Microbio
Microbiology
Bacterial Nomenclature
Group → Genus → Species → Strain
Shape names
Rod: Bacillus
Spherical: coccus
Corkscrew: spirillum
Curved Rods: vibrio
Aseptic Techniques
prevent contamination as much as possible
Examples
Complete things as quickly as possible, but don’t rush so much where you endanger yourself (knocking something over, breaking something etc.)
Disinfect work area
Pipette tips stay on the micropipette and touches nothing else
Vessels should be closed unless absolutely necessary (opened for short periods of time)
Flame the mouth of any bottle, especially glass
Work near a flame where you can feel the warmth of the flame
Close all windows and doors
Shut your mouth
Sterilise everything you can
Bacterial Growth & Culturing of Microbes
Bacterial Growth Curve
Lag phase
Cellular growth
Adapting to new environment
Cells increase in size, not increase in number
Log Phase
Rapid multiplication
Literally exponential ($2^n$)
High reproductive and metabolic rate
Stationary Phase
Death rate = Growth rate
Depletion of resources
Death Phase
Exponential cell death
Viable Cell Count
Count only viable cells (cells that can reproduce)
must know optimal culture conditions
useful for samples with a low bacterial count
requires time for incubation
spread plate method
dilute to a countable amount
30 - 300
make sure agar is dry before spreading
do repeats for reliability
incubate
count and calculate !!
Macronutrients
require large amounts
to build cell structures and help with metabolism
Micronutrients
required in small quantities
aid in enzyme function
Culture Media
contains all the nutrients required for cell growth
e.g. nutrient agar, nutrient broth etc.
defined / synthetic media
all components and concentrations are known
complex media
contains some unknown ingredients / concentrations
media components
peptones
water-soluble proteins
partial digestion of different protein sources
extracts
supply different nutrients
nucleic acid
fats
polysaccharides
vitamins
trace minerals
Classification of Bacteria
classification based on morphology
cell morphology
coccus
bacillus
spirillum
vibrio
colony morphology
form
punctiform
circular
filamentous
irregular
rhizoid
spindle
elevation
flat
raised
convex
pulvinate
umbonate
margin
entire
undulate
lobate
erose
filamentous
curled
colours
white
yellow
red
blue
classification based on cell wall
gram staining
crystal violet
soaks into the peptidoglycan layer
iodine solution
form crystal violet-iodine complex
wash with alcohol
remove the crystal violet from the peptidoglycan in the gram negative cells
counter-stain with safranin red
make the gram negative cells visible
KOH test
gram negative bacteria have a thinner cell wall
easily lysed by KOH
can see DNA strands
gram positive bacteria have a thicker cell wall
cannot be easily lysed by KOH
gram positive
90% peptidoglycan cell wall
high amounts of teichoic acids
shows up as purple when gram staining is performed correctly
gram negative
10% peptidoglycan cell wall
outer membrane of lipo-polysaccarides
shows up as red when gram staining is performed correctly
classification based on sensitivity to antibiotics
tested using kirby-bauer method
antibiotics
antibiotics are selectively toxic for bacteria
work by either
stop reproduction; or
killing cells
does not harm the patient
narrow-spectrum antibiotics only target a few bacteria
broad-spectrum antibiotics attack many different bacteria
certain antibiotics have side effects on patients
inhibition of cell wall synthesis
transpeptidase connects peptidoglycan layers to form a cell wall
penicillin binds with and inhibits the transpeptidase from cross-linking with peptidoglycan
cell wall weakens and bursts under osmotic pressure
inhibition of protein synthesis
antibiotic binds to bacterial ribosomes
targets different steps of protein synthesis
aminoacyl t-RNA binding (tetracyline)
binds to 30s unit of ribosome
blocking the binding of tRNA to mRNA
peptide bond formation (macrolides)
binds to 50s unit of ribosome
prevent peptide bond formation between amino acids
mRNA reading
translocation
inhibition of nucleic acid synthesis
quinolones
targets enzymes that unwind DNA before replication (DNA gyrase & topoisomerase)
bacterial DNA cannot unwind
rifamycin
binds to bacterial RNA polymerase
inhibit transcription of RNA
inhibition of cell membrane
polymyxin
bind to lipo-polysaccharides and phospholipids
alter structures
make membrane more permeable
leads to cell lysis
not selective
only used as last resort
can cause damage to body (e.g. kidneys)
classification based on influence of growth by environmental factors
most organisms grow is moderate conditions (e.g. pH 7, standard temperature etc.)
extremophiles grow under harsh conditions (e.g. extreme acidity, high temperatures)
pH
neutrophiles
grow best between pH 5.5 & 8.0
most bacteria are neutrophiles
acidophiles
grow best between pH 0 & 5.5
can cause serious environmental damages
e.g. can oxidize sulfides and release a lot of sulfuric acid into land and rivers
$SO_4^- + 2H^+ -> H_2SO_4$
can aid in the recovery of metals like copper, zinc, lead, and nickel
alkaliphiles
grow best between pH 8.0 & 11.5
extremophiles have methods to make the internal pH of the cell relatively close to pH 7
temperature
theres a RANGE of temperatures a microbe can tolerate
temperature where it can grow best is known as the optimum temperature
can only grow up to a certain temperature due to ezymatic and protein denaturation
unfolding of proteins
psychrophile
optimum: ~4ºC
has cold-stable enzymes
have more α-helices (flexible) than β-sheets (rigid) peptide structures
more polar amino acids
fewer weak bonds
decreased interactions between protein domains
transport processes function optimally at low temperatures due to modifications of cytoplasmic membranes
high unsaturated fatty acid content
mesophile
optimum: ~39ºC
thermophile
optimum: ~60ºC
hyperthermophile
optimum: ~88ºC
enzymes are thermo-stable
more ionic bonds
produce di-insitol phosphate, diglycerol phosphate
stabilise proteins
oxygen requirement
oxidase test
test for presence of cytochrome c
obligate aerobe
needs oxygen to grow
rely on oxygen as the electron receptor in electron transport chain
facultative anaerobe
good to have oxygen
doesn’t need oxygen
carries out aerobic respiration when oxygen in present
adopts anaerobic respiration like fermentation when oxygen is absent
aerotolerant anaerobe
does not care about oxygen
doesn’t need oxygen
strict anaerobe
must not have oxygen
generate ATP via fermentation / anaerobic respiration
inorganic compounds (e.g. sulfate, nitrate, sulfur) are used as electron receptors
less ATP generated as compared to oxygen
microaerophile
needs a little oxygen
classification based on nutrient types
autotroph vs heterotroph
autotroph
capable of making organic molecules from inorganic carbon sources (eg. $CO_2$)
e.g. plants
heterotroph
unable to synthesise organic molecules from inorganic carbon sources
feeds on organic matter produced by other organisms
e.g. animals
breakdown of compounds require energy (breaking bonds)
photo- vs chemo-
photo-
energy source: sunlight
chemo-
energy source: oxidation of inorganic compounds
photoautotroph
photosynthetic
use energy from the sun to break up $CO_2$ to make glucose
e.g. algae, cyanobacteria
chemoautotroph
able to synthesise organic molecules from $CO_2$
photoheterotroph
able to use light energy to generate ATP
takes in organic compounds
e.g. phloroflexus auratiacus
chemoheterotroph
consume pre-formed organic compounds as a carbon source
chemolithoheterotroph
utilize inorganic energy sources (sulfur, iron, manganese)
chemoorganoheterotroph
utilize organic energy sources (carbohydrates, lipids, proteins)
biochemical assays
detecting the presence of enzymes
catalase test
catalyse the decomposition of hydrogen peroxide into water and oxygen
$2H_2O_2 -> 2H_2O+O_2$
bacillius (+), colstridium (-)
starch hydrolysis test
detects for presence of anylase or glucosidase
urease test
urea → ammonia (causes pH increase)
klebsiella (+), escherichia (-)
capability to utilize nutrients
using something as a c source
will produce acid during fermentation
metabolic waste
gas formation
$H_2S$
acids