Exercise 3

STERLIZATION PROCEDURES

GEROLAGA, WINSTON JAKE C.

BS in Chemistry IV
wcgerolaga@up.edu.ph

BIO 120.1
Section 3

19th September 2018

Introduction

In every microbiology laboratory, it is vital for almost everything to be free of

contaminants and all forms of life in order for the results of the experiment to be accurate. This
can be done by sterilization. Sterilization is the complete removal of all forms of life, such as
bacteria, fungi, viruses, and microbial spores, from any surface, area, or culture media, and
equipment (Kango, 2010). In microbiology and medicine, sterilization serves to prevent the
following events: a microbial contamination of sterilized growth media, diluents, and nutrient
solutions, b entry of unwanted microorganisms into a pure culture (microbial culture containing
only one species), c the transfer of environmental microorganisms to instruments used for
medical or dental patients, and d the contamination by microorganisms of surgical incision
sites, surgical devices, and dental devices (Maczulak, 2011). This experiment focuses on
three main types of sterilization procedures: moist heat, incineration, and the use of autoclave.

Moist heat usually occurs in the form of steam, hot, or boiling water (usually ranging
from 60 to 135°C. This method can eliminate microbes by denaturation, or by the coagulation
of proteins (Parija, 2012). In this exercise, a hot water bath was set to 90°C. Another method
that uses moist heat is the autoclave. An autoclave is a piece of equipment that contains an
inner chamber surrounded by an outer chamber called a steam jacket. This jacket receives
steam from a steam supply in the autoclave and controls its entry into the inner chamber,
where the materials to be sterilized are placed. Autoclaves offer an effective and fast way to
kill microorganisms, including bacterial endospores, also by denaturation (Maczulak, 2011).

Figure 1. The autoclave and its parts. The figure also shows the flow
of steam inside the autoclave. (Illustration adapted from
Hogg, 2005).

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 Figure 1 (previous page) shows the systematic illustration of the autoclave. Usually,
autoclaves are set to operate at a temperature of 121°C and a pressure of 15 psi (103.5 kPa)
for 15 minutes for sterilizing culture plates, culture media, glassware, and even for the
decontamination of pathological waste, bacterial cultures, and biohazard containers (Estridge
et al., 2000).

Incineration, on the other hand, is a form of sterilization that uses dry heat. Dry heat
is heat with little to no moisture. The effect of dry heat on microorganisms changes the
microbial proteins by oxidation reactions and creates an arid internal environment, thereby
burning microorganisms in a slow manner (Pommerville, 2011). In this experiment, an alcohol
lamp is used as a source of dry heat. Other instruments such as Bunsen burners and ovens
can be used in sterilization using dry heat. This is commonly used in sterilizing inoculating
loops and when inoculating microorganisms.

This exercise aims to explain the principle and application of sterilization by the use
of moist and dry heat, compare the effects of moist and dry heat in sterilizing various
equipment and media, demonstrate the destruction of microorganisms by moist heat applied
under controlled conditions of time and temperature, explain the principle, procedure, and
application of autoclaving, and to illustrate the use and control of the autoclave.

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Materials and Methods

A. Materials

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

A tool used to retrieve inoculum from a

Inoculating loops culture for transferring to another
media/environment

A laboratory tool that produces an open

Alcohol lamp flame using denatured alcohol; used in
inoculation

A container filled with water and is

Water bath temperature-controlled for cooling/heating
purposes

Plates with solidified nutrient agar used in

Nutrient agar plates
cultivation of microbes

Staphylococcus aureus (S. A culture of the Gram-positive, round-shaped

aureus) broth culture bacterium in a liquid broth

Bacillus subtilis (B. subtilis) broth A culture of the Gram-positive, rod-shaped

culture bacterium in a liquid broth

A shallow cylindrical glass used in cultivating

Petri dish
microorganisms

A laboratory tool used to transport a

Glass pipette
measured volume of liquid

A finger-like tube of glass with a cover used

Culture tube
to culture microorganisms

A pressure chamber used to sterilize

Autoclave
equipment

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B. Methods

The exercise started by becoming acquainted with the autoclave and how to operate it,
then performing sterilization by inoculation, and then finally performing the procedures in moist
heat sterilization.

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. Sterilization of Culture Plates and Media and Operation of Autoclave

A single petri dish was obtained from the laboratory equipment room. Then, it was
wrapped in used paper in a burger-like manner. Then, it was labelled with the surname of the
person doing the exercise and the date when the exercise was performed.
The prepared plates were then placed into the autoclave. The autoclave was already
filled with enough distilled water, so there was no need to refill it. The door of the autoclave
was closed and tightened. The autoclave was set to operate at 121°C at 15 psi pressure for
15 minutes. After 15 minutes, the now sterile plates were set aside to cool and then stored in
a tray.

3. Incineration

A sterile agar plate was obtained. It was labeled ‘S. aureus’. It was divided in half by
marking the bottom of the plate. One section was labeled ‘control’ and the other one was
labeled ‘heated’. Another sterile agar plate was obtained, and it was labeled ‘B. subtilis’. The
plate was divided in the same manner as the plate for S. aureus, as well as the labels. All the
plates were also labeled with their respective incubation times. Figure 2 (p. 5) illustrates the
appearance of the plate after labelling.

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 control heated control heated

S. aureus B. subtilis
Incubation time: 14:40 Incubation time: 14:40

Figure 2. Label guide for incineration procedure.

Then, an inoculating loop was obtained and sterilized using an alcohol lamp. The loop
was cooled for a while and a loopful of S. aureus culture was taken from the broth culture. It
was streaked in a straight line on the ‘control’ section of the plate. The loop was heated again
and streaked on the ‘heated’ section of the plate. This whole process was repeated using the
B. subtilis broth culture. Figure 3 shows how the streaks should look like in the plates. The
plates were then sealed with tape to prevent contamination and was incubated at 35°C for 24
hours in an incubator.

control heated control heated

S. aureus B. subtilis
Incubation time: 14:40 Incubation time: 14:40

Figure 3. The correct way of streaking the inoculum in the plates. The black lines are the streaks.

After 24 hours, the observations were recorded as (+) for growth and (-) for no growth.

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 4. Moist Heat

A water bath was filled with water and was set to 90.2°C. Simultaneously, one agar
plate was obtained and was divided in two using a marking pen. It was labeled ‘control’. One
section was labeled ‘S. aureus’ and the other was labeled ‘B. subtilis’. A loopful of S. aureus
culture was streaked in a straight line to the section with the label ‘S. aureus’. The same
procedure was done to B. subtilis (Please refer to Figure 4 [p. 7] for the illustration of this
procedure).
Then, two new sterile agar plates were obtained. One plate was labeled ‘S. aureus’
and the other plate was labeled ‘B. subtilis’. These two plates were divided into four quadrants
and labeled: 5, 10, 15, 30. These numbers designate the time in minutes of the exposure of
the bacterial culture to moist heat.
The tubes with the bacterial culture were placed in a metal test tube rack and was
submerged in the boiling water bath. For both S. aureus and B. subtilis after 5 minutes, the
tubes were rapidly cooled under running tap water and then a loopful of the bacteria was
streaked onto the quadrant with the label ‘5’. The tubes were returned into the water bath for
another 5 minutes. The tubes were cooled and a loopful of bacteria was streaked onto the
quadrant labeled ‘10’. The tubes were submerged again in the water bath for another 5
minutes. After this, the tubes were cooled and a loopful of bacteria was streaked onto the
quadrant labeled ‘15’. The tubes were returned in the water bath submerged for another 15
minutes. Finally, the tubes were cooled and a loopful of bacteria was streaked onto the
quadrant labeled ’30’. Figure 5 (p. 7) summarizes and illustrates this part of the procedure.
The plates were then sealed to prevent contamination and were incubated at 35°C for
24 hours. After 24 hours, the observations were recorded as (+) for growth and (-) for no
growth.

5. Cleaning and Disinfection of Workplace

After all the laboratory work, the worktable was disinfected with 5% Lysol, followed by
70% isopropyl alcohol. All trash was disposed properly.

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 S. aureus B. subtilis

CONTROL
Incubation time: 15:39

Figure 4. A correctly-labeled and streaked plate for the moist heat sterilization procedure (controls).
Note that the incubation time is also written on the label. The black lines are the streaks of
the inoculum.

5 10 5 10

S. aureus B. subtilis
Incubation time: 16:15 Incubation time: 16:15

15 30 15 30

Figure 5. A correctly-labeled and streaked plate for the moist heat sterilization procedure (after exposure of bacteria
to moist heat). Note that the incubation time is also written on the label. The black lines are the streaks of
the inoculum.

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Results and Discussion

After 24 hours of incubation, the plates were all observed for bacterial growth. The
following are the results of the exercise.

A. Moist Heat

Between S. aureus and B. subtilis, only S. aureus exhibited growth after several
minutes of heating the broth culture in the boiling water bath. Table 1 summarizes the findings
of the exercise.

Table 1. Summary of results for the growth of S. aureus and B. subtilis after exposure to
moist heat at different time intervals. *

Exposure to moist heat

Microorganism Control
5 min 10 min 15 min 30 min
S. aureus + + + + -
B. subtilis + - - - -
* (+): indicates bacterial growth; (-): indicates no bacterial growth.
As seen in the table, both controls for S. aureus and B. subtilis exhibited growth. Also,
B. subtilis did not exhibit any growth for all time intervals as compared to S. aureus. Figure 6
shows the appearance of the plates after the 24-hour incubation. For S. aureus, it can be seen
in Figure 6 that as the time of exposure to moist heat increases, the growth of the
microorganism decreased. A study conducted by Iwasawa and Ishihara in 1967 showed that
S. aureus, after exposure to wet (moist) heat, had a lower mean death rate. S. aureus is
aerobically or anaerobically active at temperatures between 18°C and 40°C (Taylor & Unakal,
2017); since the water bath was set at 90.2°C, this implies that the microorganism’s activity
has decreased. This means that S. aureus is indeed a heat-resistant bacterium, whereas B.
subtilis is not.

B. Incineration

Both S. aureus and B. subtilis exhibited no growth after incineration. Table 2 next page
summarizes the results of the observations for bacterial growth after incineration and
incubation for 24 hours.

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Table 2. Summary of the results in the observation of bacterial growth after sterilization by
incineration. *

Growth
Microorganism
Control After incineration

S. aureus + -

B. subtilis + -
* (+): indicates bacterial growth; (-): indicates no bacterial growth.

S. aureus B. subtilis

CONTROL
Incubation time: 15:39

5 10 5 10

S. aureus B. subtilis
Incubation time: 16:15 Incubation time: 16:15

15 30 15 30

Figure 6. Appearances of the plates for the moist heat sterilization after incubation for 24 hours at 35°C.

Both S. aureus and B. subtilis did not show signs of growth after incineration.
Incineration involves heating at a higher temperature than 100°C, which is higher than the
temperatures where these two microorganisms survive. This simply shows that incineration is
a very effective form of sterilization. Furthermore, incineration can only be done in a few
seconds or minutes, depending on the amount and type of microorganism to be exterminated.

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Figure 7 shows the appearances of the plates after incineration and incubation after 24 hours.
It is pretty obvious that there was no growth in the ‘heated’ section compared to that of the
control for both plates.

control heated control heated

S. aureus B. subtilis
Incubation time: 14:40 Incubation time: 14:40

Figure 7. Appearances of the plates for the incineration method of sterilization after incubation for 24 hours at 35°C.

In summary, three methods of sterilization were done in this experiment: the use of
moist heat from a water bath, the use of dry heat by an alcohol lamp, and the use of the
autoclave (which also uses moist heat at a certain temperature and pressure). S. aureus was
found out to be moist heat-resistant after performing the exercise, and B. subtilis was not since
it did not exhibit growth after exposure to moist heat. Furthermore, the time of the exposure of
the microorganism to moist heat affected the growth. In S. aureus, the longer the time of
exposure, the lesser colonies of microorganisms survived. Incineration was more effective
than the water bath sterilization method. Also, the autoclave was familiarized and appreciated
for its ability to sterilize and eliminate microbes using fixed conditions of temperature and
pressure. Lastly, the objectives of this exercise were met, and this could be considered a
successful exercise.

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References

Estridge, B. H., Reynolds, A. P., Walters, N. J. (2000). Basic Medical Laboratory Techniques:
Medical Lab Technician Series. Cengage Learning.

Hogg, S. (2005). Essential Microbiology. John Wiley & Sons Ltd: The Atrium, Southern Gate,
Chichester, West Sussex PO19 8SQ, England.

Iwasawa, Y., Ishihara, K. (1967). Resistance of Staphylococcus aureus to Desiccation, Heat

and Ultraviolet Rays in Relation to Phage Pattern. Journal of Microbiology, 11(4),
305-309.

Kango, N. (2010). Textbook of Microbiology. I. K. International Publishing House Pvt. Ltd.:

New Delhi.

Maczulak, A. (2011). Encyclopedia of Microbiology. Infobase Learning: New York NY 10001.

Parija, S. C. (2012). Textbook of Microbiology and Immunology (2nd ed.). EIH Unit Ltd. Press:
Manesar.

Pommervile, J. C. (2011). Alcamo’s Fundamentals of Microbiology (9th ed.). Jones and Barlett
Publishers, LLC.: Barb House, Barb Mews, London W6 7PA United Kingdom.

Taylor, T. A. & Unakal, C. G. (2017). Staphylococcus aureus. In: StatPearls [Internet].

StatPearls Publishing: Treasure Island, FL.

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