Microbio Unit 2

Asexual Reproduction

A process by which organisms produce a genetically identifcal offspring without the fusion of gametes; Haploid only

Binary Fission

Single-celled eukaryote splits into two identical daughter cells

Budding

A new organism develops from some generative anomical point of the parents organism due to localized cell division

DNA replication (Replisome, Origin of Replication, Terminus Site)

Replisome- Group of proteins needed for DNA synthesis

Origin of Replication-Site at which replication begins

Terminus Site- Site at which replication is terminated, located opposite of the origin

Cytokenesis(Definition/steps of Septation, FtsZ, Min System, Divisome Formation)

The final physical process of cell division that divides the cytoplasm

Septation- Formation of cross walls between daughter cells

1. Selection of site for septum formation

2. Assembly of Z-ring which is composed of protein FtsZ

3. Assembly of cell wall synthesizing machinery

4. Construction of cell and septum formation

Min System(MinC, MinD, MinE)- prevent misplaced Z-ring formation at the cell poles

Divisome Formation- A macromolecular protein complex that constricts the inner and outer membrane to synthesize the septal cell wall

Nutritional requirements for microbial growth

Energy, Electrons, Carbon

Energy source for microbial growth

Light- Phototroph

Chemical oxidation- Chemotroph

Electron source for microbial growth

Electrons from organic material- Organotroph

Electrons from inorganic material- Lithotroph

Carbon source for microbial growth

CO2- Autotroph

Organic carbon- Heterotroph

Macronutrients

C, H, O, P, S, N

Micronutrients

Trace elements such as copper and zinc and growth factors

Prototroph

A microbe that can synthesize all necessary molecules required for growth

Autotroph

A microbe that is not able to synthesize all molecules required for growth and thus has a growth factor requirement

Influence of Environmental Factors on Growth

Salt concentration, pH, Temperature, Oxygen, Pressure, and Radiation Damage(Ionizing)

Halotolerant

The ability for an organism to grow and survive in environments with high concentrations of salt

Halophile

Grow optimally in salt concentrations about 0.2M

Extreme halophiles

Require salt concentrations of 2M to 6.2M

Acidophiles

Growth optimum between pH 0.0-5.5

Neutrophiles

Growth optimum between pH 5.5-7.0

Alkaliphiles

Growth optimum between pH 8.5-11.5

Psychrophiles

Growth optimum of temperature between 0C and 20C

Mesophiles

Growth optimum of temperature between 20C to 45C

Thermophiles

Growth optimum of temperature between 55C to 85C

Hyperthermophiles

Growth optimum of temperature between 85C to 113C

Obligate Aerobes

Oxygen is required for growth

Obligate Anaerobes

Growth ceases to exist in the presence of oxygen

Facultative Anaerobes

Both aerobic and anaerobic growth but greater growth in the presence of oxygen

Microaerophiles

Oxygen is required in low concentration

Aerotolerant Organisms

Only anaerobic growth but continues to grow in the presence of oxygen

Catalase

A enzyme that when exposed to oxygen then catalyzes the decomposition of Hydrogen peroxide into water and oxygen

2H₂O₂ → 2H₂O + O₂

Superoxide Dismutase

A antioxidant enzyme that converts toxic superoxide radicals (O2-) into molecular oxygen and hydrogen peroxide

Barotolerant

Adversely affected by increased pressure, but not as severely as non tolerant organisms

Barophilic (Piezophilic)

Require of grow more rapidly in the presence of increased pressure

Acylhomoserine Lactone (AHL)

A class of small signaling molecules used by Gram-negative bacteria for quorum sensing

Quorum Sensing

A chemical communication process that bacteria use to talk to each other, count their numbers, and coordinate group behavior based on their population density

Defined media

The exact chemical composition of the media is known

Complex media

Media contains ingredients whose chemical composition is not completely known

General Purpose Media

Support the growth of many different organisms(TSA)

Enriched Media

General purpose media supplements with special nutrients(Blood agar)

Selective Media

Encourages the growth of certain microorganisms and inhibits the growth of others(MaConkey agar)

Differential Media

Distinguish between different groups of microorganisms based on their biological characteristics(MaConkey agar)

Single-Cell Isolation

the process of separating an individual cell from a mixed population so it can be studied, cultured, or sequenced on its own. Developed by Robert Koch

Streak Plate for Isolated Colonies

a visible mound of identical microorganisms that grows from a single precursor cell after mechanical dilution

Spread Plate Method

Small volume of diluted mixture of cells is transferred and evenly spread over the surface of pre-solidified agar

Pour Plate Method

Sample is serially diluted and mixed with liquid agar and poured into a sterile culture dish

Lag Phase

The initial, preparatory stage where there is no increase in the number of living cells

Log Phase

Exponential increase in the number of living cells

Stationary Phase

Plateau in the number of living cells; the rate of cell division and cell death are roughly equal. This can be due to limitations in nutrients or oxygen, accumulation of toxic waste, or critical population density has been reached.

Death Phase

Exponential decrease in the number of living cells

Batch Culture

a closed fermentation system where all medium components and nutrients are added at the very beginning, and nothing new is added during the process except oxygen

Chemostat

The rate of incoming medium is equal to the rate of removal of medium from a vessel in a continuous culture

Direct Counting Methods

Counting Chamber, Flow Cytometry, or Membrane Filters

Indirect Counting Methods

Colony Count, Turbidity, or qPCR

Counting Chambers

An easy and inexpensive way to count both eukaryotic and prokaryotic cells. You cannot distinguish living from dead cells and cells may swim or move making it hard to count.

Electronic Counters (Flow Cytometry)

Microbes are forced through a small tube where a laser shines on them and a machine counts them. Dyes can be used to stain the cells to determine whether they are alive or dead.

Membrane Filtration (Useful for Dilute Samples)

Liquid samples are passed through a porous membrane in order to concentrate and count microorganisms

Colony Counts (Standard Plate Count)

A method used to estimate the number of live, cultural bacteria in a sample

Turbidity (Spectrophotometry)

A measure of how cloudy or murky the liquid is, a spectrophotometer is used to measure turbidity(uses light to see how crowed the liquid is)

qPCR (Quantitative Polymerase Chain Reaction)

a lab technique that copies a specific piece of DNA and measures its exact amount in real time during the process

The Great Plate Anomaly

Direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates because microscopic methods count dead cells whereas viable methods do not and different organisms have different requirements for growth

Physical Methods of Microbial Control

Heat: Dry(sterilization) and moist(sterilization/disinfection)

Radiation: Ionizing(sterlization) and non ionizing(disinfection)

Chemical Methods of Microbial Control

Gases: Sterilization/disinfection

Liquids: Animate(Chemotherapy) and inanimate(disinfection/sterilization)

Mechanical Methods of Microbial Control

Filtration: Air and liquids(sterilization)

Biological Methods of Microbial Control

Predators and Viruses: antisepsis

Toxin: Sterilization

Sterilization vs. Disinfection

Sterilization kills all living cells, spores, and viruses while disinfections kills, inhibits, or removes microorganisms that may cause disease.

Decimal Reduction Time (D-Value) and Time

Time required to kill 90% of the cells

Pasteurization

Controlled heating at temperatures well below boiling(Disinfection)

Disinfectants vs. Antiseptics

Disinfection kills, inhibits, or removes microorganisms that may cause disease while antiseptics prevent the infection of living tissue.

Bacteriostatic Agents

Inhibit bacterial growth

Bactericidal Agents

Directly kill bacteria without lysis

Bacteriolytic Agents

Kill bacteria with lysis

Disk Diffusion Tests

Disks impregnated with specific drugs are placed on agar plated and inoculated with test microbes(Kirby-Bauer Method)

E-Test

Uses strips rather than a disk impregnated with specific drugs

Paul Ehrlich (1904)

Developed the concept of selective toxicity and identified dyes that can effectively treat African sleeping sickness

Sahachiro Hato (1910)

Working with Ehrlich, identified an arsenic compound that treated syphilis

Gerhard Domagk, Jacques & Thérèse Trefouel (1935)

Discovered sulfoamides and Sulfa drugs

Ernest Duchesne & Alexander Fleming

Duchesne first discovered but it was lost and Fleming accidentally discovered it but could not purify the chemical

Florey, Chain, and Heatley

Were able to purify the compound and inject into people to cure infection

Actinobacteria (Streptomyces) as Antibiotic Producers

Streptomyces is the most important genus of antibiotic producers (~50% of all isolated Streptomyces produce antibiotics)

Semisynthetic Antibiotics modifications improve…

Toxicity reduction, absorption, stability, spectrum of activity, and resistance to modifying enzymes

Selective Toxicity

Ability of a drug to kill of inhibit pathogen while damaging the host as little as possible

Therapeutic Dose vs. Toxic Dose

Therapeutic Dose: drug level required for clinical treatment

Toxic Dose: drug level at which the drug becomes to toxic for the patient and produces side effects

Main Modes of Action of Antimicrobial Drugs

Inhibitors of the cell wall, protein synthesis, nucleic acid synthesis, and metabolic antagonists

Inhibitors of Cell Wall Synthesis

Penicillins; crucial feature is the b-lactam ring compounds (Penicillins, Vancomycin, Cephalosporins)

Protein Synthesis Inhibitors

Many antibiotics bind specifically to the bacterial
ribosome(Erythromycin, Tetracyclines), it targets:

– aminoacyl-tRNA binding
– peptide bond formation
– mRNA reading
– translocation

Nucleic Acid Synthesis Inhibitors

agents that block or interfere with the replication of DNA or the transcription of DNA into RNA in microorganisms and cancer cells(Quinolones (Ciprofloxacin, Nalidixic Acid))

Metabolic Antagonists

Act as antimetabolites(block functioning of metabolic pathways) and are structural analogs(molecules that are similar and compete with naturally occurring metabolic intermediate) (SULFA DRUGS)

Antifungal Drugs

Used to kill or stop the growth of fungi, they have fewer effective agents compared to bacteria, they target Ergosterol in cell membranes They treat superficial mycoses than systemic infections. Low therapeutic index.(Polyenes, Azoles)

Antiprotozoan Drugs

Used to kill or inhibit the growth of protozoan parasites. Mechanism of the drug is not well known

• Chloroquine & Mefloquine – Malaria

• Metronidazole – Entamoeba Infections

• Atovaquone – Toxoplasma gondii

Antiviral Drugs(Fusion Inhibitors, Nucleoside Analogs, Inhibition of release, Protease inhibitors, Interferons)

Drugs that treat, prevent, or suppress specific viral infections.

-Fusion Inhibitors: Prevents virus form fusing with host cell

-Nucleoside Analogs (Most Successful Class): Many block reverse transcriptase

-Inhibition of release: Prevents the release of influenza

-Protease inhibitors: Inhibit cleaving large viral proteins into active individual proteins

Interferons- small proteins that stimulate uninfected host cells to produce proteins that inhibit viral replication

Antibiotic Resistance Mechanisms

1. Lack of Target Structure

2. Antibiotic Exclusion

3. Antibiotic Inactivation

4. Target Modification

5. Resistant Biochemical Pathways

6. Efflux Pumps

1. Lack of Target Structure: Some Organisms Naturally Resist Certain Antibiotics

2. Antibiotic Exclusion: Inability of the Drug to Enter the Microbe

3. Antibiotic Inactivation: Enzymatic Modification or Breakdown of the Drug

4. Target Modification: Structural Changes in the Antibiotic Target

5. Resistant Biochemical Pathways: Alternative Metabolic Routes

6. Efflux Pumps: Active Removal of Antibiotics from the Cell

Biofilm

Community of microorganisms attached to surfaces and embedded in extracellular matrix(Dental plaque and Cather infections)

Turbidostat

Regulates the flow rate of media through the vessel to maintain predetermined turbidity of cell density

Sulfa Drugs

synthetic, sulfur-containing medications that stop bacteria from growing

Penicillin

Game changer in the fight against infection

Therapeutic Index

Ratio of toxic dose to therapeutic dose