Microbe Mission Science Olympiad Cheat Sheet

Microscopes: Bright Field: Light source under stage is condensed with the condenser, goes through objective lens (adjustable) and eyepiece (another lens). Focus can’t be on center and peripheral at the same time (field curvature). Low contrast (staining required) Phase Contrast: Has better contrast. Change in amplitude results in a difference in brightness when the light goes through a medium. Annular diaphragm present in the condenser, makes a ring of light on the specimen. When the light goes through the specimen, scattered light that hits the phase ring increases phase shift and shows that part as dark. Reflected Light/Inverted: Light is bounced down on a specimen then reflected back up to the eyepiece. Used to see autofluorescent cellular structures, fluorescent dyes, or fluorescent antibodies (helps see proteins). Uses high intensity UV light, bleaches photos Confocal: Like fluorescence microscopes, but uses laser instead of a Mercury Arc Lamp. Also uses pinhole so only one focal plane is seen. Laser focused in a region of the specimen by mirrors, pictures of small regions are combined to make a coherent image. Pinhole also makes it so images of each focal plane can be taken to make a 3d image Electron: Invented because light microscopes refract too much light and makes images blurred as it gets smaller. 200-250 nm is smallest possible by light microscopes. Uses electron lenses.TEM: electrons go through condenser, specimen, objective lens, then projector to see the image. SEM: electrons focused through condenser and objective lenses, then through scanning coil, then reflected off of the specimen by heavy metals onto scanners that interpret results. Estimate Size: Divide the field number (printed on eyepiece) by the magnification number to get the field of view diameter. For example, if the field number is 22 and the magnification is 400, the field of view diameter is 0.055 mm Types of Cells: Prions: Misfolded protein, causes disease (especially brain-related) Size: approx 30 nm Organelles: N/A Membrane and Cell Wall: N/A Viruses: Infectious, nucleic acid+protein coat, only lives with host cells Size: usually range from 5-300 nanometers (nm) Organelles: N/A Membrane and Cell Wall: Capsid is a protein shell that encapsulates the genetic material (not wall or membrane though) Viroids: RNA with no protein, smallest infectious agents, only in plants. Size: 246-467 nucleotides (more than 5x smaller than the smallest virus) Organelles: N/A Membrane and Cell Wall: N/A Bacteria: unicellular microorganisms with cell walls but lack organelles or an organized nucleus Size: 0.2μm in diameter and 2−8μm in length Organelles: not bound by a membrane; include chromosome and ribosomes Membrane and Cell Wall: plasma membrane; two types of cell wall: gram positive or gram negative encapsulated by peptidoglycan Archaea: A single-celled prokaryotic organism with distinct molecular characteristics; structurally similar to gram-positive bacteria, genetic processes closer to eukaryotic cells. Size: 0.7μm up to 4μm Organelles: Ribosomes and DNA Membrane and Cell Wall: plasma membrane; cell wall composed of various polysaccharides and glycoconjugates Eukaryotic Microbes: Organisms whose cells contain a nucleus and other membrane-bound organelles. Size: usually have diameters ranging from 10 to 100 μm Organelles: may include mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, and lysosomes Membrane and Cell Wall: The eukaryotic plasma membrane is a phospholipid bilayer with proteins and cholesterol embedded in it. The cell membrane is an extremely pliable structure composed primarily of two adjacent sheets of phospholipids. Cholesterol, also present, contributes to the fluidity of the membrane. Symbiogenesis: Symbiogenesis is the leading evolutionary theory of the origin of eukaryotic cells. Mitochondria, plastids such as chloroplasts, and possibly other organelles of eukaryotic cells are descended from formerly free-living prokaryotes (more closely related to the Bacteria than to the Archaea) taken one inside the other in endosymbiosis. Mitochondria appear to be phylogenetically related to Rickettsiales bacteria, while chloroplasts are thought to be related to cyanobacteria. Bacterial Cell Shapes: Bacterial Cell Shapes: Spherical (Cocci) Rods (Bacilli) Comma (Vibrios) Tightly coiled (Spirochetes) Spiral shaped (Spirilla) Gram Stain procedure: a test that checks for bacteria at the site of a suspected infection such as the throat, lungs, genitals, or in skin wounds - helps diagnose harmful bacteria 1. Applying a primary stain (crystal violet). 2. Adding a mordant (Gram's iodine). 3. Rapid decolorization with ethanol, acetone or a mixture of both. 4. Counterstaining with safranin. Gram+ vs Gram-: Gram+: thick layers of peptidoglycan Gram-: thin layers of peptidoglycan Acid fast bacteria: can resist the acid and/or ethanol-based decolorization procedures common in many staining protocols. Mechanisms of acid-fastness vary by species. Some may be partially acid-fast. Acid fastness can result in an incorrect gram stain test. Culture Bacteria: Culture methods: Broth: Bacteria are suspended in a liquid nutrient medium such as Luria Broth in an upright flask. Agar: petri dishes with agar-based growth medium, sometimes with additives, or a dipstick with the same principle (more cost effective). Long-term storage Stab: similar to agar plates, but are solid agar in a test tube. Bacteria inserted through a needle stabbed in the middle. Short-term. Culture collections: Collect and catalog lots of cultures. Solid plate culture of thermophilic: higher temps (50-70 C). Uses low acyl clarified gellan gum. Growth curves: a curve that shows various stages of the growth of bacteria. Lag Phase: bacteria prepares for reproduction - active and increasing in size. Log Phase: (or Exponential phase) This phase is marked by the doubling of the bacterial cells. Stationary Phase: rate of cell growth becomes equal to its rate of death due to limitation of nutrients. Death Phase: when the rate of death is greater than the rate of formation of new cells. Plate counts: Estimate population density of bacteria by counting the colonies of a small and dilute portion of the sample. Defined vs. complex media: Defined uses compounds and distilled water to find the exact growth medium. Complex uses digested products. Differential vs. selective media: Differential uses “indicator media” to show different microorganisms growing there. Selective kills other microorganisms. Great plate count anomaly: Estimates are that we can cultivate roughly one out of every 100 microbes Batch culture vs. chemostat growth: Growth Curves: to grow, bacteria need water, a carbon source, a nitrogen source and some mineral salts. Antibiotic Susceptibility: a test performed to cultured bacterial pathogen growth may be inhibited, or not, by exposure to specific antibiotics. Methods: Phenotypic AST detects the arrest of bacterial cell growth in the presence of static or cidal antimicrobial agents. Genotypic AST attempts to identify specific resistance genes or genetic mutations using molecular or genomic (usually DNA-based, amplification-based or sequencing-based) methods. Antibiotic resistance: Gotten through random gene mutation or transfer from one species to another. Resistance to antibiotics is encouraged by the extended use of antibiotics. Resistance transfer: genetic material is transferred between organisms, makes it very easy for antibiotic resistance to spread among many bacteria. Bacteriostatic vs. bactericidal antibiotics: Bacteriostatic slow bacteria and make it easier to kill. Need to be helped by the immune system. Bactericidal kill the bacteria directly. Genome Replication: the process by which the genome's DNA is copied in cells. Before a cell divides, it must first copy (or replicate) its entire genome so that each resulting daughter cell ends up with its own complete genome. Gene Regulation: Operons are segments of DNA that are transcribed as a group. Promoters make regulatory proteins bind to the DNA which regulates whether a gene is used. Lac operon: promoter goes to a sequence that creates a protein to inhibit enzyme production to break down lactose, but lactose breaks the inhibiting protein, allowing the enzyme to be made. Trp operon makes tryptophan, which activates the repressor, so when there is enough tryptophan, the RNA polymerase doesn’t bind and more isn’t made, but otherwise it is. Culture-Independent Methods: Metatranscriptomics: Studies what genes are active in a community of microbes, rather than just see which ones are present Metagenomics: genetic material is recovered directly from environmental samples to see what microbes are present Proteomics: Studies proteins on a large scale, studies all proteins produced by an ecosystem Metabolomics: Studies chemical processes involving the small molecule substrates, intermediates, and products of cell metabolism. Studies “the unique chemical fingerprints that specific cellular processes leave behind.” Phage Lambda: The Lambda Phage infects e-coli, and it either does the lysogeny or the lytic cycle. Depending on the circumstances, a promoter is activated and one of the two processes happens. When cl or Cro proteins are activated, the other is repressed, so only the lytic cycle or lysogeny occurs. The lytic cycle makes the cell produce more Lambda Phages and Lysogeny makes the host carry the DNA until it is activated and releases phages. Control lac and trp operons: Controlling lac operons in E. Coli has numerous applications. It can be used for metabolic engineering and recombinant protein production. Lac operons can be controlled through optogenetics, by shining blue light. This makes it easier because of factors such as easy tunability, reversibility, dynamic induction strength, and spatial control, that are difficult to obtain with chemical inducers. Direct chemicals can be used to influence the operons such as TrpR to repress Tryptonine production Functions of Microbes: Lakes & Oceans: Photosynthetic bacteria (called cyanobacteria) in lakes, ponds, and shallow oceans supply half of the world’s oxygen; break down organic matter into nitrate, phosphate, and other essential nutrients. Soil: soil microbes are bacteria, actinomycetes, viruses, fungi, nematodes, and protozoa. Production of plant growth regulators and metabolites affecting plant growth and development. Nutrient recycling, break down crop residues, and stimulate plant growth, inhibiting plant parasites and diseases. Gut Microbiome: supply essential nutrients, synthesize vitamin K, aid in the digestion of cellulose, and promote angiogenesis and enteric nerve function. Bacteroidetes and Firmicutes are the main bacteria in the metabolism of undigested food remnants. VII. Bacterial Transcription, Translation, and Genome Replication: Bacterial Transcription: Initiation: RNA polymerase recognizes the promoter sequence, facilitating the formation of the transcription initiation complex. The DNA unwinds, allowing RNA polymerase to start synthesizing RNA. Elongation: RNA polymerase moves along the DNA template, catalyzing the addition of complementary nucleotides to the growing RNA chain. Termination: Transcription concludes at a terminator sequence. Termination mechanisms include Rho-dependent termination, where the Rho factor aids in transcript release, and Rho-independent termination, involving the formation of a hairpin structure in the RNA. Bacterial Translation: Initiation: The small ribosomal subunit binds to the mRNA at the ribosome binding site. The initiator tRNA, carrying the amino acid methionine, attaches to the start codon. Elongation: The large ribosomal subunit joins, and the ribosome moves along the mRNA. During elongation, tRNA molecules bring amino acids to the ribosome, facilitating peptide bond formation. Termination: Translation concludes at a stop codon. Release factors promote the detachment of the polypeptide chain from the final tRNA, completing the translation process. Genome Replication: Initiation: DNA replication begins at the origin of replication, where DNA unwinding creates a replication bubble. DNA polymerase initiates synthesis using RNA primers. Elongation: DNA polymerase adds nucleotides to the growing DNA strands in the 5' to 3' direction. Termination: Replication concludes when the entire DNA molecule is duplicated. Termination mechanisms involve specific termination sequences. IX1. Bacterial Gene Regulation (lac Operon and trp Operon): Lac Operon: Components: The lac operon includes the promoter, operator, and three structural genes (Z, Y, A) coding for enzymes involved in lactose metabolism. Regulation: The lac operon is subject to negative regulation by a repressor protein. In the absence of lactose, the repressor binds to the operator, preventing transcription. Allolactose, an inducer, binds to the repressor in the presence of lactose, alleviating repression and allowing transcription. Trp Operon: Components: The trp operon consists of the promoter, operator, and five structural genes (E, D, C, B, A) related to tryptophan biosynthesis. Regulation: The trp operon is negatively regulated by a repressor. In the absence of tryptophan, the repressor is inactive, allowing transcription. When tryptophan is present, it binds to the repressor, activating it and inhibiting transcription. X. Culture-Independent Methods for Studying Microbial Communities: 16S/18S rRNA Amplicon Sequencing: Primer Selection: The choice of primers is crucial for targeting specific variable regions of the rRNA gene to capture microbial diversity accurately. PCR Optimization: Polymerase Chain Reaction (PCR) conditions are optimized to ensure the specificity and efficiency of amplification. Sequencing Platforms: High-throughput sequencing platforms like Illumina are commonly used to sequence the amplicons. Data Analysis: After sequencing, data analysis involves quality control, Operational Taxonomic Unit (OTU) clustering, taxonomic assignment, diversity analysis, and statistical interpretation. Metagenomics: Shotgun Metagenomics: This approach involves sequencing all genomic material in a sample, providing a comprehensive view of the microbial community. Functional Annotation: The predicted genes are annotated for their functions, allowing insights into the functional potential of the microbial community. Sample Preparation: DNA extraction, library preparation, and sequencing are crucial steps in metagenomic studies. Data Analysis: Analysis includes genome assembly, taxonomic profiling, and metagenome-wide association studies. XI. Photoautotrophic vs. Heterotrophic Metabolic Strategies: Photoautotrophic Organisms: Energy Source: Photoautotrophs derive energy from light through processes like photosynthesis. Carbon Source: They use inorganic carbon (CO2) for carbon fixation. Heterotrophic Organisms: Energy Source: Heterotrophs obtain energy by consuming organic compounds. Carbon Source: They use organic carbon compounds as a source of carbon. Metabolic Strategies of Specific Bacteria: Green Sulfur Bacteria: Photoautotrophic bacteria that use sulfur compounds as electron donors in photosynthesis. Iron-Oxidizing Bacteria: Chemoautotrophic bacteria that oxidize ferrous iron for energy. Heterotrophic Bacteria: Heterotrophs rely on organic compounds as carbon and energy sources. Cyanobacteria: Photoautotrophic bacteria capable of capturing light energy for photosynthesis. XII. Lytic and Lysogenic Virus Replication: Lytic Virus Replication: Stages: Attachment, Entry, Transcription, Translation, Replication, Assembly, Maturation, Release, Spread. Key Events: The virus attaches to the host cell, injects its genetic material, and takes over the host's cellular machinery for replication. The host cell is eventually lysed, releasing new viral particles. Lysogenic Virus Replication: Stages: Attachment, Entry, Integration, Cell Division, Lysogenic State, Induction, Entry into Lytic Cycle. Key Events: The virus integrates its genetic material into the host genome, becoming a prophage. The host cell divides, and the prophage is replicated along with the host DNA. Under certain conditions, the prophage may be induced to enter the lytic cycle. XIII. Mechanisms of Horizontal Gene Transfer and XIV. Industrial Applications of Microbes: Horizontal Gene Transfer: Transformation: Uptake of foreign DNA from the environment. Transduction: Transfer of genetic material via bacteriophages. Conjugation: Direct transfer of DNA between bacterial cells. Industrial Applications: Fermentation: Microbes are used in the production of various products, such as ethanol, organic acids, and enzymes. Kombucha: Fermented tea produced by symbiotic communities of bacteria and yeast. Algal Biofuels: Microalgae are harnessed for biofuel production. Bioremediation: Microbes are employed to clean up environmental pollutants. Phage Therapy: Bacteriophages are used to combat bacterial infections. XVI. Causes and Effects of Microbial Population Explosions: Causes and Effects: Algal Blooms: Rapid growth of algae, often due to nutrient enrichment, leading to ecological imbalances. Thrush: Fungal infection caused by Candida, often affecting mucous membranes. Enterocolitis: Inflammation of the intestine due to microbial infection, leading to diarrhea and abdominal pain. XVII. Structure and Function of Spores and Cysts: Spores: Structure: Spores have a core containing genetic material, surrounded by protective layers. Function: Spores are specialized structures for survival, resistance to adverse conditions, dispersal Cysts: Structure: Cysts are protective structures with a thick, resistant outer covering. Function: Cysts serve to protect the organism during unfavorable conditions and aid in survival. XVIII. Extremophiles and Their Characteristics: Thermophiles: High-temperature environments. Halophiles: High-salt environments. Psychrophiles: Cold temperatures. Acidophiles: Highly acidic environments. Alkaliphiles: Alkaline environments. Extension - V. Metabolic Strategies of Specific Bacteria: Sulfate-Reducing Bacteria: Chemolithotrophic, use sulfate. Purple Sulfur Bacteria: Phototrophic, use sulfur compounds. Purple Non-Sulfur Bacteria: Phototrophic or chemotrophic, no reliance on sulfur. Extension - VI. Phylogenetic Methods to Detect Horizontal Gene Transfer: Comparing Gene and Species Phylogenies. Incongruence Analysis, Phylogenetic Concordance. Phylogenetic Profiling, Phylogenomic Approaches. Network-Based Approaches, Sequence Composition Analysis. Phylogenetic Marker Genes, Statistical Methods. VII. Treatments and Vaccines for Diseases Caused by Various Organisms: Viruses: SARS-CoV-2, HIV-1, Influenza A, Hepatitis B, T4 Phage, Canine Parvovirus, Mimivirus, Poliovirus, Banana Bunchy Top Virus. Bacteria: Vibrio cholerae, Rickettsia rickettsii, Streptococcus pneumoniae, Corynebacterium diphtheriae, MRSA, Mycobacterium tuberculosis, Cutibacterium acnes, Haemophilus influenzae, Wolbachia. Fungi: Candida aureus, Alternaria solani. Protists: Plasmodium falciparum, Giardia duodenalis, Toxoplasma gondii, Alexandrium catenella. Prions and Prion-Like Proteins: PrP, Amyloid Beta, Tau Proteins, α-Synuclein. Worms: Taenia solium, Ancylostoma duodenale.