Universiti

Malaysia Sabah
Faculty of Engineering

HK03: Chemical Engineering

Semester 2 2016/2017

KC31001 Laboratory 6

BIOPROCESS PRINCIPLES

GROUP 9

EXPERIMENT 1 : PREPARATION OF CULTURE MEDIA AND ISOLATION

OF PURE CULTURE

Lecturer : Madam Hafeza Abu Bakar

Lab Assistant : Ms Nor Aemi

Date : 28 February 2017

Venue : Bioprocess Lab

NAME : ANEESCH PREETHA A/P VIDA NAIGAM

MATRIC NO : BK13110587

No. Group Members Matric No

1. ANANTH A/L MANICKAM WASH BK14110066
2. ELLVANNDHO JUNNY BK14110008
3. LAURA VITALIS BK14110141
4. NORAZRINAH BINTI RAMON BK14110179
5. ONG XIN ROU BK14110198
6. SYLVIANAH SYLVESTER BK14110053
BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

1.0 ABSTRACT

This experiment aims to prepare a broth medium. The microorganism used is

Lactobacillus sp. Plus, it is also to use an autoclave to sterilize the prepared medium and
required lab wares. The main idea is to transfer the culture using aseptic technique without
contaminating the culture or exposing ourselves to them. We transfer the culture onto the
agar plate using the streaking method.

2.0 INTRODUCTION

A microbial culture or a pure culture is actually a method used to multiply the

number of microorganism by allowing them to reproduce in a predetermined culture
media under controlled laboratory conditions. In this experiment, we have used
Lactobacillus sp. This experiment is actually to determine the type of organism and its
abundance in the sample being tested. This is one of the main diagnostic procedures in
microbiology and often used as a tool to identify the cause of infectious diseases by
allowing the agent to multiply in a predetermined medium.

The simple methods for isolation of a pure culture are spread plating on solid agar
medium with a glass spreader and streak plating with a loop. In this experiment, we
have used the streak plate technique, whereby the bacteria culture is streaked on an
agar plate. In this experiment, we have used MRS Broth Agar. The purpose of spread
plating and streak plating is to isolate individual bacterial cells, which are colony forming
units, on a nutrient medium.

The history of the modern streak plate procedure has evolved from attempts by
Robert Koch and other early microbiologists to obtain pure bacterial cultures in order to
study them, as detailed in an 1881 paper authored by Koch. Robert Koch realized the
importance of solid media and he had used potato pieces to grow bacteria and agar was
used to solidify culture media. Before the use of agar, attempts were made to use
gelatin as solidifying agent. However, gelatin had some inherent problems whereby it
existed as liquid at normal incubating temperatures (3537C) and was digested by
certain bacteria.

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

So, ever since, agar has been one of the medium used. Agar is actually prepared
from seaweeds. It has no nutritional value. The most important characteristic is that it is
not affected by the growth of the bacteria. In this experiment, we have used M.R.S.
Broth Agar. Often abbreviated to MRS, this medium is named by its inventors; de Man,
Rogosa and Sharpe. This agar was developed in the 1960s, and was designed to favour
the luxuriant growth of Lactobacilli for lab study. We have also used Lactobacillus sp. in
our experiment. It contains sodium acetate, which suppresses the growth of many
competing bacteria. This medium has a clear brown colour.

3.0 MATERIALS AND APPARATUS

Nutrient Apparatus Equipment

Peptone(Fluka) Microscope slides PH meter
Ethanol 95% (Fluka) Microscope covers Balance
MRS Broth (Oxoid) Conical flask (250 ml) Autoclave
MRS Agar (Oxoid) Graduated cylinder (1L) Incubator
Gram stain Crystal Violet Beaker (250 ml, 500 ml Oven
Gram stain iodine Loop spectrophotometer
Gram stain Safranin Pipette (100, 1000 microlitre)
Pipette tips (100,1000 microlitre)
Microcentrifuge tubes (1.5 ml)
Cotton
Aluminium foil
Sterile petri dish
Paper tape
Para-film
Filter paper 0.2 micron
Quartz cuvette
Bunsen burner

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

4.0 EXPERIMENTAL PROCEDURE

4.1. Preparation of medium and sterilization

Nutrient broth and agar was prepared in advance by our lab demonstrators.

4.2 Isolation of single colony

Firstly, the wire of inoculating loop was inserted into the bunsen burner flame
until it is red and glowing. Then, the wire was allowed to cool for a few seconds.
The tube containing the culture was picked up by free hand and it was shook
gently to disperse the culture. The tube cap was removed and the lip was flamed
in Bunsen burner flame. The sterile loop was inserted and a loop full of culture
was removed. At this point, the sides of the tube were non-touchable. The tube
lip was flamed again and the tube cap was replaced. The loop containing the
inoculum was placed into the tube. The loop was removed and the agar surface
was crossed streak three to four times and the streaking was continued into a
second adjacent area. Then, the agar plate was covered with its lid. The
inoculating loop was flamed. The streaked plate was placed into a 37 C
incubator and was incubated for 48 hours. Finally, the plates were examined and
its appearance was described. After the observation, the plates were kept in the
fridge for the following experiment.

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

Figure 4.2.1 Preparation of culture medium

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

Figure 4.2.2 The streak plate technique

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

5.0 RESULTS

Figure 5.1 Growth of Lactobacillus sp. after 48 hours in MRS Broth Agar

This observation is obtained after 48 hour of conducting the experiment. There

is a noticeable growth of bacteria colonies on the agar plate. The colonies of bacteria
that are attached together were found the most by the first streaking, and the numbers
of colonies decrease with the increasing numbers of streaking. It can be clearly seen
that the last cells which were rubbed off by the inoculating loop far enough apart can
grow into isolated single colonies. The bacteria colonies colour resembles of pale white
to the naked eye.

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

6.0 DISCUSSION

In this experiment, the pattern of the streak is not well developed. The pattern cannot
be seen. The expected streaking pattern would clearly help to identify the reducing number
of bacteria. Since this is my first time streaking, the streaking didnt go well. Yet, this
experiment is considered successful because the microbial organisms had successfully grown
on MRS Agar.

Before and after the streaking, our hands must be sanitized to wipe out the bacterial
contamination. Plus, the laminar flow must be wiped and cleaned with alcohol, which is also
for sterilization purpose. All the procedures need to be done inside the laminar flow for
safety precaution. So as to not inhaling the bacteria we are dealing with.

In the streaking procedure, a sterile loop is used so that the microbial culture is
uncontaminated. The inoculating loop is then streaked over an agar surface. On the initial
region of the streak, many microorganisms are deposited resulting in confluent growth or
the growth of culture over the entire surface of the streaked area. The loop is sterilized by
heating the loop in the blue flame of the Bunsen burner, between streaking different
sections, or zones and thus lesser microorganisms are deposited as the streaking
progresses. We have used the four quadrant streak method.

In order to obtain a well-developed culture medium, it must be stored at the specified

temperature, under specified conditions such as pH and humidity. All the prepared culture
media and their components have be stored in dark. Direct exposure to sunlight should be
avoided at all times. This is because, hot and steamy media preparation rooms are not
suitable environments to store containers of culture media; particularly containers which are
frequently opened and closed. Unopened containers should be stored at room temperature
which is in the range of 15-20C.

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

The culture media also should be sterilized in a steam autoclave at temperatures

between 121-134C for 20 minutes to make sure all pathogen is damaged. Sterilization is
actually a process that eliminates or kills all forms of life, including transmissible agents
(such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a
fluid, in medication, or in a compound such as biological culture media. Sterilization can be
achieved by applying the proper combinations of heat, chemicals, irradiation, high pressure,
and filtration. In fact, in the beginning, before streaking the bacteria culture onto the agar
plate, the inoculating loop was also sterilized to prevent foreign particle intervention.

A widely-used method for heat sterilization is the autoclave. Autoclaves commonly use
steam heated to 121134 C (250273 F). To achieve sterility, a holding time of at least 15
minutes at 121 C (250 F) or 3 minutes at 134 C (273 F) is required. Following
sterilization, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over
when the pressure is released. Proper autoclave treatment will inactivate all fungi, bacteria,
viruses and also bacterial spores, which can be quite resistant. It will not necessarily
eliminate all prions.

For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an
autoclave must not be overcrowded, and the lids of bottles and containers must be left ajar.
Alternatively steam penetration can be achieved by shredding the waste in some Autoclave
models that also render the end product unrecognizable. During the initial heating of the
chamber, residual air must be removed. Indicators should be placed in the most difficult
places for the steam to reach to ensure that steam actually penetrates there.

For autoclaving, as for all disinfection or sterilization methods, cleaning is critical.

Extraneous biological matter or grime may shield organisms from the property intended to
kill them, whether it physical or chemical. Cleaning can also remove a large number of
organisms. Proper cleaning can be achieved by physical scrubbing. This should be done with
detergent and warm water to get the best results. Cleaning instruments or utensils with
organic matter, cool water must be used because warm or hot water may cause organic
debris to coagulate.

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

Last but not the least, during the incubation, the petri dish should be inverted upside
down. It is to avoid the water vapour resulting from the respiration of the bacteria condense
and drop back to the culture of the bacteria. This may cause contamination or inhibits the
microbial growth of the desired bacteria. After incubation, the agar plate should be stored
inside a fridge in order to slow down the metabolism of the microorganism for next
experiment.

7.0 QUESTIONS

1. What can you notice in the control plate?

In this experiment, we dont have a control plate. Anyways, a control plate is a medium that
has been previously assessed for growth promotion and approved for use. Supposedly, in
this lab the control plate should have the same bacteria, same amount, and same
temperature as all the other plates. In addition, nothing is done to the control plate that will
change the bacteria.

2. Did you obtain isolated colonies on the agar plates which were streaked? If
you did not obtain isolated colonies, what changes should you make in your
technique to ensure isolated colonies?
Yes, I did obtain isolated colonies on the agar plates. If isolated colonies are not obtained,
the streaking method should be improved. By streaking the inoculum across the surface of
the agar, a spatial separation of cells is achieved. It requires some skill to achieve a plate
that reveals well-isolated colonies, however, good technique is forthcoming with experience
and practice.

1. What is subculturing?
Subculturing is made by transferring some or all cells from a previous culture to fresh
growth medium. This action is called subculturing or passaging the cells. Subculture is used
to prolong the life and/or expand the number of cells or microorganisms in the culture.

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

4. When and why do you need to apply isolation of pure culture? Support your
reason(s) with three examples.

Firstly, we apply it in the manufacturing process in the food industry and in the
pharmaceutical process. Both raw materials and final medicines and food products will
contain microorganisms unless measures are emplaced for removal. In medicines the
preparation of sterile products is an expensive procedure with skilled machines. There is
some consideration needed to ensure the manufacture of the product is such, as the
microorganism has not been contaminated. It is necessary to prevent contamination with
other bacteria since there may be competition for nutrients, the required enzyme may not
be produced as readily, and the end product may be contaminated and unsafe. The
required enzyme that is finally produced must also be isolated from the microbial cells.

5. What is the difference between streak plate and spread plate method?

For a streak plate, we streak the sample across one edge of the plate using the loop if
that is how the sample came, or a sterile loop if the sample is a liquid. It gives four different
dilutions of the sample across different areas of the plate. This is used for samples
suspected of having a high level of bacterial flora, eg faeces, sputum, skin and body cavity
swabs, and drain swabs.
For a spread plate we put a drop or a few drops of sample onto the plate and spread it
all over using the sterile loop. This is used for all of the streak plate samples except faeces
unless a very specific enrichment medium is used, resulting in fewer colonies, for samples
having a lower suspected level of bacterial flora than any of the above, and where a pour
plate cannot be done because the sample cannot be dispersed in a liquid. The spread plate
area is fully covered with bacteria and has an antibiotic disk showing moderate sensitivity of
the bacteria to the antibiotic.

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

8.0 CONCLUSION

As a conclusion , we are able to learn and conduct the appropriate methods to streak on
an agar plate. Culture media must be stored at the specified temperature, under specified
conditions such as pH and humidity. Direct sunlight have to be avoided at all times from
exposure of culture media and their component. To prevent humidity of laboratory, all
plastic containers are sealed. There are specific temperature for sterilization of culture
media. The culture media needs to be sterilized to make sure all pathogen is destroyed.
Plus, we managed to get familiar with the sterilizing method and also with the usage of an
autoclave. We proved that there is existence of Lactobacillus sp. on MRS Broth Agar.

9.0 REFERENCES

1. http://en.wikipedia.org/wiki/Growth_medium
2. www.neogen.com/Acumedia/pdf/ProdInfo/7146_PI.pdf
3. https://www.scribd.com/doc/32307482/Differences-Between-Streak-Pour-and-
Spread-Plating
4. Lab manual

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BP1 Preparation of Culture Media and Isolation of Pure Culture Semester 2 2016/2017

10.0 APPENDICES

Culture medium in incubator

Lactobacillus sp. on MRS Broth Agar

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