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MICROBIOLOGY ○ Vibrios ○ Spirochete Professor/Instructor: Danny O. Alfonso, MSc Types of Microorganisms Unit I....

MICROBIOLOGY ○ Vibrios ○ Spirochete Professor/Instructor: Danny O. Alfonso, MSc Types of Microorganisms Unit I. The Microbial World and You 1. Bacteria Prokaryotic Benefits of microbes Peptidoglycan cell wall Decompose organic waste Reproduction by binary fission Perform photosynthesis Gains energy from the use of: Produce ethanol, acetone, alcohol, cheese, ○ Organic chemicals bread ○ Inorganic chemicals Produce insulin and many other drugs ○ Photosynthesis Naming and Classifying of Organisms Nutritional types in bacterial metabolism Carolus Linnaeus Phototrophs (sunlight) Established the system of scientific Lithotrophs (inorganic compounds) nomenclature in 1739 Organotrophs (organic compounds) Binomial nomenclature 2. Archaea Each species has two names: genus + Prokaryotic specific epithet No peptidoglycan Lives in extreme environments Examples of bacteria: Includes: Staphylococcus aureus - versatile pathogen ○ Methanogens Escherichia coli - causes extraintestinal ○ Extreme halophiles illness ○ Extreme thermophiles Streptococcus pneumoniae - most common cause of community-acquired pneumonia 3. Fungi Eukaryotic Classification of bacteria based on shape: Chitin cell walls Cocci Use organic chemicals for energy ○ Coccus Yeasts are unicellular ○ Diplococci Molds and mushrooms are multicellular ○ Tetrad consisting of masses of mycelia, which are ○ Sarcina composed of filaments called hyphae ○ Streptococci ○ Staphylococci 4. Protozoa Bacilli Eukaryotes ○ Bacillus Absorb or ingest organic chemicals ○ Cocobacillus May be motile via: ○ Diplobacilli ○ Pseudopods ○ Streptobacilli ○ Cilia ○ Palisades ○ flagella Others ○ Spirilla 5. Algae ○ Corynebacterium Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025 6. Viruses Transition Period: debate over spontaneous Acellular generation Have either DNA or RNA in their core The doctrine of spontaneous generation The core is surrounded by a protein coat (Aristotle): the hypothesis that living (capsid) organisms arise from nonliving matter, a The coat may be enclosed in a lipid “vital force” forms life envelope Biogenesis: hypothesis that living organisms Viruses only replicate within a living host arise from pre-existing life cell 1668: Francesco Redi 7. Multicellular animal parasites\ Beginnings of experimental science Eukaryotes Experimented with flies and two jars with Multicellular animals one open and one sealed Helminths: Parasitic flatworms and round worms 1745: John Needham Has microscopic stages in life cycles Objected Francesco Redi Put boiled nutrient broth into covered flasks; 8. Prions there was microbial growth Three Domain Classification 1765: Lazzaro Spallanzani Bacteria Boiled nutrient solutions in flasks ○ cyanobacteria Nutrient broth was placed in a flask, heated, Archaea and then sealed; there was no microbial ○ Salt-loving microbes growth ○ Heat-loving microbes Eukarya 1865: Louis Pasteur ○ Protista Demonstrated that microorganisms are ○ Fungi present in the air ○ Plants Nutrient broth was placed in a flask, heated, ○ Animals not sealed; there was microbial growth Swan-neck flask: S-shaped flask which kept Microbiology History microbes out but let air in Confirmed biogenesis Ancestors of bacteria were the first life on Earth Golden Age of Microbiology (1857-1914) 1665: Cell theory by Robert Hooke Microbiology was established as a science Cells are the basic unit of life Louis Pasteur All living things are made of cells ○ Disproved spontaneous generation Cells come from other cells ○ Wine fermentation ○ Pasteurization: a process that kills 1673: first microbes observed by Anton van harmful bacteria Leeuwenhoek Red blood cells Pre-Pasteur: Protozoa Ignaz Semmelweis (1840s) Sperm cells ○ Hand disinfection and puerperal fever Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  Joseph Lister (1860s) Microbes and Human Disease ○ Antiseptic surgery (phenol) Normal microbiota in and on the human body Robert Koch Pathogens overcome the host’s resistance Work on anthrax proves the germ theory of which leads to infectious disease disease Antimicrobial resistance ○ the theory that certain diseases are Bioterrorism caused by the invasion of the body Re-emerging infectious diseases (EID) by microorganisms ○ WNE Developed the pure culture technique ○ Avian influenza ○ spreading bacteria thinly over a solid ○ Sars surface ○ BSE Obtained a Nobel prize in 1905 ○ HIV/AIDS Pre-Golden Age Period: The Birth of Vaccination MRSA Jenner (1796) - smallpox vaccination Methicillin-resistant Staphylococcus aureus Louis Pasteur (-100 years later) 1905: developed resistance against ○ Shows how vaccinations work: penicillin creation of avirulent strains of 1980s: developed resistance against bacteria during extended laboratory methicillin cultivation 1990s: developed resistance against vancomycin Birth of Modern Chemotherapy 1910: Paul Ehrlich developed a synthetic Bovine Spongiform Encephalopathy arsenic drug (salvarsan) to treat syphilis Caused by a prion 1930s: synthesis of sulfonamides Also causes Creutzfeldt-Jakob disease ○ an important class of antibiotic drugs (CJD) with a wide range of activity, being ○ A new variant in humans is related very effective against gram-positive to beef consumption and certain gram-negative bacteria 1928: Alexander Fleming discovered the Escherichia coli 0157:H7 first antibiotic (Penicillin) Toxin-producing strain of E. coli First seen in 1982 Modern Developments in Microbiology Leading cause of diarrhea worldwide Major Fields: ○ Bacteriology Acquired immunodeficiency syndrome (AIDS) ○ Mycology Caused by human immunodeficiency virus ○ Parasitology (HIV) ○ Virology First identified in 1981 ○ Immunology Worldwide epidemic infecting 30 million Microbial genetics and molecular biology people, 14,000 new infections every day lead to Recombinant DNA technology Sexually transmitted infection affecting (genetic engineering) males and females ○ Prokaryotic model system: Escherichia coli Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025 Unit II. Functional Anatomy of External structures of bacteria Prokaryotic and Eukaryotic Cells 1. Glycocalyx Common features of prokaryotic and eukaryotic Many bacteria secrete external surface cells layer composed of sticky polysaccharides, DNA and chromosomes polypeptide, or both Cell membrane Capsule Cytosol and ribosomes ○ Organized and firmly attached to cell wall Prokaryotes Slime layer One circular chromosome that is not ○ Unorganized and loosely attached membrane bound Allows cells to attach and is a key to No histones biofilms ○ protein that provides structural Prevents phagocytosis support for a chromosome 2. Flagella No organelles Anchored to the wall and membrane Peptidoglycan cell walls Types based on number and placement Replicates by binary fission ○ Atrichous - has no flagellum ○ Monotrichous - has a single Size, Shape, and Arrangement of Bacteria flagellum at one end Average size: O.2-1.0 micrometers x 2-8 ○ Lophotrichous - multiple flagella at micrometers one end Three basic shapes ○ Amphitrichous - a single flagellum at ○ Bacillus both ends Diplobacilli ○ Peritrichous - multiple flagella Streptobacilli surrounding the body coccobacilli Motility ○ Coccus ○ Type of bacterial movement due to Diplococci the rotation of the flagella Streptococci ○ Mechanism of rotation: “run and Tetrad tumble” Sarcinae ○ Moves toward or away from stimuli Staphylococci (taxis) ○ Spirals ○ Chemotaxis Vibrio Phototaxis Spirillum Magnetotaxis Spirochete ○ Flagella proteins are H antigens Most are monomorphic while some are 3. Axial filaments pleomorphic Endoflagella ○ Monomorphic Found in spirochetes Only has one form (Ex. Anchored at one end of a cells Escherichia coli) Its rotation causes the cell to move ○ Pleomorphic 4. Fimbria Occurs in various distinct Allow attachments forms (ex. Corynebacteria) 5. Pilus / Pili They vary in cell arrangements Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  Used to transfer DNA from one cell to ○ CV-I washes out another Mycoplasma Cell wall Bacteria with no cell wall Rigid for shape and protection Instead, they have cell membrane which ○ Prevents osmotic lysis incorporates cholesterol compounds Consists of peptidoglycan (murein) polymer (sterols), similar to eukaryotic cells of 2 monosaccharide units Cannot be detected by typical light ○ N-acetylglucosamine (NAG) microscopy ○ N-acetylmuramic acid (NAM) Linked by polypeptides (forming peptide Acid-fast cell walls cross bridges) with tetrapeptide side chain Genus Mycobacterium and Nocardia attached to NAM Mycolic acid (waxy lipid) covers thin Fully permeable to ions, aa, and sugars peptidoglycan layer ○ Gram positive cell wall may regulate Do not stain well with Gram stain and the movement of cations should be stained with acid-fast stain Gram-Positive Cell Walls Damage to cell wall Thick layer of peptidoglycan Lysozyme Negatively charged teichoic acid on the ○ Digests disaccharide in surface peptidoglycan Teichoic acids Penicillin ○ Lipoteichoic acid links to plasma ○ Inhibits peptide bridges in membrane peptidoglycan ○ Wall teichoic acid links to peptidoglycan Internal structures: cell membrane May regulate movement of cations Analogous to eukaryotic cell membrane: Polysaccharides provide antigenic variation ○ Phospholipid bilayer with proteins (Fluid mosaic model) Gram-Negative Cell Walls ○ Permeability barrier (selectively Thin peptidoglycan permeable) Outer membrane ○ Diffusion, osmosis, and transport Periplasmic space systems Lipid A of LPS acts as endotoxin Difference from eukaryotic cell membrane: O polysaccharides are antigens for typing ○ Role in energy transformation: Less sensitive to medications because outer electron transport chain for ATP membrane acts as additional barrier production Damage to the membrane by alcohols, Gram Stain Mechanism quaternary ammonium, and polymyxin Crystal violet-iodine crystals form in cell antibiotic causes leakage of cell contents Gram positive ○ Alcohol dehydrates peptidoglycan Cytoplasm and Internal Structures ○ CV-I crystals do not leave Location of most biochemical activities Gram negative Nucleoid ○ Alcohol dissolves outer membrane ○ Nuclear region containing DNA (up and ;eaves holes in peptidoglycan to 3500 genes Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  Plasmids ○ Small, non-essential, circular DNA Catabolic and Anabolic Reactions (5-100 genes) Metabolism ○ Replicate independently ○ The sum of all chemical reactions in Ribosomes an organism ○ 70S vs 80S Catabolism Inclusion bodies ○ Provides energy and building blocks ○ Granule containing nutrients for anabolism ○ Monomers ○ Transfer energy from complex ○ Fe compounds (magnetosomes) molecules to ATP Anabolism Endospores ○ Uses energy and building blocks to Stages: build large molecules ○ Dormant, tough, non-reproductive ○ Transfer energy from ATP to structure complex molecules ○ Germination ○ Vegetative cells Role of ATP in Coupling Reactions Provides resistance to: Metabolic pathway ○ UV and gamma radiation ○ Sequence of enzymatically ○ Desiccation catalyzed chemical reactions in a ○ Lysozyme cell ○ Temperature ○ Determined by enzymes, which are ○ Starvation encoded by genes ○ Chemical disinfectants Relationship to disease Collision Theory Sporulation: endospore formation States that chemical reactions can occur Germination: return to vegetative state when atoms, ions, and molecules collide Activation energy Sporulation ○ Needed to disrupt electronic The process of endospore formation configurations ○ Spore septum begins to isolate Reaction rate newly replicated DNA and a small ○ Frequency of collisions with enough portion of cytoplasm energy to bring about a reaction ○ Plasma membrane starts to ○ Can be increased by enzymes or by surround the isolated DNA, increasing temperature or pressure cytoplasm and membrane ○ Spore septum surrounds the isolated Enzymes portion, forming forespore Biological catalysts ○ Peptidoglycan layer forms between Enzyme components membranes ○ Apoenzymes ○ Spore coat forms Inactive, protein portion of an ○ Endospore is freed from the cell enzyme ○ Cofactors UNIT III: Chapter 5: Microbial Nonprotein component of an enzyme Metabolism Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  If removed, the apoenzyme Substrate concentration will not function Ex: iron, sync, magnesium Inhibitors ○ Holoenzymes Competitive inhibitors The whole, active enzyme ○ Fill the active site of an enzyme and formed by the apoenzyme compete with the normal substrate and cofactor for the active site ○ Coenzymes (NAD+, NADP+, FAD) ○ Ex: sulfanilamide Organic molecule which Noncompetitive (allosteric) inhibitors activates enzymatic activity ○ Do not compete with the substrate for the enzyme’s active site but instead interact with another part of the enzyme Enzyme classification based on type of chemical reaction catalyzed Feedback inhibition Class Type of chemical reaction Also known as end-product inhibition catalyzed Controls the amount of substance produced by a cell Oxidoreductase Oxidation-reduction Mechanism is allosteric inhibition Transferase Transfer of functional groups Energy Production: Oxidation-Reduction Reactions Hydrolase Hydrolysis Oxidation Lyase Removal of groups of atoms ○ Removal of electron without hydrolysis Reduction ○ Gain of electron Isomerase Rearrangement of atoms Redox reaction within a molecule ○ Oxidation reaction paired with reduction reaction Ligase Joining of two molecules using energy from In biological systems, electrons are often breakdown of ATP associated with hydrogen atoms Biological oxidations are often dehydrogenations Mechanism of Enzymatic Reactions a. An enzyme molecule has an active site The Generation of ATP: Phosphorylation where it binds the substrate molecule Substrate level phosphorylation b. The reaction between enzyme and ○ Transfer of a high energy PO4- to substrate molecules forms a complex and ADP may temporarily alter the active site slightly Oxidative phosphorylation c. The enzyme molecule breaks apart the ○ Transfer of electrons from one substrate molecule compound to another is used to d. Two end-products result from the reaction generate ATP by chemiosmosis e. The enzyme molecule remains unchanged and is recycled Metabolic Pathways of Energy Production: COH Catabolism Factors Influencing Enzyme Activity Cellular respiration Denaturation by temperature and pH ○ Aerobic respiration Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  Produces 34-36 ATP Step 6 ○ Anaerobic respiration ○ This step undergoes two reactions: Fermentation The enzyme glyceraldehyde 3-phosphate dehydrogenase The three steps of aerobic respiration transfers 1 hydrogen 1. Glycolysis (oxidation of glucose to molecule from pyruvate/pyruvic acid) glyceraldehyde phosphate to Multi-step breakdown of glucose into nicotinamide adenine pyruvate dinucleotide to form NADH + Glycolysis produces 2 ATP, 2 NADH, and 2 H+. pyruvate molecules Glyceraldehyde 3-phosphate Alternative pathways: pentose phosphate dehydrogenase adds a and Entner-Doudoroff phosphate to the oxidised glyceraldehyde phosphate to Steps in Glycolysis (Reference: form https://byjus.com/biology/glycolysis/) 1,3-bisphosphoglycerate. Stage 1 Step 7 ○ A phosphate group is added to ○ Phosphate is transferred from glucose in the cell cytoplasm, by the 1,3-bisphosphoglycerate to ADP to action of enzyme hexokinase. form ATP with the help of ○ In this, a phosphate group is phosphoglycerokinase. transferred from ATP to glucose ○ Thus two molecules of forming glucose,6-phosphate. phosphoglycerate and ATP are Stage 2 obtained at the end of this reaction. ○ Glucose-6-phosphate is isomerised Step 8 into fructose,6-phosphate by the ○ The phosphate of both the enzyme phosphoglucomutase. phosphoglycerate molecules is Stage 3 relocated from the third to the ○ The other ATP molecule transfers a second carbon to yield two phosphate group to fructose molecules of 2-phosphoglycerate by 6-phosphate and converts it into the enzyme phosphoglyceromutase. fructose 1,6-bisphosphate by the Step 9 action of the enzyme ○ The enzyme enolase removes a phosphofructokinase. water molecule from Stage 4 2-phosphoglycerate to form ○ The enzyme aldolase converts phosphoenolpyruvate. fructose 1,6-bisphosphate into Step 10 glyceraldehyde 3-phosphate and ○ A phosphate from dihydroxyacetone phosphate, which phosphoenolpyruvate is transferred are isomers of each other. to ADP to form pyruvate and ATP by Step 5 the action of pyruvate kinase. ○ Triose-phosphate isomerase ○ Two molecules of pyruvate and ATP converts dihydroxyacetone are obtained as the end products. phosphate into glyceraldehyde 3-phosphate which is the substrate 2. Krebs Cycle / Citric acid cycle (oxidation of acetyl in the successive step of glycolysis. CoA) Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  The transition step generates acetyl-CoA ○ The enzyme succinyl CoA from pyruvate synthetase catalyses the reaction. Acetyl group of acetyl-CoA enters TCA ○ This is coupled with substrate-level cycle phosphorylation of GDP to get GTP. Generates ATP and reducing power, and ○ GTP transfers its phosphate to ADP precursor metabolites forming ATP. Products: 2 molecules of carbon, 3 Step 6 molecules of NADH, 1 molecule of FADH2 ○ Succinate is oxidised by the enzyme and 1 molecule of ATP or GTP succinate dehydrogenase to fumarate. Steps in Krebs Cycle (Reference: ○ In the process, FAD is converted to https://byjus.com/neet/krebs-cycle/) FADH2. Step 1 Step 7 ○ The first step is the condensation of ○ Fumarate gets converted to malate acetyl CoA with 4-carbon compound by the addition of one H2O. oxaloacetate to form 6C citrate, ○ The enzyme catalyzing this reaction coenzyme A is released. is fumarase. ○ The reaction is catalyzed by citrate Step 8 synthase. ○ Malate is dehydrogenated to form Step 2 oxaloacetate, which combines with ○ Citrate is converted to its isomer, another molecule of acetyl CoA and isocitrate. starts the new cycle. ○ The enzyme aconitase catalyzes this ○ Hydrogens removed, get transferred reaction. to NAD+ forming NADH. Malate Step 3 dehydrogenase catalyzes the ○ Isocitrate undergoes reaction. dehydrogenation and decarboxylation to form 5C 3. Oxidative phosphorylation (electron transport 𝝰-ketoglutarate. chain) ○ A molecular form of CO2 is Formed by series of electron carriers released. (cytochromes) located in the mitochondria ○ Isocitrate dehydrogenase catalyzes (for eukaryotes) and plasma membrane (for the reaction. prokaryotes) ○ It is an NAD+ dependent enzyme. Oxidation / reduction reactions NAD+ is converted to NADH. ○ Electron carriers (reducing power) Step 4 from glycolysis and TCA cycle ○ 𝝰-ketoglutarate undergoes oxidative transfer their electrons to the decarboxylation to form succinyl electron transport chain CoA, a 4C compound. ○ Generates proton gradient or proton ○ The reaction is catalyzed by the motive force (pmf) 𝝰-ketoglutarate dehydrogenase ○ In chemiosmosis, pmf generated enzyme complex. energy via oxidative phosphorylation ○ One molecule of CO2 is released and NAD+ is converted to NADH. Anaerobic Respiration Step 5 Inorganic molecule is the final electron ○ Succinyl CoA forms succinate. acceptor Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  ATP yield lower because only part of the Saprophytes Krebs cycle operates under anaerobic ○ Organisms that obtains their conditions nutrients from decaying matter Parasites Fermentation ○ Rely on its host for nutrients and Any spoilage of food by microorganisms survival Any process that produces alcoholic beverages or acidic dairy products LECTURE 4: Chapter 6: Microbial Any large-scale microbial process occurring Growth with or without air Scientific definition Physical requirements - affects microbes in their ○ Uses an organic molecule as the growth and survival final electron acceptor pH ○ Does not use the Krebs cycle or Temperature electron transport chain (ETC) Gravity ○ Low energy yield Osmotic pressure Catabolism of other compounds Chemical requirements - application of Amylase pharmaceutical use ○ Digestion of starch Growth factors Cellulase Hormones ○ Digestion of cellulose (only bacteria Elements and fungi have this enzyme Microbial growth Biochemical Tests and Bacterial Identification Increase in the number of cells, not cell size Fermentation tests ○ Mannitol fermentation Physical Requirements for Growth 1. Temperature Metabolic diversity among organisms Minimum growth temperature Energy source Optimum growth temperature ○ Phototrophs Maximum growth temperature ○ Chemotrophs Types of microbes based on their Principal carbon source temperature preference ○ Autotrophs ○ Psychrophiles Organisms that can produce Cold loving their own food ○ Psychrotrophs ○ Heterotrophs Growth between 0C and Organisms that cannot 20-30C produce their own food and Organisms that can cause rely on other organisms for spoilage food Listeria monocytogenes Chemoheterotrophs Usually found in ○ Use same organic compound as meat, fish, energy source and carbon source vegetables, and ○ Ex: most medically important processed foods bacteria Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  Causes muscle ○ Some bacteria use NH4+ or NO3- aches, diarrhea, ○ A few bacteria use N2 in nitrogen nausea, vomiting fixation (food poisoning) Sulfur ○ Mesophiles ○ Found in amino acids, thiamine, and Moderate or normal biotin temperatures Phosphorus ○ Thermophiles ○ Found in DNA, RNA, ATP, and Heat loving membranes ○ Hyperthermophiles 3. Trace elements Found in extreme thermal Inorganic elements required in small environments amounts 2. pH Usually occurs as enzyme cofactors Most bacteria grow between pH 6.5 and 7.5 4. Oxygen Molds and years grow between pH 5 and 6 O2 requirements vary greatly Acidophiles grow in acidic environments (1 Types of bacteria based on their preference to 5 pH for oxygen Neutrophils ○ Obligate aerobes - lives in the ○ Grows between 6.5 and 7.5 pH presence of oxygen 3. Osmotic Pressure ○ Facultative anaerobes - can live with Hypertonic environments, or an increase in or without oxygen salt or sugar, cause plasmolysis ○ Obligate anaerobes - dies in Tonicity environment with oxygen; total ○ Hypertonic - cells shrink absence of oxygen to survive ○ Hypotonic - normal entry of water ○ Aerotolerant anaerobes - can live ○ Isotonic - cells burst with or without air Extreme or obligate halophiles ○ Microaerophiles - microbes that ○ Require high osmotic pressure or require low amount of oxygen to salt environments survive Facultative halophiles Toxic oxygen ○ Tolerate high osmotic pressure ○ Singlet oxygen ○ Do not require salt environments to ○ Superoxide free radicals survive ○ Peroxide anion ○ Hydroxyl radical Chemical requirements for growth 5. Organic growth factor 1. Carbon Organic compounds obtained from the Half of dry weight environment Structural organic molecules which are used Examples: as energy source ○ Vitamins Chemoheterotrophs use organic carbon ○ Amino acids sources ○ Purines and pyrimidines Autotrophs use CO2 2. Nitrogen, sulfur, and phosphorus Endotoxin Nitrogen Surviving mechanism of most microbes ○ Found in amino acids and proteins Released by microbes at the end of their life ○ Most bacteria decompose proteins Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025 Biofilms Slime or hydrogels formed by microbial Agar communities Complex polysaccharide Starts via attachment of planktonic bacteria Used as solidifying agent for culture media to surface structure in Petri plates, slants, and deeps Bacteria are attracted by chemicals via Generally not metabolized by microbes quorum sensing Liquefies at 100 degrees Celsius Quorum sensing Solidifies at ~40 degrees Celsius ○ Form of bacterial communication Allows communities to share nutrients and Anaerobic Culture Methods shelters them from harmful factors Reducing media Cause of most nosocomial infections ○ Contains chemicals that combine O2 (hospital acquired infections) ○ Heated to drive off O2 ○ Uses anaerobic jars Culture media ○ A novel method in clinical labs Culture medium Oxyrase is added (derived ○ Nutrients that are prepared for from the plasma membrane microbial growth of bacteria) ○ Has to be sterile Sterile Capnophiles ○ No living microbes Microbes that require high CO2 conditions Inoculum Low oxygen and high CO2 conditions ○ Introduction of microbes into the resemble those found in: medium ○ Intestinal tract Culture ○ Respiratory tract ○ Microbes grown in/on the culture ○ Other body tissues where pathogens medium grow Chemically defined media Ex: Campylobacter jejuni ○ Exact chemical composition is known Selective media Complex media Suppress unwanted microbes and ○ Extracts and digests of yeasts, meat, encourage desired microbes or plants Eosin-Methylene Blue (EMB) Agar is used Nutrient broth for Salmonella Nutrient agar Blood agar Differential media Potato broth agar - for fungi Makes it easy to distinguish colonies of Disc diffusion assay - for different microbes antibacterial properties Enrichment culture McFarland Standard Encourages growth of desired microbes used to standardize the approximate number of bacteria in a liquid suspension by Obtaining Pure Cultures visually comparing the turbidity of a test Pure culture suspension with the turbidity of a McFarland ○ Only contains one species or strain standard Colony Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025  ○ A population of cells arising from a single cell or spore from a group of Most Probable Number (MPN) attached cells Multiple tube MPN test ○ Often called a colony-forming unit Count positive tubes and compare with a (CFU) statistical table Streak plate method ○ Used to isolate pure cultures Direct Microscopic Count Preserving Bacterial Cultures Deep-freezing: -50 to -95 degrees Celsius Lyophilization (freeze-drying): frozen and Turbidity dehydrated in a vacuum Measuring Microbial Growth Reproduction in Prokaryotes Binary fission - exponential growth 1. Cell elongates and DNA is replicated Direct Methods Indirect Methods 2. Cell wall and plasma membrane Plate counts Turbidity begin to constrict 3. Cross-wall forms, completely Filtration Metabolic activity separating the two DNA copies 4. Cells separate MPN Dry weight Budding Direct microscopic Conidiospores count Fragmentation of filaments Bacterial Growth Curve 1. Lag phase Little or no cell division and can last 1 hour or several days 2. Exponential or logarithmic phase The cell begins to divide and enter the period of growth 3. Stationary phase The metabolic activities are slowed 4. Death phase Number of deaths will exceed the number of new cells Serial Dilution a series of sequential dilutions used to reduce a dense culture of cells to a more usable concentration Plate Counts an agar plate count from foods and products carried out on a specific growth medium Buenaventura, Shiloh Clarisse C. BIO 4: MIDTERMS BSBIO 2B A.Y. 2024-2025