Plugging and Wrapping of Glassware for Sterilization

Answers to Questions

1. What microbiological principle does plugging and wrapping of glassware exemplify?

Plugging and wrapping of glassware exemplify the aseptic technique which prevents the introduction of
any organisms to the laboratory (Aryal, Engida, & Adhikari, 2021). Furthermore, Malinao (2016) states
that plugging and wrapping help in eliminating or reducing the interaction of the internal environment
of the glassware with that of the external environment and may eliminate contamination caused by
microorganisms and foreign materials that are suspended in the air and help in preventing any damage
on the glassware (Malinao, 2016).

2. Can cotton plugs be reused? Explain your answer.

According to the protocol, the cotton plugs can be used repeatedly in most cases. Cotton plugs allow the
movement of air in and out of the glassware while filtering out and preventing the entry of fungal spores
or bacteria due to its fibers. Toorkey in 2015 said that cotton plugs can be recycled for use in a new
batch and re-autoclaved as long as it is not soaked with media or condensate while autoclaving. He also
stated that cotton plugs can be re-used 3 to 5 times for glassware with similar neck diameter.

However, a study in 2010 reported that using cotton plugs may have disadvantages such as the
inhibitory nature of cotton to certain microbes, the chances of contamination during handling and
accumulation of used cotton as biological waste (Muniaraj, Paramasivan, & Arunachalam, 2010).

3. What does the popping sound imply?

The popping sound implies the release of pressure. One example that can be compared to this would be
the popping sound you feel in your ear while on a plane.

Media Preparation

Answers to Questions

1. What is agar? Why is it a good support for microbial growth?

According to Zeece (2020), agar is a galactose-based heterogenous polysaccharide derived from red
algae. It is best known as the growth media used in identification and enumeration of microbial
organisms. This is because agar is ubiquitous and can be transported as dry, dissolved or gelled. It is an
ideal solidifying agent because it is stable at a wide range of temperature, has no nutritive value and is
not digested by most bacteria thus, it can support microbial growth when supplemented with
appropriate nutrients (Song, Shang, & Ratner, 2002). Furthermore, it is found to have good diffusion
characteristics, metabolically inert, and have good transparency (Basu, et. Al.,2015).

2. How will you check the sterility of the media?

To check the sterility of the media, it undergoes sterilization using the autoclave at 121℃ for 15
minutes. Furthermore, physical and chemical tests are also performed such as visual test for color, visual
test for clarity, gel strength, pH of the finished media, or damages in the containers. For a media to pass
sterility, it must demonstrate no growth (Sandle, 2014). Other methods such as filtration are used for
heat-labile or not heat-resistant components.
3. What is the difference between NA and NB?

Both NA and NB have the same components, except that NB has no agar or solidifying agent which
makes it a liquid while NA have around 1.5% to 3% agar which makes it solid. NA is commonly used for
bacterial isolation, enumeration, storage and cultivation of non-fastidious microorganisms while NB are
used to grow fastidious microorganisms and to maintain their stock (Caprette, 2015).

4. Why is gelatin not often used in the preparation of culture media in a microbiology laboratory?

Koch was the first to come across the use of gelatin in the laboratory but soon discovered that it liquifies
at temperatures above 25℃ and could be consumed by gelatinase, an enzyme produced by certain
bacteria (Bonnet, Lagier, Raoult, & Khelaifia,2019).

Preparation of Culture Media in Petri Plates

Answer to Questions

1. How can you check the sterility of the media prepared in a petri plate?

Before using the media, each batch are tested for pH, sterility, and growth promotion. To check for
sterility, the media is incubated at 30-35°C and 20-25°C for 14 days. The media that contains visible
particulate matter should not be used in tests for sterility (Media for use in sterility testing, 2006).

2. Can you store the media in petri plates at room temperature?

Media in petri plates are especially vulnerable to infection, dehydration and chemical degradation. Thus,
to protect the media from infections, aseptic preparation and storage are essential. Impermeable
wrapping and/or storing at 2-8°C will minimize water losses while storing, and protection from light,
heat and dehydration can prevent chemical degradation (Romano, 2013).

3. What is the difference between a laminar flow hood and a biological safety cabinet?

A laminar flow hood is primarily designed to provide a sterile work environment and product protection
but does not provide protection to the personnel nor the environment while a biological safety cabinet
provides protection for product, personnel, and environment (Burdg, 2016). A biological safety cabinet is
designed to protect against exposure to particulates and aerosols from biological agents. Furthermore, a
biological safety cabinet should be used for work with infectious agents or the capture of dust and
allergens from bulk operations while a laminar flow hood should be used for work with non-infectious
agents such as media preparation. Lastly, both vertical and horizontal airflow configuration are available
in a laminar flow hood while only vertical configuration is available for biological safety cabinet (Smith,
2012).

4. What is the use of UV lamp in a laminar flow hood?

The use of a UV lamp in a laminar flow hood is for effective germicide and virucide. It is efficient in
breaking up of chemical bonds, denaturing DNA and RNA and disinfectant for vegetative organisms and
viruses (Meechan & Wilson, 2006). However, series of objections have been raised to the use of UV
lamps due to its harmful effect to humans and its limits of exposure.
Ubiquity of Microorganisms

Answer to Questions

1. In what kinds of environment can microorganisms live? In what unusual environmental conditions can
they be found?

Microorganisms can live in any kind of environment such as terrestrial, aquatic, atmospheric or even in
living hosts. According to Rampelotto (2013), microorganisms that are found in unusual or extreme
environments are called extremophiles. These organisms live in environments that may be considered
as hostile or lethal to others such as in hot niches, ice, salt solutions, in acidic and alkaline conditions,
and some may grow in toxic wastes, organic solvents, heavy metals, or other habitats that are
considered intolerable for life.

2. How would you propose to reduce or eliminate aerial contamination inside the laboratory?

According to Parrett & Crilly (2000), air may contain biological agents such as plant cells, dust, parasites,
bacteria, yeasts, molds, and viruses. Pathways for these microorganisms to be released in the
atmosphere include wind, air flow, rain, and others. Moreover, Abatenh, Gizaw, & Tsegaye (2018) stated
that to prevent contamination, the use of sterile equipment and environment. To accomplish this there
are various steps and processes that can be performed. To maintain a sterile environment, it should be
exposed to fumigation and continued air sampling for microbiological evaluation. Furthermore, adjust all
laboratory physical factors such as room ventilation system. Also, it is important to clean the work area
with aseptic techniques before and after the experiment. Additionally, the use of hoods and cabinets for
work is also common when performing an experiment especially when the substance or media is
contagious, toxic or can easily spread contamination. To maintain sterile equipment, sterilization
methods such as autoclaving, hot air oven, microfiltration, flame, and others are commonly used. There
is also the importance of proper labelling, storage, and handling of substances. One should also note the
importance of wearing proper personal protective equipment (PPE). Moreover, protocols must be
formulated, implemented, and revalidated at regular intervals to maximize the reduction or elimination
of contamination inside the laboratory.

3. What is a microbial colony? How are bacterial colonies characterized?

Microbial colony is the term used when numerous or a population of individual cells grow in a solid or
semi-surface and are associated or connected to each other. A colony arises from a single
microorganism. Furthermore, bacterial colonies are characterized by colonial morphology which is
widely used in identification. There are basic elements observed to characterize a colony such as their
form, elevation, margin, surface, opacity, and chromogenesis and other distinctive characteristics such
as odor (Leung & Liu, 2020). The figure provided below shows examples of form, elevation, and margin.