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fBIOC0004 REVISIONWeek 20Microorganisms – prokaryotes (bacteria and archaea), eukaryotes (algae, fungi and protozoa), viruses are not organisms, but they are microbesMicrobes – too small to be seen clearly by naked eye, less than 0 bigZoonosis/zoonotic disease any disease or infection that is natura...

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fBIOC0004 REVISION Week 20 Microorganisms – prokaryotes (bacteria and archaea), eukaryotes (algae, fungi and protozoa), viruses are not organisms, but they are microbes Microbes – too small to be seen clearly by naked eye, less than 0.1mm big Zoonosis/zoonotic disease -

any disease or infection that is naturally transmissible from vertebrate animals to humans.

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Animals play an essential role in maintaining zoonotic infections in nature

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Zoonoses can be bacterial, viral, parasitic, may involve unconventional agents like prions (misfolded proteins with the ability to transmit their misfolded shape onto normal variants of the same protein)

Bird Flu (H5N1 Influenza) -

Influenza A viruses are divided into subtypes based on 2 proteins on their surface of the virus a) hemagglutin (H) there are 18 subtypes of this protein (H1 to H18) and b) Neuraminidase (N) there are 11 different types of this protein (N1 to N11), N is the target of some anti-flu drugs

White Death -

Tuberculosis (TB) Caused by Mycobacterium Tuberculosis (infecting humans and primates) and Mycobacterium bovis (infecting more animals like cows)

MRSA and C. difficile -

Superbugs (antibiotic resistant bacteria) Resistance acquired by genomic mutations or horizontal gene transfer

HARMFUL BUT NOT PATHOGENIC 1) Algal blooms: Cyanobacteria in Baltic sea, red tides caused primarily by dinoflagellates

CELLULAR MICROBES A) Eukaryotes -

Plans Animals Fungi Algae Protozoa

Macrobes

B) Prokaryotes -

Bacteria Archaea

NON-CELLULAR MICROBES -

Viruses Prions

Sizes of microbes Tobacco necrosis satellite virus 17nm Polio virus 28nm Influenza Virus 100nm Pox Virus 200-300nm Bacterium 1m Animal cell 30m Human cell 1.8m

The phylogenetic tree of life - defined using 16S ribosomal RNA sequencing - each domain has its own viruses

WAYS TO DISTINGUISH MICROBES AND MACROBES 1) Numbers vs Biomass 2) Importance to Biosphere (essential roles like photosynthesis, nitrogen fixation, recycling of carbon/phosphorous/sulfur) 3) Biodiversity

DOMAINS 1) Bacteria 2) Archaea 3) Eukarya

Differences between prokaryotes and eukaryotes 1) Size 2) Packaging of DNA genome Eukaryotes DNA contained in double membrane (nucleus)

Nuclear DNA divided into number of

Prokaryotes No nuclear membrane No chromosomes No Mitosis or meiosis No microtubules DNA typically circular

linear molecules DNA packaged into chromosomes

3) 4) 5) 6) 7)

Chromosomes attached to microtubules (spindles) during mitosis and meiosis Genome complexity Organelles Ribosomes Flagella Cell Wall

DNA exists as one or more copies of aggregated mass of DNA within cytoplasm – Nucleoid Plasmids often present

Exception – Gemmata obscuriglobus (planctomycetes) aquatic bacteria with life complexes have a nucleoid surrounded by a nuclear envelope

LECTURES 2A-2B Largest Bacterium – Thiomargarita Namibiensis -

0.75mm in diameter Most of volume in vacuole Chemolithiotroph (uses H2S electron donor and nitrate as an electron acceptor) Stores nitrate in vacuole since exogenous nitrate available only when the sediments by storms

Continuation of differences between eukaryotes and prokaryotes NUCLEUS Membrane bound organelle in eukaryotes cytoplasm Large well organized More than 1 chromosome DNA forms structures with histones Nucleolus and nucleoplasm are present Spherical shaped organelle Houses genetic material and provides space for transcription, DNA replication & ribosome biogenesis

NUCLEOID Nucleoid particular area in the prokaroytes cytoplasm Small poorly structured Single chromosome DNA is compact with NAPs Nucleolus and nucleoplasm are absent Irregular shape Only houses genetic material

Genome Complexity Prokaryotes

Eukaryotes

Small circular genome, 2-13 mbp 1000-5000 protein encoding genes

Larger 10-10000 mbp 6000-40000 protein encoding genes

Organelles Prokaryotes No organelles (no mitochondria, if respiration occurs it happens in the plasma membrane Sometimes have internal membrane with specialized functions

Eukaryotes Possess organelles – mitochondria (inner membrane is site of aerobic respiration and ATP synthesis), plastids (plants, algae, protists) Nucleus, endoplasmic reticulum, golgi apparatus, centrioles etc.

Mitochondria

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Mitochondria and chloroplasts are the descendents of free-living bacteria that formed an endosymbiosis with a proto-eukaryote Have retained their own genetic system with many bacterial characteristics

Endosymbiosis – process by which one of the symbiotic organisms lives inside another

Ribosomes - contain proteins and RNA and are the site of protein synthesis with RNA being the catalytic molecule - ribosomal RNA is also a useful tool for classification Prokaryotes 70S ribosomes (30S and 50S)

Eukaryotes 80S ribosomes in cytoplasm (40S and 60S) 70S ribosomes in organelles

S = svedberg unit, measure of size and density and not additive Cell Wall Prokaryotes Peptidoglycan (bacteria), various (archaea)

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Eukaryotes Cellulose in plants and most algae Chitin in fungi

Bacterial cell wall very important for characterizing bacteria (ex. Gram positive/negative) Diatoms, algae living in houses made of glass, only organism with cell walls made out of transparent opaline silica

White death – hereditary Anthrax - highly infectious animal disease that causes skin pustules, fever, nausea - Due to bacillus anthracis - Endospores are very resistant and can survive in soil and animal products for hundreds of years

Landmarks in microbiology -

Lucretius: roman philosopher suggested that disease was caused by invisible living creatures (98-55 BC) Antony Van Leeuvenhoek: first to observe and describe microbes using simple microscopes (1670) Louis Pasteur: disproves the notion that bacteria rise by spontaneous generation Robert Kock – germ theory of disease. Bacterial culture isolates from the blood of a diseased animal can induce the disease in a healthy animal

Koch’s Postulates – rules for conforming a causal link between a microorganism and a disease 1) Microorganism must be found in abundance in all organisms suffering from the disease, should not be found in healthy animals 2) Microorganism must be isolated from a diseased organism and grown in pure culture 3) Cultured microorganism should cause disease when introduced in healthy organism 4) Microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the specific causative agent Exception in Koch’s Postulates -

The cause of leprosy cannot be ‘grown in pure culture’ in the lab There may be pathogens for which there are no animal models

Isolating, growing and characterizing bacteria -

Originally very difficult to isolate bacterial strains, because bacteria were grown only in liquid culture

Joseph Lister (1878) – Dilution method Koch – developed the use of solid medium for spatial separation of individual cells

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Solid medium now usually prepared from culture medium with a gelling agent Originally this was gelatin but Fannie Hesse suggested the use of agar Richard Petri developed the petri dish

Isolation of Clones (strains using solid medium) -

Mixed culture, spread on petri-dish Then pick single colony and incubate to obtain a pure culture

Each colony is a clone or strain 1) 2) 3)

Strains can be maintained by Regular sub-culture to fresh medium Frozen storage Freeze-drying

Many culture collections around the world which supply known strains -

ATCC: American type culture collection PCC: Pasteur culture collection NCTC: National collection of types cultures NCIB: National collection of Industrial Bacteria

LECTURES 3A-3B Classifying and Naming Bacteria Example – Escherichia Coli (genus, species) -

Always italics

Taxonomy Classical approach – identify a lot of features like size, shape, nutrient requirements, metabolism, cell wall and membrane components, host range (for pathogens) Molecular taxonomy – evolutionary relationships established by DNA/RNA/Protein sequences like the gene for 16S ribosomal RNA or use a set of stanfarf genes or WGS (whole genome sewuencing) Species – on a basis of overall DNA sequence, similarity >70% Exception – M. tuberculosis and M. bovis are >99% identical but are considered 2 distinct species

CHARACTERIZING NEW ISOLATES Single colony = clone = strain = isolate Categories of tests 1. 2. 3. 4. 5. 6. 7.

Morphology (colony and single cells) Physiology Biochemistry Serology Bacteriophage Pathogenicity Chemistry and macromolecular analysis

1. MORPHOLOGY A) Colonial Morphology – appearance of colony on specific agar under specific conditions -

Shape Color Texture Size

B) Cell Morphology – appearance of cells when viewed under microscope

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4 typical classes of cell shape: Cocci (spherical), rods, vibrio (comma, shaped), spirillum (spiral)

Observing Bacteria under microscope

- Samples are typically fixed and stained Fixation – kills and preserves sample on microscopic slide 1) Heat fixation – gentle flame heating of air-dried sample (preserved overall morphology but not delicate structures within cell) 2) Chemical fixation – Chemicals like ethanol, formaldehyde that penetrates cells and react with cellular components, inactive and insoluble. Preserves fine cell structure

STEPS 1. Preparing a smear - Spread culture in thin film over slide and dry in air 2. Heat fixing and staining – pass slide through flame to fix, flood slide with stain, rinse and dry 3. Microscopy – place drop of oil on slide, examine with 100x objective Simple staining using dyes All dyes - Have chromophore groups (conjugated double bonds) that gives the dye its color - Bind to cell components (either ionic, covalent or hydrophobic bonds) 2 Types of ionizable Dyes (pH may affect effectiveness of staining) 1) Basic Dyes – methylene blue, crystal violet, chalachute green 2) Acid Dyes – rise Bengal, acid fuchsin

Simple stains first used by Koch 1877 -

Dried bacterial smears and stained them with methyl violet

Differential staining procedures – allow the separation of bacteria into different groups based on their staining properties Paul Ehrlich – carries out studies on dyes in 1877-91 and developed acid-fast staining (staining is due to high lipid content of the cell walls as seen with mycobacteria) 1. 2. 3. 4. 5.

Smear + hot carbol fuchsin Wash with water 10 min 20% H2S04 Wash with water Counterstain with methylene blue

The Gram Stain – 1884 distinguishes 2 classes of bacteria on the basis of their cell wall structure (the more peptidoglycan in the walls the higher the retention of the pigment) 1. 2. 3. 4. 5.

Heat-fixed smear + Crystal Violet (basic dye) Iodine/KI added [CVI] formed in cells Wash with solvent (like 95% ethanol) Stop with water Counterstain (carbol fuchsin, safranin)

Gram positive bacteria

Gram negative bacteria

Dark purple Layers of cell wall (outside to inside): peptidoglycan and membrane

Cocci shape

Pink or red Layers of cell wall (outside to inside): outer lipopolysaccharide and protein membrane, periplasm, peptidoglycan, periplasm, membrane Rod shape

C) Cell Cytology – Light and electron microscopy Features -

Capsule (and slime) – made of polysaccharides, capsules are more organized, harder to remove Polyphosphate Slime Pilus Spore Polyhydroxybutyrate – inclusion bodies for storage of metabolites Flagellum

2)Physiology a) Nutritional requirements Examples -

Source of C and N (gaseous or fixed) Bacteria that can fix nitrogen are termed diazotrophs Growth factor requirements (vitamins, amino acids, etc) these are called auxotrophs

b) Light -

Important for photosynthetic microbes (some obligate, some facultative phototrophs) Most bacteria are sensitive to UV light – however some are particularly resistant (deinococcus species and novel species recently found in the stratosphere)

c) Temperature -

Microbes can be classifies as psychrophiles, mesophiles, thermophiles depending on their optimum growth temperature

Salinity (salt concentration) -

Freshwater < coastal areas < oceans < salt lakes All marine organisms are moderate halophiles (3.5% salt) All extreme halophiles are found in salt lakes (solar lake, sinai)

Oxygen -

Essential for respiration for also very toxic and a highly reactive gas Bacteria can be classified according to their requirement for/tolerance of oxygen

a) b) c) d) e)

Obligate aerobes: need oxygen for growth Obligate anaerobes: oxygen is not needed, it is toxic Facultative aerobes: can grow without oxygen but will still use it when it is available Aerotolerant anaerobes: can grow with oxygen present but do not need to use it Microaerophiles: are damaged by normal atmospheric levels of oxygen, grow at low oxygen levels

Pressure -

Any microbes inhabit deep lakes, oceans, every 10m, pressure increases by 1atm Deep water contain lower concentrations of bacteria Most deep sea bacteria are barophiles – grow best at high pressures Few extreme barophiles – will not grow at normal atmospheric temperature Pressure affects enzyme-substrate binding, membrane transport

Toleration of chemical inhibitors a) Respiratory inhibitors

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Gram negative bacteria are sensitive to sodium azide (NaN3) and inhibits cytochrome c oxidase. Enterococcus and other gram positive bacteria are resistant

b) Chaotropic agents - Pseudomonas resistant to phenol c) Antibiotics - Mycoplasma lack a cell wall therefore resistant to penicillin Experiment 1 – inoculation of nutrient agar plates and selective medium plates Experiment 2 – bacteria and their antibiotic sensitivity Experiment 3 – enumeration of bacteria using serial dilution and enrichment of enterobacteria plating on selective media EXPERIMENT 4 – Isolation of skin bacteria and determination of their antibiotic sensitivity

3) BIOCHEMISTRY Biochemical tests for specific enzymes or metabolic pathways

a) Sugar metabolism Fermantation: anaerobic breakdown of the sugar into smaller molecules with the release of waste products (sometimes ethanol/organic acids/gases). Different waste products characteristic of different species Oxidation: breakdown of sugar to carbon dioxide and water, an aerobic process

Tests for sugar fermentation -

Inoculate: liquid medium + sugar + pH indicator + durham tube Peptone media with phenol red indicator One uninoculated tube e. coli, one glucose germenter with gas production, glucose fermenter without gas production, non fermenter

Oxidation/fermentation test 1. Tube with soft agar + glucose + pH indicator 2. Incubate and oxidative growth only (orange with a bit of yellow on top) pseudomonas 3. Fermentation is yellow (e. coli)

Catalase test

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Cell suspension, add hydrogen peroxide Bubbles of oxygen: catalase + eg. Staphylococci No bubbles = catalase – eg. Streptococci

Urease test -

Urea to ammonia, pH rises and color change occurs Test strips contain dehydrated substrates + indicators in mictrotubes Just add the bacterial suspension and incubate Different strains give characteristic color patterns

4. Serology

Detection of bacterial antigens using specific antibodies 1. Inject antigen into an animal, harvest specific antibodies from the blood stream 2. Test in vitro for recognition of the cell surface antigen by agglutination (process that occurs if antigen is mixed with corresponding antibody called isoagglutinin) -

Serology tests well in 96-well microtiter plates Antibacterial antibody labeled with fluorescent dye

5. Bacteriophage typing Bacteriophages – bacterial viruses, most have a specific host range -

Lawn of bacteria on agar Drops of different phages Phages that can infect and lyse this bacterium produce a clear done on the plate

6. Pathogenicity Pathogens – specific symptoms, definite host range -

Symptoms can be enough to indicate identity sometimes

1. Diphtheria – formation of a pseudomembrane in the throat (Corynebacterium diphtheriae) 2. Leprosy – loss of fingers and toes (mycobacterium leprae) 3. Plague – buboes, skin darkening (yersinia pestis)

7. DNA Analysis a) -

Genome sequencing Numerous bacterial species now completely sequenced Sequences do not give the complete answer to how a bacterium functions Do produce a lot of information about the biochemistry, physiology, phylogenetic relationships and evolution Determining the sequence and gene content of a bacterial genome is still a major undertaking, complete genome sequences can be used as reference points when characterizing new isolates

b) Polymerase Chain Reaction (PCR) -

Simple in vitro technique to amplify short, specific DNA sequences Now used to regularly diagnose bacterial or viral infections PCR is incredibly sensitive, can be used to detect a single bacterial cell

With DNA based tests, we can identify the bacteria without culturing them first, making clinical diagnosis much faster

1. 2. 3. 4.

Swab and culture Plate-out Re-culture clones Carry out tests

c) Metagenomics – mass sequence analysis of genomic DNA within microbial populations without isolation/culturing

LECTURE 5A-5B Media - a culture medium produces nutrients for growth and multiplication of the microorganism Macronutrients – C, H, O ,N, P, S, K, Mg, Ca, Na, Fe Micronutrients (trace elements) – Co, Zn, Mo, Cu, Mn, Ni, Se, W

Defined media – assembled from a specific list of chemicals Undefines (complex) media – include undefined things like meat broth, yeast extract, blood products etc. can be easier to grow bacteria on this medium if the growth factors are not known The great plate count anomaly – hundreds times more cells seen compare to colonies found -

Most of our knowledge of bacteria comes from pure strains in culture, there are many important species of bacteria (some present in huge quantities in the environment) that cannot be grown in pure culture. Many have only been recently recognized, many still unknown

Growth – increase in biomass

- Typically accompanies by cell division a) Binary fission b) Budding

Binary fission of bacteria -

Cell division and separation Filamentous growth Whilst rods divide in one place, cocci can divide in 1,2 or 3 planes With binary fission, the population doubles at each division Under constant conditions, doubling occurs at regular intervals = exponential growth Incredibly large numbers of bacteria aren’t produced because the growth conditions don’t remain constant

Batch culture (closed system) – Simple way to grow cells in the lab -

Growth medium Inoculate with small quantity of culture Incubate by shaking

Lag phase – inoculum adapting to new conditions, synthesis of new cellular components (enzymes for nutrients in medium)

Exponential phase – maximum growth, population doubling at regular intervals, doubling time depends on organism, medium and conditions. G...