Introduction to Microbiology

Introduction to Microbiology

• Microbiology studies microbes or microorganisms too small for the human eye to see (less than 0.1mm).
• Microbes include prokaryotes (bacteria & archaea), eukaryotes (algae, fungi, protozoa) and viruses.
• Viruses are microbes, but not organisms.
• Microbes were the first organisms on Earth.
• Prokaryotic microbes thrive in diverse environments, including the digestive system, mouth, hot springs, bacterial biofilms, and acidic drainage water.

Algal Blooms and Red Tides

• Algal blooms occur in the Baltic Sea.
• Cyanobacteria are photosynthetic bacteria and prokaryotes often referred to as blue-green algae.
• Cyanobacteria produce metabolites toxic to humans and animals, causing green-blue swirls in water.
• Red tides are blooms of Dinoflagellates, eukaryotic algae/microbes, forming red clusters in water.
• Dinoflagellates metabolites enter shellfish, which when consumed by humans, leads to food poisoning.

Viral Pathogens

• SARS-CoV-2, the virus responsible for COVID-19, has four main groups: alpha, beta, gamma, and delta.
• SARS-CoV-2 sometimes evolves into more serious versions.
• It jumped from bats to other mammals, then to humans, making it a zoonotic disease primarily affecting animals but transmissible to humans.
• Viral pathogens, bacterial pathogens, parasites, or prions can cause zoonotic diseases.
• Influenza A includes bird flu and swine flu.
• Influenza A viruses are segmented, negative-stranded RNA viruses.
• Influenza is categorized by hemagglutinin (H) (18 types) and neuraminidase (N) (11 types) proteins on its surface.
• Rhino virus and Corona virus can cause the common cold.
• Ebola, found in Africa, kills the host so quickly that the virus does not have a chance to spread.
• Ebola is an enveloped, non-segmented, negative-stranded RNA virus.
• Smallpox was completely eradicated through a global vaccination campaign.

Bacterial Pathogens

• Yersinia pestis causes the black death.
• Mycobacterium tuberculosis/bovis causes tuberculosis, still a major problem.
• Superbugs like MRSA and C. difficile, are difficult to treat because of antibiotic resistance due to genomic mutations or horizontal gene transfer.

Parasites

• Parasites always require a host to survive.
• Plasmodium falciparum causes malaria, affecting red blood cells and causing liver damage.

Microbes vs. Macrobes

• Microbes process half of CO2, while macrobes process around half of O2 fixation.
• Fixation is done by macrobes and microbes.
• Microbes are a source of food, medicine, and play an essential role in causing diseases.
• Microbes drive nutrient cycling and provide energy.source
• Microbes are a source of industrial products, building materials, clothing, entertainment, companionship, and inspiration.
• Microbes makeup larger biomass than macrobes.
• Microbe colonies, are can be visible to the naked eye only.
• Animals, fungi, and plants are not microbes.

Microbial Properties

• All cells have a cytoplasmic membrane, genome, and ribosomes.
• All cells have a metabolism, and can grow and evolve.
• Some cells can differentiate to form new cell structures such as a spore.
• Cells interact with each other using chemical messengers.
• Self-propulsion is possible due to flagellum.
• Microbes can exchange genes using mechanisms such as horizontal gene transfer.

Prokaryotes and Eukaryotes

• Eukaryotic cells are larger than prokaryotic cells.
• Some bacteria, like Thiomargarita sulfide, can be huge (0.75 mm in diameter), where most of the cell volume is in the vacuole, where NO3 is stored.
• Eukaryotic cells have many more organelles than prokaryotic cells.
• The cell wall is made of cellulose carbohydrates (plants, algae), chitin N-acetylglucosamine (fungi), peptidoglycan (bacteria), or pseudo-IP68 5-layer (archaea).
• Pseudo-IP68 prevents amoeboid movement and phagocytosis; some cells will not have peptidoglycan.
• Peptidoglycan, composed of N-acetylglucosamine and N-acetylmuramic acid (a GP-1,4 linkage), can be degraded.
• There is a short peptide side chain attaching to the N-acetylmuramic acid which joins the subunit together.
• The subunit together forms tiltal glycan tetrapeptide.
• The peptide sidechains of UU adjacent peptidoglycan chains are cross-linked by covalent peptide bonds (W) to form a giant peptidoglycan.
• Gram-positive bacteria have thick peptidoglycan and an interbridge made of glycine cross-linked to lysine.
• Gram-negative bacteria have thinner peptidoglycan and a short cross-link from DAP to D-alanine.
• Pseudo-peptidoglycan or pseudomurein is composed of N-acetylglucosamine and N-acetyltalosaminuronic acid joined by B-1,3 linkages attached to a small peptide chain with L amino acids ONLY.
• Archaea contains an S-layer in contain S-layer.
• Diatoms algae contains transparent cell walls which contains silica - "house of glass".

Cell Envelope Structures

• Gram-negative bacteria contain an inner membrane, a thin PG layer, and an outer membrane containing porins and LPS lipids with attached sugary (very immunogenic).
• Gram-negative bacteria Outer membrane Protective contains MDR - multidrug resistant proteins that force out toxins/antibiotics.
• Gram-positive bacteria have no outer membrane, but a thick PG layer, with LTA lipo-teichoic acid embedded in the cell wall.
• In gram-positive bacteria antibiotics target the PG layer.
• Mycobacteria have a thin PG layer with an arabino-galactan layer on top, making the cell wall thicker.
• The outer membrane of mycobacteria contains mycolic acids which are immunogenic and provide protection by forming an impenetrable, waxy coat.

Organelles and Genome Packaging

• Bacteria that are cyanobacteria contain thylakoid membranes (site of photosynthesis & respiration) in contact with cytoplasm and have no membranes.
• Eukaryotic genomes contain DNA inside a double membrane structure that is wrapped around histones to form nucleosomes, then packaged into chromosomes.
• They attach to microtubules during cell division & histones = nucleosome.
• Prokaryotic genomes have circular DNA that exists as one or more copies per cell which is dependent on growth rate, condensed DNA and NAPS, also known as nucleoid which is DNA stacked on top of each other.

Genome Complexity and Ribosomes

• Prokaryotic genomes are small and circular, containing 1000-5000 coding genes.
• Eukaryotic genomes are larger, containing 16000-40000 coding genes.
• Prokaryotic ribosomes are usually cytoplasmic 70S (30S & 50S).
• Prokaryotic ribosomes could be localised to membrane.
• Eukaryotic ribosomes can be cytoplasmic or ER associated, 80S (40S & 60S), and 70S ribosomes in organelles.
• The differences in ribosome types causes different antibiotic sensitivity, as antibiotics are specific to different ribosome types.

Flagella and Archaea

• Eukaryotic flagella feature a bundle of microtubules surrounded by a membrane sheath.
• Prokaryotic flagella contain a single filament.
• Archaea flagella contains a single filament archaellum.
• Archaea can be isolated from hot springs in Yellowstone and inhabit extreme environments (salt, temperature, pH, pressure).
• Archaea are major producers of methane, enabling cows to digest cellulose grass.
• No archaea are known pathogens.
• Euryarchaeota (halophiles & methanogens) are usually found in high-salt environments.
• Euryarchaeota have high GC content in their DNA, which provides stability.
• Euryarchaeota release methane (ME) as a waste product.
• Crenarchaeota (hyper-thermophiles & sulfur lovers) are isolated from sulphuric mud ponds.
• Asgard archaea, named after Norse gods, encode many genes unique to eukaryotes and are found in Loki's castle in microbial communities.
• Some require a mate (not the same species), and have long filaments to grab things.

Microbial Growth and Division

• Media, or culture medium, provides nutrients for the growth and multiplication of microorganisms, including energy sources and building blocks.
• Media can be liquid with dissolved nutrients or solid with agar plates.
• Bacteria can swim through soft surfaces or sit on hard surfaces.
• Nutrients are acquired through diffusion and must contain N, P, S
• Heterotrophs acquire carbon by eating organic matter.
• Autotrophs fix CO2 into organic matter.
• Energy sources for heterotrophs get from organic compounds like glucose, which provides C for biomass and energy for respiration or fermentation.
• Energy sources for autotrophs obtain its energy by generating C from inorganic compounds and either use light (phototrophs) or oxidation of electron donors (chemotrophs).
• Defined media have simple components with a specific list of chemicals.
• Defined media is sometimes difficult to grow bacteria on, but controls all factors affecting growth and helps in metabolism investigation.
• Undefined media includes meat broth, yeast extract, blood products, and calf serum.
• Undefined media is easier to grow bacteria on it , however all growth factors are not known.

Growth

• Growth in microorganisms is an increase in biomass on a single-cell level through the construction of new cellular material.
• Membranes pinch off and cells divide.
• The rate of population growth determined by division rates.
• In binary fission, a newly divided cell grows to maturity, forms a septum, cell walls form, and the cell divides or the first three steps do not happen, thus the cell do not split.
• In not splitting the cell will form long chains of stitched-together cells, and filaments grow from the middle.

Exponential Growth

• Cn = Co * 2^n
• n = number of divisions
• Ct = Co * 2^(t/Td) = how long it takes for a new cell to mature and split by 2
• Td = doubling time rate (t/n)
• Plotting time (hours) on the x-axis and Ct / Co on the y-axis produces a straight line, indicating exponential growth.
• Exponential growth occurs for a limited number of generations before nutrients are consumed.
• Growth rate equals division rate minus death rate.

Growing Microorganisms

• Simplest culture: Batch culture
• Batch Culture provides all the nutrients needed for growth.
• The culture is inoculated with aliquot of bacterial cells and closed system.
• In batch culture At first, there will be exponential growth until all nutrients are consumed, so growth then will slow and eventually halt.
• Incubate the culture.
• The graph of "Time" (x-axis) vs. "log N" (y-axis) shows four phases: lag, exponential, stationary, and death.
• Lag phase: inoculum adapts to new conditions, synthesizing new cellular components.
• Death phase: cells start dying due to toxic waste and starvation.

Counting Methods

• A counting chamber can be used to count both live and dead cells.
• Serial dilution and plating on agar can also be used, providing larger colonies with more dilutions due to access to nutrients.
• Concentration of original culture = (dilution factor * number of colonies) / volume plated.
• Pick a plate with several hundred colonies.
• Only live cells grow colonies.
• Counts CFU (colony-forming units) like clusters.
• A coulter counter can also be used to count, but cannot distinguish live and dead cells.

Measuring Growth Rate

• Optical density can measure growth rate.
• A specific wavelength is chosen to determine how much light has been absorbed when shone through a cuvette containing the culture.
• To measure biomass, harvest cells by centrifugation/filtration and measure wet or dry weight.

Most Probable Number

• Most probable number uses a carry out dilution.
• Most probable number, count "growth" or "no growth".
• Most probable number, uses tables to estimate cell number.

Other Measures

• Electrical impedance measures the change in electrical conductivity.
• Electrical impedance provides quality control in the food industry.
• Microbial metabolism produces ionic byproducts that alter the electrical conductivity of a liquid medium.
• The growth rate is given by dC/dt = μCo * e^(ut) = με, where μ is the specific growth rate.
• If the specific growth rate (μ) is constant, the graph will be a straight line.
• Growth rate is related to substrate concentration.

Specific Growth Rate (μ)

• μ = µmax (S / Ks + S)
• µmax is never reached/attained.
• Ks is the substrate concentration where the specific growth rate is half of the max specific growth rate
• When S >>> Ks, μ = μmax
• When S << Ks, μ = (µmax / Ks) * S affinity , which is tangent when the substrate conc = 0
• The graph with two carbon sources in growth medium
• In diauxic growth/double growth, carbon source shows up in growth medium and is easier to metabolise, graph steeper.
• In diauxic growth/double growth, 2 phases of exponential growth occur.
• The graph, with "time" on the x-axis and "number of bacteria" on the y-axis, shows a curve with two distinct exponential growth phases.
• First phase in diauxic growth is as Glucose Used" and increases quickly.
• Second phase in diauxic growth the number of bacteria labeled is "Lactose Used", rises more slowly after a lag phase.

More Ways to Grow Bacteria

• Chemostat culture provides nutrients continuously: a continuous system.
• Drip in limiting reagent in a chemostat over time into the reaction chamber.
• Chemostat microbes grow
• Chemostat culture remove the old medium at the same rate it is added with the new medium in: with substrate.
• Chemostat culture wants a steady-state of exponential growth as conditions in chemostat remain constant.
• Fout / V = Fin / V = D.
• dC / dt = D(Co - C) + μC
• Characterizing Prokaryotes.
• Microbes live in communities sometimes cooperatively and sometimes in conflict.
• Some microbes need others' waste products to live while others are killed by bacteria released after death.

Identifying Microbes

• Shape
• Shape (coccus, rod, spirillum, spirochete.
• Shape also includes: stalk, hypha, budding & appendaged, filamentous unseparated rods).
• Size (eukaryotic cells are much larger than prokaryotic cells).
• The smallest known eukaryote is Ostreococcus, a green alga, measuring 1 micron in diameter.
• The sulfide oxidizer Thiomargarita grows and gets energy by oxidizing sulfur, measures 100 micron in diameter.
• The larger the bacteria, the smaller the ratio of surface area to volume which is limited by nutrients that can be acquired
• Clustering
• Clustering includes streptococcus forming strings of cells and staphylococcus, forming bunches like grapes.
• Stains
• Staining includes : gram+ & gram- bacteria.
• Staining will stain with crystal violet; fix stain with iodine; decolorize with alcohol; and counterstain with safranin.
• Gram-positive stains purple and Gram-negative stains pink.
• Other stains includes natural fluorescence, DAPI stain, and FISH probes.
• Look at conditions they like to grow in as different organisms have different optimums like temperature, salinity, pH, pressure.
• Fix CO2?
• Are given organic matter?

Tolerance Tests

• Tolerance tests involves tolerance to chemical inhibitors (respiratory inhibitors and chaotropic agents), and tests for antibiotics.
• Minimal inhibitory concentration (MIC) the smallest amount of an antimicrobial substance that prevents the growth of a target microorganism.
• On a graph, the x-axis is where each next well's concentration of antibiotic is halved, and the y-axis contains different strains.
• Select antibiotic that will cause inhibition of microbial growth with tests: Biochemical tests, sugar metabolism, and fermentation.
• Fermentation involves the breakdown of sugar into smaller molecules and produces different waste products amongst different species in anaerobic conditions.
• Oxidation involves the breakdown of sugar into CO2 + H20 takes place in aerobic conditions.
• Catalase test involves all suspension + H2O2 forming bubbles: = catalase.
• Perform a Test testing for pH also known as the urease Test.

Serology Tests

• Serology tests detects bacterial antigens using specific antibodies.
• Can make antibodies fluorescent and if cells light up then they have the required antigens.

Bacteriophage Tests

• Bacteriophage tests drip different bacteriophages across lawn to see what response is.
• Either cell will be infected and lyse and become clear on lawn or not.
• Resistance could be developed, as bacteria or phages can evolve.
• Bacteriophage tests is unreliable over time through symptoms.

Pathogencity Testing

• Pathogenicity through symptoms can indicate identity.
• Genome sequencing is possible with DNA analysis.
• If there are no culturing of cells is required and diagnosis is much faster PCR analysis.
• Use of specific growth media to favor the growth of a specific microorganism.
• Select a metagenomics when species diversity is preferrably low.

Extremophiles/Bacteria

• Extremophiles thrive at extreme pH, salinity, pH & pressure such as salt contents.
• Psychrophiles are < 15 degrees C
• Mesophiles are 15-50 degrees C
• Thermophiles are 50-80 degrees C
• Hyperthermophiles are 80-115 degrees C
• Thermophiles contains an extreme temperature on proteins
• More a helix is flexible and can retain a taQ Polymerase
• More sheets is stiff and extracts and runs hyperthermophile experiment increases speed of.
• Unsaturated fats is more liquid at room that that of saturated fats.
• An organism that is high temp. adapted will contain more saturated lipids.
• Acidophiles lives from 0.7-4 pH
• Alkaliphiles lives from 8-12.5 pH.
• Aquire powerful proton pumps to maintain neutrall PH by pumping them out and helps resist osmotic imbalance on cell
• halophile = 15-37.5% naCL
• PIEZOPHILES/baropiles UP to 130 MPA
• exist in deep subsurface
• different forms of energy released are also extremes

Microbiome and Microbiota

• Microbiota a collection of micro organisms in a particular environment including bacteria, archaea, fungi ect
• Micro biome collective genomes present in microbiotia
• Human Microbiota collective genomes living on and in body includes nose mouth genitalds
• contain 3 by 10^13 cells Probing the MICORBOIME
• is this bacteria even located there
• take microbial community then streak onto plate for individual species and identify
• maconkey agar and stay yellow when comes into contact with non lacotorse bacteria but goes back to turning pink after comes in contact after is is yello
• only vibro species can grow on Tcbs which results in pH chang e the turn yellow
• imaging location is there
• label and image cells thru and then imaging thru microscope Sequence
• isolare dna next sequence after genomic rna
• next genomic rna and actual genes being expected
• lack strictueral understanding or community and behavior. need a color. label bacteria
• expressed gene with light and wavelength require oxygen bioloumense can see with out the light with one color and enzyme exitation ###ex vivo- ex vivo - picture in side tissue by cuttting oppen and thin sections
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• can fix multiple species
• bacteria communities one fixed. solid surface
• microbal increases
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• bacteria cannot grow because it takes space to get killed
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In general - the process of replication where virus enters cell - acute/ lysis

• replicates but is unable to do further function - latency (prophage insertion in lysogeny) Once it can, it becomes virus- producing again What causes infections? Antigentic drift - mutations to virus that lead to new strain
• could be closely related to progenitor
• if recognized as strain then responds through protection
• if not - infection happens Antigentic shift - different viruses of combine or reassert genome stages of viral infection

1. entry

Antigenic Shift occurs among influenza A viruses when different viruses infect the same cell and begin a process called genetic re assortment

There's a specific site inside the bacterium where DNA replication begins. DNA is made from two strands

Bacterial Pathogenesis

There's the direct mode of transmission- occurs when when infected host transmits pathogen directly to a susceptible host without an intermediary.

Then there's the indirect mode of transmission-When transmission in facilliated by aliving a Nonliving intermediate the vehicle- food fluids air living vectors

Basic reproduction number

• Average number of people that one infected person will spread the virus too
• if the reproduction number is less than one it declines and.

Endemic- constantly present at low numbers in. an geographic areas the reservoir. Epidemic- unusually high numbers in. A graphic areas are population are carriers Pandemic- global epidemic will become epidemic Emergent disease suddenly prevalent again Re emerging disease under control that become prevalent again Case fatility percent of infected who does total death / total

Active immunity

antigen specific limits infection apoptosis isolates Deceased cells

Natural and artificial

Passive immunity Natural maternal antibodies in uterus throughcenta after birth breast-feeding fades within

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