SY 10401

PRACTICAL OF MICROBIOLOGY

LABORATORY REPORT 3

MEDIA PREPARATION, ISOLATION OF PURE CULTURE AND BACTERIAL

GROWTH

19/4/2012

DIVAKARAN AL/ CHANDRAHASAN

BS11110710

MISS. HARTINIE BT MARBAWI

MISS ARYUNNI ABU BAKAR

BIOTECHNOLOGY PROGRAMME

SEMESTER 2, SESSION 2011/2012

Title
Media preparation, isolation of pure culture and bacterial growth

Objectives
1. To learn how to make sterile microbiological agar plate media and pour agar plates.
2. To learn how to perform the aseptic technique to inoculate and grow a pure culture in
agar plates.

Introduction
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. Slices of sterilized potatoes became the first
solid media employed on which to grow bacteria. This process was a procedure that worked
only for a few organisms and only until the bacteria decomposed the potato surface. A
search for other materials led to experimentation with the suitability of gelatin and agar-agar
as solidifying agents. Gelatin was difficult to prepare and difficult to use at room
temperature, let alone at the higher temperature of an incubator, and many bacteria digest
the protein. Agar, because of its characteristics of melting only when boiled, rarely being
digested by bacteria, and providing a substance in which other nutrients could be dissolved,
proved to be a suitable material on which to grow bacteria.
An agar plate is a sterile Petri dish that contains agar plus nutrients (media), used to
culture microorganisms. Selective growth compounds may also be added to the media, such
as antibiotics. Individual microorganisms placed on the plate will grow into individual
colonies, each a clone genetically identical to the individual ancestor organism (except for
the low, unavoidable rate of mutation). Thus, the plate can be used either to estimate the
concentration of organisms in a liquid culture or a suitable dilution of that culture, using a
colony counter, or to generate genetically pure cultures from a mixed culture of genetically
different organisms, using a technique known as streaking.
Like other growth medium, the formulations of agar used in plates may be classified
as either defined or undefined; defined medium being synthesized from the individual
chemicals as required by the organism so that the exact molecular composition is known,
while undefined medium is made up of natural products such as yeast extract, where the
precise composition is unknown.

Materials
1. LB Agar (Bacto Tryptone 10g/L, Bacto Yeast Extract 5g/L, NaCl 10g/L and Agar 10g/L)
2. Culture of bacteria
3. Nutrient agar plates
4. Inoculating loop
5. Bunsen burner
6. Marking pen

Methods
A. Agar Plates Preparation

1. A 250 ml conical flask was filled with 50 ml distilled water. A large stir bar was placed in.
2. The media was added with ingredients without agar (the weight of ingredients needed
to prepare 100 ml was calculated). One ingredient was let to be dissolved first before
the next was added.
3. The volume was adjusted to about 80 ml and pH to 7 using NaOH.
4. Distilled water was added to the final volume (100ml) in a graduated cylinder.
5. The solution was transferred back to conical flask and 1% agar powder was added.
(Example : If volumes of 100 ml are made, 1g of agar will be used)
6. The flask was covered with tin foil and a piece of autoclave tape.
7. The medium was autoclaved for 15 minutes and cooled down to 50 ˚C before pouring
into Petri dishes.

B. Streaking Agar Plates

1. The LB plates were labeled with the name of the bacteria and date.
2. The inoculating loop was sterilized in the Bunsen burner flame until red hot. It was
allowed to cool in 10 seconds.
3. The lid of the plate with E.coli given was lifted and the loop was touched to an area of
the plate without any growth to be made sure the loop was cool. Then a colony was
selected and the colony was touched with the loop. The lid was replaced.
4. The lid of the sterile LB plate prepared was raised and the loop was streaked across
approximately one-quarter of the plate. The loop was hold loosely like a pencil and the
loop was lightly flicked across the surface of the agar. It was streaked rapidly rather
than drawing on the plate. The lid was hold just above the plate while streaking to
minimize airborne contaminants.
5. The lid was replaced on the Petri dish and the loop was resterilized in the Bunsen
burner. At an angle to the original streaks, the sterile loop was streaked five or six times,
across the original streaks into a second quarter of the plate. The second quarter was
streaked without passing the loop in the previous streaks.
6. Step 5 was repeated with a third and fourth quarter of the plate and the lid was
replaced. All of the plate’s area was covered virtually with streaks.
7. The E.coli strains were incubated at 37˚C in inverted position and were observed after
24 hours. The results were recorded.
Results
Data
Picture below shows the agar medium with E. coli after incubated for 24 hour.

Discussion
Streaking is a technique used in microbiology to isolate a pure strain from a single
species of microorganism, often bacteria. A microbiological culture can be grown so that the
organism can be identified, studied, or tested. A sterile cotton swab or inoculation loop is
sterilized and dipped in a broth or patient specimen containing many species of bacteria.
The loop is then spread across one quadrant of an agar plate containing a growth medium
which has been sterilized in an autoclave. This introduces a solution of the bacteria or fungi
to a substrate which provides them nutrients. Choice of which growth medium to use
depends on which microorganism is being cultured and which are being selected for, if any.
Growth media are usually based on agar, a gelatinous substance derived from seaweed.
The loop is re-sterilized and dragged across the inoculated quadrant of the streak plate. This
is done to collect some bacteria on the loop.
The loop is spread around another fourth of the plate much like the previous step.
The loop is sterilized and the procedure is repeated. Each time the loop gathers fewer and
fewer bacteria until it gathers just one single bacterial cell that can grow into a colony.The
streak plate is then incubated, usually for 24 to 36 hours, to allow the bacteria to reproduce.
At the end of incubation there should be enough bacteria to form visible colonies in the
areas touched by the inoculation loop. From these mixed colonies, single bacterial or fungal
species can be identified based on their morphological (size/shape/colour) differences, and
then sub-cultured to a new media plate to yield a pure culture for further analysis.
 An autoclave is a pressurized device designed to heat aqueous solutions above their
boiling point to achieve sterilization. Under ordinary circumstances (at standard pressure),
liquid water cannot be heated above 100 °C in an open vessel. Further heating results in
boiling, but does not raise the temperature of the liquid water. However, when water is
heated in a sealed vessel such as an autoclave, it is possible to heat liquid water to a much
higher temperature. As the container is heated the pressure rises due to the constant
volume of the container. The boiling point of the water is raised because the amount of
energy needed to form steam against the higher pressure is increased. An autoclave is ideal
laboratory equipment used to prepare a standard solution, at 121˚C for 15 minutes. An agar
plate container is labeled with autoclave tapes to determine whether the container is
completely sterilized after autoclaving. The exhaust of autoclave is then closed to build up
pressure. If a little of the exhaust is opened, the pressure inside the autoclave will drop
gradually and may affect the autoclave process.

Conclusion
As a conclusion, the agar plate can be prepared in the laboratory for the purpose of
microorganism culturing. The well isolated colony is necessary for further microorganism
study. This is done by streaking, enhance by the sterilized inoculation loop. For the optimal
reproduction of microorganism, some ideal conditions must be prepared, such as incubation
for the temperature of 37˚C.

References
1. Karp, Gerald. 2005. Introduction to the Study of Cell Biology. In: Fitzgerald, Patrick,
Osnato, Geraldine, Russiello B, editors. Cell and Molecular Biology. 4th Edition. Hoboken,
New Jersey: John Wiley and Sons, Inc.
2. Madigan, Micheal T. Martinko, John M. Dunlap, Paul V. and Clark, David P. (2009).
Brock: Biology of Microorganisms. Nutrition, Culture, and Metabolism of Microorganisms.
12th Ed. San Francisco:
3. Pearson Education, Inc. Madigan, Micheal T. Martinko, John M. Dunlap, Paul V. and
Clark, David P. (2009). Brock: Biology of Microorganisms. Microbial Growth Control. 12th
Ed. San Francisco.

Questions
1. Define the following terms:
a) Selective media
Selective media allow certain types of organisms to grow, and inhibit the growth of
other organisms. The selectivity is accomplished in several ways. For example,
organisms that can utilize a given sugar are easily screened by making that sugar the
only carbon source in the medium. On the other hand, selective inhibition of some
types of microorganisms can be achieved by adding dyes, antibiotics, salts or specific
inhibitors which affect the metabolism or enzyme systems of the organisms. For
example, media containing potassium tellurite, sodium azide or thallium acetate (at
concentrations of 0.1 - 0.5 g/l) will inhibit the growth of Gram-negative bacteria.
Media supplemented with penicillin (5-50 units/ml) or crystal violet (2 mg/l) will
inhibit the growth of Gram-positive bacteria. Tellurite agar, therefore, is used to
select for Gram-positive organisms and nutrient agar supplemented with penicillin
can be used to select for Gram-negative organisms.
b) Differential media
 Differential media or indicator media distinguish one microorganism type from
another growing on the same media. A differential medium distinguishes between
different types of bacteria based on some characteristic of the bacteria that is
growing on it. Typically there is a colour change that results from certain bacterial
metabolic products reacting with substances or chemicals that have been added to
the media. This type of media uses the biochemical characteristics of a
microorganism growing in the presence of specific nutrients or indicators such as
neutral red, phenol red, eosin or methylene blue added to the medium to visibly
indicate the defining characteristics of a microorganism. This type of media is used
for the detection of microorganisms and by molecular biologists to detect
recombinant strains of bacteria. Examples of differential media include eosin
methylene blue (EMB) which is differential for lactose and sucrose fermentation,
MacConkey (MCK), which is differential for lactose fermentation, mannitol salt agar
(MSA) which is differential for mannitol fermentation and X-gal plates which are
differential for lac operon mutants.

2. What is the importance of getting a ‘pure culture’ from a mix culture?

A pure culture ensures that only one type of bacteria is present. It is usually derived
from a mixed culture by transferring a small sample into new, sterile growth medium in
such a manner as to disperse the individual cells across the medium surface or by
thinning the sample many fold before inoculating the new medium. Both methods
separate the individual cells so that, when they multiply, each will form a discrete colony
which may then be used to inoculate more medium, with the assurance that only one
type of organism will be present. Pure cultures are used to determine the type of
organism and its abundance in the sample being tested. It is one of the primary
diagnostic methods used as a tool to determine the cause of infectious disease by letting
the agent multiply in a predetermined media. The pure culture also provides a source of
reference to enable to verify more easily the organisms. It also provides a readily
accessible source of cultures of known organisms and a means of preserving genetic
resources of such organisms. Pure culture collections have gained an important role as
resources for authentic, reliable microbial cultures for both research and practical use.

3. Why does the streaking method you used to inoculate your plates result in isolated
colonies?
The streak plate method is a way to physically separate the microbial population and is
done by spreading the inoculate back and forth with an inoculating loop over the solid
agar plate. Then, single cells will have been isolated from the biomass. Streaking is like
diluting. The idea is to spread the bacterial cells thinly so as to get individual cells spread
over the agar surface.