SBT 202 PRACTICAL One

SBT 202 – Principles of Bacteriology
PRACTICAL 2 – PURE CULTURE TECHNIQUES

• Culture: growth in media containing living MOs.
Culture media: solution which contains nutrients
needed for bacterial growth.
• In this case we will be using nutrient or nutrient
broth agar to culture or grow bacteria
• Nutrients in media Water. Source of C. Source of N.
Buffer system.
• To dissolve materials to be transported across
cytoplasmic membrane. Source of C.
Required for the construction of all organic
molecules. Usually glucose.
• Source of N. - Examples: Proteins: broken
down into amino acids. Beef extract: broken
down into nucleic acids.
• Buffer system.Most bacteria grow at pH
7.0.This is achieved by using a buffer
system;Carbonate buffer.Phosphate
buffer.Source of minerals.Required in small
amounts (Iron, Sulfur, phosphorus… etc)
• Types of media: - Media can be classified
according to any of the following criteria:
Composition. Content / percent of agar.
Purpose. Agar: The usual solidifying agent for
media. A hydrocolloid derived from red algae
(Gellidium species).
• Properties of agar: Melting point: 100 degrees.
Remains liquid until cooled to 40 degrees. Cannot
be metabolized by most bacteria.
• Types of media Culture media can be
classified into two categories according to
their composition; Chemically-defined
(Synthetic).The exact chemical composition is
known. Prepared from biochemicals off the
shelf. Example: Minimal Salt Medium.
• Complex (Undefined).The exact composition
is not known. Usually contain complex
materials of biological origin, such as; Blood.
Milk. Yeast extract. Beef extract.
• Types of media According to percent of agar: Broth
media (liquid).
No agar is added.Example: Trypticase soy broth
media.Semisolid media.<1% agar.Example:
Motility media. Solid media.% agar.Example:
Trypticase soy agar.
• Solid media Solid media are divided into three
forms:
Slanted agar position. Deep agar position. Agar
plate media.
• Agar plate Provide large surface area.
Used for isolation and subculture purposes.Always
incubated in inverted position to avoid water
condensation that forms on the surface of
petridishes.
• 9Slant & Deep Agar Slant agar:
Used for maintenance and preservation of pure
cultures for subculture purposes. Deep agar: Used
for storage. Also used for studying the gaseous
requirements for MOs.
• A pure culture: is a laboratory culture
containing a single species of organism. A pure
culture is usually derived from a mixed culture
(one containing many species) by transferring
a small sample into new, sterile growth
medium.
Bacterial colony appearance
• Bacteria can be seperated from each other by
one of the following methods: Streak plate or
pour plate. Remember the use of aseptic
technique to prevent contamination from the
environment.
Aseptic technique
• Aseptic technique or sterile technique is used
to avoid contamination of sterile media and
equipment during cell culture.
Aseptic technique
• When working with cultures of living organisms,
it is extremely important to maintain the
environments in which cells are cultured and
manipulated as free of other organisms as
possible. This requires that exposure of
containers of sterilized culture media to outside
air should be minimized, and that flame is used to
"re-sterilize" container lids and rims.
Aseptic technique
• This means passing rims and lids through the flame
produced by a Bunsen burner in order to kill
microorganisms coming in contact with those surfaces.
• Heat is an excellent means of killing microorganisms,

and the Bunsen burner is the sterile technician's best
friend.
•
• The bacterial sample can be transferred into
new, sterile growth medium in such a manner
as to disperse the individual cells across the
medium surface or by thinning the sample
manyfold 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.
• Isolation of a pure culture may be enhanced
by providing a mixed inoculum with a medium
favouring the growth of one organism to the
exclusion of others.
• METHODS TO ISOLATE THE BACTERIA• Streak
plate culture• Stroke• Stab• Pour plate•
Liquid culture• Specific methods for anaerobic
cultures.
• LIQUID CULTURING - Liquid cultures are done
in• Tubes• Bottles• Flasks• Blood culture•
used in Water analysis.
• STAB CULTURE • Puncturing suitable medium
such as nutrient agar, gelatin, • Observe
gelatin liquefaction.
• ANAEROBIC BACTERIAL ISOLATION
• Anaerobic bacterial isolation needs specified
conditions, E.g. use of:
• i) use of dessicator
• ii) Candle jar
• iii) Gas pack
• iv) Mac in tosh Fildes Anaerobic jar
OBJECTIVES:
• The student should, at the end of practical, be
able to use methods of pure culture to isolate
a desired microorganism (bacteria0 from
mixed populations.
Streak plate method
• Procedure: Bacillus subtilis, Escherichia coli
and Staphylococcus aureus.
• 1. Flame the loop wire until it is red hot.
Remove the cap from the culture tube and
flame the mouth of the tube. Do not
contaminate the cap or the loop during this
procedure.
Procedure
• 2. Remove a loopful of the bacteria. Flame the
mouth of the tube again and replace the cap on
the tube. Spread the organisms (bacteria) over a
small region on the edge of the plate as in the
diagram.
• 3. Flame the loop and let it cool for a few
seconds.
• Streak from the end of region 1 across the edge
of the plate forming region 2, - do the same for
region 3 and 4.
Streak plate procedure
Spread plate method
• The spread plate technique is a method for
transferring bacteria to an agar plate and
distributing it evenly. The technique makes it
easier to quantify bacteria in a solution.
• Spread plates allow for the even spreading of

bacteria onto a petri dish; allowing for the
isolation of individual colonies, for counting or
further experiments.
• Using aseptic techniques, take a loop of stock
culture, transfer to the center of the agar
plate, and spread it evenly over the surface
with a glass spreader – do this while rotating
the plate on the table.
• Return the glass spreader to the alcohol.
• Incubate for 48 hours in an inverted position.
Look for isolated colonies.
Results
• The streaked plates will be incubated in an
inverted posited (place plates up-side down),
for 48 hrs.
• Why is it important to invert the petri plates
during incubation?
• Because moisture will condense on the
surface of the agar and cause confluent
growth instead of isolated colonies. Moisture
drops down from the lip to the agar surface.
RESULTS:
• 1. Examine each of the streak plates and make a sketch
indicating the distribution of both the streak and spread
plate methods.
• 2. Describe the colonies that appear on the plates i.e size,
color and elevation.
• QUESTIONS:
• You expose an agar plate to the environment and get a
good bacterial growth. A number of colonies overlap and
you wish to isolate the organisms in pure culture. Which of
the two techniques would you use? Explain your answer.
• Can a pure culture be prepared directly from a mixed broth
or a mixed agar slant culture. Explain your answer.

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SBT 202 – Principles of Bacteriology

PRACTICAL 2 – PURE CULTURE TECHNIQUES

• Heat is an excellent means of killing microorganisms,

• Spread plates allow for the even spreading of

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