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Introduction to Microbiology and BacteriologyMicrobes vs “Macrobes” Numbers of organisms It is impossible to get real numbers, but nearly all organisms on our planet are microbes. Many live within us Biomass Some recent estimates suggest that total bacterial biomass is greater than the total biomass...

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Introduction to Microbiology and Bacteriology Microbes vs “Macrobes” 1. Numbers of organisms It is impossible to get real numbers, but nearly all organisms on our planet are microbes. Many live within us 2. Biomass Some recent estimates suggest that total bacterial biomass is greater than the total biomass of macroorganisms (nearly all plants). One estimate - 5 x 1030 bacteria on our planet!! 3. Importance to the biosphere In terms of “primary productivity” – photosynthetic carbon dioxide fixation – about half is done by macrobes (plants) and half by microbes (photosynthetic bacteria and microalgae, mainly in the oceans). Microbes also play an essential role in processes such as nitrogen fixation and the recycling of carbon, sulphur, phosphorus, etc. 4. Biodiversity (“species richness”) As stated earlier, macroorganisms form only a few small twigs on the tree of life. However, despite being very closely related, they can be very diverse in appearance. Whereas, microbes can be similar in appearance while being very distantly related. Microorganisms are much more diverse because they’re much older so have more time to diverse. Differences between prokaryotes and eukaryotes 1. Size Eukaryotes are much larger 2. Packaging of DNA genome  In eukaryotes... DNA (except for organellar DNA) is contained with a double membrane structure - the nucleus Nuclear DNA is divided into a number of linear molecules DNA packaged into elaborate proteinDNA structures termed chromosomes, which are attached to microtubules.  In prokaryotes..... (“pro-karyote” means before nucleus) No nuclear membrane No chromosomesNo mitosis or meiosis No microtubules DNA is typically circularExists as one or more copies per cell as an aggregated mass of DNA within the cytoplasm - the nucleoid Plasmids (smaller circular DNAs) often present. (Plasmids rare in eukaryotes)  (Gemmata obscuriglobis - one of the Planctomycetes, aquatic bacteria with complex life-cycles. The nucleoid is surrounded by a nuclear envelope! So beware of simple generalisations in biology, there are nearly always exceptions) 3. Genome complexity  Prokaryote

Small, circular genome (2 – 10 Mbp). 1000–5000 protein coding genes  Eukaryote Much larger (10 – 10,000 Mbp). 6,000–40,000 protein coding genes 4. Organelles  Eukaryotes Possess organelles:Mitochondria, Plastids (plants, algae and some other protists) also...nucleus, endoplasmic reticulum, Golgi apparatus, centrioles, etc..  Prokaryotes No organelles But do sometimes have internal membrane systems with specialised functions Mitochondrion  surrounded by a double membrane. Inner membrane is site of aerobic respiration and ATP synthesis.  Mitochondria are (almost) universal in eukaryotes.  Prokaryotes do not have mitochondria. If respiration occurs, it is usually in the plasma membrane.  Chloroplasts  eukaryotes, absent in prokaryotes.  Site of photosynthesis - light absorption, oxygen evolution, carbon dioxide fixation  Cyanobacterium  Bacteria do sometimes have specialised internal membrane systems.  Thylakoid membranes - Site of photosynthesis and respiration. Thylakoids are in contact with the cytoplasm, not bounded by envelope membranes  A bacterial origin for the mitochondrion and the chloroplast organelles  Mitochondria and chloroplasts are the descendents of free- living bacteria that formed an endosymbiosis with a proto- eukaryote ~2 bya and ~1 bya, respectively.  Chloroplasts and mitochondria have retained their own genetic system, with many prokaryotic characteristics 5. Ribosomes Ribosomes contain proteins and RNA and are the site of protein synthesis  Prokaryotes: 70S ribosomes (S = Svedberg unit, a measure of size and density)  Eukaryotes: 80S ribosomes in the cytoplasm, 70S ribosomes in the organelles (this is evidence of prokaryotic origin)  Different antibiotic sensitivity:e.g. chloramphenicol (antibiotic) inhibits only 70S ribosomes, cycloheximide inhibits only 80S ribosomes 6. Flagella Not all cells have flagella, however....  Eukaryotes: Bundle of microtubules surrounded by a membrane sheath. They have a 9+2 pattern (universal) in the microtubules within the flagellum. It is a whip like structure helping to propel the cell.  Prokaryotes: a single filament made from flagellin. They have a solid region of flagellum attached to a rotary motor that is driven by ATP hydrolysis. 

7. Cell wall Cell walls (where present) is made of a rigid polymer layer outside the cell membrane  Advantages: Rigidity, defined shape, protection, helps with specificity and antibiotics  Restrictions: no amoeboid movement, no phagocytosis, etc.  Eukaryotes: Animals do not have a cell wall. Cellulose cell wall in plants and most algae Chitin cell wall in fungi  Prokaryotes: Peptidoglycan cell wall in bacteria Archea have various cell walls Bacteria can be split into two, gram positive bacteria have a thick cell wall, whilst gram-negative have a thinner call wall with a membrane outside.

Prokaryotes are all around us, they are the dominant organisms on the planet, and inhabit every environment...  Canary Spring, Yellowstone. 101–102°C (extreme tempreture)  Bacterial biofilm on basalt from 1500m depth (extreme pressure), Columbia River Basin, USA  Extrememly acidic, iron-rich mine drainage- home of specialized archaea  The human digestive tract. ...however, it is only in the last few hundred years that we have become aware of bacteria.  e.g. The Black Death or Bubonic Plague first struck Europe in 1347 and over the next century wiped out ~50% of the population. [now known to be caused by Yersinia pestis].  Anthrax. A highly infectious animal disease that causes skin pustules, fever, nausea, etc. Due to Bacillus anthracis. The endospores are very resistant and can survive in soil and animal products for hundreds of years. Some landmarks in microbiology: 1. The Roman philosopher Lucretius (circa 98–55 B.C.) first suggested that disease was cause by invisible living creatures.  2. In 1670’s, Antony van Leeuvenhoek was the first to observe and describe microbes (“animalcules”) using simple microscopes (magnification ~50–300x).  3. 1861 Louis Pasteur convincingly disproves the notion that bacteria arise by “spontaneous generation” by carrying out an experiment. 4. 1880’s Robert Koch – the germ theory of disease. A bacterial culture isolated from the blood of a diseased animal can induce the disease in a healthy animal.  Koch’s postulates - rules for confirming a causal link between a microorganism and a disease. a. A specific organism must invariably be associated with all cases of the disease. b. The organism must be isolated in pure culture and then sub-cultured over repeated generations.

c. When inoculated into a healthy susceptible animal, the organism must again cause the disease. d. The organism must again be isolated in pure culture from lesions of the disease. Isolating, growing, and characterising bacteria  Originally very difficult to isolate bacterial strains, because bacteria were grown only in liquid culture.  Joseph Lister (1878) – Dilution method Used to try and isolate bacteria, if you carry on diluting you should end up with a with one that is pure. This is very difficult to achieve.  Robert Koch developed the use of solid medium for spatial separation of individual cells. Solid medium now usually prepared from culture medium + gelling agent. Originally, gelatin, but Fannie Hesse (wife of one of Koch’s assistants) suggested use of agar.  Richard Petri, developed the Petri dish  Isolation of clones (strains) using solid medium:  Mixed culture  Spread on Petri dish  Inoculate (pick single colony and incubate)  Pure culture  Each colony is a clone or strain. Strains can be maintained by e.g.: 1. regular sub-culture to fresh medium 2. frozen storage 3. freeze-drying  There are many culture collections around the world, which supply known strains:  ATCC - American type culture collection  PCC - Pasteur culture collection  NCTC - National collection of type cultures  NCIB - National Collection of Industrial Bacteria Media  A culture medium provides 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  Different microbes get their carbon in different ways:

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The media must also supply other substances essential for bacterial growth. e.g.

And ‘trace elements’ in μM or nM amounts (e.g. Cu, Mn, Co, Zn...) Some bacteria require specific growth factors (vitamins, amino acids..)  This makes it difficult to grow bacteria as we don’t know what they require.  Defined media – assembled from a specific list of chemicals  Undefined (complex) media – include undefined things like meat broth, yeast extract, blood products...  Sometimes much easier to grow bacteria on complex media, especially when you don’t know all the growth factors required. 

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

Classification and nomenclature of bacteria  Classification - process of arranging organisms into named groups.

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Nomenclature - process of naming genera, species, etc. A species is a “group of similar strains”  note that the concept of “species” as applied to animals, etc. doesn’t apply to bacteria in the same way. Latin name is underlined or in italics.The genus name can be abbreviated e.g. E. coli. Note also higher levels of classification: (families, orders, classes, phyla, domains) informal lower level of classification - strains

Taxonomy  How do you tell one bacterium from another? How do you decide what order, family, genus etc. it belongs in?  The classical approach (biology): Identify lots of features - size, shape, nutrient requirements, metabolism, cell wall and membrane components, etc. etc.  Molecular taxonomy (molecular biology): Evolutionary relationships established by DNA/RNA/Protein sequences (e.g. gene for 16S ribosomal RNA).  “Species” definition on the basis of overall DNA sequence similarity (>70% DNA sequence similarity = same species). Characterising new isolates 1. Morphology A. colonial morphology Appearance of colony on specific agar under specific conditions  Shape  Colour  Texture  Size B. Cell morphology Appearance of cells when viewed under microscope 4 typical classes of cell shape:  Cocci (spherical)  Rods  Vibrio (comma-shaped)  Spirillum (spiral)  Cell arrangement depends on pattern of cell division 

Observing bacteria under the microscope Although living bacteria can be observed under the microscope, samples are typically fixed and stained.  Fixation - kills and preserves sample on microscope slide. 1) Heat fixation: gentle flame heating of air-dried sample (preserves overall morphology but not delicate structures within cell) 2) Chemical fixation: Chemicals (e.g. ethanol, formaldehyde) that penetrate cells and react with cellular components –> inactive and insoluble. Preserves fine

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cell structure. Simple staining using dyes Many types of dyes, but all...  have chromophore groups (conjugated double bonds) that gives the dye its colour.  bind to cell components (either ionic, covalent or hydrophobic bonds).  Two types of ionisable dyes  Basic dyes(e.g. methylene blue, crystal violet, malachite green)  Acid dyes(e.g. rose bengal, acid fuchsin)  pH may affect effectiveness of staining  Simple stains first used by Koch 1877 Dried bacterial smears and stained with methyl violet. He could see shape, size, number and how they divide/separate. But he could not differentiate cells that are the same shape. Differential staining procedures allow the separation of bacteria into different groups based on their staining properties: Paul Ehrlich carried out studies on dyes (1877-81) and developed acid-fast staining (staining is due to high lipid content of cell walls) 1. Smear (e.g. sputum)+ HOT CARBOL FUCHSIN 2. Wash with water 3. 10 min 20% H SO 2 4 4. Wash with water 5. Counterstain with methylene blue  Some bacteria will retain the dye and are acid-fast, they stay dyed even in acid washed conditions. E.g. mycobacteria. This can be used as an initial diagnosis of TB The Gram Stain (Christian Gram 1884) distinguishes two classes of bacteria on the basis of their cell wall structure 1. Heat-fixed smear + CRYSTAL VIOLET (Basic dye) 2. Iodine/KI added [CVI] formed in cells 3. Wash with solvent (e.g. 95% ethanol) 4. Stop with water 5. Counter-stain (e.g. carbol fuchsin)  Gram-positive = dark purple, cocci  Gram-negative = pink or red, rods

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Cell cytology (Light and electron microscopy)

2. Physiology  Nutritional requirements e.g. Source of C and N (gaseous or ‘fixed’)[bacteria that can fix N are termed diazotrophs]. 2  We can select for diaztrophs by eliminating the nitrogen source from the medium, so only those bacteria that can use atmospheric nitrogen can grow. Growth factor requirements (vitamins, amino acids, etc.). These are auxotrophs.  Light  Important for photosynthetic microbes (some obligate can only grow by photosynthesis, some facultative phototrophs, these can choose to grow photosynthetically or hetreotrophically).  Most bacteria are sensitive to UV light, this can be used to reduce bacteria load – however, some species are particularly resistant (e.g. Deinococcus species and novel species recently found only in the stratosphere).  Temperature  Microbes can be classified as psychrophiles, mesophiles or thermophiles depending on their optimum growth temperature. 

Salinity (salt concentration)  Freshwater < coastal areas < oceans < salt lakes

 All marine organisms are moderate halophiles (about 3.5% salt), extreme halophiles found in salt lakes (e.g. Solar Lake, Sinai). These can grow under high pressure and have developed features to aid survival under different osmotic gradients 

pH Range  acidophiles (low pH)  alkaliphiles (high pH)

 Oxygen Essential for respiration, but also a very toxic, highly reactive gas. Bacteria can be classified according to their requirement for/tolerance of oxygen.  Obligate aerobes: need oxygen for growth  Obligate anaerobes: oxygen not needed, it is toxic  Facultative aerobes: can grow without oxygen but will use it when available  Aerotolerant anaerobes: can grow with oxygen present, but do not need it or use it  Microaerophiles: are damaged by normal atmospheric levels of oxygen, but grow at low levels of oxygen 

Pressure  Many microbes inhabit deep lakes, oceans - every 10 m depth increases pressure by 1 atmosphere. 90% of ocean water is >1000 m down (therefore > 100 atmospheres). 3 5  Deep waters contain lower concentrations of bacteria (but still 10 - 10 per ml) Most deep-sea bacteria are barophiles - they grow best at high pressures. A few are extreme barophiles - they will not grow at normal atmospheric pressure.  Why? Pressure affects enzyme-substrate binding, membrane transport, etc.  Most barophiles are also psychrophiles and are adapted to low tempretures (deep ocean water at a constant 2-3 °C)

 Toleration of chemical inhibitors for example: 1. Respiratory inhibitors Enterococcus (e.g. E. faecalis) and other Gram +ve bacteria resistant to sodium azide (NaN ), but Gram –ve bacteria are sensitive, it is toxic to them. Can be used to test 7 3 classes of bacteria. 2. Chaotropicagents Pseudomonas resistant to phenol. Chaotrphic agents (e.g.phenol) cause protein denaturation. 3. AntibioticsMycoplasma (e.g. M. pneumoniae) lack a cell wall therefore resistant to penicillin. Antibiotics can be used as a diagnostic test for different species.  Can characterise bacterial isolates using MICs for range of antibiotics. This is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism.  Alternatively, antibtiotic impregnated discs  Or graduated strips

3. Biochemistry i.e. Biochemical tests: tests for specific enzymes or metabolic pathways  Sugar metabolism: Fermentation: anaerobic breakdown of the sugar into smaller molecules - release of waste products (sometimes ethanol, sometimes organic acids, sometimes gases). Different waste products characteristic of different species. Oxidation: breakdown of sugar to CO and water – aerobic, requires oxygen.

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Test for products of sugar fermentation  Inoculate: liquid medium + sugar+ pH indicator + Durham tube Oxidation/fermentation test

 Catalase test Hydrogen peroxide + cells = bubbles of oxygen  Urease test Urea  NH (pH rises) 3 4. Serology Detection of bacterial antigens (typically a cell-surface protein or carbohydrate) using specific antibodies 1) Inject antigen into an animal, harvest specific antibodies from blood serum. 2) Test in vitro for recognition of the cell- surface antigens e.g. by agglutination 5. Bacteriophage typing Bacteriophage – bacterial viruses. Most have a specific host range

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6. Pathogenicity  Pathogens:specific symptoms and a definite host range  Sometimes symptoms alone are enough to indicate identity e.g:  Diphtheria - formation of a pseudomembrane in the throat Corynebacterium diphtheriae   Leprosy - loss of fingers and toes. Mycobacterium leprae  Plague - buboes, skin darkening. Yersinia pestis 7. DNA analysis Genome sequences  Numerous bacteria now completely sequenced  Sequences do not give the complete answer on how a bacterium functions, but they do provide a huge amount of information on biochemistry, physiology, phylogenetic relationships and evolution. Note: Determining the sequence of a bacterial genome is still a major undertaking not what you would do initially to characterise a new isolate. However, complete genome sequences can be used as reference points when characterising new isolates.

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DNA hybridisation  A physical technique that measures the overall sequence similarity between an unknown strain and a reference strain.  Used in the definition of a bacterial species: >70% hybridisation = same species.

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Polymerase chain reaction (PCR). Simple in vitro technique to amplify short, specific DNA sequences. Now used regularly to diagnose bacterial/viral infections.

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Labelled nucleic acid probes. Short DNA sequences that hybridise (=bind) to specific chromosomal sequences. Used clinically to detect specific species of bacteria.  PCR and labelled nucleic acid probes are incredibly sensitive - could be used to characterise single bacterial cells.  With DNA-based tests, we can identify bacteria without culturing them first - makes clinical diagnosis much faster  Furthermore, we finally have a way to understand the true diversity of the bacterial world. Less than 1% of bacteria in the environment can be cultured.  Metagenomics - mass sequence analysis of microbial populations without isolation/culturing.

Growth an increase in biomass, typically accompanied by cell division  binary fission, more typical in the bacterial world.  Cell division is also accompanied by separation, then filamentous growth (i.e. organisms grow in a thread like fashion and so they can exchange material between cells)  Whilst roads can divide in one plane, cocci can divide in one two or three plane.  With binary fission, the population doubles at each division. Under constant conditions, doubling occurs at regular intervals, and this gives exponential growth.  budding, mother cell buds off daughter cell 

1 bacterium has a mass of roughly 10-13g, so 10 million million bacteria = 1g. Growth conditions do not remain constant so exponential growth is limited and this number can never be reached.

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A simple was to grow cells in a lab is by batch culture (...