legend is found in Table 1.

8
 The results imply that 0.5% bleach and 5% bleach are the most effective disinfectants in
terms of inhibiting bacterial growth after 24 and 48 hours. It also implies that the time of incubation
greatly affects bacterial growth: the longer the incubation time, the more bacterial growth.

In summary, this exercise has proven that disinfectants are very crucial in a
microbiological laboratory, because it has the ability to inhibit the growth of bacteria. However,
disinfectants do not actually eliminate all the bacteria; there would always be a minute amount
left. Hydrogen peroxide (1%) is quite a good disinfectant, as seen in the results of this exercise.
Inoculation of the microorganisms should be done neatly and orderly so as to prevent the
propagation of errors in the results. This exercise also has proven that time is directly proportional
to bacterial growth: the longer the incubation time, the more bacterial growth. Lastly, proper
inoculation techniques were performed and realized that it should be enforced in every laboratory
session.

9
References

Dreyer, M. S. (2007). Pharmacology for Nurses and Other Health Workers. Pearson: South Africa.

Kingzett, C. T. (2012). How to Disinfect: A Guide to Practical Disinfection in Everyday Life, and
During Cases of Infectious Illness. American and Continental "Sanitas" Company:
Michigan.

Reichert, M., & Young, J. H. (1997). Sterilization Technology for the Health Care Facility. Jones
& Bartlett Learning.

Sandle, T. (2015). Pharmaceutical Microbiology: Essentials for Quality Assurance and Quality
Control. Woodhead Publishing.

Tortora, Funke, & Case. (2013). Microbiology: An Introduction (11th ed.). Pearson Education, Inc.:
Glennview, Illinois.

10