FACULTY OF FOOD SCIENCE AND NUTRITION

NT20203 FOOD MICROBIOLOGY

TITLE:

1. PRACTICAL 3 - TECHNIQUES FOR THE

OBSERVATION OF LIVE ORGANISMS: HANGING
DROP
2.PRACTICAL 4 – CULTIVATION TECHNIQUES:
TRANSFER AND SELECTION OF COLONY
Activity 2: Streak plate technique

NAME: IZWANA AINA NAZIRA BINTI SHUHAIMI

MATRIC NUMBER: BN20110125
LECTURER NAME: PROF. HO AI LING
PRACTICAL DATE: WEDNESDAY (10 MAY 2023)
DATE OF SUBMISSION: 24 MAY 2023
TECHNIQUES FOR THE OBSERVATION OF LIVE
ORGANISMS: HANGING DROP

Introductions
Both alive and dead bacteria can be distinguished using microscopic
preparations (Erkmen, 2021). For determining a microorganism's morphological
appearance, which includes its size, shape, mobility, and reactions to various
chemicals and immunological antisera, direct observation of a living sample is
essential. There are two widely used techniques for identifying living microorganisms:
hanging drop and wet mount. Both of these procedures normally don't require the
use of any stains and can be used to detect whether or not an organism is motile.
The ability of an organism to move without restriction is referred to as motility.
Motile organisms move either fast or slow in one direction.

Objectives
1. To examine living, unstained, very small organisms.
2. To determine an microorganisms motility.

Materials
Depression slides, cover slips, Vaseline, nutrient broth culture (pond water sample),
Pasteur, pipette or dropper, toothpicks

Methods
1. Vaseline is applied to the surface edges of cover slip using a toothpick.
2. 1 drop of culture is placed in the center of the cover slip.
3. A depression slide is pressed slowly onto the cover slip, with the concave part over
the drop.
4. The slide is then turned upside down with the cover slip on top of the slide.
5. The prepared slide is observed under the microscope with low magnification
power and then with high magnification power.
RESULT

40x magnification
Description: type- volvox, motility- brownian

DISCUSSION
In this experiment, hanging drop method has been applied to identify the
type and motility organisms in pond water sample. The shape of microbial are volvox
and brownian motility which later identify as chlamydomonas. Living microbes are
frequently examined using the hanging drop technique. A drop of fluid containing the
microorganisms is placed on a depression slide with a circular concavity in the centre
as part of the technique, and the slide is then suspended from the cover slip
(Crawford, n.d.). This techniques don't require staining because microbial cytoplasm
is typically translucent, making them visible under a light microscope (CliffNotes,
2022). Reducing the light source intensity can assist in finding the microorganisms
because observation without staining can make it challenging to detect them. Both
techniques can be applied to observe motility of the microorganisms. The techniques
of hanging drop and wet mount enable the viewing of live things. It is easier to
conduct than the wet mount but is only good for short-term observation because the
wet mount has a tendency to dry out fast when exposed to the heat of the
microscope light. Although the hanging drop technique is more difficult, it enables
longer-term observation and more accurate motility monitoring.
The hanging drop method offers several advantages for studying bacteria.
Firstly, it is an essential instrument for examining bacterial locomotion, as well as the
shape, size, and organization of bacteria. Importantly, this method does not alter the
form and arrangement of the cells, allowing for accurate observations. Compared to
the wet mount method, the hanging drop approach provides a better view of
bacterial movement, enabling researchers to study motility in detail. It is also
valuable for classifying bacteria based on their motility characteristics, distinguishing
between motile and immobile species.
Furthermore, the hanging drop method allows for the study of Brownian
motion, which is the erratic movement of bacterial cells caused by the bombardment
of water molecules. This phenomenon can provide insights into cell behavior and
interactions. Additionally, the use of petroleum jelly to bond the coverslip to the
hollow slide facilitates repeated examination of the tested specimen, enabling
researchers to analyze bacterial behavior over time.

However, there are some disadvantages to consider. Working with harmful

microorganisms in a live environment can pose safety risks, requiring appropriate
precautions to prevent the spread of infectious agents. Moreover, while the
depression slide used in the method is cost-effective, the coverslip is delicate and
requires careful handling, as it can break or become damaged.

CONCLUSION
Wet mount and hanging drop techniques are the two techniques most frequently
employed in the microbiology lab to identify the bacterium. As the sample is hanging
on the cover slide rather than being forced down like in the wet mount approach, the
microorganisms can move freely for a longer amount of time, making the hanging
drop method preferable to the wet mount method for studying the motility of the
microorganisms.
PRACTICAL 4 – CULTIVATION TECHNIQUES: TRANSFER
ANDSELECTION OF COLONY

Activity 2: Streak plate technique

INTRODUCTION
Most culture media are prepared either as a solid (agar) or a liquid (broth)
depending on the analysis goals. Culture media come in a variety of forms, including
general-purpose, selective, and differentiated media. Nutrient broth and nutrient
agar are examples of general-purpose media. These media are frequently used in the
growth of bacteria. It slows the development of undesirable organisms while
promoting the growth of desired species in selective medium like brilliant green agar
and eosin methylene blue agar. Differential media, like violet red bile agar, offer
conditions that allow specific colonies to be separated from other species (CliffsNotes,
n.d.). But some of the media employed in this exercise have both selecting and
differentiating qualities.

OBJECTIVES
1. To produce isolated colonies of an organism on an agar plate.

MATERIALS
Sterilized nutrient broth and agar medium, E.coli culture, Bunsen burner, inoculating
loop

METHODS
a) The mixed culture tube was shaken gently.
b) The inoculating loop was flamed to redness and the cap of the culture tube
removed with the free fingers of the hand holding the inoculating loop, and the lip of
the tube flamed.
c) A loopful of the mixed culture was removed after the loop has cooled for at least 5
seconds.
d) The lip of the tube is flamed again, the cap was replaced and put in a rack or
another appropriate place.
e) The petri dish was held so that the bottom rest on the palm. The petri dish cover
and the inoculum was placed at the edge of the agar surface farthest from us.
f) The inoculum was streaked on the agar surface from side-to-side in parallel lines
covering approximately one quarter of the plate. Do not dig into the agar.
g) The inoculating loop was flamed again and the petri dish is rotated one quarter of
a full turn.
h) Step f) was repeated with the second set of streaks.
i) Step g) and h) were repeated. The inoculating loop is flamed before putting it
down. Theplate is incubated in an inverted position.

RESULTS

Petri dish 1 Petri dish 2 Petri dish 3

Petri dish 4 Petri dish 5

E. coli colony morphology: Have circular form, medium size, convex elevation,
smooth surface, white colour, entire margin, opaque structure

DISCUSSION
The most often used technique for separating particular bacteria from a
mixture of microorganisms is the streak plate technique. To ensure that, after
inoculation, individual cells will be suitably far apart on the surface of the agar
medium, this method demands that the number of organisms in the inoculums be
lowered through dilution. When the inoculum was streaked across the agar's surface,
either using the three-sector T streak method or the four-quadrant approach, the
bacteria were diluted to the point where, on the final few streaks, single cells
dropped out of the loop and formed distinct colonies.The streak plate method also
has an advantage over the pour plate because it is easier to use and takes less time
to complete. Because picking the colonies below the agar's surface would disrupt
those on the surface, serial dilution of the original sample in the pour plate technique
is too time-consuming. In addition, the temperature of the agar medium needs to be
carefully controlled to prevent killing the microorganisms in the sample. Additionally,
the causative agent of a bacterial disease can be found when bacteria are streaked
and isolated using the streak plate approach.
When the cultivation is intended to produce distinct bacterial colonies from a
mixed population, the streak plate approach is favoured over the pour plate method.
It creates isolated, pure bacterial colonies, allowing for the qualitative examination of
the many bacterial species present. Contrarily, the pour plate can only be used to
count the amount of colony-forming bacteria that are present in a liquid specimen.
Additionally, if the bacteria to be cultured are known to be extremely heat-sensitive,
this procedure is also used. Similar to the previous example, even though the pour
plate method of counting bacteria is more accurate than the streak plate approach, it
will typically result in a lower count because heat-sensitive organisms may perish
when they come into contact with the hot, molten agar media. As a result, the
streak plate method is frequently preferred to the pour plate.
Bacteriological incubators are essential equipment used in laboratories to
create a controlled environment that promotes the growth of bacteria under stable
conditions. These incubators provide an insulated chamber where bacterial culture
plates or other culture vessels can be placed. The incubators maintain a constant
supply of hot air, ensuring a consistent temperature throughout the chamber.
Bacteriological incubators have a range of important applications in microbiology and
research. Firstly, they are used for growing bacterial cell cultures, providing the
optimal conditions required for bacteria to multiply and thrive. This is crucial for
various experimental and diagnostic purposes. Moreover, bacteriological incubators
are used in the isolation and examination of bacteria. Furthermore, bacteriological
incubators offer the flexibility to control and adjust the temperature settings. This
allows researchers to grow bacteria at different temperatures, thereby studying their
growth patterns and requirements. It provides valuable insights into the
environmental conditions necessary for optimal bacterial growth and metabolism.

CONCLUSION
Microorganisms can be multiplied by being grown in culture media under carefully
regulated laboratory conditions. This process is known as microorganism cultivation.
Microorganisms can be isolated, cultured, and identified using the cultivation process.
In this experiment, two different types of media—nutrient broth medium and
nutrient agar medium—were used to cultivate E. coli. To prevent contamination from
other germs, both processes must be conducted using aseptic technique. When the
colour of the nutrient broth medium changes from clear to hazy (turbid), it means
that E. coli is growing there. We also used the streak plate approach to observe E.
coli colonies from the nutrient agar medium. The experiment is deemed successful
because the observations of the colonies are consistent with the references.
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