Microbiology Exam Two

Besides the use of NADH and enzymes, what do aerobes use to protect themselves against toxic oxygen products?

Vitamins C and E

Contrast RNA with DNA and delineate the three types of RNA.

(Messenger) mRNA: They bring information from the chromosome in nucleotide triplets (codons) that code for a specific amino acid. (Transfer) tRNA: See figure 7.14 - the tRNA has an anitcodon region that recognizes its complementry mRNA codons at its 3' end. on the acceptor stem binds to a specific amino acid (OH). Many tRNAs can recognize more than one codon. Ribosomes and (ribosomal) rRNA BACTERIA: Bacterial 70S ribosomes with 50S and 30S subunits 50S = two rRNA molecules, 34 peptides 30S = one molecule of 16S rRNA and 21 ribosomal polypeptides Ribosomes and rRNA for EUKARYOTES: 80 S with 60S and 40S subunits in cytosol and RER 70S in chloroplast and mitochondria There are three sub units to the rRNA 1. The A site: accommodates the tRNA with an amino acid 2. The P site: where the rRNA holds the tRNA and the growing peptide 3. The E site where the tRNA is discharged

Tell why bacteria methylate certain bases in their genome.

1. Control of genetic expression: Methylated genes are turned off (not transcribed) or turned on (transcribed). 2.Initiation of DNA replication: methylated nucleotide sequences initiate DNA replication 3.Protection against viral infection: Methylation allows for cells to recognize their DNA vs viral DNA. 4. Repair of DNA

Chart the steps of DNA replication in Prokaryotes. Include the following elements: gyrase, helicase, primase, ori region, DNA Polymerase I and III, leading and lagging strands, ligase and the role of an RNA Primer

1. DNA replication begins at a specific sequence of nucleotides called the origin. DNA helicase comes and unzips the DNA strand by breaking hydrogen bonds between the base pairs. 2.DNA polymerase binds replicated DNA from the 5' to 3' end. Each strand is replicated this way but since they are antiparallel there are two strands the leading strand is synthesized continuously and the lagging stand is joined with segments. SYNTHESIS of the LEADING STRAND Synthesis occurs toward the replication fork in a series of five steps 1. Primase makes an RNA primer complementary to the DNA strand. The RNA primer has the 3' hydroxyl group needed by DNA polymerase III. 2. Triphosphate deoxynucleotides form hydrogen bonds to their complementary bases. A-T and G-C 3.Using the high energy bonds DNA polymerase III covalently links the Triphosphate deoxynucleotides 500 to 1000 nucleos per second 4. DNA polymerase has a proofreading function to remove any mismatched bases. proofreading exonuclease function. errors are one in a billion 5.DNA polymerase I comes in and replaces the RNA primer with DNA SYNTHESIS of the LAGGING STRAND 1. Primase adds RNA primers in multiple spots (every 1000-2000 DNA bases) from the 5' to 3' end (Since the 5' end is closest to the replication fork) 2.Nucleotides pair up with their complements 3.DNA polyermase III joins the nucleotides and proofreads them. The lagging strand is built with segment called Okazaki fragments. 1000-2000 nucleotides 4. DNA polymerase I replaces the RNA primers of Okazaki fragmentswith DNA and proof reads the DNA it added. 5.DNA ligase joins the Okazaki fragments into a continuous DNA strand GYRASE Too keep the DNA from being overstraind ahead of the relpication fork Gyrase cuts DNA and bends it and rejoins it.

Describe a barophile

A barophile can live in extreme pressure. Pressure keeps it alive. Can not cause disease in humans

Drugs that kill bacteria by altering there cell membrane

A cell with a damaged membrane dies from disruption in metabolism or lysis. Detergent-like or surfactant But often damage our membranes too Polymixin example: interacts with phospholipids and cause leakage, particularly in Gram-negative bacteria. Best use is as a Topical can cause kidney & CNS damage Used for some drug resistant bacteria

Define genome

A genome is the entire genetic complement including genes, the sequences that code for RNA or polypeptide molecules, and the sequences that connect polypeptides together.

What is the catalase test?

Adding hydrogen peroxide to bacteria and seeing if there are bubble or not

What kind of components would you expect to find in a chemoheterotrophs agar?

Amino acids, glucose...

Describe Nitrogen fixation.

Bacteria are able to use N2 gas and reduce it to ammonia. This makes nitrogen available for other organisms. Essential for lifere

Describe bacterial growth phases

Bacterial growth and reproduction: Lag phase: Bacteria are getting used to their environment by producing enzymes necessary to survive. Reproduction is at a stand still Log Phase: Bacterial chromosome replication, growth and reproduction are at its peak. Bacteria are the most susceptible to antibiotic drugs at this stage. Stationary Stage: nutrients are depleted and waste accumulates slowing reproduction. The number of cells dying equals the ones being created Death Phase: Cells die faster than they are reproduced. Some cells may survive here especially those with endospores.

Explain why bacteria can respond quickly to make what proteins and enzymes that are needed.

Bacterial translations can occur almost simultaneously with translation because BOTH happen in the cytosol. It doesn't need to wait for the products of transcription like eukaryotes.

Why is DNA replication considered semiconservative?

Because the daughter DNA molecules each contain a new and old strained of DNA

Why don't antimicrobial drugs influence a patient?

Because the drugs only effect 70S ribosomes and not 80S

Know the drugs that kill bacteria by messing with their cell walls

CELL WALL A major group are the Beta-lactam antibiotics Penicillins and cephalosporins are themain beta-lactams Act on peptidoglycan synthesis in young, growing cells Penicillium chrysogenum fungus- major source The drug has a beta-lactam ring that binds bacterial enzymes that build the cell wall. Penicillin-binding proteins or transpeptidases will bind this drug in the active site instead of making the cross links in the peptidoglycan Cephalosporins Isolated from Cephalosporium acremonium mold Acts in a similar way to penicillins Fewer allergies & less resistance Two R groups to modify, four generations Non Beta-lactam Cell Wall Inhibitors Vancomycin Binds to ends of NAG-NAM precursor most effective in treatment of Staphylococcal infections in cases of penicillin and methicillin resistance (MRSA) or if patient is allergic to penicillin; toxic and hard to administer; restricted use BUT: seeing VRSA Bacitracin - narrow-spectrum produced by a strain of Bacillus subtilis; used topically in ointment, blocks peptidoglycan elongation (part of Neosporin) Isonaiazid (INH) and Ethambutol block an enzyme in mycolic acid synthesis...gram negatives!

Explain the role of candle jars and what microbes require or benefit from low oxygen culture.

Candle jars uses candles to decrease oxygen and increase CO2. This helps mircoaerophiles,areotolerant anaerobes and capnophiles grow and inhibits strict anaerobes. Use for the identification of Gonorhoeae

Explain the types of mechanisms by which antimicrobial drugs affect the growth of pathogens.

Cell wall synthesis

For each drug covered in class, be able to tell how it acts (blocks folic acid pathway, binds 30S subunit, etc.) and any major side effects or issues that were noted in class. Explain why the natural forms of penicillin had a narrow range for one group of bacteria.

Cell wall synthesis Drugs that are only effective on growing cells. ie NOT effective on dormant cells beta-lactams: Irreversibly bind to enzmyes that link NAM to NAM in the cell wall. This causes the cell membrane to bulge out during growth and eventually lyses. Penicillins and cephalosporins are the main beta-lactams

Explain the roles of Ffactor, F+cells and Hfr cells in bacterial recombination. Contrast what happens in these two types of conjugation.

Cells with the F factor are F+ cell that have the genetic sequencing for being a DNA donor. In normal conjugation the cell donates a free plasmid and both become F+ donors. The Donor sends a single strand over and both replicate it. Both plasmids are FREE in cytosol. Hfr cells (High frequency recombination) cells are much the same as regular conjugation but the F plasmid is integrated with the chromosome. When the donor joins only a portion of the F plasmid and extra DNA enter the F- cell. Since only a portion is donated the F- STAYS F- but has new DNA.

What kind of organisms use CO2 and chemical compounds for energy? What is the classification?

Chemoautotrophs: Hydrogen, sulfur, and nitrifying bacteria, some archaea

What kind of organisms use Organic compounds and chemical compounds for energy? What is the classification?

Chemoheterotrophs: Aerobic respiration: most animals, fungi (saprobes), and protozoa, and many bacteria. Anaerobic respiration: some animals, protozoa, bacteria and archaea. Fermentation: some bacteria, yeasts, and archaea

Define and apply the following terms: Chemotherapeutic agent, antimicrobials, prophylaxis, antibiotic, synthetic, and semi-synthetic. Contrast to common usage of the term antibiotic. Explain, using R groups, how semi-synthetic drugs arise (some examples will be given below).

Chemotherapeutic agent or drug: drugs that act on diseases Antimicrobics: drugs for treating microbial infections Prophylaxis: treatment or action to prevent infection Antibiotic: natural compounds from microbes against bacteria

Review what plasmids are (previous in course) and now name four of the many types of plasmids in terms of their function or role.

Circular small molecules of DNA the replicate independently of the chromosomes. Don't usually contain information for normal cell growth and reproduction but are sometime advantageous. There are four main types of plasmids 1. Fertility (F) plasmids: carry instructions for conjugation (the transfer of DNA to other bacteria. 2.Resistance (R) plasmids: carry genes that resist antimicrobial drugs or heavy metals. 3.Bacteriocin plasmids: carry genes for proteinaceous toxins called bacteriocins that kill similar species that lack the plasmid...out competes other similar bacteria 4.Virulence plasmids: carry instructions for bacteria to produce toxins that make them pathogenic. E. Coli are good in the gut until they carry this plasmid that then causes diarrhea.

Describe methods for collecting clinical samples. and explain the isolation methods used to obtain them.

Clinical samples are taken from blood, CSF, stomach, urine,lungs,and diseased tissue. Two isolation methods used are the use of a streak plate and pour plates. The only difference between the two is that the pour plate utilizes dilutions and CFUs can be seen in the agar.

Augmentin

Combines amoxicillin with clavulanic acid Clavulanic acid blocks the microbe's pencillinase

Contrast transcription and translation.

DNA sequences are transcribed to RNA sequences. RNA sequences are translated into proteins.

Contrast the terms degerming, sanitation, and pasteurization (see Table 9.1).

Degerming: The mechanical removal of germs using methods such as hand washing and alcohol rubs Sanitation: Removal of pathogens and microbes up to a governments standards Pasteurization: Use of heat to destroy pathogens and reduce the amount of spoilage causing microbes in food.

Selective Media

Encourage the growth of wanted microbes and inhibit the unwanted ones. For example a high salt agar could be made to encourage halophiles (staphylcoccus A.) and inhibit others. Or leaving out glucose selects for organisms that can obtain carbon from amino acids.

How does eukarryotic mRNA differ from those in bacteria?

Eukaryotoes make pre mRNA to make mRNA Eukayotic mRNA only carries instructions for ONE polypeptide Simultaneous transcription and translation of mRNA can NOT happen

Identify when filters might be used and the advantage and disadvantage of their use

Filters can be used with heat sensitive things such as ophthalmic products. Also used to count the amount of microbes in a fluid. HEPA filters used in rooms of people with infectious diseases such as TB and immunocompromised patients like those with AIDS and burn victims

Describe Transformation gene transfer

Gene transfers were discovered when living bacteria where seen to take DNA from dead bacteria and use it. A cell that has this ability is said to be competent. Cell wall and membrane alternations allow for DNA entry

Describe five types of physical methods of microbial control.

Heat, Refrigeration/Freezing, Desiccation(dehydrating) and lyophilization(dehydrating and feezing), Filtration, Osmotic Pressure, Radiation

Discuss the use of osmotic pressure in control and give an example. Do the same with desiccation and cold.

High concentration of salt or sugar to inhibit bacteria via osmotic pressure. When put in a hypertonic solution water is removed from the bacteria and renders in enzymes useless (bc they need an aqueous environment to function) Fungi are more able to grow in hypertonic environments

Transport Media

Hospital staff use this so microbes in samples (feces, urine etc..) stay alive for analysis. Also ensures other people are not infected. Anaerobic samples have less than an hour.

Contrast the regulation of an inducible operon with that of a repressive operon using lactose and tryptophan. Explain CAP in the lactose operon.

INDUCIBLE (CATABOLIC) The Lac Operon is inducible because a repression protein blocks the operator (structural genes can't be made). Note that this repression protein is constantly made by the regulatory gene. It can be taken off by an inducer (allolactose) that allows for transcription of mRNA for the production of lactase.Once lactase levels fall from catabolism, the proteins reattach to the operator. ***When Glucose is present lac operon is still shut off (cell like glucose way better) Glucose levels are high there is low cAMP. when Glucose is low cAMP is high. cAMP binds to CAP which binds to the promoter allowing RNA polymerase to do its thing (as long as there is still allolactose present) (E. Coli) REPRESSIBLE (ANABOLIC) Don't want too much Inactive repressors are being made by the regulatory gene. The operon is on transcribing mRNA to build trytophan. Once tryptophan levels are high they bind to the repressor making them active. Repressors bind to the operator shutting down transcription.

Fluconazole- Azole example

Interfere with ergosterol synthesis which are in fungi cell membrane instead of cholesterol in ours Example- use in vaginal yeast infections Side effects as well but a little less Many related drugs: Example: Miconazole for skin ringworm (dermal fungal infections)

Describe Transduction gene transfer

Involves the transfer of DNA from one cell to another via viruses. Can occur on both prokaryotes and eukaryotes. Think of viral replication steps, attachement, injection of DNA, host DNA dissolves, virus replicates and incorperates its DNA AND HOST DNA. The virus then infects another cell with HOST DNA with is introduced to the genome via recombination.

Describe several ways that development of resistance can be thwarted.

Make sure the drug is administered for a long enough time Use synergism to enhance the effects of the drugs. (Penicillin makes it easier for streptomycin to get into the cell to inhibit protein synthesis. Clavulanic acid inhibts B-lactamases Also using more than one drug kills off bacteria that may be resistant to one off the two. Limit the use of antimicrobial's to necessary cases Creating second generation drugs by adding side chains to defeat MDR strains

Anaerobic Media

Media that contains low or no oxygen for obligate anaerobes. The depth of a container could be sufficeint or the addition of reducing media that reduces oxygen and removes it from the media.

Differential Media

Media used to tell the difference between certain microbes. Can certain microbes grow in salty conditions? Do they metabolize lactose?

How is heat used to control microbes? List each type and their possible substituents.

Moist Heat More effective than dry heat because water is a better conductor of heat than air. The moist heat denatures proteins and destroys cytoplasmic membranes. Endospores, cysts, and some viruses can survive this. Autoclaving As pressure increases the heat required to boil water increases making an autoclave more effective than boiling. 121 Celsius and 15 psi for 10 minutes is the "gold standard" killing all microbes. Remember that a test tube with Bacilus Geothermo. may be present to determine if the rest a sterile Pasteurization Heating the food the destroy pathogenic microbes are used. Thermoduric organisms survive this process but do not cause disease in humans. Time and temperature varies with each food product. Batch method runs milk through tubes heated to 63 C for 30 minutes. Flash method runs milk through tubes heated to 72 for 15 seconds Ultra hight temperature uses tubes at 135 for 1 second Ultra-High-Temperature Sterilization Same process as above...kills all microbes creating an indefinite shelf life...creamer Dry heat Substances that would be destroyed with steam such as powders use dry heat ie ovens. Because air is less effective at being a conductor of heat it needs longer times and higher temperatures to promote oxidation (combustion) and protein denaturization.

Define mutation. Contrast spontaneous and induced. Define the major types

Mutation is a change in nucleotide base sequences particularly its genes. Point Mutations: A single nucleotide base pair is affected. i.substitutions - adding a base for another one ii. Frameshift mutations: changes proteins the most because it shifts all the triplets changing the meaning a. Insertion b. Deletions Inversion, duplication (all three base pairs repeted too many times) or transposition (moved around)

Linezolid (Zyvox)

Newer type of drug- blocks the mRNA from binding with both subunits Completely synthetic, may limit resistance or at least slow it! Able to act on MRSA and some other resistant species But very expensive- can cost several thousand for a course of treatment

Explain how osmotic pressure affects microbes

Obligate Halophiles: Microbes that survive in high osmotic pressure ie salt lakes Facultative halophiles: do not require high salt concentrations but can tolerate them. Staphylcoccus Aureus

Distinguish among aerobes, facultative anaerobes, anaerobes, aerotolerant and obligate versions and micoraerophiles. Describe them with tubes in lab

Obligate aerobes : need oxygen for the final electron acceptor in the ETC ( Only concentrated at the top of the tube) obligate anaerobes: Use something else for the final electron acceptor in the ETC (only concentrated at the bottom of the tube) Facultative anaerobes: (E Coli) - aerobic organisms that maintain life via fermentation or anaerobic respiration though efficiency is reduced in the absence of oxygen. (Dense at top of tube and less dense as you reach the bottom the the tube) Aerotolerant anaerobes - (lactobacilli) do not use aerobic metabolism but tolerate oxygen because they posses the enzymes that detoxify O2 poisonous forms. (Evenly dispersed through the tube) Microaerophiles : (Helicobacter pylori) require oxygen in 2%-10% concentration in the stomach. They have a limited ability to detoxify hydrogen peroxide and superoxide radicals.

What kind of organisms use CO2 and light for energy? What is the classification?

Photoautotrophs: Plants, algae, cyanobacteria use H2O to reduce CO2, producing O2 Green sulfur bacteria and purple sulfur bacteria do not use H2O or produce O2

What kind of organisms use organic compounds for carbon and light for energy? What is the classification?

Photoheterotrophs: Green non sulfur bacteria and purple non sulfur bacteria some archaea

Explain how oxygen can be deadly. What are the four toxic forms? Tie into catalase and Superoxide dismutase.

Oxygen can be deadly because it takes electrons from other compounds (oxidizes them). This causes a cascade effect which can be very harmful to a cell. O2 - Singlet oxygen -produced in aerobic metabolism- used by human white blood cells to oxidize pathogens. Caratonoids in plants are used to remove the excess energy from the singlet 02- superoxide radical - produced from the incomplete reduction of O2 during ETC in aerobes. Also produced during metabolism by anaerobes in the presence of oxygen- Superoxide radicals must be converted into hydrogen peroxide via superoxide dismutases. 2 O2- + 2H+ ==> H2O2 O2-2 (peroxide anion)- Formed from superoxide dismutase. aerobes detoxify the peroxide anion via catalase the converts 2 H2O2 into 2H2O and O2. Test is done by simply adding hydrogen peroxide to a colony of bacteria and seeing bubbles. Peroxidase uses NADH to reduce hydrogen peroxide into H2O and NAD+ OH- - hydroxyl radical - MOST reactive - formed from the incomplete reduction of H2O2. because of catalase and peroxidase the threat of this is minimal.

contrast the genomes of Prokaryotes, Eukaryotes and viruses

PROKARYOTIC GENOME Genomes can be found in a chromosome or plasmid Found in a haploid (one copy) which contains DNA and molecules associated with protein and RNA No membrane surrounds the chromosome Loops are 50,000 to 100,000 base pairs long archaea have DNA wrapped in histones EUKARYOTIC GENOMES Found in nuclear and extranuclear DNA. Chromosomes are diploid (two copies of DNA) They have linear DNA (prokaryotes have circular) DNA is in nucleus which is made from the nuclear envelope Much more complex than prokaryotes The DNA is negatively charged and wraps around a positively charged histone forming beads called nucleosomes. Nucleosomes clump to form chromatin. When genes are active less dense chromatin are called euchromatin. When genes are inactive the chromatin is more densely packed to form heterochromatin Before cell division chromosomes are highly dense.

Compare and contrast the major types of antimicrobial chemicals that were covered in class, and discuss the positive and negative features of each and their applications.

Phenols: Rings structures used for antimicrobial agents. Halogens or organic side chains are added. These chemicals denature proteins and disrupt the cell membrane of most pathogens. Negative effects include foul smells, skin rash, and nerve damage in infants. Alcohols Effective against everything besides fungal spores and bacterial endospores. Considered intermediate disinfectants. Tinitures are alcoholic mixtures with other antimicrobial chemicals in it. Alcohol must be with water to act on microbes. Swabbing alcohol is physical not chemical removal. Halogens iodine: Can't destroy protozoan cysts=iodophore is an organic molecule that slowly releases iodine. chlorine: used to treat water, bleach, Chlorine gas can be used to disinfect large spaces, chloramines used as long lasting anitseptics bromine: used in hot tubes because it evaps more slowly than chlorine at high temps fluorine used in water and mouthwash that disrupts the biofilm of bacteria Oxidizing agents Peroxides, ozone, and peracitic acid. These release radical oxygen species that are the most effective against anaerobes. Surfactants Soaps and detergents are used to decrease the surface tension between hydrogen bonds of water. QUATS quaternary ammonium compounds that distrput ion concentrations in cells. Not effective against mycobacteria, nonenveloped viruses or endospores. Psedumonas loves QUATS so its classified as low level disinfectants Heavy Metals attach to sulfur atoms in cysteine which inhibits protein functions. Low level STATIC Aldehydes terminal CHO which makes them highly reactive. Its expensive and can sterilize. Gaseous Agents Used to kill organisms in things such as pillows Enzymes lysozymes in tears degrade the peptidoglycan walls of bacteria

Identify the relative resistance of different microbial groups. Top being the most resistant...

Prions Endospores mycobacteria cysts of protozoas active protozoans Gram negatives Fungi Non enveloped Viruses Gram Positives enveloped viruses

How does RNA polymerase differ from DNA polymerase?

RNA polymerase.... unwinds and opens DNA ---helicase is NOT required does NOT need a primer transcribes only ONE of the DNA strands is slower than DNA polymerase III incorporates robnucleotides rather than deoxyribo..obviously has Uracil rather than thymine has less efficient proofreading capabilities error=1 per 10,000

Discuss the factors you consider in choosing an antimicrobial drug.

Readily available Cheap Chemically stable Easily administered Non toxic and non allergenic Selectively toxic against a wide range of pathogens

Know the purpose beginning point and end point for replication, transcription and translation.

Replication: Purpose is the copy the genome Beginning Point: Origin Ending Point: Origin or end of linear DNA Transcription: Purpose: make DNA Beginning Point: Promoter Ending Point: terminator Translation: Purpose to make polypeptides Beginning Point: AUG codon End Point: UAA,UAG,UGA stop codon

Drugs That Inhibit Nucleic Acid Synthesis

Rifampin Also made by Streptomyces sp. Inhibits RNA Polymerase Note drug interactions: birth control Turns fluids orange Quinolones (Fluoroquinolones) Broad spectrum effectiveness Concerns have arisen regarding the overuse of quinolone drugs CDC is recommending careful monitoring of their use to prevent ciprofloxacin-resistant bacteria

Identify three main categories of side effects of antimicrobial therapy.

Safety and Side Effects Toxicity: what are the negative side effects? Allergies: anaphylatic shock due to penicillin Disruption of Normal Micrbiota: antagonism described above

Describe Bacterial Conjugation of gene transfer

Sex pilus is used to transfer DNA from one bacteria to another

Describe the relative frequency of mutations and their effect on bacteria.

Silent mutations occur because of redundancy in coding. The same phenotype is expressed therefore its silent Missence mutations changed an amino acid. If the change is in a critical area of a protein there is an adverse effect (making the protein useless). If it is in a unimportant space nothing will happen (protein is functional) Nonsense mutations put a stop codon where a protein codon should be. This makes nearly all proteins nonfunctional. Only if there is a mutation at the end of the gene all frameshift mutations make the protein nonfunctional These only happen for every 1 in 10 Billion times mutangens can increase this to every 10^4 or 10^6 genes

What should be considered when treating and controlling microbes?

Site to be treated: The fact that harsh chemicals can not be used on living tissues and surgical equipment should be sterilized. Relative susceptibility of microorganisms: How susceptible are the organisms to chemicals and harsh environments? Bacilius and Colstridium (D.Dif) can form spores which are the most resilient of the life forms. Mycobacteria have the waxy layer making them resistant to heat and water based chemicals Cysts on protozoa protect against the entry of chemicals and radiation Environmental conditions: High temperatures and low pH with chemical agents work the best.

Compare the effects of -static versus -cidal control agents on microbial growth. Apply and use these terms correctly.

Static refers to the inhibition but not complete destruction of a microbe - Refrigeration and freezing Cidal refers to the destruction of a specific microbe - Germicides, aldehyde's, propylene and ethylene oxide

How does transcription differ between bacteria and eukaryotes?

Transcription occurs in nucleus,mitochondia, and cholorplasts with Eukaryotic cells where is occurs in the cytosol in bacteria. Eukaryotes have five different RNA polymerases while bacterial cells only have one sigma factor. Eukaryotes must have mRNA to begin translation

Contrast Sterilization, disinfection, and antisepsis, and describe their practical uses.

Sterilization: Destruction of all living microbes usually with pressure heat and moister. Disinfectants: Desturction of most living organisms with concentrated harsh chemicals on inanimate objects Antisepsis: Reduction in the number of microorganisms on living tissue. Less concentrated chemicals are used to protect the living tissues.

Drugs that block metabolic pathways

Sulfonamides Competitive inhibitor of the Folic Acid Pathway: looks like PABA Trimethoprim Looks like folic acid, so inhibits the next step from DHF to THF (what we call folic acid) SXT: Trimethoprim with sulfonamides SYNERGY UTIs, Pneumocystis in AIDS patients

Amphotercin B

Surfactant that disrupts membranes by binding ergosterol in fungal membranes (there is a loss of selective permeability and pores are created in the membrane)

Drugs that kill bacteria by messing with protein synthesis

Target the differences in Procaryote ribosomes; can also damage the mitochondria both have 70S Aminoglycosides Inhibit 30S subunit Streptomycin,Gentamicin,Tobramycin Tetracycline Block 30S subunit so tRNA can't enter A site,Broad spectrum Erythromycin & Macrolides Prevents release of tRNA on 50S subunit Fairly non-toxic Broad spectrum Used in atypical pneumonia, chlamydial infections

Explain how temperature affects microbes. Explain minimal, maximal, and optimal growth temperature.

Temperature: High temperatures denature proteins making them lose their functions. This is due to the hydrogen bonds breaking. Lipids become fragile at low temps and too fluid at high temps. Minimal growth temperature is the lowest temperature that a cell can undergo metabolism. maximal = highest temp until death. Optimal = best temp for metabolism

Describe three steps in RNA transcription, mentioning the following: DNA, RNA polymerase, promoter, 5' to 3' direction, terminator and Rho.

The three steps of RNA transcription in BACTERIA include the following: 1.Initiation of Transcription: RNA polymerase bind to specific DNA nucleotides called promoters that are on the beginning of a gene that initiates transcription. The sigma factor on the RNA polymerase recognizes the promoter. Then it unzips the DNA helix to form a bubble. Different sigma factors vary greatly in strength and determine the amount of gene transcription 2.Elongation of RNA transcript 5' -->3' Transcription does NOT occur at promoter but 10 sequences away. triphosphare ribonucleotides align with their complementary DNA bases. molecules are linked with phosphate bond. With ONLY ONE strand of DNA the RNA polymerase moves down the DNA elongating the RNA transcript. Like DNA polymerase RNA polymerase only links nucleotieds to the 3' end. 3. Termination of Transcription There are two ways termination happens here... i. self-termination- RNA polymerase encounters a region rich in GC slowing it down (making it harder to unwind) because of hydrogen bonding. This causes a change in RNA polymerase and it reaches A rich areas (which lack hydrogen bonding) causing it to break off the DNA. ii. Rho-dependent Termination- A Rho protein travels down to the 3' end of RNA and pushes between the polymerase and DNA breaking the attraction.

Explain what is meant by the generation time of bacteria

The time required for a bacterial cell to grow and divide. Or the time for a sample of cells to double in number. E. Coli and S. aureus have a generation time of 20 minutes or less (food poisoning)

Explain the principle of selective toxicity and apply it to what we have learned. Note how the whole course so far has led up to understanding this.

The toxin must be more toxic to the pathogen than to the host. Because protozoans and fungi are the most similar to use the toxin is less effective and can cause humans harm. Also viruses must use our cells and enzymes to reproduce making the toxin ineffective. General mechanisms of action the drug may stop the synthesis of a cell wall destroying bacteria and fungi Drugs that effect 70S ribosomes and not 80S Drugs that effect the cytoplasmic membrane Drugs that inhibit differences in metabolic pathways Nucleic acid synthesis Blocking of recognition of host cell

Compare the four basic categories of organisms based on their carbon and energy sources. Be able to tell where saprobes and parasites fit in.

There are Four main types of organisms 1. Ones that utilize an inorganic source of carbon as their sole source of carbon: Autotrophs 2. One that utilize an organic source of carbon as their sole source of carbon (catabolize reduced organic molecules such as proteins, carbs, amino acids, and fatty acids) : Heterotrophs 3. Organisms that acquire energy from redox reactions involving inorganic and organic chemicals are called :Chemotrophs 4. Organisms that use light as their energy source are called: phototrophs

Differentiate between ionizing and non-ionizing radiation as they relate to microbial control. Note concerns and issues involved with these.

There are two types of radiation, Particulate radiation which uses high speed subatomic particles,protons Electromagnetic radiation uses energy released from atoms that have undergone internal changes Ionizing radiation This kind of radiation uses wavelengths shorter than 1nm that are able to create ions that disrupts hydrogen bonds, oxidizes covalent bonds and creates highly reactive oxygen species. This damage causes DNA damage ie fatal mutations. The waves are produced by cathod ray machines. Used on food Nonionizing radiation Wavelengths greater than 1nm that cannot force electrons out of orbit and therefore do not create ions. UV light, visible light, infared radiation, and radio waves and microwaves. UV light creates pyrimidine dimers distorting the DNA destroying the cells abilities to replicated DNA and transcribe protein

Contrast Thermal Death time and Thermal death point

Thermal Death Time: The time it takes to sterilize a volume of liquid at a set temperature Thermal Death Point: The lowest temperature required to kill a bacteria in 10 minutes

Describe how populations of resistant microbes can arise. Tie this in with R plasmids and means of recombination.

They are usually out competed by normal bacteria because they spend more energy up keeping their resistance proteins. when the others die they are selected. These bacteria may have aquired these genes via horizontal gene transfer, transformation, transduction, or conjugation.

Why are anaerobes sensitive to oxygen?

They lack superoxide dismutases

Define genetic recombination and identify the three kinds. Tell why these are often called horizontal gene transfer.

This involves the exchange of nucleotide sequences between two DNA molecules between two homologous sequences. These are called horizontal gene transfers because microbes in the same generation transfer genes (rather than being passed down in vertical) very rare less than 1% The three types are transformation transduction bacterial conjugation

Describe the synthesis of polypeptides in translation, including steps of Initiation, elongation and termination.

Three steps of translation and there componets: INITIATION: Requires energy from GTP Initiation complex is formed from ribosomal subunits,protein factors, and tRNA(meth). NOTE initiation can occur in prokaryotes while mRNA is being made from DNA 1. The small ribosomes unit attaches to the bottom of the mRNA so that its P unit attaches to the start codon. 2. tRNA attaches to its complentry codons (start is meth) and attached using GTP-->GDP 3.The large subunit attached from the top forming the initiation complex ELONGATION 1. The next amino acid is carried in by another tRNA to the A site at the ribosome. The bond is stabilized by elongation factors and GTP 2.a ribozyme connects the last amino acids and the newly added one together (peptide bond). The polypeptide is now attached to the tRNA in site A. 3.GTP shifts the robosome down the mRNA and moves each tRNA into a different subunit. **Remember the units go in this order EPA (reverse ape) the tRNA in A moves to P and so on. 4.The tRNA in unit E is Ejected into the cytosol and another is Added to A (based off of the codons of the tRNA matching to mRNA) 5.Polyribosomes follow eachother producing multiple copies of the same polypeptide TERMINATION 1.Release factors halt elongation 2.Rybozymes activated by the stop codon cut the polypeptide chain from the final tRNA and causes the ribosomal subunit to dissipate from the mRNA

List six ways by which microbes can be resistant to drugs. Identify several major resistant strains today and what MDR means. Identify what beta-lactamases or penicillinases do. Identify efflux pumps.

Via beta-lactamases (penicillinase) that breaks the ring structure making it useless Change in structure or electrical charge of the cell membrane proteins (porines of Gram negatives) These mutations result from mutations in the chromosomes Resistant cells may change the target of the drug or cause it to bind to its substrate less effectively. This is seen with sulfonamides and erythmycin Cells may change their metabolic pathways that the drug acts on. For example a bacteria can obtain Folic acid from the environment instead of synthesizing it to gain resistance against sulfonamides. Multi drug resistance due to efflux pumps (ATP) that pump the drug out before it has had the chance to work Bacteria in Biofilms retard the diffusion of the drug into the cell. Also cells in the biofilm have slower metabolisms making drugs less effective Mycobacterium tuberculosis attaches a postively charged potein to DNA gyrase to block the target site of fluororquinolone

Enrichment Culture

What happens when the microbe of interest is too small to observe amongst many other microbes? The medium is selective for this microbe. Then samples are taken and incubated until that specific type is concentrated. Cold enrichment: E.Coli grows slowly in cold temperatures where the vibrio cholerae grows at the same rate. The more its processed the more cholerae will be present.

Describe binary fission as a means of reproduction.

When a cell grows twice its size and splits into two cells. This happens in four steps: 1.Replication of the chromosome (attached to cell membrane in prokaryotes and microtubules in eukaryotes) 2. The cell elongates and growth pushes chromosomes apart. (mitosis in Eukaryotes) 3. New cell membrane and wall across the midline 4. The cells either stay together forming chains or separated and divide in planes to form clusters

Defined Media

all the nutrients that a microbe needs are provided (and known) in the media.

What is a fastidious organism

an organism that needs many nutrients and growth factors to grow in a lab environment

Define antagonistic, synergistic, and symbiotic relationships

antagonistic - the relationship kills or harms one of the microorganism Synergistic - even though each could live independently, the relationship benefits both more than being alone Symbiotic - the two rarely can live without each other. They must be together to live

Contrast narrow and broad spectrum

broad spectrum are those that work on a wide variety of pathogens= tetracycline narrow spectrum are those that work only on a few pathogens=penicillin can NOT get into Gramnegatives and only work on Gram positives remember its not always good to use broad spectrum's because it disrupts microbial antagonism and could cause a worse secondary infection

Identify what capnophiles are and how you might provide the conditions they need. Are they important in the clinical lab?

capnophiles are organisms that require high CO2 and low O2. You could use candle jars to grow them. They are important because they include Gonorehoeae and need to be ID.

Contrast methods of counting bacteria and measuring their concentration and reproduction.

counting bacteria: DIRECT METHODS NOT requiring incubation: Microscopic Counts: cell counter in which the number of cells are counted and average in a unit measurement. Advantageous when a quick count is needed but its difficult to distinguish between dead and alive cells. also hard to count mobile cells Electron Counters: a coulter counter counts cells as they interrupt the electrical flow. Only works well on large yeast and protozoans. Not useful for bacteria because of debris and clumps Flow cytometry: same as electron counters but uses light with florescence for counting DIRECT METHODS requiring incubation: Serial Dilution and Viable Plate Counts: Stepwise dilutions that allow for the concentration of cells to be enough to count and be statistically significant. Calculates the CFU Membrane Filtration: Large samples with low concentrations of bacteria are poured through a membrane that traps them. They are then put on solid medium and the colonies are counted. in this case, the CFU is equal to the original large sample. Most Probable Number: Test tubes are diluted until zero is reached. The number of tubes that test positive from the dilutions are compared against a chart and can estimated the amount of bacteria in the sample. INDIRECT METHODS Turbidity: Using a spectrophotometer to see how cloudy the sample is. The more cloudy the more concentrated. This is the fastest way possible. However, the solution must be 1 million per liter or it is not turbid enough for accurate readings. It will also not work if the solution forms a sediment or accumulates at the top Metabolic Activity: metabolic rates helps determine how many microbes are in a sample by measuring pH, nutrient utilization and waste production. Dry weight: filamentous microorganisms are filtered dried and weighed Genetic Methods: The unique DNA sequences are isolated using Polymerase chain reaction (PCR) and hybridization of DNA are utilized. This can estimate the amount of bacteria and archaea in a sample.

Autotrophs that acquire electrons from the same inorganic source that gaven them carbon are called what?

lithotrophs

Complex Media

media is made from nutrients of more complex things like soy, yeasts, beef or protein. Exact chemical composition is unknown. Used to grow a wider variety of microbes and is used on organisms whose nutrient needs are unknown. Enriched complex media has blood added to provide heme and growth factors like NADH.

Heterotrophs that acquire electrons from the same organic molecule that gave them carbon are called what?

organotrophs

Explain how pH affects microbes

pH changes can interfere with hydrogen bonding of the microbe Neutrophiles: pH 6.5 - 7.5 Acidophiles: pH survive in pH of zero Obligates: require acidic environments and die if it approaches pH 7 Tolerant: Survive but do not prefer low pH Alkalinophiles: pH11.5 (Cholera)

Describe DNA's structure

phosphate sugar base. DNA strand run "antiparelle" in that one end runs 5' to 3' and the other strand runs 3' to 5'. The 3 terminates with the hydroxyl and the 5 terminates with a phosphate at carbon 5 of the sugar.

Define psychrophiles, mesophiles, psychrotrophs, and thermophiles.

psychrophiles: Thrive is temperatures below 15 C and even below 0 C. mesophiles: 20C to 40C these cause human disease and food spoilage psychrotrophst: above 15 and 20°C thermophiles: live in temperatures above 45 and 80 degrees C

Describe how quorum sensing can lead to formation of a biofilm

the process of which cells respond to the density of nearby cells. quorum sensing molecules sense how many and what type of cells are in the biofilm. An increase in cells increases quorum sensing molecules that bind to the cells. this causes new genes to be expressed changing the characteristics. this causes a synergystic relationship. The increases the ability of cells to build and maintain bio-film.

Define therapeutic index and therapeutic range or window.

therapeutic index - the ratio of the dose of the drug that is toxic to humans as compared to its minimum effective dose Therapeutic window or range is the range of concentrations you can use safely and effectively

Explain with transposons are and what they do. Why are the flanked with inverted repeats. Contrasts insertion sequences and complex transposons.

transposons are segments of DNA that can move from one location on the same or different molecule. It can go to any chromosome or plasmid (or between the two) plaindrome sequnces are at the ends (they are the same read forward and backwards) otherwise known as inverted repeat (IR) Complex transposons are responsible for antibiotic resistance. They contain more than just the transposition gene insertion sequences are the simplest of these- they just code for the enzyme that lets it move about and inserts it in a certain region somewhere else

Describe how you can determine what antibiotics to use on an isolate from a patient.

weather to use broad or narrow spectrum drugs The effectiveness of the drug Kirby-Bauer diffusion susceptibility test is used to see how far a drug inhibits growth and then its molecular weight is compared to see which is the most effective Minimum Inhibitory concentration test uses the effective antibiotic above to see at what minimum dose it impair the growth and reproduction of the microbe. The MIC is determined with the broth dilution test. Another test called the Etest uses a strip of continuously diluted drug to observe the results. An alternative to the MIC is the Minimum Bactericidal Concentration Test. Instead of the concentration needed to inhibit the growth, the MBC is used to determine the concentration to KILL the microbes. Both are measured in ug/ml Safety and Side Effects Toxicity: what are the negative side effects? Allergies: anaphylatic shock due to penicillin Disruption of Normal Micrbiota: antagonism described above