Practical 1: Sterilisation Techniques and Culture Methods

Aim:
1. To learn the importance of sterilisation in the laboratory.
2. To acquire the skill of aseptic technique in microbiology.
3. To identify and differentiate between true motility and Brownian motion among
Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and
Escherichia coli.

4. To be able to perform and evaluate different microbial culture methods.

5. To observe and distinguish colony morphology of various type of bacteria and its
growth in different culture media.

Introduction:
Sterilization is a process to eradicate all forms of live microorganisms from a
substance. This procedure is done for articles that have direct application in humans
and animals. These materials include drugs, nutraceuticals, surgical equipment, food
etc. Sterilization is done to preserve the substance for a long time without decay.
Secondly, if a substance is not sterilized, any microbes in it may cause infections to
the people who consume it. So sterilization is essential. The microbes are invisible to
naked eye and even those like bacteria have a protective sheath on their surface
making them resistant to sterilization. For this effective sterilization techniques are
designed and studied in microbiology. The methods of sterilization include heat
methods, chemical sterilization and filtration. The heat method of sterilization is again
of two types based on the type of heat used. The heat method of sterilization is again
of two types such as moist heat methods and dry heat methods based on the type of
heat used. There are three types of filters used for sterilization which are membrane
filters, Seitz filters and candle filters. [1]
Microbiological culture is a method of producing or multiplying microbial
organisms by letting them reproduce in a culture media under laboratory conditions.
[2] There are few types of culture methods can be used for reproducing particular

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bacteria. Normally, broth culture and plate culture will be used to culture the bacteria.
Culture media is a liquid or gel designed to provide necessary nutrient requirements
for supporting the growth of the microorganisms. This culture media preparation
requires aseptic technique to prevent foreign bacterial contamination. It is usually
prepared from protein by acid or enzymatic digestion. The standard solid medium is
nutrient agar, a gelatinous substance derived from seaweed. The basic liquid medium
is nutrient broth, typically a mix of water, meat extract peptone, and sodium chloride.
[3]
Normally, the motility of the bacteria and the types of motion of the bacteria
are studied under microscope. Some bacteria with flagella or some special structure
enables them to move or “swim”. There are some bacteria have Brownian movement.
These bacteria with Brownian movement cannot swim or move, but they vibrate at
fixed place. However, flagella are not possessed by all microorganisms. Pseudomonas
Aeruginosa and Staphylococcus Aureus are used for evaluation of bacterial motility in
this practical session. There are three methods used in determining the motility of
microorganisms; these include the hanging drop method, soft agar stab and swarm
agar plate.

Method:
Refer to laboratory manual PP141/4 Microbiology For Pharmacy, Practical 1, page 8-
15.

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Results：
Part A: Sterilisation
i. Dry Heat Sterilisation
 Heating

Observation: Both the inoculating loop and inoculating needle turned red hot
when hold vertically towards the flame of Bunsen burner. The red glow faded
immediately and retained to their original colour once they were removed from
the Bunsen burner.
 Flaming

Observation: The scalpels and forceps are heated by swaying them gently across
the flame of Bunsen burner. The scalpels and forceps have no visible changes. In
addition, the cultural tubes are heated by placing the mouth of the cultural tubes
near to the flame. No significant is observed.

ii. Moist Heat Sterilisation

Figure 1 shows the observable change to the nutrient broth in both autoclaved and
non-autoclaved McCartney bottles.

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 McCartney bottle Observation
Autoclaved Nutrient broth remained clear.
Non-autoclaved Nutrient broth becomes cloudy.

iii. Filtration

Observation:
The colonies in yellowish color are formed
on the surface of the membrane filter after
the bacteria is incubated for 24 hours.

Part B: Culture Methods

Type of Culture Media Observations
i. Broth Culture The nutrient broth
E.Coli grown in a liquid medium appeared to be turbid
and a whitish layer was
observed at the bottom.
This indicated a
sediment growth
pattern.

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 ii. Agar Slope Culture Escherichia coli
E.Coli grown in a test tube of growth occurred on the
solid medium surface of the slanting
part of nutrient broth
with beaded growth
pattern.

Table 2 shows the observations of the growth patterns of E. coli in two different types
of culture media.
iii. Stab Culture

Types of Observation
bacteria Before incubation After incubation Growth pattern
on the culture

Escherichia Liquefied nutrient Liquid gelatin solidified after put Effuse (spreading)
coli gelatin into ice water.
Cream Escherichia coli colonies
appeared throughout the gelatin.

Bacillus Liquefied nutrient Lipuid gelatin remains the same Effuse (spreading)
subtilis gelatin after put into ice water. Pale
yellow colony of Bacillus subtilis
appeared throughout the gelatin.
Table 3 shows the growth pattern of Escherichia coli and Bacillus subtilis in nutrient
gelatins and the changes in nutrient gelatins.

iv. Plate Culture

 Quadrant Streaking
Observation:

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 The inoculums (Escherichia coli) grow along the four segments of parallel
streaked line has higher concentration of colonies but the colonies lessen after
the second segment. The forth segment has the least concentration of
colonies, it has been streaked apart.

Bacteria Size Colour Form Elevation Margin Texture

(mm)
Escherichia 2.0 Creamy Circular Raised Entire Smooth
coli yellow /Shiny

Table 4 shows the observations of colony morphology and growth of Escherichia

coli in plate culture.

 S-shaped Streaking

Observation:

The inoculums (Escherichia coli)

grow along S-shaped streaked lines
in Petri dish .The line is occupied
with high concentration of the
colonies. No single isolated colonies
can be seen in this streaking
method.
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Part C: Motility Evaluation
i. Hanging Drop Method

Bacteria Observation
Pseudomonas The bacteria swim around. Motility movement was
aeruginosa observed.
Staphylococcus aureus The bacteria vibrate in its own position. Brownian motion
was observed.
Table 5 shows the motility of Pseudomonas aeruginosa and Staphylococcus aureus
observed under a microscope.

ii. Soft Agar Stab

Bacteria Observation
Pseudomonas aeruginosa

Bacteria Pseudomonas aeruginosa swim away from the

line of inoculation. The growth pattern of the inoculum
shows that it spread away from the line of inoculation.
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 Staphylococcus aureus

Bacteria Staphylococcus aureus grow along the line of

inoculation. No spreading of the growth of the bacteria
can be seen.
Table 6 shows the growth pattern of Pseudomonas aeruginosa and Staphylococcus aureus
iii. Swarm Agar
in a soft agarPlate
culture.

Bacteria Size Colour Form Elevation Margin Texture

(mm)
Pseudomonas 6-7 Creamy Circular Convex Entire Rough
Aeruginosa white
Staphylococcu 4-5 Creamy Irregular Raised Undulate Smooth
s Aureus white /Shiny
Bacteria Observation
Pseudomonas
aeruginosa Three yellowish single
colonies are found on the
surface of the swarm agar
plate. The spreading of these
three single colonies can be
seen.

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Staphylococcus
aureus Three yellowish single
colonies are found on the
surface of the swarm agar
plate. There are no spreading
of these three single colonies
can be seen.

Table 7 shows the observation of colony morphology of Pseudomonas aeruginosa and

Staphylococcus aureus in swarm agar plate cultures.

Discussion:
Part A: Sterilisation

Sterilisation can be divided into two methods which is physical and chemical
methods. Physical method used in this practical which the sterilisation is done by heat
and filtration. Chemical sterilisation includes using chemicals such as alcohols to
remove traces of microbial life. In part A of this practical, three types of sterilisation is
carried out to sterilise different kind of apparatus.
In dry heat sterilisation, inoculating loop and straight wires are burnt over the
flame until turn red. This is a simple method of effective sterilization of such articles
but it is limited for those articles that can be heated to redness in flame. Besides that,
flaming was carried out. The articles such as scalpels, needle, forceps and mouth of
culture tubes are sterilized by passing through the flame of Bunsen burner without
allowing them to become hot. Even though most vegetative cells are killed, there is no

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guarantee that spores too would die on such short exposure. [7]This method is limited
to those articles that can be exposed to flame. Cracking of the glassware may occur.
Another form of dry heat sterilization was hot air oven sterilization. In this type of dry
heat sterilization, the tools used were placed in an oven with a high temperature for
nearly two hours as the heat in water was more readily transferred to a cool body
compared to when they were heated in air. These three types of dry heat sterilization
are carried out before the culture process so that we can prevent any unnecessary
contamination on the culture.
The second type of sterilization is moist heat sterilization which involved the
instruments being autoclave under a temperature above that of boiling water
temperate. Different time is required for various container sizes and volume of liquid.
In this part of experiment, two McCartney bottles are filled with nutrient broth and
one of the bottles is placed inside the autoclave unit at 15 psi for 20 minutes, at
121˚C. The bacteria only grew in the non-autoclave nutrient broth. The nutrient broth
turns turbid. The other bottle that has been autoclaved remain unchanged. This is
because when the nutrient broth is autoclaved, the high temperature and the pressure
in the autoclave unit kill virtually all vegetative cells present and the endospores. [8]
In comparison, moist heat sterilization was more effective than dry heat sterilization
as the hydrated protein molecules were denatured with lower energy spent than
dehydrated semi-crystalline structure protein.
The third type of sterilization is filtration. Filtration doesn't kill any
microorganisms but only remove them from the solution. In the filtration method, a
membrane filter is used. It was made up of cellulose acetate, cellulose nitrate,
polycarbonate, polyvinylidene fluoride, or other synthetic materials with a thickness
of 0.1mm, had very small pores sizes with diameters of 0.2 µm which small enough to
trap bacteria or absorb the microbes are used. Thus, it known as a high efficiency
particulate air filters as it can remove almost all the microbes larger than 0.3µm in
diameter. Filtration was conducted by placing the heat-sensitive sucrose solution into
the upper chamber and then was forced through the membrane filter using a vacuum

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located at the lower chamber. The membrane was then removed and placed on the
nutrient agar plate. Consequently, bacterial growth was observed, as many colonies
were seen formed and eventually stained the whole membrane filter with a yellowish
shade. This shows that contaminants and bacteria is filtered out from the sucrose
solution.

Part B: Culture Method

A culture medium is a nutrient material prepared for the growth of

microorganisms in s laboratory. Four culture methods such as broth culture, agar slope
culture, stab culture and plate culture were used in this experiment. Aseptic techniques
were carried out at the start of all procedures to ensure complete sterilization as a very
important preventative measure from contaminations.
First, broth culture is which bacteria grow in liquid medium. Liquid medium is
easier to use because it saves time and wide range of bacteria are suitable for this
medium. The broth solutions are clear before bacterial inoculums are dipped into it.
Escherichia coli is used in this experiment. Based on the observations, it was recorded
that Escherichia coli grew in a sediment pattern as a whitish layer was observed at the
bottom of the test tube and the colony had spread throughout the whole test tube,
causing the nutrient broth appeared to be turbid. This shows that Escherichia coli
would grow and multiply forming colony under favourable condition which is in
broth culture that contains suitable nutrients and a suitable temperature which is 37˚C.
Secondly, agar slope culture is used to preserve bacteria culture. As for the agar
slope culture, the solid agar was solidified into slant form so that we have more
surfaces to sweep the bacteria. For the agar slope culture with E. coli, colonies of E.
coli are seen on the surface of the agar in a beaded form. This result shows the E. coli
was creamy yellow and showed a beaded growth pattern.
In stab culture, Escherichia coli and Bacillus subtilis were the two bacteria
cultured. For Escherichia coli, the gelatine remained as solid. However, for Bacillus

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subtilis, the gelatine turned into liquid form on the top portion of the gelatine. Hence,
this proves that Bacillus subtilis produce gelatinase enzyme which can liquefy the
gelatine. Escherichia coli do not produce this enzyme.
Lastly, which is the plate culture, two ways of streaking were done – quadrant
streaking and S-shaped streaking. Quadrant streaking was carried out to obtain a
single colony. It was observed that this method do enable us to reduce the bacterial
colonies to a single colony thus we were able to observe its colony morphology on the
plate culture. Based on the cultural characteristics, Escherichia coli’s colonies were
found to be in circular form with an average diameter of 2.0mm, creamy yellow,
raised elevation and an entire margin with smooth and shiny texture. When S-shaped
streaking method was done, the circular form of growth and size of single
Escherichia coli’s colony could not be identified. It grew along the streaking line only
and there is no any single colony to be seen.

Part C: Motility Evaluation

Motility evaluation is the part C of this experiment. Hanging drop method has
been carried out by the first. It is used for investigating the motility of Pseudomonas
aeruginosa and Staphylococcus aureus. True motility is a movement in some
consistent direction, although there may be many twists and turns. Brownian motion
is not a true form of motility which the bacteria only vibrate at the same place. This is
because microscopic particles suspended in a liquid or gas, caused by collisions with
molecules of the surrounding medium. Based on the result, Pseudomonas aeruginosa
showed true motility, swimming forward and backwards, reversing rapidly by
changing direction of flagella rotation whereas the immotile Staphylococcus aureus
showed Brownian motion.
For soft agar stab, the results showed that Pseudomonas aeruginosa grow in the
way of spreading whereas Staphylococcus aureus bacteria grow along the line with no

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spreading. This shows that Pseudomonas aeruginosa is motile bacteria because it
moves and spreads from the line. The reason for the movement can be for the survival
purpose such as looking for nutrients, causing turbidity throughout the medium. For
the Staphylococcus aureus, the stab line remains. This shows that the bacteria only
stayed at fixed place.
Lastly is the swarm agar plate method. Both the bacteria Pseudomonas
aeruginosa and Staphylococcus aureus are once again dipped vertically into the
swarm agar plate at three areas using a straight wire and are incubated. After
incubating, Pseudomonas aeruginosa is presence in three creamy white colonies
formed on the agar which spreading occurred on each colony whereas Staphylococcus
aureus is presence in three creamy white colonies but there is no branching nor
spreading of the colony occurs on each colony. This proved that Pseudomonas
aeruginosa is a motile bacterium whereas Staphylococcus aureus is a non-motile
bacterium. Besides, some of the key morphology can be determined. From the results,
Pseudomonas aeruginosa are circular in form, convex elevated, entire in margin, and
have rough surface whereas Staphylococcus aureus appeared in irregular form,
convex elevated, undulate, and smooth and shiny texture.

Questions and Answers:

1. Give the principles of various sterilisation methods you have studied.
In dry heat sterilisation, heat kills microorganisms by destructive oxidation.
Dry heat sterilization is accomplished by conduction. The heat is absorbed by the
outside surface of the item, then passes towards the centre of the item, layer by
layer. The entire item will eventually reach the temperature required for
sterilization to take place. Dry heat does most of the damage by oxidizing
molecules. [4]
In moist heat sterilization, heat kills microorganisms by denaturing protein in
autoclave. Autoclaving is a process of steaming under pressure in an autoclave at
15 psi, 121°C. Under theses extreme conditions, all microbes including endospores

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 can be killed in 15 minutes. [5] 3 aspects to be concerned, which are thermal death
point (TDP), thermal death time(TDT) and decimal reduction time. Thermal death
time(TDT) is the length of time to be rendered sterile, thermal death point(TDP) is
the degree of heat resistance of bacteria must be considered while for decimal
reduction time, it is the time taken for 90% of bacterial population to be killed at a
given temperature. Medium with higher volume will need a longer time for
sterilization since heat need more time to go into the medium.
Main usage of filtration is to sterilise liquid suspension and trap
microorganisms with a membrane filter. Bacteria which have larger size than the
membrane filter pores will be trapped as the residue on the membrane filter.

2. Name the items in your laboratory that can be sterilised using the autoclave.

Petri dishes, beakers, and forceps, boiling tubes, pipettes, test tubes, McCartney
bottles, laboratory glassware, some solutions and water can be sterilized using
autoclave.

3. What are the precautions taken when you operate an autoclave?

The bottles should not close too tightly when bottles are sent for autoclave. We
have to make sure the container is occupied not more than 75%. This is to ensure
that the fluid has room for expansion when heat applied. Also, exhaust valve must
be closed. It is necessary to wear gloves when we want to take out the bottles and
open the autoclave unit after cooling for some time. [6]

4. What are the essential requirements of a culture medium?

The culture medium must be sterile first so that no other microbial growth was
found on the medium. It must contain the right nutrients for the optimal growth of
specific microorganisms. The essential requirements of a culture medium should
include a suitable pH, an appropriate concentration of oxygen, sufficient moisture

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 and nutrients in the correct amount and types for the particular species of bacteria
to be cultured. In addition, the culture medium needs to be sterilised to ensure a
pure culture will be obtained at the end. Then, the growing culture should undergo
incubation at a suitable temperature.

5. What are the advantages of liquid medium?

Liquid medium is more effective on culturing microorganisms as the liquid form

causes less damage to the spores. A large number of pure culture microorganism
can be grown in the medium. The bacteria is suspended in a liquid broth. It is
water-based and does not solidify at temperature above its freezing point. It can
flow when the container is tilted. The growth rate of microorganisms is also
markedly faster in a liquid medium as compared to a solid medium. In addition,
several growth patterns can be observed in liquid medium, which are
uninoculated, pellicle, sediment, turbidity and flocculent.

6. What are the advantages of solid medium?

By using solid medium, number of different microbes appear on the sodium

medium can be identified and counted easily and the mixed bacteria can be
separated easily. Furthermore, there will be less contamination when a solid
medium is used for culture. A solid medium can also be incubated at a higher
range of temperatures approaching 100oC before it liquefies. Hence, this is useful
for culturing thermophilic bacteria.[7]

Conclusion:
In conclusion, aseptic techniques and sterilisation methods are essential initial
steps before carrying out any laboratory procedures to ensure that all techniques are
done meticulously in order to fully eliminate unwanted microorganisms from the tools
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and apparatus being used in any experiments. These processes are also important for
us to get a more accurate and reliable results. Apart from that, different culture
methods are used to investigate the growth pattern of bacteria in broth and slants and
the colony morphology in agar plate. Based on the result, the growth pattern of E.coli
in liquid medium is sediment whereas it is grown in beaded growth pattern in solid
medium. In addition, soft agar stab and swarm agar plate culture is important in
determining the motility of microbes cultured. True motility (Pseudomonas
aeruginosa, Escherichia coli, Bacillus subtilis)and Brownian motion (Staphylcoccus
aureus) are identified and differentiated by hanging drop method, soft agar stab and
swarm agar plate.

References:

1. Study Read.What is Sterilization? 9 Sterilization Methods in Microbiology.

Available at: http://www.studyread.com/what-is-sterilization-methods
(accessed 24th September 2016)

2. Greenwood D, Slack R, Peutherer J, Barer M. Medical Microbiology: A Guide

to Microbial Infections: Pathogensis, Immunity, Laboratory Diagnosis and
Control. 17th ed. UK: Churchill Livingstone; 2007.

3. Willey JM, Sherwood LM, Woolverton CJ. Prescott's Principles of

Microbiology. 1st ed. USA: McGraw-Hill; 2009.

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4. Microbe Online.Dry-Heat Sterilization: Principle, Advantages and
Disadvantages. Available at: https://microbeonline.com/dry-heat-sterilization-
principle-advantages-disadvantages/ (accessed 24th September 2016)

5. Pharmainfo.net. PRINCIPLES OF STERILIZATION. Available at:

http://www.pharmainfo.net/siriki-praveen-kumar/blog/principles-sterilization
(accessed 24th September 2016)

6. Willey JM, Sherwood LM, Woolverton CJ. Prescott's Principles of

Microbiology. 1st ed. USA: McGraw-Hill; 2009.

7. R.S. Setty. Biotechnology-li: Including Cell Biology, Genetics, Microbiology.

New Age International.1 January 2007. Pg 262.

8. R. Vasanthakumari. Textbook of Microbiology. BI Publications Pvt. Ltd. 1

January 2007. Pg. 74

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