Microbiology Test 2

DNA polymerase

Synthesizes, proofreads, and repairs DNA.

Terminator

The site one a DNA strand at which transcription ends.

Autotrophs.

Uses an inorganic carbon source for energy (CO2).

Preservation Of Microbes

* Short term: Refrigeration from 0 to 70°C * Long term: Deep-freezing is from -50°to -95°C & Lyophilization (freeze-drying) is from -54° to -72°C then dehydrated in a vacuum. The liquid stage is skipped in lyophilization.

Phases of bacterial growth.

* A growth curve for an exponentially increasing population, plotted logarithmically by a dashed line and arithmetically by a solid line. The knowledge of the bacterial growth curve is critical in understanding the population dynamics and the population control of infectious disease. Bacterial populations follow a specific series of growth phases from lag, log, stationary, and death. Example: Staphylococcus spp. * Lag Phase: Intense activity when preparing for population growth, but no increase in population. * Log Phase: Logarithmic, or exponential, increase in population. The logarithmic growth in this phase is due to reproduction by binary fission (bacteria) or mitosis (yeast). * Stationary Phase: Period of equilibrium; microbial deaths balance production of new cells. * Death Phase: Population is decreasing at a logarithmic rate.

Modes of action of microbial control agents

* Alteration of membrane permeability - Cellular contents may leak into surrounding medium, interfering with the growth of the cells. Meaning cells are lost that are needed. * Damage to proteins - Damage to enzymes vital to cellular metabolic activities. * Damage to nucleic acids - Damage to DNA that carries genetic information will stop cell replication and cellular metabolic function such as enzyme synthesis.

Reproduction In Prokaryotes

* Binary Fission - Cell divides into two. * Budding - Parent and donor cells separate. * Conidiospores - By certain filamentous bacteria (actinomycetes or streptomyces). * Fragmentation of filaments.

Media Of Microbes

* Chemically Defined Media - The exact chemical composition is known. Where chemoautotrophs, photoautotrophs, and microbial assays are grown. * Complex Media - The exact chemical composition is unknown, so its not chemically defined. Example: extracts and digests of yeasts, meat, or plants. Nutrient broth (NB), Nutrient agar (NA), Tryptic Soy broth (TSB), and Tryptic Soy agar (TSA). * Reducing Media - When organisms are stored in tightly closed tubes. Mainly used to grow or maintain obligate anaerobes. Contains chemicals such as thioglycolate or oxyrase (enzyme from a bacterial enzyme to remove O2). No oxygen. * Liquid Media - Can be heated to drive off O2. No oxygen. * Selective Media - Suppression of unwanted microbes and encouraging desired microbes to grow. * Differential Media - Differentiation of colonies of desired microbes from others. * Enrichment Media - Similar to selective media, but designed to increase the number of desired microbes to detectable levels. * Streak Plate Method - Used to isolate pure cultures.

Bacterial genetic materials

* Expression - Genetic information is used within a cell to produce the proteins needed for the cell to function. Cell metabolized and grows. * Replicated - Genetic information can be transferred between cells of the same generation. Recombinant cell. * Replication - Genetic information can be transferred between generations of cells. Daughter cells.

DNA

* Gene Libraries - Made of pieces of an entire genome stored in plasmids or phages. Collection of clones containing different DNA fragments. Essential for maintaining and retrieving DNA. * cDNA - Made from mRNA by reverse transcriptase to replace the intron because they don't do anything to help with making DNA. * Synthetic DNA - Made by synthesis machine. The only way to predict the DNA sequence is by knowing the amino acid sequence of the genes protein product from the genetic code.

Applications of genetic engineering

* Hormones - Such as Insulin and Somatostatin, and other products. * Subunit vaccines - Consists of only the protein portion of a pathogen. Is a lot safer because its almost impossible to get the disease because only a small amount of the virus is used in the dose. * Nonpathogenic viruses - Carrying genes for a pathogen's antigens are used as vaccines. * Gene therapy - Replaces defective or missing genes. May provide cure for some genetic diseases. * Gene silencing - A new technology. Involves RNA interference (RNAi) uses short interfering RNAs, siRNA binds to mRNA causing its enzymatic destruction. Gene expression silenced. * Human Genome Project - Nucleotides have been sequenced. Useful for diagnoses of diseases and treatments.

Temperature Ranges For Microbes.

* Minimum, optimum, maximum. * -30 - 0 degrees Celsius: There is no significant growth below freezing. * 0 - 10 degrees Celsius: Refrigerator temperatures may allow slow growth of spoilage bacteria. Very few pathogens. * 10 - 20 degrees Celsius: Bacteria survive and some may grow. * 20 - 50 degrees Celsius: Causes a rapid growth of bacteria and some may produce toxins. (aka danger zone). Example: Range at which bacillus cereus multiplies in rice in about 30-60 minutes. * 50 - 60 degrees Celsius: Very slow bacterial growth * 60 - 130 degrees Celsius: Destroy most microbes, however, the lower temperatures take more time.

PH Requirements For Microbes.

* Most bacteria grow best between 6.5-7.5. * PH is the -log hydrogen ion concentration (H+). * Fungi (molds and yeasts) grow between pH 5-6. * Acidophiles grow in acidic environments that grow well below 5.5. * Neutrophiles grow in "neutral" environments that prefer between 5.5-8.5. * Alkalophiles grow in alkaline environments and live above pH 8.5.

Direct Measuring Methods Of Growth.

* Plate Counts - Perform serial dilutions of a sample. Each dilution goes from 10^-1 then 10^-2 and so on in decreasing order. Used to ensure the right number of colonies are counted by the pour plate method or spread plate method. Normally includes 30 to 300 colonies. * Filtration - Low concentration of bacteria can be counted. * MPN - Used to estimate the number of microbes by dilutions with a statistical table. * Direct Microscopic Count - Breed method is used for milk by spreading a sample on a marked square that contains a known volume on a slide. Petroff-Hausser cell counter is a spread sample on a marked square that contains several squares of known volumes.

Nutrients For Microbial Growth.

* Required for growth and development. * Water - Makes up 80%-90% of microbes. * Carbon - The organic structural backbone of living matter used for energy. * Nitrogen - Synthesizes cellular materials (amino acids and proteins) by decomposing proteins. * Sulfur - Synthesizes cellular materials (amino acids and vitamins) by decomposing proteins. * Phosphorus - Synthesizes cellular materials (DNA, RNA, ATP, and phospholipid membranes).

Methods of transfer of genetic materials

* Transposon - Segments of DNA that can move from one region of DNA to another. * Transposase - Contain insertion sequences for cutting and resealing DNA. * Complex Transposons - Carry other genes such as the gene for kanamycin resistance. * Transformation - {rocess in which genes are transferred from one bacterium to another as "naked" DNA in solution. Example: Streptococcus pneumoniae. * Conjugation - Process of transfer of genetic material from. one cell to another involving cell to cell contact * Transduction - Process of transfer of DNA from one cell to another by a bacteriophage. Genetic recombination - The process of joining pieces of DNA from different sources.

Indirect Measuring Methods Of Growth.

* Turbidity - Cloudiness is used to measure the degree of microbes. * Metabolic Activity - By measuring CO2/ATP. * Dry Weight - Used in filamentous bacteria and molds where the fungus is removed from the growth medium, filtered to remove extraneous material, dried in a desiccator, then weighed.

Colony-forming units (CFU).

A colony is a population of cells arising from a single cell or spore or from a group of attached cells.

Solidifying Agent For Culture Media

Agar is a complex polysaccharide, from marine alga, that is used as solidifying agent for culture media in Petri plates, slants, and deeps. Generally not metabolized by microbes. Liquefies at 100°C. Solidifies ~40°. Agar has long been used as a thickener in foods such as jellies and ice cream.

Genome.

All of the genetic material in a cell.

Forensic microbiology

Application of methods in the field of microbiology especially nucleic acid techniques or DNA technologies to answer microbiological questions for the legal system Employs microbiological techniques such as: Hybridization with DNA probes, RFLP in DNA fingerprinting, DNA, microarrays, and PCR (for rapid identification of microbes that causes an amplification in DNA). Useful for tracing the source of infection and similar DNA methods are used in rape conviction.

Obligate Anaerobes.

Can grow and are maintained in a reducing media. Only anaerobic growth, where growth is best without the presence of oxygen. Lacks enzymes to neutralize harmful forms of oxygen and can't tolerate oxygen.

Facultative Anaerobes.

Can grow in the absence of oxygen by fermentation or anaerobic respiration. Both aerobic and anaerobic growth, but grow better in the presence of oxygen. Presence of enzymes catalase and SOD allows toxic forms of oxygen to be neutralized that can use oxygen.

Halophiles.

Can grow under high solute concentration. Extreme/obligate require high osmotic pressure. Facultative tolerate high osmotic pressure.

Capnophiles.

Candle jar or CO2 packet is used for anaerobic organisms that require high CO2. Example: Neisseria meningitidis.

Mutation

Change in genetic material that can have a positive, neutral, or negative effect. A mutagen is an agent that causes mutations by increasing the rate to 10^-5 to 10^-3 per replicated gene. Chemical mutagens are nitrous acid (alteration of adenine pairs with cytosine instead of thymine). Ionizing radiation mutagens cause the formations of ions that can react with nucleotides and the deoxyribose-phosphate backbone causing the mutation by binding to DNA to repair mutations by lygase. UV radiation mutagens to separate thymine dimers. Different types: * Spontaneous Mutation - Occur in the absence of a mutagen. Rate is one in 10^9 replicated base pairs or one in 10^6 replicated genes. * Base Substitution - AKA point mutation. The most common mutation that is the change in one base and can result in missense mutation (change in amino acid) or nonsense mutation (introduces a stop codon). * Frameshift Mutation - Insertion or deletion of one or more nucleotide pairs, when the codons change because of amino acids. * Positive Selection - Direct mutant cells because they grow or appear differently. * Negative Selection - Indirect detects mutant cells because they don't grow. * Replica Plating - Used to isolate mutants.

Disinfectants

Chemicals used for disinfection. Very few disinfectants achieve sterility. No single type is appropriate for all circumstances. Types: * Phenol. * Phenolics - Derivative of phenols to increase antimicrobial activity or reduce irritating qualities. Example: Lysol. * Bisphenols - Disrupts plasma membranes by the use of phenols that includes two groups, triclosan and hexachlorophene. * Biquanides - Disrupts plasma membranes by chlorhexidine. * Halogens - May damage enzymes and other proteins by iodine (tincture, iodophor, betadine, isodine).

Disinfection of water by chlorination

Chlorine (Cl2) forms hypochlorous acid (HOCl) when added to water. HOCl is a strong oxidizing agent and germicidal by inhibiting cellular enzyme systems at equilibrium. The acid (bleach) is the most effective form of chlorine because it is neutral in electrical charge and diffuses as rapidly as water through the cell wall. Because of its negative charge the OCl- ion can't enter the cell freely making it ineffective on bacteria.

Types of plasmids

Circular pieces of DNA that exists independent of the chromosome. About 1-5% the size of bacterial chromosome. Self replicating. Gene containing. * Conjugative Plasmids - Carries genes for sex pili and transfer of the plasmid. * Dissimilation Plasmids - Encode enzymes for catabolism of unusual compounds. * R Codes - Encodes for antibiotic resistance. The resistance transfer factor is the code for replication and conjugation. R-determinant is the code for enzymes that inactivate certain drugs or toxic substances. Example: Bacteroides fragilis.

Sources of genes for cloning

Colony hybridization is used to select a cloned gene. Procedure: * Make replica of master plate on nitrocellulose filter. * Treat filter with detergent (SDS) to lyse bacteria. * Treat filter with sodium hydroxide (NaOH) to separate DNA into single strands. * Add radioactively labeled probes (short segments of single-stranded DNA). *Probe will hybridize with desired gene form bacterial cells. * Wash filter to remove unbound probe and expose filter to x-ray film. * Developed film is compared with replica of master plate to identify colonies containing the gene of interest.

Nucleotides

Composed of adenine, thymine, cytosine, guanine, and uracil.

Ionizing radiation

Damages DNA by highly reactive radicals from the ionization of water. Has a short wavelength and more energy. Example: Z rays, gamma rays, electron beams.

Principles of effective disinfection

Depends on concentration of disinfectant, organic matter, PH, and time.

Photoautotrophs.

Derive their energy from carbon dioxide (CO2). Organisms that use light for energy. Examples: Cyanobacteria, some Purple and Green Bacteria.

Disinfection.

Destruction of vegetative pathogens on living tissues. Treatment is almost always by chemical antimicrobials. Removal of pathogens.

Hosts for expression of cloned gene products

E. coli is used because it is easily grown and its genomics are known. It is easy to lyse E. coli, to get product. The disadvantages need to eliminate endotoxin from products and need to lyse cells to recover products.

Psychrotrophs.

Normally grow between 20-30 degrees Celsius. Grow slowly at very low temperature. Can slowly degrade food stored in a refrigerator (food spoilage).

Mesophiles.

Normally grow between 30-40 degrees Celsius.

Organic Growth Factors.

Essential chemical organic compounds that are unable to synthesize that are obtained directly from the environment. Some include vitamins, coenzymes, amino acids, purines, and pyrimidines. Some microbes synthesize all of their vitamins, but some can't so they depend on external sources of vitamins (biofilms example pseudomonas aeruginosa). Other chemical requirements include carbon, nitrogen, sulfur, and phosphorus.

Gene regulation

Genetic control mechanisms that regulates transcription of mRNA by regulating synthesis of enzymes. Codes for the repressor protein that stops gene transcription by blocking RNA polymerase from binding to the promoter or blocking its progress on the DNA. * Repression - Inhibits gene expression and decreases synthesis of enzymes mediated by regulatory proteins called a repressor. Before transcription. * Induction - Turns on transcription of genes. A substance that mediates this process is called the inducer. Before transcription. * Operon Model - A series of genes regulated by one regulatory protein that consists of the promoter and operator sites and structural genes they control. Example: Lac operon. The lac operon is for lactose metabolism, where lactose is digested by a catabolic pathway catalyzed by inducible enzymes (b-galactosidase, permease, and transacetylase). When the repressor is active the allolactose is the inducer that binds to the repressor protein and inactivates it making it not able to bind to the operator, where the regulated enzymes are made. When the repressor is inactive, tryptophan binds to the inactive repressor protein and activates it, which turns off the enzyme expression involved in tryptophan biosynthesis that prevents transcription. Before transcription. * microRNAs (miRNAs) - Inhibit protein synthesis in eukaryotes that contains approximately 22 nucleotides. The mode of action is similar to siRNA. After transcription. * Similar short RNAs in bacteria - Help cells cope with environmental stress by low temperature adn oxidative damage. After transcription.

Agricultural applications of recombinant DNA techniques

Genetic engineering using agrobacterium that contains Ti plasmid that can induce tumor-like growth (crown gall) when integrated into plant DNA. The plant is simultaneously made to produce nutrients (carbon and nitrogen sources) for the bacterium. It can be used to introduce foreign genes in plants. Examples: Bt toxin, Herbicide resistance, Suppression of genes using Antisense DNA (used to produce MacGregor tomatoes, which stay firm). Drawback - the bacterium does not naturally infect grasses and cannot be used to improve grains.

Chemoheterotrophs.

Get most of their carbon from the source if their energy by organic materials such as proteins, carbohydrates, or lipids. Examples: Most Bacteria and some Archaea.

Hypertonic Environment.

Higher concentration of solutes (salt or sugar) than in the cells, cause plasmolysis. High osmotic pressure remove necessary water from cell, which kills many bacteria. Can be very useful in food preservation. Causes the cell to shrink.

Competent cells

Hosts that easily take up DNA. Many host do not naturally take up DNA, thus competent cells have to be prepared for transformation by soaking in a solution of calcium chloride.

Applications of restriction fragment length polymorphism

Hybridization with DNA probes.

Bacteriostasis.

Inhibiting (not killing) microbes.

Glucose effect

Inhibition of metabolism of alternate carbon sources by glucose. Unlike lactose, the enzymes for glucose utilization are constitutive. Bacteria growing on a medium containing glucose and lactose first consume the glucose and then, after a short lag time, the lactose. During lag time the intracellular cAMP increases, the lac operon is transcribes, more lactose is transported into the cell, and b-galactosidase is synthesized to break dow nlactose.

Low Temperature.

Inhibits microbial growth by refrigeration, deep freezing, and lyophilization because the metabolic rate is reduced so they can't reproduce or synthesize.

Trace Elements For Cultivation.

Inorganic elements required in small amounts. Act as enzyme cofactors needed for enzyme function. These include iron, copper, molybdenum and zinc.

Recombinant DNA technology

Insertion or modification of genes to produce desired proteins. AKA Genetic Engineering. Typical procedure: * Vector, such as a plasmid, is isolated. * DNA containing gene of interest from a different species is cleaved by an enzyme into fragments. * Desired gene is selected and inserted into the plasmid. * Plasmid is taken up by a cell, such as a bacterium. * Cells with gene of interest are cloned with either of two goals in mind.

Transcription

Involves decoding the language of nucleic acids and converting it into the language of proteins. Steps of this process: * RNA polymerase binds to the promoter and DNA unwinds at the beginning of the gene. * RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA. * The site of synthesis moves along DNA. DNA that has been transcribed rewinds. * Transcription reaches the terminator. * RNA and RNA polymerase are released and the DNA helix reforms.

DNA sequencing methods

Is the determination of the exact sequence of the nucleotides. Different methods: * Random Shotgun Sequencing - Isolate DNA. Fragment DNA with restriction enzymes. Clone DNA in a bacterial artificial chromosome. Sequence DNA fragments. Assemble sequences. Edit sequences by filling in the gaps. The study of genetic material taken directly from environmental samples called metagenomics. * Primer Walking. * The human genome project - Recombinant DNA technology has made possible sequencing of genomes of organisms notable, the human genome. Contains about three billion nucleotide pairs and 20,000-25,000 genes. * Nucleotide sequence of cloned genes can also be determined. * DNA Fingerprinting - Can be used in identification. Can track source of an infectious disease outbreak. Tracking includes isolating E.coli from patients, obtaining DNA, digesting DNA with restriction enzymes, run on a gel to separate fragments then stain the bands, and banding patterns will be different.

Dry Heat Sterilization.

Kill endospores by oxidation. Can be done by flaming, incineration, or hot-air sterilization (oven).

Disinfection with alcohols

Kills bacteria and fungi by destroying the membranes by denaturing proteins and dissolving lipids, but does not kill endospores and nondeveloping viruses. Examples: Ethanol and Isopropanol. Pure ethanol (100% alcohol) is not effective because it can't enter the cytoplasm because it requires water to denature proteins. The optimum concentration is 70%.

Moist heat sterilization.

Kills microbes by coagulation of proteins (denaturation) by breaking hydrogen bonds which hold them together. * Boiling - Kills only vegetative cells of bacterial pathogens. Endospores are not destroyed by boiling. * Autoclaving - Achieved by applying steam under pressure in an autoclave that is usually conducted at 15 psi for 15 min (temperature rises to 121 degrees Celcius). Heavily contaminated materials are autoclaved for longer time. Labile media are autoclaved at lower heat treatment and shorter holding time. If glucose is present the temperature and time need to be lowered. Endospores are killed.

Sanitization.

Lower microbial counts on eating and drinking utensils. May be done with high temperature washing or by dipping into a chemical disinfectant. Commercial sterilization is the application of enough heat to kill the endospores of c.botulinum.

Differential Media.

Make it easy to distinguish colonies of different microbes. Example: Streptococcus pneumonia, which is used in a blood agar plate to differentiate bacteria which destroy the red blood cell (hemolysis - s.pyogenes) by producing enzymes called hemolysins. Staphylococcus aureus on a tellurite-glycine medium. E.coli on EMB medium with black centered colonies surrounded by characteristic metallic green sheen. E. aerogenes on EMB medium showing characteristics with dark colored colonies.

DNA ligase

Makes covalent bonds to join DNA strands, okazaki fragments, and new segments in excision repair.

Microbes In Anaerobic Jar

Microbes grown on petri-dishes to observe individual colonies are incubated in anaerobic jars.

Pasteurization.

Mild heat treatment to reduce spoilage or organisms and pathogens. Used extensively to preserve milk. However, thermoduric organisms survive pasteurization because they are heat resistant.

Decimal reduction time (DRT)

Minutes to kill 90% of a population at a given temperature.

Psychrophiles.

Normally grow at 15 degrees Celsius.

Genomic.

Molecular study of genomes.

Order of resistance of microbes to chemical agents

Most resistant to least resistant. * Prions. * Endospores of bacteria. * Mycobacteria. * Cysts of protozoa. * Vegetative protozoa. * Gram negative bacteria. * Fungi (including most fungal spores). * Viruses without envelopes. * Gram positive bacteria. * Viruses with lipid envelopes.

Mutagenesis and mutagens

Mutations are responsible for much of the diversity of life. Site-Directed Mutagenesis - AKA targeting genes. Change a specific DNA code to change a protein.

Safety and ethical issues concerning "genetically modified microorganisms

Need to avoid accidental release. Genetically modified crops must be safe for consumption and for the environment.

Disinfectants with oligodynamic action.

Oligo means few. Very low concentrations can inhibit bacteria. Denature proteins by combining with SH groups. Examples: (metals) Ag, Hg, Cu. Silver nitrate may be used to prevent gonorrheal opthalmia neonatorum. Silver sulfadiazine used as a topical cream on burns. Copper sulfate is an algicide.

Semiconservative DNA replication.

One original strand and one new strand.

Obligate Aerobes.

Only aerobic growth because they require oxygen in high concentrations. The presence of enzymes catalase and SOD allows toxic forms of oxygen to be neutralized that can use oxygen.

Microaerophiles.

Only aerobic growth, where oxygen is required in low concentrations and diffused in a medium. Produce lethal amounts of toxic forms of oxygen if exposed to normal atmospheric oxygen.

Aerotolerant Anaerobes.

Only anaerobic growth, however growth continues in the presence of oxygen. Growth occurs evenly and oxygen has no effect on it. Presence of one enzyme, SOD, allows harmful forms of oxygen to be partially neutralized and tolerates oxygen.

Plasmid as vectors

Plasmids are an antibiotic resistance genen and restriction site. Have beta-galactosidase gene gives you the opportunity if a blue and white selection. Steps: * The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant. * All treated bacteria are spread on a nutrient agar plate containing ampicillin and b-galactosidase substrate, then incubated. * White colonies have a plasmid with insert that must contain a foreign DNA. * Blue colonies ave a plasmid without insert and must not contain a foreign DNA.

Desiccation.

Prevents metabolism of microorganisms by water, however the still remain viable for years after.

Transfer of recombinant DNA

Procedures require that DNA molecules be manipulated outside the cell then returned to living cells. * Transformation - Transfer of naked DNA in solution from one bacterium to another. * Electroporation - Insertion of DNA into a cell using an electrical current. * Protoplast fusion: Joining of two cells by first removing their cell wall. Hybrid cell (recombinant cell). Example: Algal Protoplasts Fusing. * Microinjection: Introduction of DNA directly into a cell usually with a glass pipette. * Gene gun: Inserts DNA coated "bullets" (particles of gold or tungsten) into the cell.

Sepsis.

Refers to microbial contamination.

Degerming.

Removal of microbes from a limited area. Mostly a mechanical removal by alcohol swabs.

Sterilization.

Removal or destruction of all microbial life by heating or filtration for liquids and gases like in fermintation. Commercial is the application of heat to kill endospores of C botulinum.

Filtration.

Removes microbes by the passing of liquid or gas through a screen like material with pores small enough to retain organisms (HEPA >0.3um) (membrane >0.22um)

Auxotrophs and uses of auxotrophic mutants.

Replica plating is very useful in mutant selection. An auxotroph mutant needs a specific nutrient to grow. The ames test shows if a chemical can cause cancer by the certain mutant causing a histidine growth (contains a auxotrophic mutant). A plate lacking a histidine will have a missing colony. Example: Salmonella.

Genes.

Segment of DNA that encodes a functional product. Usually a protein.

Microbial Detoxification Of Toxic Oxygen.

Since oxygen is the final electron acceptor in an anaerobic atmosphere, it turns toxic from H2O2. Contains singlet oxygen (1^O2^-1) that has been boosted into a higher energy state and extremely reactive, super-oxide free radicals (O2^-1) that have an enzyme SOD (super oxide dismutase) to neutralize the toxins in cellular components, peroxide anion(O2^-2) that has a catalase to reduce toxins in respiration, and hydroxyl radical that is an intermediate from of oxygen being the most reactive (OH).

Effect of soap on microbes

Soap has little value as a disinfectant, but plays a major role in cleaning by emulsification (mixture of water and oil). Decreases the surface tension. Acid anionic detergents and quaternary ammonium compounds (cationic detergents).

Detection of specific DNA. sequence or cloned genes

Southern blotting detects specific DNA fragment by hybridization. Uses DNA probes to detect specific DNA sequence in restriction fragments separated by electrophoresis.. The fragments are called RFLPs (restriction fragment length polymorphisms). The probe binds to the specific DNA sequence (hybridization).

Topoisomerase (DNA gyrase)

Supercoiled DNA is relaxed ahead of replication fork by enzymes.

Selective Media.

Suppression of unwanted microbes and encouraging desired microbes. Example: Bismuth sulfite agar, which is bismuth sulfite inhibits gram negative bacteria. Brilliant green agar, which is brilliant green dye selectively inhibits gram positive bacteria. Phenyl alcohol agar, which is selectively inhibits gram negative bacteria.

Asepsis.

The absence of significant contamination. Aseptic surgery techniques prevent microbial contamination of wounds. Examples: Nosocomial infections caused 10% of deaths in surgical cases. Also, deaths of delivering mothers were as high as 25%.

Microbial growth.

The increase in number of cells (aka colonies), not cell size. Colonies are groups of cells large enough to be seen with the naked eye. Microbial growths requires: * Optimum temperature. * Certain PH levels. * Osmotic pressure. * Contain specific chemicals. * Oxygen present. * Trace elements.

Genetic code

The language of mRNA is in the form of codons, groups of three nucleotides. The sequence of codons on an mRNA molecules determines the sequence of amino acids that will be in the protein being synthesized. Each codes codes for a particular amino acid.

Stop codon

Translation ends. AKA nonsense codon. Contains only three. Example: UAA, UAG, UGA.

Translation

The overall goal is to produce proteins using mRNAs as the source of biological information. Steps of this process: * Components needed to begin come together. * On the assembled ribosome, a tRNA carrying the first amino acid is paired with the start codon on the mRNA. The place where this first sits is called the P site. A tRNA carrying the second amino acid approaches. * The second codon of the mRNA pairs with a tRNA carrying the second amino acid at the A site. The first amino acid joins to the second by a peptide bond. This attaches the polypeptide to the tRNA in the P site. * The ribosome moves along the mRNA until the second tRNA is in the P site. The next codon to be translated is brought into the A site. The first tRNA now occupies the E site. * The second amino acid joins to the third by another peptide bond, and the first tRNA is released from the E site. * The ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide. *When the ribosome reaches a stop codon, the polypeptide is released. * The last tRNA is released and the ribosome comes apart. The released polypeptide forms a new protein.

Thermophiles.

The specific group is hyperthermophiles, that normally grow at 80 degrees Celsius or above. Theses are usually Archaea members, which can be found in hot springs. Other groups normally grow at 50-60 degrees Celsius.

Promoter

The starting site on a DNA strand for transcription of RNA by RNA polymerase.

Genetics.

The study of what genes are, how they carry information, how information is expressed, and how genes are replicated. The study of heredity.

Start codon

Translation of mRNA begins here. AKA sense codons. Contains 61 with 20 amino acids (sometimes 22 amino acids). Example: AUG.

Generation time.

The time required for a cell or population to double in number. This is found by multiplying the total number of hours by sixty minutes then dividing by the number of generations. Then in order to find the number of generations, take the log of the number of cells at the end minus the log of the number of cells at the beginning divided by 0.301 (log 2). Or logNt = logNo + nlong2.

Transfer RNA (tRNA)

The type of RNA molecule that brings amino acids to the ribosomal site where they are incorporated into proteins.

Osmotic Pressure.

The use of high concentrations of salts and sugars to preserve food creates a hypertonic environment then causes plasmolysis. Denies the cell moisture it needs in order to grow.

Tools of biotechnology

The use of microorganisms, cells, or cell components to make a useful product. Example: Foods, antibiotics, vitamins, and enzymes. * Selection - Culture a naturally-occurring microbe that produces desired product. AKA natural selection. * Mutation - Might result in a microbe with a desirable trait. * Restriction Enzymes - A major technical tool of genetic engineering that cuts specific sequences of DNA and can't digest its host bacterial DNA with methylated cytosines. Contains EcoRI (digestion "sticky ends" to make things join easier) and Smal (restriction enzyme cleavage "blunt ends"). Bacteriophage cells have a restricted range and can be destroyed by the presence and absence of specific restriction enzymes. Example: Bacillus amyloliquefaciens, E.coli, Haemophilus aegyptius, and Haemophilus influenzae. * Vector - A self-replicating DNA used to carry the desired gene to a new cell. Carry new DNA to desired cell. Plasmids and viruses can be used as vectors. Shuttle vectors: can exist in several different species. Example: Bacterial, yeast and mammalian cells or bacterial, fungal and plant cells. * Clone - A population of cells arising from one cell (genetically identical). * Polymerase Chain Reaction - Makes multiple copies of a piece of DNA enzymatically. Need a primer. Used to clone DNA for recombination, amplify DNA to detectable levels, sequence DNA, diagnose genetic disease, and detect pathogens.

Thermal death time (TDT)

Time to kill all cells i a culture at a given temperature.

DNA helicase

Unwinds double stranded DNA at the replication fork creating two anti-parallel DNA single strands.

Evaluating a disinfectant.

Use-dilution test (AOAC method). Test organisms are Salmonella choleraesuis, Staphylococcus aureus and Pseudomonas aeruginosa. (disk diffusion method where chlorine is more effective). * Metal rings dipped in test bacteria are dried at 37°C for a short time. * Dried cultures are placed in disinfectant (at use concentration) for 10 min at 20°C. 3. Rings are transferred to culture media to determine whether bacteria survived treatment.

Organic acids

Used for the control of molds and bacteria in foods and cosmetics (along with nitrates and antibiotics). Inhibit metabolism. Example: Sorbic acid, benzoic acid, and calcium propionate.

Selective & Differential Media.

Used to detect the presence of specific microorganisms. Those are associated with disease, poor sanitation, and screening for production of specific products. Useful in clinical and public health microbiology. Also useful in other applied aspects of microbiology. Example: Manitol salts agar, when the indicator changes color if manitol is fermented to acid, and salt (7.5% sodium chloride) inhibit competing bacteria. Differentiational Example: Staphylococcus epidermis. Selective Example: Staphylococcus aureus (survive best in salty environments).

Heterotrophs.

Uses organic carbon sources for energy (oxidize).

Effectiveness of antimicrobial treatment

Usually depends on the number of microbes, the environment (organic matter, temperature, biofilms), time of exposure, or characteristics of microbes.

Culture.

Where microbes are growing in or on a culture medium. When microbes are introduced into a culture medium to initiate growth its called inoculation. Culture medium is when nutrients prepared for microbial growth in the laboratory. Sterile culture medium contains no living microbes, where the media must initially be sterile. Pure culture only contains one species or strain.

DNA synthesis

Which is transcription and translation that helps convert genetic information on a gene to a functional product.