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Culture Documents
from mother
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)
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
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:
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
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
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
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
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:
Flame sterilize the Remove tube caps and After the inoculation loop
inoculation loop flame sterilize the tops cools down, immerse it into
of the tubes rapidly 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: