Isolation, Cultivation, and Cultural Characterisation of Microorganism

BASIC LABORATORY TECHNIQUES: ISOLATION,

CULTIVATION, AND CULTURAL CHARACTERISATION OF
MICROORGANISM
Clarissha Vallerie Widjaja (0340155), Farah Ula Nabilah (0340810), Helena Wiranata
(0338467), Tan ShuYue (0341056)

Culinology, School of Food Studies and Gatronomy, Taylors University, Jalan Taylors, 47500
Subang Jaya, Selangor, Malaysia

OBJECTIVES

Some objectives had achieved after completing the practical session were using aseptic
techniques for the transfer and handling of microorganisms and instruments including sterilizing
and maintaining sterility of transfer instruments, performing aseptic transfer using variety plate
techniques (streak-plate, pour plate, spread plate), and obtaining microbial samples.

INTRODUCTION

Microorganisms grow in certain range of resources and condition. In particular, Eschericia coli is
a Gram-negative organism but is not strictly rod shape. The cell cycle models in this bacterium are mostly
grown in dispersive manner and the zonal growth is involved either as semiconservative symmetrical or
as asymmetrical growth [CITATION Koc82 \t \l 1033 ]. Whereas other kingdom of microorganism,
Saccharomyces cerevisae is a type of yeast that commonly use in food production as a leavening agent.
Saccharomyces cell has oval, cylindrical, or spherical form and the cell division is typically by
budding[CITATION TMa14 \n \t \l 1033 ]. There is report about this organism that “Unicellular organisms
growing in or on solid media normally remain together to form discrete units visible to the unaided eye
called colonies”[ CITATION Gar97 \l 1033 ]

Inoculation techniques in a standard microbiology laboratory used in cultivation of

microorganism as transferring a tiny sample of microorganism into a container of nutrient medium, which

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 Isolation, Cultivation, and Cultural Characterisation of Microorganism

provides an environment for them to grow and multiply. Media has a significant purpose in bacteria,
yeasts, and mould growth. A media should contain nutritional content in order for the inoculum to grow
and expand. One of the most media used in cultivation is agar which is a good gelling agent contained
glucose as a convenient carbon source, minerals, and trace elements[CITATION Gar971 \t \l 1033 ]. There
are some procedures that used to run inoculation process. Streak plate technique is used to obtain
individual, well-separated colonies in large number[CITATION Sin04 \t \l 1033 ]. This method yields to
different sizes of inoculum in certain area. Streak plate technique proved that the possibility of
microorganism reproduction is higher due to less competition for nutrients with other colonies. As with
spread plate technique is employed with expectation for isolation of pure culture from mixed
colonies[CITATION Dub06 \t \l 1033 ]. It results to evenly distributed and visible individual colonies
which can be count easily. While pour plate technique is technically mixing the culture with liquified
nutrient agar which makes it countable [CITATION Dub06 \t \l 1033 ]. This method does not require much
skill which is easier and faster than streak plate technique. Inoculated tubes are poured into sterile Petri
dishes and are allowed to solidified. By doing so, the procedure effectively dilutes the sample, and the
number of cells per volume is so decreased that cells have ample space to form separate colonies o the
second or third plate.

MATERIALS AND METHODS

Streak Plate. Material that is used were bunsen burner, Saubaraud dextrose agar, inoculation
loop, sterile swab, and nutrient agar. The tabletop was prepared and has been decontaminated
with ethanol 70% using paper towels. Then, the first primary inoculum across the corner of the
culture plate was streaked with a heavy loopful of cultures. Hold the charge with the surface of
the agar; inoculum, smeared backwards and forward across a small area of the medium, the loop
removed, and the Petri dish closed. A flamed loop that has been chilled was turned through 90ᴼ
anticlockwise. Afterwards, the plate that has been streaked with chilled loop; from area A across
the surface of the agar in three parallel lines, again, the loop removed, and the Petri dish closed.
The flamed loop that has been chilled was turned through 90ᴼ anticlockwise again. Move to next
step, from area B was streaked across the surface of the agar in three parallel lines, the loop
removed and the Petri dish closed. For the third time, the flamed loop that has been chilled was

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 Isolation, Cultivation, and Cultural Characterisation of Microorganism

turned through 90ᴼ anticlockwise. Proceed to the next step that loop was streaked across the
surface of the agar from C into the center of the plate. The loop removed and the Petri dish
closed. Finally, the plate was incubated inside an inverted position at 37ᴼC overnight, therefore
the agar plates that show the single colonies are captured on the next day.

Streaking with a sterile swab. Streaking with sterile swab was using the same procedure as
streaking with wire loop, yet the difference was sterile swab bud directly streaked on the surface
of solid agar medium. The shoes were swabbed with sterile swab that has been moisten with
sterile water prior to swab the surface. Meanwhile, mucosal surface was swabbed directly using
dry sterile swab. Further, after the selected surface was swabbed; with the swab, the primary area
on nutrient agar were streaked in three parallel lines, the swab discarded, and using a flamed loop
the streak procedure was continued. The result was recorded on the next day.

Pour Plates. Petri dish, Molten agar, 200µl Pipette and tips, Bacterial cell suspension were
equipment that were used to make pour plate. At first, 100 µl of bacterial cell suspension were
inoculated into the molten nutrient agar using micropipette. Immediately, contaminated pipette
tip was discarded in Biohazard container. In the meantime, swirled gently the bottle between the
hands, until the culture and the medium mixed thoroughly, yet avoid making air bubbles. The
bottle was held in the left hand; the lid removed with the little finger of the right hand, and
flamed the neck of the bottle. With right hand, the lid of the Petri dish was lifted, and the mixture
poured into the Petri dish and the lid replaced. After the dish rotated gently and the medium
ensured, covered the plate evenly. Eventually, the agar medium at the plate solidified, and
incubated in an inverted position at 37ᴼC overnight. Result was recorded on the next day.

Using a spreader “hockey stick” and spread plates. Materials that used were nutrient agar,
200µl Pipette and tips, Glass spreader, 70% ethanol in beaker, Bacterial cell suspension. Once
the lid of microcentrifuge tube that contain the bacterial culture were opened, a sterile pipette
was held in the right hand and the microcentrifuge tube that contain the bacterial culture in the
left. With the pipette, a small amount of bacterial culture was aliquoted. The lid closed using the
index of our left hand. Lifted partially the lid of the Petri dish containing the solid nutrient agar
with left hand, and drop the culture in pipette tip dropped onto the surface. Replaced the lid of

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 Isolation, Cultivation, and Cultural Characterisation of Microorganism

Petri dish, and the pipette were discarded. The lower end of the spreader dipped into a small
volume of 70% alcohol. Through a Bunsen burner flamed to ignite the alcohol passed quickly.
The alcohol sterilized the glass rod, by kept the distance between the alcohol and the burner.
Lastly the spreader removed from the flame and the alcohol were allowed to burn off.

Using a wire loop and flaming. Inoculation loop and bunsen burner were tools that applied on
this technique. Placed the end of the wire in the light of blue cone of the flame. Continue by rest
of the wire drawn upwards slowly into the hottest region of the flame. 3. It has been held until
red. Used when it was cooled already without put it down or wave it around. Last, re-sterilized
the loop immediately after use.

Flaming the neck of the bottles and test tubes. The lid of the bottle was loosened and
removed. With left hand, the bottle was lifted. With little finger of the right hand, the lid of the
bottle removed, and the neck of the bottle were flamed by passing the neck forward and
backward through a hot Bunsen flame, after that, the lid replaced using little finger.

RESULTS AND DISCUSSION

Streak-plate Technique. Output of streak-plate methods are shown in the Figure 1. Streak plate
method was used by spreading inoculum on a plate with good spacing among each other so the
growth possibility of bacteria increased. It has been reported that for many downstream
applications it is imperative to start with either a single colony or a pure bacterial culture
generated by inoculating media with cells from a single colony [ CITATION Jam01 \l 1033 ] . Based
on experiment result, growth of saccharomyces cerevisiae colony on streak plate 1 and 2 were
invisible, yet the 3rd and 4th streak plate were showing observable growth of s. cerevisiae colony.
There was some hypothesis among our group members on reasons why colonies on streak-plate
1 and 2 were not appear, it could be the wire loop that too hot while taking the inoculum, or not
enough time for the inoculum to grow into visible. This hypothesis is supported by a journal
which stated that with knowledge of the basic concepts of medium composition and of the
physical conditions which may limit microbial growth, one can enhance their ability to grow
bacteria and fungi in pure culture and to enrich for, isolate, and culture many microorganisms of

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 Isolation, Cultivation, and Cultural Characterisation of Microorganism

interest from the environment[CITATION Tan07 \t \l 1033 ] . The colony between streak plate 3 and
4 presented different distribution, result of relative amount that taken using wire loop and the
pressure given when spreading the inoculum. Streak plate 3 (Figure 1-a) reveals line-like s.
cerevisiae colony distribution with yellowish white to translucent color with convex elevation,
while streak plate 4 (Figure 1-b) presents sizes of colonies that differ from line-like form to
medium and pinpoint form. Shape differentiation were result of certain available spaces needed
to grow, for instance, at Figure 1-b the colonies which grew on the left side were having
punctiform to small circular dots, compare to the right-side colonies were developing bigger
circular dots. Pinpoint to small sizes were formed because bacteria colonies didn’t have enough
space to grow as big as the colonies that had larger surface area. Overall s. cerevisiae colonies
had the same characteristics on convex elevation, smooth appearance and entire margin.

Figure 1. Picture of Saccharomyces cerevisiae colonies in streak plate 3 (a) and 4 (b)

Swab-plate technique. Figure 2 displays the results of swab-plate techniques which divided into
two kinds of swab surface that are mouth and shoes. Swab mouth plate showed few growths of
bacteria colonies that have specific characteristics as forming white and circular shape, vary from
pinpoint to small dots, also appeared as slightly raised on the surface of agar medium (Figure 2-

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 Isolation, Cultivation, and Cultural Characterisation of Microorganism

a). Meanwhile, the growth of bacteria from shoes are concentrated on one area with one big
circular white colony and surrounds with approximately seven pinpoint dots which having
similar traits as presents on Figure 2-b. All the bacteria grew only on the surface area with
slightly raise elevation.

Figure 2. Pictures of bacteria colonies by swabbing mucosal area (a) and shoe soles surface (b

Spread-plate Techniques. The usage of hockey stick during inoculation, made the Escherichia
coli bacteria spread over the surface of solid agar medium, therefore the form of bacteria is
spreading as a thin translucent layer as shown on Figure 3. The irregular size of colony
distribution with one to some holes at the middle of the colony was result on uneven pressure
used to spread the inoculum. Growth of Escherichia coli on the surface also showed flat
elevation. The appearance of the outer edge was described as lobate which had marked
indentations.

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 Isolation, Cultivation, and Cultural Characterisation of Microorganism

Figure 3. Picture of Escherichia coli colony by spreading the inoculum on the surface solid agar
medium

Pour-plate Techniques. From analysis sample one to four, each of the plates are giving the same
results of bacteria growths as shown on Figure 4. Escherichia coli colony was framed both on
the surface area and in the medium of solid agar medium with effuse form and flat elevation. The
colony optical feature’s presented white opaque to translucent color. Holes that formed were
product of even distribution throughout the agar medium. Result from the experiment of pour
plate techniques were supporting the statements from introduction that the method will
effectively diluted the sample so making it easier to count.

Figure 4. Picture of Escherichia coli colony that inserted inside semi-solid agar medium and
solidified.

CONCLUTION

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 Isolation, Cultivation, and Cultural Characterisation of Microorganism

In brief, each methods for isolating bacteria has their own purpose: streak plate technique is more
effective for organism growth due to availability of sufficient spaces, as with spread plate
making the organism easier to count for quantification of microbial population, while pour plate
procedure grows the organism not only at the surface but also within the medium.
Saccharomyces cerevisiae colony as a fungi has different appearance compare to Escherichia coli
bacteria cells due to different kingdom of organism. Saccharomyces cerevisiae colonies can be
described as a yellowish white color that forming convex smooth appearance. Likewise, dented
leaves outer edges and translucent thin spreading layer are the traits for Escherichia coli bacteria
colonies. Although studies of isolation techniques by growing these microorganisms at the agar
medium found evidence of highly success percentage to isolate them from mixed culture, from
the data some of our plate, especially streak-plate techniques were not producing any sign of
microbial growth. As analyzed before it could be the wire loop that too hot while taking the
inoculum, or not enough time for the inoculum to grow into visible. Further studies are therefore
necessary to determine clearer results convinced by reliable sources.

ACKNOWLEDGEMENT

We thank Dr. Kuan Yau Hoong and Dr. Benjamin Wong Tziak Ze for assistance with the
project.

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