Biol 2103 Biological Laboratory Course

Module of Basic Microbiology Techniques

Dr. Aixin Yan

7S-01, Kadoorie Biological Sciences Building

Contact info: ayan8@hku.hk

Include “BIOL2103” in the subject when you E-mail me

Outline for Lecture 1-2

I. General introduction of the module

II. The diversity of the microbial world

III. Safety measurements in microbiology labs

IV. Aseptic techniques used in the sampling, handling,

storage, transfer etc. of microorganisms
 Why basic microbiology techniques?
found everywhere
Microorganisms are ubiquitous and important:

• Most populous group of organisms and are found

everywhere on the planet. contain 50% of the biological
carbon and 90% of the biological nitrogen (ubiquity)
• Play a major role in recycling essential elements, source of
nutrients and some carry out photosynthesis ecosystem
dominant
hot spring, skin, intestinal tract, etc.
(environment, biosphere, ecology)

• Benefit society by their production of food, beverages,

antibiotics and vitamins (Biotechnology)
pathogens
• Causative agents of some infectious diseases (Medicine)
• Microbiomes are closely related to human health, physiology,
and metabolism (Human physiology)
 don’t have to memorize all

Emerging frontier in Biomedical Sciences: Microbiota

100 trillion (1014) microbial cells are harbored in the human body
how do mothers pass bacteria to offsprings

stabilize in human body

-> differentiate

from mother

“The role of the infinitely small in nature is infinitely large”

so many microbial cells in human body! Louis Pasteur
 Microorganisms 3 keys messages
viruses (cannot survive w/o host)
Organisms and acellular biological entities that are too
small to be seen clearly with the naked eye, and lack highly
differentiated cells and distinct tissues. organization
many of them are singular
Include: Fungi, protists, bacteria, archaea, and virus

nanometre micrometre
 Basic Microbiology Techniques 3 labs

Three Experiments:
Experiment I: Aseptic Techniques
Experiment II: Visualizing of Microorganisms and Isolating
Microorganisms from Living Environments
Experiment III: Enumeration of Microbes count the no. of microbes (quantification)

Why these three experiments:

Escherichia coli Fundamental techniques for
Bacillus subtilis collecting, maintaining, and (Lab 4)
Staphylococcus epidermidis transferring microorganisms
Pseudomonas aeruginosa To visualize and quantify
Saccharomyces cerevisiae microorganisms
rmb these names !!!

With the assistance of special Allow the microorganisms

instrument --- Microscope (Fixing, grow to sufficient biomass
Staining, and Microscopy) to observe and quantify
(Lab 5) (Lab 6)
 Learning outcomes
1. Be able to perform basic microbiology procedures including
Bunsen burner, why?
pouring plates, aseptic transfer of microbes, spread plate,
simple stain, microscopy, and counting of microbes

2. Be able to understand principles of the basic

microbiological techniques learned in this module

3. Be able to plan and prepare basic supplies to carry out an

experiment work in clinical biology lab

4. Be able to critically evaluate experimental results

open questions, no standard ans

5. Be able to maintain scientific records and communicate results

6. Be able to understand the team dynamics needed to work
cooperatively in a research team
 Members of the Microbial World
Including:
Cellular: fungi, protists, bacteria, archaea
Acellular: viruses,smallest
viroids,infectious
satellites, prions
pathogens known

Bacteria and Archaea

eukaryotic: The nucleolus is a condensed
classification based on chromatin region where ribosome synthesis occurs.
molecular and physiological with two phospholipid bilayers
properties
Eukaryotic Prokaryotic
cellular MO cellular MO

8
 Bacteria
• Usually single-celled
• Majority have cell wall with peptidoglycan
• Most lack a membrane-bound nucleus
• Ubiquitous and some live in extreme environments
• Some cause infectious diseases
distinctive colour under gram stain technique
its organization is distinctive only in bacteria, not in archaea bc of peptidoglycan
 Bacteria
Diverse shape, size, and organization:
 Archaea after invention of sequencing technique
• Distinguished from Bacteria by unique rRNA gene sequences
• Lack peptidoglycan in cell walls form hydrophobic part
• Have unique membrane lipids (ether bond)
• Many live in extreme environments
• Some have unusual metabolic characteristics
more stable in acidic/ alkaline environment

Bilayer of C20 lipid

Archaeal Bacterial
membrane membrane
Methanogens:

CO2 + 4H2 CH4 + 2H2O

in their stomachs for grass digestion

methane (greenhouse gas) as by-product
 Protists and Fungi (Eukaryotic Microorganisms)
A prokaryotic (bacterial) cell: Eukaryotic cell:
 Fungi
• Fungi (decomposers) Mycology – study of fungi
– yeast - unicellular • Have many important applications:
– fermentation – yeast used in making
– mold – multicellular
bread, wine, beer, cheese, soy sauce
– Mushroom – organic acids – citric and gallic acid
– certain drugs – ergometrine, cortisone
v Chitin as their cell – antibiotics – penicillin, griseofulvin
wall component

Edible mushrooms
Compressed yeast
Molds

Granulated dried
active yeast
 Protists
• Protists – generally larger than Bacteria and Archaea; Most
exist in unicellular form; Lack the level of tissue organization
present in higher eukaryotes
• No typical definition
– algae – photosynthetic (“plant like”)
– protozoa – may be motile, “hunters, grazers”
eat grass
(Plasmodium: cause of malaria) (“animal like”)
– slime molds
(“fungus like”)
– water molds
Bacteria and Archaea
 Virus (Acellular Agents)
• Viruses – protein and nucleic acid
• Viroids – only RNA Can infect all cell types:
• Satellites – only nucleic acids • Bacterial viruses
• Prions – proteins only – bacteriophages (phages)
• Few archaeal viruses
• Most are eukaryotic viruses
– plants, animals, protists, and fungi
 Why some viruses are so contagious ?
The number of people that one sick person will infect (on average) is called R0.
Maximum R0 values for the following virus:

replication of viruses itself

2019
COVID-19

A graph of responses
versus time in surface
plasmon resonance
the smaller the value, studies
the higher the binding affinity to surface SPR sensorgram
more infectious

2003
SARS

(for your information)

Deadly pandemics occurred in human history, but
many were stopped

(for your information)

Lab Safety
 Lab Safety
Purposes:
To ensure: • The success of experiments
• Safety of the experimenter
1). Personal Habits:
Dress: Long pants, Close-toed shoes, Lab coats
Hair: For long hair, take necessary steps to prevent it from dangling
near lit burners or cultures.
Behavior: • No eat, drink, smoke, or apply cosmetics in the laboratory
• Wash your hands thoroughly with soap and water
before leaving the laboratory and any time they
become contaminated with culture material.
• Clean up your bench as you work, disposing used items
properly.
2). Labeling:
Label all cultures and solutions properly with your name
or initials, date, lab section, and necessary experiment
information.
3). Biological Materials
- Anything that comes in contact with a living culture (slides,
tubes, flasks, petri dishes).
- Prevent transmission of microbes (a potential source of
disease) to unsuspecting individuals and sites.
• Discard all biologically contaminated materials to indicated
places, not regular trash !
• Never remove any cultures from the laboratory.

4). Chemicals
• Understand the properties of the chemicals used.
• Keep volatile, flammable liquids (e.g. ethanol) away from flames.
5). Glassware
• Handle with extreme care
• Clean and report if you break them

6). Equipments
• Make sure you know how to operate them before your use
• Handle with extreme care
• Making the area clean and neat
• Report if you find any problem or it is broken

A safety video clip during the lab session

 Experiment I: Aseptic Techniques
Background:
Why aseptic techniques in microbiology study:
• Ensure only one type of microorganism is present and being
investigated in the experiment (pure culture).
Pure culture: a growth of microorganisms (a culture) that contains one
cell type.
• Ensure that the microorganisms do not escape from the container,
contaminating the laboratory, and possibly causing diseases.
Bunsen
Benson Burner:
I. Collect microorganisms
1. Flame sterilization
Procedures:
Inoculation loop
Hold the loop
at a steep angle to ensure the
larger area of loop being sterilized
Open flame is a safety hazard and
must not be left unattended !
if the loop is too hot, it’ll kill microorganisms
Move the loop with
the tip in the hottest
part of the flame
the tip of the
inner blue cone
hottest area

Q: If the loop has just liquid

been dipped in culture Allow the loop to
before you want to use it, cool a few seconds
what would you do to
flame sterilize it? one extra step: dip the liquid and dry up
in the tip of the inner blue cone
II. Isolate and maintain microorganisms
2. Streaking plates: streak for several times to draw the lines here

Purposes:
To obtain a pure culture of an organism --- single colonies
have largest area
almost half of the plate area
Procedures:
Rotate Rotate
plate plate
The 3-phase
streaking: Sterilize Sterilize
inoculation inoculation
loop loop

flex and extend the fingers during streaking

 Following overnight (~14-16 hrs)
incubation : Confluent growth
37 degree celcius
agar side up since the diameter of lid is larger at the beginning of
larger surface area as protection streak
to avoid contamination
Phase I

Phase II
Phase III
Isolated colonies at
the end of streak
II. Isolate and maintain microorganisms
3. Pouring agar plates:
transparent
A solid support allowing the growth, observation,
storage, transfer, and differentiate microorganisms
Developed during the establishment of the bacterium
Bacillus anthracis being the causing agent of the
disease anthrax by Robert Koch.
Purpose: to isolate suspected bacterial in pure culture
Recipe of the LB medium:
Assortment of peptides formed by the
10 g tryptone digestion of casein by the protease trypsin
10 g NaCl Sources of amino acids, carbohydrates,
inorganic elements (Ca, P)
5 g Yeast Extract
15 g agar Water-soluble portion of the yeast
1 liter dd H2O autolysis product
very slow hydrolysis of yeast
Sources of vitamins, nitrogen, amino
Autoclave 60min acids, carbon
under high temp. and pressure
3. Pouring agar plates: Chemical structure of
a single agarose unit
Single polymer
Agar: form H-bonds with more compact structure
Helix formation

Cooling
Higher order structure

Agar plate enclosed

in a Petri dish
Below 45 °C
Procedures:
Bring petri dishes within your arm reach

Take one bottle of melted agar broth

Remove the cap, flame the mouth of the

bottle, and pour the plate (15/20ml per plate)
 Functional Types of Agar Plate (for your reference)
By supplying different nutrients in agar plates, they can be
utilized for both isolation and identification of microorganisms:

Mannitol salt agar to

E. coli on blood plate differentiate some gram
Eosin methylene blue (EMB) agar
positive bacterial species to differentiate some gram
negative bacterial species

α, β, and γ
Lactose and non-lactose
Haemolytic bacteria
fermenting gram negative Blood agar culture of bacteria
on blood agar
bacilli on MacConkey plate from the human throat
clear zones means bacteria digested blood
4. Spread plates:
Purposes:
To separate microorganisms and obtain a pure culture of an
single colonies are evenly distributed
organism --- single colonies
Procedures:

Pipetting 200ul cell suspensions onto

the surface of the LB agar plate.

Sterile an L-shaped glass rod

How?
dip the glass rod to ethanol on the top of Bunsen Burner

While rotating the plate using your left

hand, move the spreader across the
entire surface of the agar plate.
III. Transfer microorganisms one hand to hold two test tubes

5. Tube transfer [from liquid sample source to culture medium (inoculation)]

Purposes: Transfer of culture from plates to tubes, or from tube to tube

Flame sterilize the Remove tube caps and
inoculation loop flame sterilize the tops
of the tubes rapidly
After the inoculation loop
cools down, immerse it into
the broth culture to obtain a
loop of inoculum and imerse
into the other tube

Flame
sterilize the
Flame sterilize the tops inoculation
of the tubes rapidly loop
Return the caps
Results observation and recording of the
practical include:

ü Smoothness of pouring plates, presence of

bubbles, contaminations etc.

ü Pattern of microbial growth on streaking and spread

plates

ü Size, color, morphology of the colonies

ü Count the numbers of colonies on spread plates

ü Any other phenomenon