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Laboratory Activity # 4
Isolation of Bacteria into Pure Culture

Specific Objectives:

After this activity, the student is expected to:

1.isolate colonies from previous cultures into pure cultures using the streak plate technique
2. characterize the colonies morphologically as to form, elevation, margin and
Color
3.test the biochemical properties of the isolated colonies
a. Gram-staining reaction
b. hemolysin test
c.IMVIC Tests

Introduction

Microbial populations rarely occur singly in nature. In order to study the characteristics and
behavior of a microorganism, it should be first separated from the contaminating species by
isolation techniques. The procedure results into a pure culture presumably resulting from a
single parent cell. A pure culture is composed of cells arising from a single progenitor. The
word Axenic is also used to refer to a pure culture. The progenitor (also termed as colony-
forming unit- CFU) from which a particular pure culture is derived may be either a single cell or
a group of related cells Scientists use several techniques to isolate organisms into pure cultures.
These techniques require that:

1.all apparatus and culture media must be previously sterilized or freed from microorganisms.
2. the desired bacterium must be separated from naturally occurring microbial populations into
the sterile culture medium
3. aseptic techniques which prevent the entry of microorganisms must be followed.

The size of the colonies depends upon the type of organisms, the growth medium used and the
number of colonies per unit area. The colony size usually remains quite constant after 24 to 48
hours of incubation. However, if the microorganism has a long generation time, it may continue
to grow beyond 48 hours. Some organisms are motile and may spread over the entire surface and
produce a confluent mass of cells.

Methods of Isolation
1.Streak Plate- the most commonly used isolation technique. In this technique, a sterile
inoculating loop is used to spread an inoculum across the surface of nutrient agar. The loop is

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also used to lightly streak a set of pattern that gradually dilutes the sample to a point that
appropriate period of time called incubation, colonies develop from each isolate. The loop is
sterilized between streaks. The various types of organisms present are distinguished from one
another by differences in colonial characteristics.

a.The Interrupted Streak Method

The interrupted streak method, the loop is not heated because a few remaining bacteria in the
loop will give isolated colonies in the 2nd half of the plate

b.Multiple Inoculation

This is not a very reliable method from the standpoint of getting isolated colonies , but
sometimes it is resorted to for purposes of economy or when the available media are not
sufficient. A plate may be seeded or streaked with as many 6 to 8 different organisms.

c.Multiple Streak Method

c.1.Overlap Streak Method:

a. Start the inoculation at the further side of the plate tracing a zigzag course from side to
side until you finish inoculating approximately one third of the plate.
b.rotate the plate 90 degrees counterclockwise and heat the loop.
c.continue the inoculation from the further side of the plate again making sure that the
loop touches portions of the plate previously streaked . Continue the inoculation until you have
covered another third of the plate
d.rotate the plate another 90 degrees counterclockwise and heat the loop.
e. Proceed with the last phase of the inoculation in a manner similar to the second set of
streak;, making sure that this last set of streaks overlap a portion of the second set. Make certain
that you do not touch portions of the plate which have been inoculated with the first set of
streaks.

If the inoculum is heavy. Proceed as given above but if the inoculum is rather thin , the
heating of the loop may be dispened with. The Main purpose of inoculating plates, in general, is
to be able to grow isolated colonies . In the overlap streak method, the overlap zones provide
isolated colonies in the second and third areas of inoculation.

c.2 Radial Streak Method

a.place a loopful of the broth culture of the organism near the edge of the plain agar plate
(about cm away from the periphery)
b, from this source of organisms, make radial straight lines streaking towards the other
side at about 10 0 angles. Start from the side of the plate until the whole plate has been inoculated
The loop is lifted every time the other end of the plate is reached and streaking again started at
the source of organisms
c.Incubate at 35 0 C for 24 hours.

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 c.3 Multiple Interrupted Streak Method

a.Divide the plain agar plate into 4 quadrants

b.with a loopful of the broth culture of the organisms, inoculate one quadrant in a zigzag
manner startibg near the periphery towards the center
c.pass the wire loop with organisms across a section just previously streaked and
inoculate the next quadrant. Do the same for the 3rd and 4th quadrants
d.Incubate at 35 0 C for 24 hours

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2.Pour Plate- the CFU are separated from one another using a series of dilutions. There are
various ways to perform pour plate isolations. In one method, an initial 1 ml sample is mixed
with 9.0 ml of a nutrient broth in a test tube. After mixing, a new 1m sample from this test tube is
then used to inoculate a 2nd test tube. The process is repeated to establish a series of dilutions.
Samples from the test tubes are transferred into sterile nutrient agar in Petri dishes. After
incubation, isolated colonies form in the Petri dishes from CFUs that have been separated via
dilution series.

3.Spread Plate- this techniques utilizes an L-shaped glass rod in spreading the inoculum on the
surface of sterile solidified agar.

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4.Swab Method- this is used to pick up microflora from surfaces, especially from overly smooth
surfaces like metals, plastic and glass on which ordinary loop would be ineffective. It can be
used to qualify and quantify the flora on a given surface area.

Materials:

1.previous cultures
2. test tubes, Petri dishes
3. sterile nutrient broth and nutrient agar
4. inoculating loop
5. alcohol lamp
6. autoclave
7. gram stains
8. microscope
9. incubator

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10. blood agar plate
11. IMVIC reagents

Procedure: Get a colony from plates exposed to hospital ,food establishment and comfort room

1.Streak plate (Multiple Interrupted Streak Method)

a. Sterilize wire loop over the flame. Cool by holding still for 5 seconds.
b. Get a small portion of the colony from previous Petri dishes (cough, nose, canteen, CR) .
Streak repeatedly overn an area of the new sterile nutrient agar in a Petri dish and test tube
c. Heat the loop to kill all residual cells.
d. Rotate the plate so that your next streaks will be perpendicular to the first. Getting your
inoculum from near the end of your streaks, make streaks with long singular and unidirectional
strokes with the loop flat on the agar.
e. Rotate the plate so that your 3rd streaks will be perpendicular to the 2 nd. Again, taking your
inoculum from near the ends of the 2nd streak, make long strokes toward one direction with the
loop. Heat the loop.
f. Rotate the plate again and streak. Flame and cool loop. Make the final streak towards the
center of the plate. Heat the loop.
When making streaks, be sure not to cut the agar with the wire loop.
g. Incubate the plates inverted for 24 to 48 hours.
h. Study the morphological characteristics after the incubation period based from the chart
provided.
i. Test the gram staining affinity and hemolytic activity of the isolate.

2.Slant Growth (test tube slant)

a. From the same colony used for streak plate, heat the mouth of the test tube slant with nutrient
and gently inoculate in a zigzag manner the obtained colony starting from the bottom towards
the upper part of the slant portion.
b. Incubate the test tube for 24 to 48 hours.
c.study the slant growth characteristics (refer to the given chart)

3.Test the hemolytic activity of the isolates either from the slant growth or plate growth

4. Perform IMVIC Test of the isolates

IV. Observation
1.illustrate the growth form (slant growth/plate growth) of the different pure cultures and give
their cultural characteristics.
2. indicate below the illustration the biochemical properties of the isolates (Gram staining
affinity and hemolytic activity, IMVIC Tests)
3. give the possible identification of your isolates

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Morphological Characteristics:
1.Shape/form: circular, punctiform, rhizoid, irregular, filamentous, spindle
2. Margin: entire, undulate, lobate, curled, rhizoid, filamentous
3. elevation: flat, raised, convex, pulvinate, umbonate
4. size: punctiform, small, moderate, large
5. texture: rough, smooth
6. pigmentation: nonpigmented ( cream, tan, white); pigmented ( red, yellow, green)

REPORT SHEET (Attach plate/document) refer to page 108 to compare your

obtained type of bacterial growth.

CULTURAL CHARACTERISTICS

A.Slant Growth Characteristics

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B.Plate Growth Characteristics

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