exam1 – Microbiology – Flashcards
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Unlock answersFeatures common to all cell types |
Bounded by a plasma membrane Contain cytoplasm Utilize energy and raw materials through metabolism Have both DNA and RNA Reproduce by cell division processes |
Prokaryotic Cells |
Have: no (or few) internal membranes Many processes that are associated with organelles in eukaryotes (e.g. Respiration, photosynthesis) are mediated by specialized regions (folds, etc.) of the plasma membrane in prokaryotes There is no membrane-bound nucleus in prokaryotes. Instead the DNA is located within a specialized region of the cytoplasm of the cell called the nucleoid region. There is no nuclear membrane surrounding the nucleoid. Includes: the bacteria & archaea the terms “prokaryotic cell” and “bacterial cell” often are used interchangeably Shapes & Arrangements: See shapes handout Sizes Typically ~ 0.1 - 20 m (with some exceptions) Typical coccus: ~ 1 m (e.g. Staphylococcus) Typical short rod: ~ 1 x 5 m (e.g. E. coli) Barely within the best resolution of a good compound light microscope |
Prokaryotic Cell Structures |
Plasma membrane The cytoplasmic matrix Ribosomes Cytoplasmic inclusions Nucleoid Prokaryotic cell walls Capsules, slime layers, and S-layers Bacterial flagella and motility Bacterial spores |
Functions of Prokaryotic Cell Structures |
Maintain Cell Integrity Regulate Transport Specialized Functions in Bacteria |
Prokaryotic Plasma membrane Structure |
Phospholipid (know the structure of a phospholipid) Bilayer with Associated Proteins Cholesterol is absent (except in the mycoplasma group) Hopanoids are often present in place of cholesterol. Similar in molecular structure to cholesterol. Some archaea have plasma membranes with unusual lipids and monolayer structures |
Internal Prokaryotic cell membranes “Mesosomes” |
folds of the plasma membrane Respiratory and Photosynthetic folds |
The cytoplasmic matrix |
Composition: Viscous aqueous suspension of proteins, nucleic acid, dissolved organic compounds, mineral salts Network of protein fibers similar to the eukaryotic cytoskeleton |
Prokaryotic Ribosomes |
Sites of protein synthesis Typically several thousand ribosomes per bacterial cell, depending on the state of its metabolic activity Smaller than eukaryotic ribosomes |
Cytoplasmic inclusions |
Glycogen Granules similar in structure to starch Poly--hydroxybutyrate granules Lipid droplets – mostly di and triglycerides Gas vacuoles - Metachromatic granules – common in chorynebacterium (Phosphate crystals or volutin granules) Sulfur Granules |
Nucleoid |
Chromosomal DNA Typically, one chromosome per bacterial cell Consists of double-stranded, circular DNA A few recently discovered groups have >1 chromosome per cell and linear chromosomes Plasmid DNA – smaller dbl stranded molecule that encode extra functions R-Plasmids – encodes antibiotic resistance in a cell. Antibiotic = substance secreted by one type of microbe to inhibit or kill another type of microbe Ex. PCN inhibits cell wall production F-Plasmids – F for fertility. Carry the genes for the process of conjugation (transfer of DNA from one cell to another) |
Prokaryotic cell walls |
Cell wall is a polymer found outside the plasma membrane that increases the structural strength of the cell and helps prevent osmotic lysis. |
Gram Staining |
Method developed by Gram in 1888 Gram-positive cells stain purple Gram-negative cells stain pink Later, it was discovered that the major factor determining Gram reactions is the bacterial cell wall structure “Gram-positive” & “Gram-negative” These terms can mean either: Staining results, or Types of cell wall structure |
Peptidoglycan Structure |
Composition A Polysaccharide Composed of alternating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) both are 6C sugars Peptide crosslinking between NAM units: Tetrapeptide or pentapeptide chains attached to NAM may “crosslink” adjacent PG strands This gives PG (pentaglycine) a net-like or mesh-like structure that contains the cell wall. Indirect cross linking: Found in Gram-positive bacteria TP chains of adjacent PG strands are linked by pentapeptide chains Direct crosslinking: Found in both Gm + and Gm - bacteria TP chains are directly attached to each other Proteins always contain the L-alanine amino acids |
Gram-positive Cell Wall |
Thick layer of Peptidoglycan 20-80 nm in thickness Extensively crosslinked, both with indirect & direct links Teichoic Acids
Periplasmic Space
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Gram-negative Cell Walls |
Outer Membrane 7 - 8 nm in thickness Bilayer of lipopolysaccharide and phospholipid, with outer membrane proteins Lipopolysaccharide contains: *
Lipid A is the bacterial endotoxin: triggers inflammatory effects and hemorrhaging Outer Membrane Proteins:
Periplasmic Space
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Variations on Cell Wall Architecture |
Acid-fast Cell Walls Many genera in the ;High GC gram-positive; bacterial group contain mycolic acids (very hydrophobic molecule, long aliphatic chain), embedded in the peptidoglycan Mycolic acids are a class of waxy, extremely hydrophobic lipids due to long hydrocarbon chains. Certain genera contain very large amounts of this lipid, and are difficult to gram stain These genera may be identified by the ;acid-fast; staining technique
Bacteria that naturally have no cell walls,
Archaea Have archaea cell walls with no peptidoglycan, often found in extreme environments. Many have cell walls containing pseudomurein, a polysaccharide similar to peptidoglycan but containing N-acetylglucosamine and N-acetyltalosaminuronic acid |
Capsules, slime layers, and S-layers |
Species and strain specific Structure of capsules ; slime layers
Structure of S-layers
Functions of capsules & slime layers
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Fimbriae and Pili |
Short, hair-like filaments of protein on certain bacterial cells Believed to function in attachment In a few species, specialized pili (sex pili, encoded by genes on the F plasmid. F pili are responsible for conjugation, F+ E. coli have F pili) enable the transfer of DNA from one cell to another (conjugation) |
Bacterial flagella and motility Function |
Motility Almost all motile bacteria are motile by means of flagella Motile vs. nonmotile bacteria
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Bacterial flagella and motility Structure |
Bacterial flagella are completely different in structure and mechanism than eukaryotic flagella. Filament Composed of the protein flagellin. Hook & Rotor Assembly Permits rotational "spinning" movement. Protein shaft mounted in a protein bushing. True wheel and axle movement, possibly the only example in the natural world. Energy comes from a H ion gradient across the plasma membrane via cellular respiration down the e- transport chain. See fig on slide 7. Know fig. on slide 6. |
Bacterial flagella and motility Mechanism of Motility |
“Run and Tumble” Movement controlled by the direction of the flagellar spin
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Bacterial flagella and motility Chemotaxis |
Response to the concentration of chemical attractants (nutrient molecules) and repellants As a bacterium approaches an attractant: the lengths of the straight runs increase As a bacterium approaches a repellant (nitrogenous waste products): the lengths of the straight runs decrease Mechanism of chemotaxis: Stimulation of chemotactic receptors in the PM: this triggers a “cascade” of enzymatic activity that alters the timer setting of the flagella rotors |
Bacterial spores Function |
To permit the organism to survive during conditions of desiccation, nutrient depletion, and waste buildup Bacterial spores are NOT a reproductive structure, like plant or fungal spores. Some of the most resistant and durable structures in biology. |
Bacterial spores Occurrence |
Produced by very few genera of bacteria Major examples
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Bacterial spores Significance in Medicine & Industry |
Spores are resistant to killing Cannot be killed by moist heat at 100°C (boiling) Killing spores by moist heat requires heating to 120°C for 15-20 min (autoclaving or pressure cooking) |
Bacterial spores Sporulation |
see fig on slide 43. The process of spore formation
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Bacterial spores Spore Germination |
When a spore encounters favorable growth conditions The spore coat ruptures and a new vegetative cell is formed |
Eukaryotic Cells |
Have: complex internal membrane system compartmentalization membrane-enclosed organelles DNA is enclosed in a membrane-bound nucleus Includes: animal & plant cells, fungi, & protists (protozoa & algae) |
Eukaryotic Cell Structures Nucleus |
Location of the cell’s DNA Major processes: DNA replication DNA expression (transcription) |
Eukaryotic Cell Structures Ribosomes |
Thousands are located suspended in the cytoplasm and attached to the rough endoplasmic reticulum Major process: Protein synthesis (translation) Ribosomes in the eukaryotic cytoplasm are larger than prokaryotic ribosomes Ribosomes are also found within mitochondria and chloroplasts; the ribosomes of these organelles are very similar in structure & size to prokaryotic ribosomes |
Eukaryotic Cell Structures Cytomembrane system |
Folded sacks of membranes within the cytoplasm Carry out processing and export of the cell’s proteins Major components:
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Eukaryotic Cell Structures Mitochondria |
Located in the cell’s cytoplasm Major process: cellular respiration The mitochondria oxidize nutrient molecules with the help of oxygen Some of the energy is conserved in the form of chemical energy (energy-containing chemical compounds) that can be used for biological processes Evolved from bacteria by a process called endosymbiosis |
Eukaryotic Cell Structures Chloroplasts |
Located in the cytoplasm of plant cells, algae cells, and certain protozoan cells Major process: photosynthesis Using the energy from light, CO2 is converted into carbohydrates such as glucose Evolved from bacteria by endosymbiosis |
Eukaryotic Cell Structures Cytoskeleton – |
maintain structure and shape, and allow for cytoplasmic streaming. Microfilaments – actin monomers, involved in muscle contractions. Microtubules – tubulin monomers, involved in intracellular transport. Intermediate filaments – Keratin for ex. |
Eukaryotic Cell Structures Vacuoles |
mostly involved in nutrient storage, predominantly found in plant cells. |
Eukaryotic Cell Structures Peroxisomes |
contain enzymes that break down peroxides. |
Structure of a “Virus Particle” |
Noncellular Biological Entity Contains either DNA or RNA (not both) Nucleic Acid is surrounded or coated by a protein shell (capsid) Some viruses possess a membrane-like envelope surrounding the particle |
Viral Replication |
No independent metabolism or replication Replicate only inside an infected host cell Do not replicate via a process of cell division Replicate via a process of: Attachment and Penetration Disassembly (uncoating) Synthesis of Viral Protein and Nucleic Acid Reassembly of new viral particles Release of new viral particles |
Nutrient Requirements Energy Source |
Phototroph Uses light as an energy source Chemotroph Uses energy from the oxidation of reduced chemical compounds |
Nutrient Requirements Electron (Reduction potential) Source |
Organotroph Uses reduced organic compounds as a source for reduction potential Lithotroph – ex. Thiobacillis – reduces sulfur to sulfuric acid. Uses reduced inorganic compounds as a source for reduction potential. |
Autotroph |
Can use CO2 as a sole carbon source (Carbon fixation). Thiobacillis. |
Heterotroph |
Requires an organic carbon source; cannot use CO2 as a carbon source |
Organic nitrogen |
Primarily from the catabolism of amino acids |
Oxidized forms of inorganic nitrogen |
Nitrate (NO3-) and nitrite (NO2-) |
Reduced inorganic nitrogen |
Ammonium (NH4+) |
Dissolved nitrogen gas (N2) |
(Nitrogen fixation) 80% of the atmosphere |
Inorganic phosphate |
H3PO4 titrate NaOH to increase the pH of the solution. (pH:6.8 = H2PO4- and HPO42-) |
Phosphate |
important buffering agent. |
Phosphoester linkage |
links 2 organic molecules. This is how phospholipds link in cellular membranes. |
Phosphate |
important regulatory molecule. Can cause proteins to undergo conformational changes that affect its function. |
Oxidized inorganic sulfur |
Sulfate (SO42-) |
Reduced inorganic sulfur |
Sulfide (S2- or H2S) |
Special requirements |
Amino acids Nucleotide bases Enzymatic cofactors or “vitamins” Some bacteria are able to manufacture all of their own essential biomolecules with only simple carbohydrates as a nutrient source. Thiobacillis is able to do this with only CO2. |
Prototroph |
A species or genetic strain of microbe capable of growing on a minimal medium consisting a simple carbohydrate or CO2 carbon source, with inorganic sources of all other nutrient requirements. Wild-type E. Coli is a prototroph that is able to subsist on only glucose. |
Auxotroph |
A species or genetic strain requiring one or more complex organic nutrients (such as amino acids, nucleotide bases, or enzymatic cofactors) for growth. |
Simple Diffusion |
Movement of substances directly across a phospholipid bilayer, with no need for a transport protein Movement from high low concentration No energy expenditure (e.g. ATP) from cell Small uncharged molecules may be transported via this process, e.g. H2O, O2, CO2 |
Facilitated Diffusion |
Movement of substances across a membrane with the assistance of a transport protein Movement from high low concentration No energy expenditure (e.g. ATP) from cell Two mechanisms: Channel & Carrier Proteins |
Active Transport |
Movement of substances across a membrane with the assistance of a transport protein Movement from low high concentration Energy expenditure (e.g. ATP or ion gradients) from cell Active transport pumps are usually carrier proteins |
Active transport systems in bacteria ATP-binding cassette transporters (ABC transporters): |
The target binds to a soluble cassette protein (in periplasm of gram-negative bacterium, or located bound to outer leaflet of plasma membrane in gram-positive bacterium). The target-cassette complex then binds to an integral membrane ATPase pump that transports the target across the plasma membrane. |
Active transport systems in bacteria Cotransport systems: |
Transport of one substance from a low high concentration as another substance is simultaneously transported from high low. For example: lactose permease in E. coli: As hydrogen ions are moved from a high concentration outside low concentration inside, lactose is moved from a low concentration outside high concentration inside |
Active transport systems in bacteria Group translocation system: |
A molecule is transported while being chemically modified. For example: phosphoenolpyruvate (found in the next to last step in glycolosis): sugar phosphotransferase systems (PTS) PEP + sugar (outside) pyruvate + sugar-phosphate (inside) |
Active transport systems in bacteria Iron uptake by siderophores: |
Low molecular weight organic molecules that are secreted by bacteria to bind to ferric iron (Fe3+); necessary due to low solubility of iron; Fe3+- siderophore complex is then transported via ABC transporter |
Liquid medium |
Components are dissolved in water and sterilized |
Semisolid medium |
A medium to which has been added a gelling agent Agar (most commonly used) doesn’t melt until it reaches 100o C Gelatin – is easily broken down by bacteria. Silica gel (used when a non-organic gelling agent is required) |
Chemically defined media |
The exact chemical composition is known e.g. minimal media used in bacterial genetics experiments |
Complex media |
Exact chemical composition is not known Often consist of plant or animal extracts, such as soybean meal, milk protein, etc. Include most routine laboratory media, e.g., tryptic soy broth |
Selective media |
Contain agents that inhibit the growth of certain bacteria while permitting the growth of others Frequently used to isolate specific organisms from a large population of contaminants |
Differential media |
Contain indicators that react differently with different organisms (for example, producing colonies with different colors) Used in identifying specific organisms |
“Growth” |
generally used to refer to the acquisition of biomass leading to cell division, or reproduction |
A “batch culture” |
a closed system in broth medium in which no additional nutrient is added after inoculation of the broth. |
Typically, a batch culture passes through four distinct stages: |
Lag stage Logarithmic (exponential) growth Stationary stage Death stage |
The mean generation time |
(doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in units of minutes. |
Growth rate (min-1) |
= 1/mean generation time |
Mean generation time |
= ln(2)/specific growth rate |
Mean generation time |
can be determined directly from a semilog plot of bacterial concentration vs time after inoculation |
Microscopic cell counts |
Calibrated “Petroff-Hausser counting chamber,” similar to hemacytometer, can be used Generally very difficult for bacteria since cells tend to move in and out of counting field Can be useful for organisms that can’t be cultured Special stains (e.g. serological stains or stains for viable cells) can be used for specific purposes |
Serial dilution and colony counting |
Also know as “viable cell counts” Concentrated samples are diluted by serial dilution The diluted samples can be either plated by spread plating or by pour plating |
Serial dilution (cont.) |
Diluted samples are spread onto media in petri dishes and incubated Colonies are counted. The concentration of bacteria in the original sample is calculated (from plates with 25 – 250 colonies, from the FDA Bacteriological Analytical Manual). A simple calculation, with a single plate falling into the statistically valid range, is given below: Serial dilution (cont.) If there is more than one plate in the statistically valid range of 25 – 250 colonies, the viable cell count is determined by the following formula: Where: C = Sum of all colonies on all plates between 25 - 250 n1= number of plates counted at dilution 1 (least diluted plate counted) n2= number of plates counted at dilution 2 (dilution 2 = 0.1 of dilution 1) d1= dilution factor of dilution 1 V= Volume plated per plate |
Membrane filtration |
Used for samples with low microbial concentration A measured volume (usually 1 to 100 ml) of sample is filtered through a membrane filter (typically with a 0.45 μm pore size) The filter is placed on a nutrient agar medium and incubated Colonies grow on the filter and can be counted |
Turbidity |
Based on the diffraction or “scattering” of light by bacteria in a broth culture Light scattering is measured as optical absorbance in a spectrophotometer Optical absorbance is directly proportional to the concentration of bacteria in the suspension |
Mass determination |
Cells are removed from a broth culture by centrifugation and weighed to determine the “wet mass.” The cells can be dried out and weighed to determine the “dry mass.” |
A “continuous culture” |
is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat. Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained. |
“-phile” |
is often used to describe conditions permitting growth |
“tolerant” |
describes conditions in which the organisms survive, but don’t necessarily grow |
“Obligate” (or “strict”) |
means that a given condition is required for growth: obligate thermophile requires heat in order to grow. an obligate thermophile requires elevated temperatures for growth |
“Facultative” |
means that the organism can grow under the condition, but doesn’t require it The term “facultative” is often applied to sub-optimal conditions A facultative thermophile may grow in either elevated temperatures or lower temperatures |
Temperature |
Most bacteria grow throughout a range of approximately 20 Celsius degrees, with the maximum growth rate at a certain “optimum temperature” |
Psychrophiles: |
Grows well at 0ºC; optimally between 0ºC – 15ºC |
Psychrotrophs: |
Can grow at 0 – 10ºC; optimum between 20 – 30ºC and maximum around 35ºC |
Mesophiles: |
Optimum around 20 – 45ºC |
Moderate thermophiles: |
Optimum around 55 – 65 ºC |
Extreme thermophiles (Hyperthermophiles): |
Optimum around 80 – 113 ºC |
Acidophiles: |
Grow optimally between ~pH 0 and 5.5 |
Neutrophiles |
Growoptimally between pH 5.5 and 8 |
Alkalophiles |
Grow optimally between pH 8 – 11.5 |
Halophiles |
require elevated salt concentrations to grow; often require 0.2 M ionic strength or greater and may some may grow at 1 M or greater; example, Halobacterium |
Osmotolerant (halotolerant) |
organisms grow over a wide range of salt concentrations or ionic strengths; for example, Staphylococcus aureus |
Strict aerobes: |
Require oxygen for growth (~20%) |
Strict anaerobes: |
Grow in the absence of oxygen; cannot grow in the presence of oxygen |
Facultative anaerobes: |
Grow best in the presence of oxygen, but are able to grow (at reduced rates) in the absence of oxygen |
Aerotolerant anaerobes: |
Can grow equally well in the presence or absence of oxygen |
Microaerophiles: |
Require reduced concentrations of oxygen (~2 – 10%) for growth |
Quorum Sensing |
A mechanism by which members of a bacterial population can behave cooperatively, altering their patterns of gene expression (transcription) in response to the density of the population In this way, the entire population can respond in a manner most strategically practical depending on how sparse or dense the population is. |
Quorum Sensing Mechanism: |
As the bacteria in the population grow, they secrete a quorum signaling molecule into the environment (for example, in many gram-negative bacteria the signal is an acyl homoserine lactone, HSL) When the quorum signal reaches a high enough concentration, it triggers specific receptor proteins that usually act as transcriptional inducers, turning on quorum-sensitive genes |
Peptide crosslinking between NAM units: |
Tetrapeptide or pentapeptide chains attached to NAM may “crosslink” adjacent PG strands This gives PG (pentaglycine) a net-like or mesh-like structure that contains the cell wall. |
Indirect cross linking: |
Found in Gram-positive bacteria TP chains of adjacent PG strands are linked by pentapeptide chains |
Direct crosslinking: |
Found in both Gm + and Gm - bacteria TP chains are directly attached to each other Proteins always contain the L-alanine amino acids |