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BIOL 22100 Lab Practical 2 Study Guide

by: Gayatri

BIOL 22100 Lab Practical 2 Study Guide BIOL 22100

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About this Document

Contains all of the learning objectives, explained in detail from Labs 14-19, including vocabulary and major lab procedures
Thomas Walter
Study Guide
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This 5 page Study Guide was uploaded by Gayatri on Saturday April 23, 2016. The Study Guide belongs to BIOL 22100 at Purdue University taught by Thomas Walter in Spring 2016. Since its upload, it has received 171 views. For similar materials see Microbiology in Biology at Purdue University.


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Date Created: 04/23/16
BIOL 22100 Lab Practical 2 Study Guide Exercises 14 – 15: Bacterial Conjugation and Phages 1. Know the difference between transduction, transformation, and conjugation; and how these processes can increase a genetic variety in bacterial cells a. Transduction: phage transfers DNA between cells b. Transformation: bacterial cell accepts a naked DNA c. Conjugation: direct cell-cell contact, Steps: i. Copy of F (fertility) factor will be transferred from a donor cell to a recipient cell 2. Know the basic structure and the gene organization of typical F factor a. F-factor: a circular DNA molecule that carries genes responsible for: i. Pili production ii. Self-replication of F-factor iii. Transfer of a F-factor’s copy (tra-operon) b. Can have additional, non-essential for conjugation, genes (Ab-resistance, resistance to heavy metals, etc.) c. Medical significance of conjugation – can lead to an appearance of cells with multiple Ab- resistances 3. Understand the principles of selection and counter selection used in the conjugation experiment a. MacConkey agar will be used to differentiate Lac+ and Lac- cells b. MacConkey agar will be supplemented with one (Kan OR Cat) or both (Kan AND Cat) antibiotics to select different types of cells (see the table below): c. Cat is used to select cells with F-factor (donor cells and transconjugants) d. Kan is used as a counterselective marker – it kills KanS donor cells e. Cat+Kan combination selects for transconjugants only! 4. Define bacteriophage, plaque, pfu, lysogeny, lysogen, lysate, temperate phage, prophage, and host specificity for viruses a. Bacteriophage: bacterial viruses, can be (1) DNA containing – T coliphages or (2) RNA containing – M12 (those that infect E.coli are studied the most) b. Plaque: clear spot on an agar where bacterial cells have been lysed by a phage c. PFU: plaque forming units d. Lysogeny: process where viral DNA can persist in the bacterial host for generations without causing lysis e. Lysogen: the bacterial host in a lysogeny situation containing the viral DNA f. Lysate: preparation containing lysed products of cells g. Temperate phage: the phage in a lysogenic situation that is inside the bacterial host h. Prophage: the latent noninfectious form in which the temperate phage is carried by lysogenic bacteria i. Host specificity for viruses: 5. Know the basic information on M13 and T7 phages (genome structure and host specificity) a. M13 bacteriophage i. Filamentous, rod shaped capsid ii. ssDNA genome iii. Ff phage: uses sex pili of E. coli and other gram negative bacteria for attachment, can ONLY infect F+ cells b. T7 bacteriophage i. Lytic phage, binal capsid ii. dsDNA genome iii. Infects enteric bacteria by attaching to cell adhesion receptors and infects BOTH F+ and F- cells 6. Know the 3 shapes of phages. Understand the functions of the parts of a binal-shaped phage a. Helical b. Polyhedral/icosahedral c. Binal: combo of polyhedral tail and helical structure i. All of the viral nucleic acid is located within the head. ii. Tail functions as an organ of attachment to susceptible host cells 7. Recall the 5 stages of the infectious cycle of bacteriophages a. Adsorption/attachment: phage binds via weak bonds to specific receptor sites on bacterial cell wall (recall host range and specificity) b. Penetration: phage makes enzymes that penetrate cell wall, viral genetic material enters cell (ONLY in T phages: tail inserts DNA from head à bacteria host while protein coat stays outside) c. Biosynthesis of viral components: DNA of phage directs bacterial cell’s metabolism to make viral components (proteins, DNA) d. Maturation: intracellular development and assembly into new virions e. Cell Lysis: bacterial cells are lysed by phage enzymes (holins and lysins) and infective phages are released to go infect more cells 8. Understand the process of lysogeny a. Viral DNA can stay in the host without causing lysis, host functions normal and viral DNA is transmitted through generations b. Phage is temperate phage, bacterial host is lysogenic strain or lysogen c. Temperate phages can become lytic phages under certain conditions such as starvation, temp change, UV light à cell is killed/lysed 9. Know the difference between two cycles of viral reproduction: the lytic cycle and the lysogenic cycle. Understand that same phage can be lytic or temperate. Know how the lysogenic cycle can be converted into the lytic cycle (prophage induction) a. The lytic cycle (main cycle) is one of the two viral cycles of reproduction, the other one being the lysogenic cycle i. Lytic cycle à cell is destroyed ii. Lysogenic cycle à remaining latent in the cell, eventually going into lysogenic cycle b. The two are connected, and diversion to either occurs at the penetration stage c. Steps: Attachment à Penetration of viral material into cell à Biosynthesis OR integration the biosynthesis à Maturation à Release à Repeat 10. Understand the application of phage specificity through phage typing a. Phage typing = identifying unknown bacteria according to its susceptibility to bacteriophage based on host specificity of phage type 11. Know how to calculate the phage Titer a. T = N x 1/DF x P, where b. N = # of plaques appeared on the spot after incubation; DF = dilution factor; P = plating factor = the reciprocal of the volume of the phage drop. Exercises 16 and 17: UV Radiation and Nitrogen Fixing Bacteria 1. Know which nucleotide bases are purines and which are pyrimidines a. A and G are purines (2 rings) b. C, T, and U are pyrimidines (1 ring) 2. Define genotype and phenotype; wt and mutant; mutagenic factors; know different types of mutations a. Genotype: genetic composition of an organism, all organisms have more genes than are actually expressed, can be manifested as a single phenotype at certain times b. Phenotype: observable property/appearance of an organism c. Wild type: normal d. Mutant: change in the genetic code, has different effects on the cell/organism e. Types of mutations: i. Harmful/lethal (most of them) ii. Silent (affect genotype but not phenotype) iii. Beneficial (very rare) 3. Understand the multiple effects of UV light on bacterial cell. Be able to explain a. 240-300 nm is effective for killing bacteria but the optimum is 260 nm (bacterial DNA is disorganized) b. Effects of UV light: i. Deamination of cytosine: Cytosine à Uracil (leads to destabilization) ii. Formation of thymine dimers: occurs between adjacent thymines on the same strand à DNA cannot be replicated or transcribed, but damage may be repaired 4. Know 2 methods to repair thymine dimers and the difference between mechanisms a. Repairing thymine dimer: i. Photo reactivation (PR): enzymatic cleavage of thymine dimers by PR enzyme photolyase, activated by visible light, template is repaired and resumes normal functional template ii. Biochemical excision repair: Cut and patch” repair that uses exonuclease, DNA polymerase I, and DNA ligase. (difference is main source of energy) 5. Understand the advantage that spore-forming bacteria have in relationship to UV light a. Bacillus bacteria can produce spores from their vegetative cells, which are highly resistant to heat, chemicals, (disinfectants), and even radiation 6. Understand although UV light is damaging, why it is not always the best choice for sterilization a. UV light is not very penetrative, good for surface sterilization only 7. Define symbiosis and symbionts. Know the 3 types of symbiotic relationships. a. Symbiosis = living together b. Symbionts = organisms that live together in a particular environment 8. Provide the examples based on the human host – microbe interactions a. Commensalism: the microorganism benefits from the association, whereas the host is neither benefited nor harmed. i. Example: remora fish and sharks b. Mutualism: a state in which both the host and the microorganism derive benefit. i. Example: rumen bacteria and cows c. Parasitism: a state in which the microorganism benefits at the expense of the host; the host may suffer or has severe disadvantages. i. Example: any microbial infection 9. Know the definition for the normal microflora a. Normal microflora are beneficial to the body, work in symbiosis with us 10. Explain the advantages and potential disadvantages of having it a. Good because they fight off bad bacteria, bad because can affect us negatively when immunocompromised 11. Know what plants and bacteria can undergo nitrogen fixation a. Plants i. Leguminous plants (e.g. peas, beans, soybeans, alfalfas, clovers). b. Bacteria i. Genera Rhizobium and Bradyrhizobium [Gram(-) rods]. This plant-bacteria association is an example of mutualism), nitrogen fixation is only possible if these organisms are co-existing [nitrogenase is made by the bacteria, transaminase is made by host plant] 12. Know what type of symbiotic relationship between Rhizobium and the leguminous plants a. The nitrogen-fixing bacteria (Rhizobium) infect root cells and excrete metabolites which trigger cell divisions in the root cortex leading to nodules formation b. After such an invasion, rod-shaped motile Rhizobium cells become pleomorphic (of undefined shape) non-motile bacteroids – this is an adaptation to a new lifestyle 13. “Oxygen dilemma “and how it is solved a. Nitrogen fixation requires a lot of energy that can be produced through aerobic respiration only b. Nitrogenase is very sensitive to oxygen c. Dilemma is solved by the co-production (plant – the globin part; bacterium – the heme part) of leghemoglobin. Leghemoglobin removes oxygen outside the nodules, and nitrogenase can function properly. Exercise 18 – 19: Antiseptics and Disinfectants 1. Define antiseptic and disinfectant a. Antiseptic = a chemical agent that can be safely used externally on living tissues to destroy microorganisms or to inhibit their growth. b. Disinfectant = a chemical agent used on inanimate objects (i.e. NOT living tissues) to destroy microorganisms or to inhibit their growth. 2. Know 3 methods to evaluate the effectiveness of chemical agents and how each works a. Phenol coefficient b. Use-dilution method c. Filter paper 3. Understand the concept of a phenol coefficient and why the larger the coefficient - the higher the effectiveness a. Phenol is the standard disinfectant used to compare others b. The larger the coefficient, the higher the effectiveness c. Calculation: find the highest dilution that killed all organisms, find the ratio of that to phenol i. E.g. a 1:1000 dilution of an agent has the same effect as a 1:100 dilution of phenol ⇒ phenol coefficient of that agent is 10 (1000:100) 4. Use-Dilution method: how it works- a. Bacteria are coated on toothpicks, are treated with different dilutions of an agent or at a constant concentration with varying length of time à toothpicks will be washed and incubated with broth à broth is observed for growth à agents that prevent growth at the greatest dilutions are considered the most effective. 5. Filter Paper method: how it works – a. Filter paper disk is soaked with chemical agent and placed on agar with organism à clear area around disk = agent can inhibit or kill cells/ growth à diameter is measured and compared to standard list 6. Understand the concept of how organisms are considered resistant to antimicrobials even though there is some inhibition. Know what an antibiogram is a. Some organisms are considered to be resistant even though there is some inhibition because they are NOT inhibited by therapeutic concentration. i. Higher concentration would inhibit growth but would also cause serious side effects. b. Antibiogram = to determine the pattern of resistance in order to chose a therapeutic dose of a particular antibiotic that is both effective and has minimal side effects. 7. Understand the Kirby-Bauer disk method, how important it is to be standardized, and how to implement such a standardization. Know the factors affecting the size of the zone of inhibition a. Used to determine bacterial susceptibility to various chemical agents, easy to use b. Does NOT determine if an agent is bacteriostatic or bacteriocidal! c. Must be standardized in order to obtain consistent and reproducible results. It uses a standardized inoculum and a nutrient agar of standard composition (Mueller-Hinton Agar). 8. Understand the difference between antimicrobial drugs and antibiotics. Know organisms that produce antibiotics. a. Antimicrobial drugs are used to treat infectious diseases by killing microorganisms or inhibiting their growth. b. Antibiotics are antimicrobial drugs produced/derived by/from living organisms (fungi, bacteria, etc.). c. Not all the antimicrobial drugs are antibiotics (e.g., sulfa drugs)! 9. Understand the broth dilution method and its use (expensive and hard) a. Is used to determine: i. Minimal inhibitory concentration (MIC) = lowest concentration that prevents visible growth but NOT killing. ii. Minimal bactericidal concentration (MBC) = lowest concentration that prevents visible growth and induces killing. iii. MBC > MIC 10. Know the different effects of antimicrobial agents on growth (bacteriostatic, bacteriocidal, bacteriolytic) a. Bacteriostatic = Inhibit growth, NO killing, NO cell lysis, binding to cellular targets is NOT tight. Becomes free when [drug] ↓ b. Bacteriocidal = Inhibit growth, Induce killing, NO cell lysis, binding to cellular targets is tight. Do NOT become free when [drug] ↓. c. Bacteriolytic = Inhibit growth, Induce killing, Cause cell lysis 11. Define synergism and antagonism a. Synergism = a combination of 2 drugs has more antimicrobial activity than of either agent given alone. i. To provide optimal therapy or to lessen the toxicity of individual drugs by reducing the dosage of each drug. b. Antagonism: combination drugs will interfere with each other’s action mechanism.


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