Microbiology Exam #1 Notes PDF

Protozoa - Single-celled eukaryotes - Find them anywhere with sufficient moisture - Can enter a dormant stage with no water - Classified through locomotion - Pseudopod = fake feet - Will grab a substrate and pull themselves forward - Glidin...

Protozoa - Single-celled eukaryotes - Find them anywhere with sufficient moisture - Can enter a dormant stage with no water - Classified through locomotion - Pseudopod = fake feet - Will grab a substrate and pull themselves forward - Gliding motility - Never seen freely swimming around; always needs a substrate - Cilia = hair-like projections - Flagella = larger and longer than cilia but less numerous - Apicomplexa = non-motile (no pseudopods, cilia or flagella) - but contain a lot of the medically relevant parasites and protozoa - (eg. malaria, toxoplasma = can cause miscarriage) - ALL DISEASE CAUSING Algae - Unicellular or multicellular - Photosynthetic - Classified on the basis of pigmentation - AGAR - Solidifying agent, like jello - Nutritionally, theres nothing - Culture bacteria in a media (liquid broth) - Add agar (petri dish) to form colonies - You can see thousands of cells visually - Diatom: a significant contribution of algae, use petroleum Parasites - Worms (microscopic → 23ft) - Liver or blood fluke, tape worms (in GI tract) Viruses - Non living - Infectious particle - By itself, it cannot replicate; needs a host (needs your cell) GOLDEN AGE OF MICROBIOLOGY Does Microbial Life Spontaneously Generate? Spontaneous generation = rises out of nowhere - Asexual reproduction = add branch to soil and it will sprout - Sexual reproduction - Non-living matter = WRONG Aristotle = spontaneous generation - Living things can arise from non-living matter - Just need a little bit of air (only microscopic) Francisco Redi - Used jars and a piece of meat - Decaying meat in open jar - Maggots on meat arose from eggs laid there by flies - No spontaneous generation - Flask covered with mesh (air can get in and out) - Flies are attracted - No maggots/larvae on the meat John Needham - Believe that microbes could spontaneously generate - Boiled mutton in corked flask - Had beef broth and boiled it = became cloudy = spontaneous generation - Incorrectly sterilized Spallanzani - Did needham’s experiment - Boiled mutton in airtight flask - No microbes arose - Concluded that there is no spontaneous generation * notes that spallanzani contradicts needham Louis Pasteur = swan-necked flasks - Disproved spontaneous generation - “Swan-necked flasks” never sealed; not contaminated (sterile) - When tilted, dust seeped into broth = cloudy - MICROBES PRESENT/FLOATING IN THE AIR What Causes Fermentation? Fermentation - Led to the process of pasteurization - Intentional use of microbes for making products - 2 colonies in grape: - Bacteria = vinegar - Yeast = wine - Saved the wine industry Pasteurization - Boiling until sterile (pathogens are killed) - Set up the scientific method - Observation - Question - Hypothesis - Experiment (including control groups)  - Observations What Causes Disease? Pasteur = Germ theory of disease (pathology) - Pathology = study of disease - Germs = pathogens Robert Koch = etiology - Studied etiology of disease (what causes a disease) - Studied microorganism from solid surface (agar) - Anthrax What causes disease = Robert Koch - Looked at etiology - How is it caused; the process - This organism causes this disease; these are the signs and symptoms Koch’s postulates (hypothesis/suggestion) - Eg. identified a patient with strep throat - Tracked signs and symptoms: fever, pus pockets in back of tonsil area, difficulty swallowing - Take a swab of the individual’s throat - Try to isolate the pathogen - Isolated pathogen - Put in a healthy individual - Signs and symptoms appear again - Then isolate bacteria again - Proves that the signs and symptoms are the same from a sick individual and a healthy individual that just received the pathogen 1. Got a disease; signs and symptoms 2. Try to isolate the pathogen 3. Then put pathogen in healthy individual 4. Got the same signs and symptoms 5. Isolate pathogen again Media or broth = liquid to grow bacteria Agar = solidify - Identify bacteria through looking at morphology (color, texture) Hans Gram - Classified bacteria based on their cell wall - Developed gram-stain - Find if gram positive or gram negative Gram-positive = purple Gram-negative = pink How can we prevent infection and disease? - HAI = healthcare-associated infections (nosocomial) - 6 practitioners 1. Ignaz Semmelweis = handwashing - Required medical students to wash their hands in chlorinated lime water - Purpural/Childbirth Fever - Severe infection of the uterus - Happens postpartum, after childbirth; fatal - Individuals did not wash their hands - After washing hands, the incidence decreased - Strep throat, Norovirus 2. Joseph Lister = antiseptic technique (sterilization) (Father of Antiseptics) - Listerine - Saw a high incidence of post operative infections - Used antiseptic (phenol / carbolic acid) - Not used anymore; causes skin irritation - Now betadine and iodine are used - Sprayed wounds, surgical incisions, and dressings 3. Florence Nightingale = nursing - Policies to prevent HAIs into nursing practice 4. John Snow = epidemiology - Epidemiology (where it starts, where it spreads) - Cholera (1854); sewage/pump 5. Edward Jenner = Father of Immunology - Developed vaccine against smallpox; symptoms = pustules (fluid-filled), scars, disfiguration - Via respiratory route or through skin-to-skin contact - Milk maids (milk cattle) a. Saw pustules/lesions on their skin but did not die (cowpox) b. Similar to small pox; same disease but caused by a different virus c. Scratched material/crust into someones skin d. The child got pustules and did not die e. Scratched chickenpox f. The child was protected and did not develop the disease (chickenpox) - Pasteur’s Vaccine: Cholera, rabies, anthrax, - chickenpox (?) 6. Paul Ehrlich = magic bullets - Seek out pathogen - Used arsenic to cure syphilis (worked) - Chemotherapy = bad connotation ( kills all cells ) How do we defend against disease? - Serology = Antibodies - Uses blood serum - Lymph, plasma (yellowish portion of the blood) - Gives immunity - Von behring and Kitasato: presence in the blood of chemicals and cells that flight infection - Immunology - The study of the body’s defense against specific pathogens - Combining T-cells and B-cells and how they collaborate and protect you - Chemotherapy - Alexander Fleming - First antibiotic discovered (penicillin) - 1923 - Zone of inhibition (mold prevented growth of the bacteria staphylococcus) - Was not used to treat - Sulfonamide - First antibiotic used to treat infection Monkeypox - Still around 1-3 recalls a year Cucumbers were infected with salmonella Parvovirus: affects humans and dogs (can be fatal) EEE (easter equine encephalitis) - Massachusetts - Spread through mosquitoes West nile virus - 1999 - New york epicenter - Causes meningitis - Through mosquito Listeria and nitrates - Crossing the placental barrier - Will cause a spontaneous miscarriage Trichinella - Parasitic worms found in undercooked meat Pneumovirus - Kids and elderly get this Pertussis - Whooping cough  - 2-3 month long - May choke or break their ribs Bird flu - Identified in cattle, birds, chickens, and humans - Not easily transmissible from person to person (H5N1 virus may change) CHAPTER 3 - Function and parts will definitely be in the exam Prokaryotes Eukaryotes - No nucleus or membrane-bound - Have nucleus and membrane-bound organelles organelles - only have 1 chromosome (all the - Have smooth and rough endoplasmic genes needed to survive) reticulum Bacteria & archaea Fungi - Unicellular = yeasts - Multicellular = molds Protozoa - Single-celled Algae - Unicellular or multicellular Animals and plants Parts Parts Nucleoid Smooth endoplasmic reticulum - Nuclear material or DNA is here - Lipid metabolism (tucked away in a part of the cells) Golgi apparatus - Free in the cytoplasm - Post office; sorts things out that are Ribosomes used from the reticulums - Translate mRNA Cell wall Flagella (motility) - Some have this, some do not. Cell wall Cell membrane - Composition may differ (gram-positive - Determines what goes in and what or gram-negative) goes out of the cell Glycocalyx - Outermost covering Cytoplasmic membrane - Eukaryotes also have this Plasmid - Extra chromosomal piece of DNA - Not part of the normal chromosome - Some bacteria have it - Can cause disease Glycocalyx - Not all bacteria have this - Gelatinous, sticky substance surrounding the outside of the cell - Capsule → slime layer → glycocalyx - Made of polypeptides, polysaccharides (sticky sugar) or both - Composed of sugar and proteins - Subdivide into: Capsule = Immune Evasion Slime Layer - firmly attached to the cell wall - loosely adhered, water soluble - Organized repeating units - Looks like a gel - Immune evasion - Involved in biofilms holding  - Antiphagocytotic (only - Biofilm formation solids) - Community of bacteria living - Phagocytic cell cannot together eat the bacteria - Usually antibiotic-resistant because it is slippery - Eg. plaque in teeth - Strep pneumoniae capsule Flagella - Movement - Not present on all bacteria - Long structures that extend beyond cell surface - Anchored to the cell wall - Composed of: - Filament: string-like structure - Hook: anchors flagella onto cell’s surface - Basal body: anchors the filament and hook to the cell wall String less organ (?) = does not have a flagella Monotrichous = one flagellum Amphitrichous = both poles have 1 flagella Peritrichous = most motile, multiple flagellas everywhere Lophotrichous = many flagella at both poles Circular = coccus / cocci Rod = bacillus/bacilli Spiral = spirilla - Lyme disease (borrelia) - Syphilis - Leptospirosis - Very motile bcus they have endoflagella Spiral-shaped bacteria / spirochetes Endoflagella / axial filament - Endo = within - Found under the cell wall; between the cell wall and the cell membrane - flagella wrap around the bacteria - Corkscrew shape - Corkscrew-like motility BACTERIA USUALLY HAVE A ROTATING MOTION Fimbriae - Like cilia (hair-like projections) - Sticky, bristle-like projection, shorter than flagella - For motility, forms biofilms and is used for adhering to surfaces  - Used by bacteria to adhere to one another (biofilms), to move - Often causes disease Pili - Modified fimbriae - Hollow tube - DNA conjugation/transfer - Exchange genetic material from 1 bacterium to another bacterium. - Can exchange plasmid and dna - like antibiotic resistance - Can only have 1 donor (pili) and 1 receiver Klebsiella pneumoniae - Treated with every antibiotic - Became antibiotic resistant Bacterial Cell Walls - Protect the bacteria from osmotic rupture/forces - Give structure and shape - Target of antibiotics - Gives bacterial cells characteristic shapes - Composed of peptidoglycan (protein - sugar) Gram - Used to classify bacteria - Peptidoglycan is very crucial - Gram-positive or gram-negative - Composed of alternating sugars NAM - NAG - N-acetylglucosamine (NAG) - N-acettylmuramic acid - Produced into fibers, strung together like an onion - Derivatives of glucose (base) Tetrapeptide (cross bridge / connecting chain) - No matter which one, it always involved peptides (peptidoglycan) - Involves at least 4 amino acids that attach - ALWAYS NAM-NAG - How it’s produced: - From all the NAM, there’s a tetrapeptide crossbridge which only hangs off the NAM residue. You can connect both NAM and NAG residue using the crossbridge like a connecting chain (Pg.3) Gram-Positive Bacterial Cell Wall (Purple) Gram-Negative Bacterial Cell Wall (Pink) Thick layer of peptidoglycan Thin layer of peptidoglycan Teichoic acid (polyalcohol) Found in Cytoplasmic Membrane - Bacterial cell replication - Outer Membrane - Helps in cell adhesion and cell - Composed of LPS division lipopolysaccharide (lipid, - Gives bacteria a negative protein, sugar) charge, which helps it adhere - (ENDOTOXIC SHOCK) to other cells - Causes fever, - Can form a biofilm vasodilation, inflammation, shock, - Presence of 60% mycolic acid (lipid) and blood clotting in acid-fast bacteria - If a large amount of - Waxy gram-negative bacteria - Helps survive (antibiotic is introduced inside a resistance, slow growth body desiccation) - Mimics stabilize - Causes disease patients before - Only found in pathogenic they die bacteria - Blood pressure tanks, organs Example: Mycobacterium tuberculosis / shut down, high leprae fever - Stain light purple - IV fluids may - Gram positive stabilize them - Have an outer layer composed of and metabolize mycolic acid on their own Lipid LPS = ENDOTOXIN Gram-negative infections - LPS is released when bacteria are destroyed (prescribed wrong antibiotic) - Macrophages can release LPS - Example: Gram-negative bacteria = e coli Phagocytosis 1. Endocytosis - Things go in; they get eaten by the cell - Macrophage can release LPS - Depends on the quantity of LPS - Too much bacteria is destroyed 2. Exocytosis  - things go out Bacteria Without Cell Walls - No gram = mycoplasma - Mycoplasma pneumoniae / walking pneumoniae - Think that they have a cold; seems mild at first - Can go away on its own - Found through X-ray; diffused throughout the lungs - Fever, persistent cough, sweating at night - Highly contagious - Lack a cell wall completely - Prone to osmotic rupture / protection Structure: Phospholipid bilayer - Composed of lipids (amphipathic) and associated proteins - Amphipathic (One part is changed and the other part is not) - Inner - Hydrophobic (not charged) - Polar fatty acid chains / lipids Outer - Hydrophilic - Phosphate groups (charged) - Will interact out of the cell (extracellular) or inside of the cell (intracellular) Integral proteins (amphipathic) Peripheral proteins - Make up the channels - Either the inside or the outside - A section that spans the extracellular - Only on one side environment and intracellular - Cannot stand the lipids inside environment - On both sides of the cell - Is a membrane itself - Thing can come in or leave - Active or passive transport - Protein carriers - Can stand the lipids  inside Function: - Maintain environment that is separate from the external environment - Giving the cell autonomy, can exist on its own - Selectively permeable: membrane determines what goes in and what goes out - Integral protein will determine this Active transport Passive processes of movement across a cytoplasmic membrane - Uses ATP, requires energy in order to move it - Requires no energy - Likes to move things against the - Goes from higher to lower membrane 1. Diffusion 1. Group translocation - Through the phospholipid - Bacteria wants glucose for bilayer respiration 2. Facilitate diffusion - Bring glucose molecule in - Helps it to get in and out of the - Gets phosphorylated cell (chemically altered) into the - Through a nonspecific channel cell and becomes glucose-6- protein phosphate - Through a permease specific - Trapped in the cell, energy for one chemical, binding of reserve substrate causes shape - Only in prokaryotes change in the channel protein - Does not occur in eukaryotic 3. Osmosis (movement of water) cells - The diffusion of water through a specific water channel protein or through the phospholipid bilayer - Aquaporin - Water has its own carrier - The specific protein which is the movement of water - Determines the movement of water / osmosis - Water cannot move freely against the membrane because water has a polar charge  - Big particles: through the integral protein or channel Effects of isotonic, hypertonic, and hypotonic solutions on cells “Water follows salt” Isotonic - Equal water solute distribution between the inside and the outside of the cell Hypertonic - High solute concentration on the outside of the environment of the cell Hypotonic - High solute concentration on the inside of the environment of the cell No cell wall Hypertonic Hypotonic - Mycoplasma - Water rushes out of - Will explode, no Protozoan, animal the cell structural support = cells - cremates / shrivels up death (plasmolysis) - Can still recover if the cell was not in that environment for a long time - Can also be used as a preservative (honey, salt) Cell wall Hypertonic Hypotonic - Bacteria (except - Cell wall remains - Will stay intact, can mycoplasmas), fungi, intact; however, the resist these stressors algae, and plants plasma membrane can peel off the cell wall / pull away Cytoplasm of bacteria Cytosol - Liquid portion of cytoplasm, mostly water - Nucleoid, ribosome floats around in the cytosol or bathed in this Inclusions - Storage vesicles / energy reserve - May include reserve deposits of chemicals - Polyhydroxybutyrate - Stored in inclusions - Biodegradable plastic; degrades over time Endospores - Bacillus - Bacillus anthracis which causes anthrax - Clostridium - Tdap vaccine - Tetanus = paralysis - Diphtheria = upper respiratory infection - Pertussis = whooping cough - Both can turn into spores during environmental stressors - Resistant to extreme conditions such as heat, radiation, chemicals - Increase the compound dipicolinic acid - Decreases moisture content - preserve DNA - Rest of the cell disintegrates - Can germinate again with the right environment —---------------------------------------------------------------------------------------------------------------------------- EUKARYOTIC CELL STRUCTURE Composed of various polysaccharides - Cellulose is found in plant cell walls - Fungal cell walls are composed of chitin - Algal cell walls are composed of variety of polysaccharides Cells walls and Cytoplasmic Membranes - All eukaryotic cells have cytoplasmic membrane - Are a fluid mosaic of phospholipid and protein - Contain steroid lipids to help maintain fluidity - Control movement into and out of cell - Cell membrane on eukaryotes functions in endo and exocytosis Eukaryotic flagella and cilia - Flagella undulate (whip-like motion) - Cilia are more numerous than flagella (adhesion, movement) Nonmembranous organelles - Ribosomes: protein synthesis - Prokaryotes = 70S - Always in the cytosol - Eukaryotes = 80S - In the cytosol or bound to the endoplasmic reticulum - Bacteria have a ribosome called 70S - S = sedimentation - Ribosome size - Reason why we can target protein synthesis for making antibacterial drugs - Can make antibacterial drugs which target certain bacterial ribosomes (targets prokaryotes) - 2nd largest class of antibiotics (protein synthesis/ribosome) - 1st class (cell wall) —---------------------------------------------------------------------------------------------------------------------------- ****he did not discuss these parts during lecture, i think you should just refer to the picture below******** - Cytoskeleton - Extensive network of fiber and tubules - Anchors organelles - Produce basic shape of the cell - Made up tubulin microtubules, actin microfilaments and intermediate filaments KNOW TABLE 3.5 = know organelles and its general function Other Nonmembranous Organelles Centrioles - Are composed of nine triplets of microtubules - Located in region of cytoplasm called centrosome Centrosomes: play a role in mitosis Membranous Organelles Nucleus - Largest organelle in cell, houses DNA - Semiliquid portion is called nucleoplasm - Contains chromatin - Nucleolus synthesizes rRNA - Surrounded by nuclear envelope - Contains nuclear pores Endoplasmic reticulum - Netlike arrangement of flattened, hollow tubules continuous with nuclear envelope - Functions as transport system - Two forms - Smooth endoplasmic reticulum (SER) - Rough endoplasmic reticulum (RER) Golgi body - Receives, processes, and packages large molecules for export from cell  - Packages molecules in secretory vesicles that fuse with cytoplasmic membrane - Composed of flattened hollow sacs surrounded by phospholipid bilayer Lysosomes, peroxisomes, vacuoles, and vesicles - Store and transfer chemicals within cells - May store nutrients in cell (vacuole) - Lysosomes contain catabolic enzymes - Peroxisomes contain enzymes that degrade poisonous wastes - Catalase —---------------------------------------------------------------------------------------------------------------------------- Membranous Organelles - Mitochondria: powerhouse of the cell - Produces most of the cell’s ATP - Have two membranes composed of phospholipid bilayer - Found in eukaryotes - Interior matrix contains 70S ribosomes - Has its own circular molecule of DNA Example: Chloroplast (eukaryotic) - Has DNA and 70s ribosome - Light-harvesting structures found in photosynthetic eukaryotes - Use light energy to produce ATP - Phospholipid bilayer membranes Endosymbiotic Theory - Eukaryotes formed from union of small aerobic prokaryotes with larger anaerobic prokaryotes - Smaller prokaryotes became internal parasites - Parasites lost ability to exist independently - Larger cell became dependent on parasites for metabolism - Fusion of two cells - Suggest that mitochondria used to be bacteria and created a symbiotic relationship - Similar scenario for origin of chloroplasts - Retained its characteristics - Not universally accepted Nucleolus - Within the nucleus - Contains rRNA - Ribosomal RNA = rna and protein - In eukaryotes - Responsible for printing out other ribosomes (?) - Can be used in taxonomy  - Will tell you the species of bacteria will help classify the species Active Transport Processes Found Only in Eukaryotes: Endocytosis and Exocytosis - Endocytosis - Things come in - Phagocytic cells - Digest from lysosome (feed it) - Lysosome - Digestive enzymes - Phagolysosome - Bacterial cells can now digest - Heart of the immune response - Macrophage will take the proteins of the bacteria it absorbed and put it on the cell’s surface - Will interact with a T-cell and B-cell and instruct that there is an infection in the body - T-cell and B-cells will kill the infection - Exocytosis - things leave - LPS (lipopolysaccharide) also leaves CHAPTER 5 Microbial Metabolism Alternatives to Glycolysis - Yield fewer molecules of ATP than does glycolysis - Yield different metabolites needed in anabolic pathways Glycolic metabolism - Glycolysis - Take glucose and break it down - Extracting energy, alternate ATP - Kreb’s cycle, electron transport chain  - Occurs in bacteria and eukaryotic cells - Two pathways - Entner-Doudoroff pathway - Pentose Phosphate Pathway - Ribulose and ribose from glucose 6 phosphate Pentose phosphate pathway - Associated with anabolism - Runs side by side with glycolysis - Ribulose 5-phosphate = nucleotides or ribose RNA - Bacteria can take any one of those compounds and extract it from the pathway to use it for something else - Used to make nucleotides, pentose, ribose (5 sugar) Anabolism - Builds up - Anabolic pathway - Take two nucleotides and make DNA; take 2 glucose molecules to make starch Catabolism - Breaks down - Take starch to make 2 glucose molecules Respiration - Produces 36 and 38 ATP molecules - 3 cycles: Cells require constant source of NAD+ - Glycolysis (NAD becomes NADH) - Kreb’s Cycle (NAD becomes NADH) - Electron Transport Chain (NADH becomes NAD) - Oxygen—final electron acceptor (if you dont have oxygen, the FEA is pyruvic acid) - Monitors level of NAD to ensure cells should have enough NAD  - Not enough NAD - Electron transport chain and Krebs cycle shuts down - Glycolysis still functions = make pyruvic acid (end product of glycolysis) Fermentation - Unique to bacteria and yeast, but not all bacteria undergo fermentation - Not producing 36 ATPS but only 2 ATPS - Temporary process: When O2 becomes available again, fermentation stops - Only functions with glycolysis - Sometimes cells cannot completely oxidize glucose by cellular respiration (glycolysis only) - Fermentation pathways provide cells with alternative source of NAD+ - Cells need a constant supply of NAD - Pyruvic Acid: - It is the end product of glycolysis - It is an electron acceptor (takes the place of oxygen in fermentation) - Partial oxidation of sugar to release energy using an organic molecule from within the cell as the final electron acceptor - If O2 is limited, pyruvic acid will take the place of O2 to stay alive 1. Alcohol Fermentation (ethanol) - Lose CO2 → acetaldehyde → ethanol - May also create ethanol which is alcohol or wine 2. Lactic Acid Fermentation - Pyruvate → lactic acid  - When O2 is deprived = soreness that occurs in muscles (working out) CHAPTER 7 Microbial Genetics Gene Recombination and Transfer Recombinant - Cells with DNA molecules that contain new nucleotide sequences - Pick up DNA from the outside and assimilate DNA into mine Transferring: Gene Transfer Among Prokaryotes Vertical gene transfer - Passing of genes to the next - (mother and father: first generation) → fertilization of egg and sperm → (children; second generation) Horizontal gene transfer - Within the same generation - Donor cell contributes part of genome to recipient cell - Taking my DNA and assimilating it into your DNA - 3 types 1. Transformation - Recipient cell takes up DNA from the environment  - When bacteria takes DNA from the outside and puts it inside its gene - Involves the uptake of extracellular DNA - External DNA/naked DNA (outside of the cell) - Cell floats around in broth and then close to you is DNA - You can take that DNA into your cell - Whatever genes that DNA you take up codes for, you can make proteins - If you bind to the DNA, you can express the genes Additional notes from JJ (suriano said to skip this but i put it here just in case) 2. Transduction - Transfer of DNA from one cell to another via replicating virus - Requires a virus that targets or is specific to a bacteria - Bacteriophage or phage - Viruses specific to bacteria + cant infect animal or human cells only bacteria - Bacteria - has 1 chromosome and is circular - Virus must be able to infect both donor and recipient - Can transfer antibiotic resistance and toxin genes - Originally, the bacteria did not have those abilities but can now express it after infection  - Phage injects dna - New phages grow into the cell - Phages will build itself inside the cell; however it will inadvertently take DNA from the bacteria on accident 1. Make the shell 2. Make more of the genome 3. Assemble - Phages cannot distinguish DNA; just needs to put itself together - Leave host then infect another cell - Specialized transduction - Only certain donor DNA sequences are transferred - Toxins associated with scarlet fever Examples E.coli O157:H7: - Can cause enterohemorrhagic e coli - Colon bleeding diphtheria (corynebacterium diphtheria) - Did not have the genes to make toxins (originally) - Bacteriophage had toxins and transferred it over to the bacteria - Bacteria now has the ability to make the toxin strep throat - Originally could not produce a toxin which caused erythema toxin (rashes) - Probably the most elaborate way to transfer DNA 3. Bacterial conjugation - Bacterial mating: need 2 bacteria - Genetic transfer requires physical contact between the donor and recipient cell - Donor cell remains alive - Mediated by conjugation (sex) pili Involves the pilus/pili - Encoded on the plasmid of the bacteria - Transfers the plasmid - Can only happen between a cell that has the gene to produce the pilus and a cell that does not - Not all cells can make a pilus - Now both cells have the same plasmid - Can contain antibiotic resistance, toxins, phenotypes F+ cell = has pilus F- cell = does not have pilus F = means fertility factor CHAPTER 4 Microscopy, Staining, and Classification Microscopy - The use of light or electrons to magnify objects - Science of microscopy begun by Antoni van Leeuwenhoek General principles of microscope 1. Wavelength of radiation 2. Magnification - Increase in size MAGNIFICATION Magnification (multiplication of 2 numbers) Ocular x Objective = Magnification Ocular = 10x (constant) Objective = - Scanning = 5x - Low power = 10x - High power = 40x - Oil = 100x Example: give me the total magnification of something being viewed under high power 10x * 40x = 400 3. Resolution - Ability to distinguish objects close together - Can distinguish that there are 2 separate cells 4. Contrast - How do you stain things with different dyes - Bacteria cells will stain, glass does not - Bacterial cells are colorless - Differences in intensity between two objects or between an object and its background - Important in determining resolution - Staining increased contrasts Light Microscopy Simple Microscope - Contain a single magnifying glass (one lens) - Similar to a magnifying glass - Leeuwenhoek use simple microscope to observe microorganisms - Made microscopes for each specimen and recorded it - Originally did not have light; needed to go outside to see Compound Microscope - Series of lenses for magnification (10x,40x,100x) - Light passes though specimen into objective lens - Oil immersion lens increases resolution - Becomes part of the lens system - Objective gets closer to the sample - Light waves will scatter and will not go into the objective = cant see image - Oil will increase surface area and light rays will concentrate into the objective so the sample can be seen - Have one or two ocular lenses - Total magnification = magnification of objective lens x magnification of ocular lens - Most have condenser lens Florescent Microscope (UV light) - Direct UV light source at specimen - Specimen radiates energy back as a longer, visible wavelength  - UV light increases resolution and contrast - Some cells are naturally fluorescent; others must be stained (fluorescein isothiocyanate, auramine o) - Used in immunofluorescence to identify pathogens and to make visible a variety of proteins Example: Tuberculosis - This bacteria grows very slowly due to mycolic acid - Mycobacteria = multiplies every 48-72 hrs - Take a sputum sample and add specimen to the slide - React the slide with antibodies specific to the bacteria Antibody - Y shaped - Add dye to the stem; have something that fluoresces at the end of the stem - GF/PF = fluoresces green - Specific to mycobacteria - Antibody will bind to bacteria then it fluoresces under microscope Immunofluorescence - Immuno = tool is antibody Electron Microscope - Light microscopes cannot resolve structures closer than 200 nm - Electron microscopes have greater resolving power and magnification - Magnifies objects 10,000x to 100,000x - Detailed views of bacteria, viruses, internal cellular structures like organelles or membranes, molecules, and large atoms - Use gold particles for staining - Two types - Transmission electron microscopes (sectioned) (TEM) - Have a sample and you can make sections of it (like a MRI or CAT scan) - Detailed images of organs (inside cell) - - Scanning electron microscopes (not sectioned) (SEM) - Only looks at the surface Staining - Helps to increase resolution  - Most microorganisms are difficult to view by bright-field microscopy - Coloring specimen with stain increases contrast and resolution - Specimens must be prepared for staining Additional notes from JJ Principles of Staining - Dyes used as stains are usually salts - Chromophore is the colored portion of the dye - Acidic dyes (neg charged) stain alkaline structures - Basic dyes (pos charged) stain acidic structures - More common because most cells are negatively charged Simple stains - composed of single dye Differential stains - use more than one dye 1. Gram stain 2. Acid-fast stain (Niehl-Neelsen) 3. Endospore stain (Schaeffer-Fullton) 4. Histological stains - Two common stains used for histological specimens  1. Gomori methenamine silver (GMS) stain a. Detects fungi in tissue b. See if person has fungal infection of the lungs 2. Hematoxylin and eosin (HE) stain a. Histology (overall picture) b. See biopsy of a tissue; irregularities of a tissue c. Show structure and type of cell, like to study the cell of a tumor Example: thyroid biopsy - Fine needle aspirin (?) Special stains - reveal specific structures 1. Negative and Anthony’s stain Capsule stain Staining for Electron Microscopy - Chemicals containing heavy metals are used for transmission electron microscopy - Stains may bind molecules in specimens or the background Classification and Identification of Microorganism - Taxonomy consists of classification and identification - Similarities between organisms - Physical (phenotypic) - Can often be used to identify microorganism - Protozoa, fungi, algae, and parasitic worms can often be identified based only on their morphology - Some bacterial colonies have distinct appearance used for identification - Biochemical - Fermentation vs non-fermentation - (can produce acid during fermentation, or gas (H2S) or not) - - Phenol Red Lactose - pH indicator - Has a sugar lactose within  - Fermentation of sugars - Bacteria will produce acid and/or gas (CO2) - Neutral = red - Acidic = yellow - Durham tube - Inverted tube within tube - Determine if bacteria will produce gas - You will see bubbles - The third tube is not inoculated or has no bacteria - Or it is a bacterium that does not ferment lactose Additional notes (JJ) Gas - never produce on its own, make acid byproducts firist Sim - it contains ferrous sulfate - Good biochemical test of determining what type of bacteria you have - Serological (antibodies) - Serum/Serological tests - 1. Albumin = Most abundant protein - 2. Immunoglobulin/Antibodies (IGg) (2nd most abundant protein) - Specific against certain pathogens - Serology—study of serum (liquid portion of blood after clotting factors removed) - Many microorganisms are antigenic - Trigger immune response that produces antibodies - Serum is an important source of antibodies - Antibodies can be isolated and bind to the antigens that triggered their production - - Basis of many tests (covid, flu, drug, pregnancy tests) - Interacting with an antibody to give something visible - Binds to whatever substance you are looking for - Nucleic acid analysis - DNA and RNA (rRNA) - Like fingerprints  - Prokaryotic taxonomy now includes the G + C content of an organism's DNA - Bacterial DNA will have a higher G + C (guanine and cytosine) content compared to all other organisms - To identify if bacterial or not - To classify if a bacteria is high G+C or low G+C - Ribosomal RNA = rna and protein - In eukaryotes - Responsible for printing out other ribosomes (?) - Can be used in taxonomy - Nucleic acid sequence can be used to classify and identify microbes - Will tell you the species of bacteria will help classify the species Carolus Linnaeus and Taxonomic Categories - Current taxonomy system began - His system classified organisms based on characteristics in common - Group organisms that can successfully interbreed into categories called species 1. Proposed only 2 kingdoms a. Animals and plants 2. Present classification is five kingdoms a. Animalia, plantae, fungi, protista, and Prokaryotae i. Protista = single celled eukaryotes ii. Prokaryotae = bacteria and archaea  - Protozoas used to be grouped with animals - Fungi used to be grouped with plants - Greater emphasis on comparisons of organisms’ genetic material led toa addition of domains. - Monera = bacteria Domains: Carl Woese - Compared nucleotide sequences of rRNA subunits - Proposal of three domains as determined by ribosomal nucleotide sequences - Eukarya (fungi, plants, algae), bacteria, archaea - Cells in the three domains also differ with respect to many other characteristics - Archaea do not have peptidoglycan - 1st mechanism to categorize things  - Know genus and species - The first name is always genus, and the second name is species (binomial nomenclature) - Genus can be abbreviated - Always italicized - Within a genus, you can have multiple species - S. aueres - S. epidermis Dichotomous keys - Series of paired statements where only one of two "either/or" choices applies to any particular organism - Key directs user to another pair of statements or provides name of organism  - Always starts with a gram stain Additional notes (JJ)