LUCERO, Abigail Ray T.

BSBIO 3 – 2MB

LABORATORY ACTIVITY #2

LABORATORY RULES AND SAFETY GUIDELINES

1. Enumerate at least three protective gears needed to be worn when working inside a
microbiology laboratory.

A person needs to dress appropriately when using the laboratory. They

need to wear a lab coat, face mask, safety glasses and disposable gloves for
protection and to prevent contamination. Wearing closed shoes are also
important to reduce any accidental injuries.

2. Differentiate general waste from pathological waste and infectious waste. Provide some
examples.

General waste are materials that are non-hazardous and does not pose any
particular hazards. Examples are paper towels, plastic bags and cling wraps.
Pathological waste includes human and animal tissue samples, amputated organs
or body parts. Infectious waste includes fluids, solids or materials that have
the potential to carry infectious microorganisms and transfer diseases.
Examples are syringes and needles, bandages, and scalpel blades.

3. What the difference between a biohazard and biorisk?

Biohazards are any substances or conditions that poses a threat or

hazardous to the health of humans and other living organisms. Biorisks are the
risk (function of likelihood and consequences) that a particular biological
event (accidents, unexpected discovery, misuse of biological agents) may occur.

BIOSAFETY LEVELS

1. What is the difference between biosafety and biosecurity?

Biosafety is the containment principles and practices implemented to

prevent unintentional exposure to biohazards. Biosecurity is the protection,
control and accountability for valuable biological materials within
laboratories to prevent unauthorized access, loss, theft, misuse, diversion or
intentional release.

2. Describe the 4 levels of biosafety using the table provided.

Biosafety level BSL – 1 BSL – 2 BSL – 3 BSL – 4

High risk; Very high
Typical risk Low risk Moderate risk potentially risk; life-
lethal threatening
 Sample
Escherichia HIV, Mycobacterium
organism (1-2 Ebola virus
coli Influenza tuberculosis
examples)

Agents
associated Exotic and
with human indigenous
Dangerous and
diseases; may microbes that
Agents that exotic agents
transmit cause serious
are minimal that pose
through or
Pathogen type to no threat high risk of
percutaneous potentially
to cause aerosol-
injury, lethal
diseases transmitted
ingestion and diseases
infections
mucus through
membrane inhalation
exposure

STERILIZATION METHODS

Describe the different sterilization methods that can be applied in the microbiology laboratory.

Type of
Sterilization
Name sterilization Description Application
method
(Physical/Chemical)

A machine
that uses
steam under Used for
Moist
pressure to decontamination
heat/steam
Autoclave physical kill and sterilizing
(121-124°C
pathogenic lab instruments
for 15 mins.)
microbes and media
(vegetative
and spores)

A process
where
packaged and Used by dairy
non-packaged and other food
Moist
foods are processing
Pasteurization heat/steam physical
treated with industries to
(<100°C)
mild heat to prolong shelf
eliminate life
vegetative
bacteria
 Used for
sterilizing lab
A machine
materials that
Dry heat that is
withstand high
(>150°C for primarily
Hot air oven physical temperature
30 mins. to 1 used for dry
(glasswares,
hr) heat
metal
sterilization
equipments,
oils, powders)

Used with
Bunsen burners
or alcohol lamp
A quick,
for sterilizing
simple method
petri dishes,
Flame that uses
Dry heat Physical inoculating
sterilization open flame to
loop or needle;
sterilize the
used in aseptic
instrument
technique and
culture
transfer

An instrument
that
sterilizes
inoculating
For sterilizing
loops and
loops and
Infrared needles
Bacti cinerator physical needles, as
sterilization without using
well as glass
an open
pipettes
flame; ideal
for use in
anaerobic
chambers

ASEPTIC CULTURE TECHNIQUES

1. Provide three reasons why the use of aseptic technique is essential when handling microbial
cultures in the laboratory?

Proper and appropriate use of aseptic technique is important to prevent

contamination of certain microbes a person is handling with. This technique
aims to prevent contamination of the room and the personnel we are working
with, as well as possible infections through the transfer of microbes to
susceptible areas of the body.
2. Why are culture media sterilized before use?

Culture media must be sterilized before use because they are exposed to
microbial contamination from preparing and handling them, and those microbes
could potentially affect experimentation and observation of cultures. Since
the medium contain nutrients, the microbes will reproduce and start using the
nutrients for growth, and the medium itself will be turbid and becomes
contaminated.

3. Why is culture medium cooled to about 48⁰ to 50⁰C before it is poured into petri plates?

The culture medium is cooled firstly to save your hands from too much
heat. Second, it is done to minimize steam condensation and prevent microbial
samples from damaging especially when using pour plate technique.

4. What is the purpose of flaming in the aseptic technique?

Flaming in aseptic technique ensures to remove any unwanted

microorganisms that are exposed when preparing cultures. Most microbes are
killed when exposed at high temperatures, so flame sterilization is an essential
procedure.

5. In subculturing, when do you use the inoculating loop and the inoculating needle?

Inoculating loops are used for transferring the inoculum from liquid
medium (broth) to another or to a petri dish for streaking. Meanwhile,
inoculating needles are used in solid medium and are suitable for removing
microorganisms with a single colony.

6. What is the purpose of the ethanol in the spread-plate technique?

The ethanol is used for sterilizing the L-shaped spreader by dipping,

before it is ignited into small flame.

7. Why is it important to invert the petri plates during incubation?

Petri dishes must be placed upside down during incubation to prevent the
accumulation of water condensation dripping from the media that could disturb
or compromise the culture. The rate of evaporation also decreases when the
petri dishes are inverted, preventing the media from drying up.