Linnaeus: Binomial Nomenclature: scientific name for organism = genus + species names (examples: Homo sapiens, Staphylococcus aureus (bacteria that live on the skin), Escherichia coli (bacteria found in the colon))
Types of microorganisms:
Bacteria: unicellular; prokaryotic
- 3 common shapes: coccus (spherical or ovoid); bacillus (rodlike); spiral (corkscrew or curved)
- also star-shaped (genus Stella) or square (Archaea) genus Haloarcula) forms
- cell wall with peptidoglycan
- cell division by binary fission
- autotrophic & heterotrophic forms
- some motile (flagella or axial filament)
Archaea: unicellular; prokaryotic
- cell walls in some (lacks peptidoglycan)
- often found in extreme environments
- 3 main groups: methanogens (release methane as a waste product of respiration); extreme halophiles (live in extremely salty environments); extreme thermophiles (live in hot sulfurous water)
Fungi: unicellular (yeasts) & multicellular forms; eukaryotic
- cell wall with chitin
- molds: form mycelia (branching, filamentous hyphae)
- reproduce sexually or asexually
- heterotrophic; saprophytic
Protozoa: unicellular; eukaryotic
- move by flagella, cilia, or pseudopods (cytoplasmic extensions)
- heterotrophic; free-living or parasites
- sexual or asexual reproduction
Algae: unicellular, colonial & multicellular forms; eukaryotic
- unicellular forms most important in microbiology
- cell wall with cellulose
- autotrophic; found in fresh & salt water, soil & in association with plants & fungi (lichens)
Viruses: abiotic (nonliving); acellular
- nucleic acid core (DNA or RNA) surrounded by protein coat
- some have a lipid envelope
- parasitic (need host to reproduce)
Multicellular Animal Parasites:
- helminths: flatworms & roundworms
Classification of Microorganisms:
3 domains proposed by Carl Woese:
Bacteria: cell wall with peptidoglycan
Archaea: cell wall without peptidoglycan
Eukarya: protists, fungi, plants & animals
Historical Perspectives:
Robert Hooke (1665): first to observe cells under a microscope
- cell theory: all living things composed of cells (Schleiden and Schwann, 1830s)
Antoni van Leeuwenhoek (1673-1723): observed live microorganisms (³animalcules² later found to be bacteria & protozoa)
Spontaneous Generation vs. Biogenesis:
Spontaneous generation: the idea that living things can arise from nonliving matter
- Francisco Redi: maggots appeared on decaying meat only after flies were able to lay eggs on the meat
- John Needham: claimed microorganisms could arise spontaneously from heated nutrient broth
- Lazzaro Spallanzani: suggested Needham¹s results were due to microorganisms from the air
- Rudolf Virchow: Biogenesis: living things only arise from preexisting cells/organisms
- Louis Pasteur: repeated Needham¹s experiments using flask with bent neck – air was able to enter the flask, but bacteria from the air were not able to travel through the neck of the flask – no contamination proof for biogenesis.
Pasteur¹s results were used to develop aseptic techniques to prevent contamination by unwanted organisms.
Fermentation: Pasteur found that yeast ferment sugars to alcohol, & bacteria can convert (oxidize) the alcohol to acetic acid (spoilage).
Pasteurization: Pasteur also found that one could heat alcoholic beverages & milk just enough to kill most of the bacteria that cause the spoilage.
Germ Theory of Disease: microorganisms may cause disease
- Ignaz Semmelweis: showed physicians that did not disinfect hands could transmit infections (childbirth fever) to patients
- Joseph Lister (1760s) introduced the use of disinfectants to clean surgical wounds & control human infections
- Agostino Bassi (1835): proved a silkworm disease was caused by a fungus
- Louis Pasteur: using Bassi¹s data, showed that another silkworm disease was caused by a protozoan
- Robert Koch: using Bacillus anthrax in cows, proved that microorganisms can cause disease the experimental steps used form the basis of Koch¹s postulates
Vaccination: confers immunity to a specific disease by inoculation with a vaccine a preparation of living avirulent microorganisms or killed pathogens, isolated components of pathogens, or recombinant DNA
- Edward Jenner (1798) showed that inoculation with material from the cowpox virus provides humans with immunity to smallpox
- Pasteur later showed that avirulent bacteria could be used as a vaccine for fowl cholera
Chemotherapy: the chemical treatment of disease chemotherapeutic agents can be in the form of synthetic drugs and antibiotics
- Paul Ehrlich: used salvarsan (an arsenic-containing chemical) in the treatment of syphilis
- Alexander Fleming: isolated penicillin as a component of the mold Penicillium that inhibited growth of a bacterial culture
- Microorganisms can relatively rapidly develop resistance to a chemotherapeutic agent by mutation
Modern Developments in Microbiology:
Bacteriology: the study of bacteria
- began with van Leeuwenhoek¹s first observation of bacteria under the microscope
- new pathogenic bacteria are still being discovered
- Heide Schulz: discovered a bacterium that could be seen without a microscope (Thiomargarita namibiensis)
Mycology: the study of fungi
- rising fungal infection rates have led to the need for new techniques for diagnosing & treating fungal infections
Parasitology: the study of protozoa & parasitic worms
- previously unknown parasitic diseases are being discovered through clearing rain forests and developing in the immunosuppressed (cancer patients; AIDS patients)
- genomics (the study of all genes of an organism) has allowed the classification of bacteria & fungi according to their relationships with other bacteria, fungi & protozoa
Virology: the study of viruses
- Dmitri Iwanowski (1892): showed the agent that caused mosaic disease of tobacco passed through a filter fine enough to stop bacteria
- the agent, smaller than bacteria, was found in 1935 by Wendell Stanley to be a virus (tobacco mosaic virus (TMV))
- the development of the electron microscope in the 1940s has allowed virologists to observe the structure of viruses in detail
Immunology: the study of immunity
- began in Western culture with the development of Jenner¹s vaccine in 1796
- vaccines are now available for numerous diseases and new vaccines are under development
- interferons inhibit viral replication; can be used in treatment of viral infections (but generally not in the cure of these infections)
- Rebecca Lancefield (1933): proposed classification of streptococci by serotypes (variants based on differences in cell wall components)
Recombinant DNA Technology: microorganisms can be genetically engineered to manufacture necessary substances (e.g.: hormones)
- Paul Berg (1960s): showed fragments of human DNA could be attached to bacterial DNA to form recombinant DNA
- molecular biology studies how genetic information is carried in DNA and how DNA directs protein synthesis
Microbes and Human Welfare
- microorganisms degrade dead plants & animals and recycle their chemical elements
- bacteria used to decompose organic matter in sewage
- bioremediation: uses bacteria to clean up toxic wastes
- bacteria that cause insect disease are used as biological controls of insect pests
- biotechnology uses microbes to make foods & chemicals
- in gene therapy, viruses are used to carry replacements for defective or missing genes into human cells
- genetically engineered bacteria are used in agriculture to protect plants from frosts & insects and improve shelf life
Microbes and Human Disease
- normal microbiota (flora): microbes that are normally present on the skin & in the body
- an infectious disease is one in which pathogens invade a susceptible host
- an emerging infectious disease is a new or changing disease showing an increased incidence in recent past or a potential future increase in incidence
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Elements are composed of atoms
Atoms are composed of subatomic particles:
- protons (+ charge)
- neutrons (no charge)
- electrons (- charge)
The atomic number of an atom = the number of protons in its nucleus
- the periodic table is grouped according to atomic number (Hydrogen (H) has an atomic number of 1, Helium (He) has an atomic number of 2)
The atomic mass (mass number) of an atom is the number of protons + the number of neutrons in its nucleus
- the mass of electrons is negligible
- Hydrogen (H) has a mass number of 1 (no neutrons), Helium (He) has a mass number of 4
The atomic weight of an element is the average of the relative weights of all the isotopes of that element (the atomic weight of Hydrogen is 1.008).
Isotopes are atoms of an element that have the same number of protons (atomic number) but different mass numbers (different numbers of neutrons).
- Examples: 12C, 13C, 14C
- Radioisotopes are unstable isotopes that spontaneously decay into more stable forms (it can take up to thousands of years for half the atoms in an element to decay to the stable state)
- Radioactivity can be detected with scanning devices, & radioisotopes can be incorporated into biological moleculesthis makes radioisotopes useful tools for biological research & medicine).
Molecules: 2 or more atoms held together by chemical bonds
- when 2 or more atoms of the same element bind, they form a molecule of that element
- when 2 or more different atoms bind, they form one molecule of a compound
Chemical Bonds:
Electrons of an atom differ in amount of potential (stored) energy
- electrons closest to the nucleus have the least potential energy (nonbonding electrons)
- electrons farthest from the nucleus have the greatest potential energy (valence or bonding electrons)
First energy level can contain a maximum of 2 bonding electrons
Second energy level, and all additional energy levels, can contain a maximum of 8 bonding electrons
Octet rule: except for the first energy level, the outermost energy level is most stable when it has 8 bonding electrons (the first energy level is most stable with its maximum of 2 bonding electrons)
Bonding:
Ionic Bonding: transfer of electrons from one atom to another
- results in ions: charged particles resulting from charge imbalance (greater or fewer electrons than protons) due to electron transfer
- Examples: NaCl, MgCl2, Na2O
- Chemical formulas of compounds based on # of valence electrons (example: from above: MgCl2, Mg has 2 valence electrons to donate, while Cl can only accept 1, so two Cl atoms are needed to accept the 2 valence electrons donated by one Mg atom)
Covalent bonding: sharing of electrons between 2 or more atoms
- each atom acquires an octet of valence electrons (electrons in outermost shell). Examples: CH4, O2, H2, C6H12O6
Polar Covalent bond: unequal sharing of electrons between atoms in a covalent bond (e.g.: water, H2O)
- due to difference in electronegativities of atoms in bonds
- more electronegative atom has slight negative charge, less electronegative atom has slight positive charge
- asymmetrical differences lead to polar molecules
Hydrogen Bonding:
- bond between a slightly positive hydrogen atom of one molecule, and a slightly negative atom (usually oxygen or nitrogen)of the same or another molecule
- weak bonding compared to ionic and covalent bonding, but many bonds increases strength
- good example is water molecules
Chemical Reactions:
Synthesis (combination) reaction: atoms or molecules combine to form a larger molecule
- metabolic synthesis reactions are termed anabolic reactions
Decomposition reaction: a molecule is broken down into smaller molecules, or its constituent atoms
- metabolic decomposition reactions are termed catabolic reactions
Exchange (displacement) reaction: components of the reactant molecules change partners, resulting in different molecules as products
- example: neutralization reactions (strong acid + strong base -> salt + water) HCl + NaOH -> NaCl + H2O
Oxidation-Reduction reaction (redox reaction): an electron donor is oxidized while an electron acceptor is reduced
Oxidation: loss of electrons or H atoms
Reduction: gain of electrons or H atoms
- example: cellular respiration glucose is oxidized to CO2 and oxygen is reduced to water
Exergonic reactions: release energy
Endergonic reactions: require (absorb) energy
In cells, the energy released by ATP hydrolysis (an exergonic reaction) is used to fuel endergonic reactions such as metabolism & muscle contraction
All chemical reactions are, in theory, reversible however, many biological reactions show little or no tendency to go in the reverse direction
- chemical equilibrium: neither the forward nor the reverse reaction is reversible (for each product molecule formed, one product molecule breaks down)
The rate of a chemical reaction is influenced by:
1. temperature: molecules move faster as the temperature is increased (increases collisions) moderate temperature is best; high temperatures often denature (inactivate) enzymes
2. particle size: small molecules move faster (more (forceful) collisions)
3. concentration: usually increased reactant concentrations increases rate (more collisions)
4. catalysts: increase rate of chemical reactions without themselves being changed in the reaction enzymes are biological catalysts
Biochemistry:
Organic Molecules: Carbon-based molecules
- Carbon atoms are bonded mainly to atoms of hydrogen, oxygen, and nitrogen, as well as some other atoms
- Always contain carbon and hydrogen
- Always covalent-bonding
Inorganic Molecules: Molecules that do not contain carbon and hydrogen (e.g.: salts, strong acids and bases, metal compounds)
- usually ionic-bonding
Properties of Water:
1. resists changes in temperature (in part due to hydrogen bonding)
2. Water has a high heat of vaporization
- high boiling point (100 degrees Celsius)
- heat of vaporization (energy required to convert water to steam) is 540 calories (very high)
- energy needed to break hydrogen bonds
3. Water is the universal solvent:
- many compounds dissolve in water (separate into ions)
a. ionic compounds : salts
b. polar covalent compounds
- Water is a polar molecule: the negative ends of water molecules are attracted to positively charged ions, and the positive ends of water molecules are attracted to negatively charged ions
4. Reactivity: water is an important reactant in many chemical reactions, used in the buildup & breakdown of organic macromolecules
- Dehydration synthesis (condensation) reactions: formation of a bond with removal of water
- Hydrolysis reactions: breaking of a bond by the addition of water
pH scale (power of hydrogen): indicates acidity or basicity of solution
- ranges from 0 (strong acid) to 14 (strong base); pH=7 is neutral
- water ionizes to release hydrogen ions and hydroxide ions
Acid: molecules that release hydrogen ions (H+) when dissolved in water
- acids are hydrogen ion (proton) donors
Base: molecules that release hydroxide (OH-) ions , or increase the number of hydroxide ions available, when dissolved in water
- bases are hydrogen ion (proton) acceptors
Buffers: maintain stable pH of solution (resist changes in pH)
- Buffers can take up excess hydrogen or hydroxide ions
- Buffers have acidic and basic components
- Blood uses carbonic acid (acidic) – bicarbonate ion (basic) buffer system
- normal pH of blood is between 7.35 & 7.45
- Bicarbonate ions take up added hydrogen ions, and carbonic acid takes up excess hydroxide ions
Carbohydrates: (contain carbon, hydrogen, and oxygen atoms)
Monosaccharides: simple sugars with a backbone of 3 to 7 carbon atoms
- Glucose is a 6-carbon sugar (hexose) found in the blood of animals, and Fructose is a hexose found in fruits
- Ribose is a 5 carbon sugar (pentose) found in RNA (in DNA, the pentose sugar is deoxyribose)
Disaccharides: 2 monosaccharides joined by condensation
- Maltose (a disaccharide in the digestive tract) = glucose + glucose
- Lactose ( a disaccharide in milk) = glucose + galactose (another hexose)
- Sucrose (a disaccharide in fruits & vegetables) = glucose + fructose
Polysaccharides:
1. Glycogen is a highly branched polymer of glucose, and is the storage form of carbohydrates in animal cells (stored in liver cells)
2. Starch is a more moderately branched polymer of glucose, and is the storage form of carbohydrates in plant cells
3. Cellulose is an unbranched polymer of glucose, with adjacent chains held together by hydrogen bonds, giving it a very rigid structure. It is the major structural component of plant cell walls
Lipids:
In the form of neutral fats (fats or oils)
One triglyceride = Glycerol + 3 fatty acids
- Glycerol has 3 carbon atoms and 3 hydroxyl groups
- Fatty acids have a long hydrocarbon (carbon + hydrogen) chain with a carboxylic acid group at one end
- Condensation joins a fatty acid to each of the hydroxyl groups in glycerol
- The condensation reaction removes the ionizable functional groups from fatty acids and glycerol; hence, these molecules are very hydrophobic
Saturated fatty acids: each carbon atom in the fatty acid molecules have the maximum number of bonded hydrogen atoms (each carbon is saturated with hydrogen atoms); there are no C=C double bonds
Unsaturated fatty acids: one or more carbon atoms in the fatty acid molecule has less than the maximum number of bonded hydrogen atoms; there are one or more C=C double bonds
In animal cells, neutral fats are in the form of fats
- fats are solid at room temperature
- fats contain more saturated fatty acids
In plant cells, neutral fats are in the form of oils
- oils are liquid at room temperature
- oils contain more unsaturated fatty acids
Phospholipids = Glycerol + 2 fatty acids + 1 polar (phosphate-containing) head group (instead of third fatty acid in triglyceride)
- allows molecules to have hydrophobic end (2 fatty acids) and hydrophilic (phosphate) end
- these molecules are the subunits of biological membranes in cells (e.g.: plasma membrane): the polar head group is in contact with water on the inside and outside of the cell, and the hydrophobic fatty acid chains are buried in the center of the membrane
Bacteria of the genus Mycobacterium contain waxy complex lipids in their cell walls composed of mycolic acids (long hydroxylated branched-chain fatty acids)
Steroids are composed of 4 fused carbon rings plus some variable functional side group
- Cholesterol is a structural component of the plasma membrane in animals, and is used in the synthesis of vitamin D and bile salts
- Cholesterol is a precursor form of steroid that is modified to produce several other types of steroids
- Steroids function as hormones in animal cells
- Sterols (steroids with an attached hydroxyl group) are important constituents of the plasma membrane in animals & mycoplasmas they prevent packing of the fatty acid chains
Proteins:
Proteins are composed of chains of amino acid monomers
- There are 20+ different amino acids in cells of living organisms
- Amino acids have a basic core structure plus an additional functional side chain
- Each amino acid has a central carbon bonded to an amino group, a carboxylic acid group, a hydrogen atom, and the remaining side chain (R group); it is the R group that differs in different amino acids
- Condensation of two amino acids in a growing polypeptide chain results in the formation of a peptide bond; the peptide bond joins the amino group of one amino acid to the carboxylic acid of the previous amino acid in the polypeptide the R groups do not normally bond between amino acids (the exception is cysteine, which forms disulfide (S-S) bonds within and between polypeptide chains for added strength
- Hydrolysis of peptide bonds occurs between specific amino acids in a protein by the activity of specific protease enzymes (e.g.: pepsin)
- R groups can be nonpolar & hydrophobic, or polar & hydrophilic, depending on the atoms present
- most amino acids are stereoisomers... they can exist in 2 forms (L-isomers and D-isomers)
- proteins contain L-isomer amino acids
- D-amino acids are rare... but do occur in the peptidoglycan layer of bacterial cell walls
Polypeptide: a chain of many amino acids joined by peptide bonds
- a protein can be composed of one or several polypeptide chains
Protein Structure
Primary Structure: the sequence of amino acids in a polypeptide chain
Secondary Structure: the formation of discrete structures involving several amino acids within a polypeptide chain (held together by hydrogen bonds)
a. Alpha helices
b. Beta pleated sheets
Tertiary Structure: the conformation of the polypeptide chain following interactions of regions of secondary structure
- interactions can involve hydrogen bonds, ionic bonds and covalent bonds (disulfide bonds)
- polypeptide folds into a specific, consistent, and reproducible structure
Quaternary Structure: structure following interaction and bonding between two or more (the same or different) polypeptide chains
- hydrogen or ionic bonding between polypeptide chains
Denaturation: disruption of specific 3D structure of a protein by increasing temperature (boiling) or changing pH
- may be reversible (remember: the structure of a given polypeptide is specific as well as consistent and reproducible)
Nucleic Acids:
Nucleic Acids are polymers of nucleotide monomers
- a nucleotide = a pentose sugar + a phosphate + a nitrogenous (nitrogen-containing) base
- In RNA (Ribonucleic Acid), the pentose is ribose
- In DNA (Deoxyribonucleic Acid), the pentose is deoxyribose (missing a hydroxyl group at carbon # 2 relative to ribose)
DNA:
DNA is the genetic material of the cell (inherited from parents)
- Composed of a sequence of four different nucleotides
- The 4 nucleotide subunits of DNA are named after the nitrogenous base each
contains; the 4 bases are : adenine (A)
cytosine (C)
guanine (G)
thymine (T)
- Adenine and Guanine are purine bases, and have very similar structures
- Cytosine and Thymine are pyrimidine bases, and have very similar structures
- DNA forms a double-helical structure (DNA is double-stranded), in which two chains bond together; the sugar and phosphate groups are on the outside, and the nitrogenous bases interact by hydrogen bonding in the middle of the double helix
- A pairs with T through 2 hydrogen bonds
- C pairs with G through 3 hydrogen bonds (stronger)
- The 2 strands (nucleotide chains) of the double helix are complementary:
each base always pairs with its complement, so that the second strand of the double helix can be deduced, and synthesized in the cell, by simply pairing complementary bases
RNA:
- RNA is synthesized from 1 strand of DNA
- RNA does not form a double helix (no pairing of complementary bases between 2 strands); RNA is single-stranded
- RNA also uses 4 nucleotide subunits; however, uracil (U) replaces thymine in RNA
- *Sometimes RNA molecules pair with complementary bases within the single RNA strand, forming loop structures which may be necessary for some function in the cell (e.g.: transfer RNA (tRNA))
- *Some RNA molecules are structural in the cell (ribosomal RNA), and some have enzymatic activity
- Noting the above exceptions, the major function of RNA in the cell is carrying the genetic information for proteins from genes in the nucleus to ribosomes in the cytoplasm
- This RNA intermediate between genes and proteins is called messenger RNA (mRNA)
ATP (Adenosine Triphosphate)
ATP is a nucleotide that provides energy for most of the chemical reactions occurring within cells
Energy is released when the terminal phosphate is hydrolyzed (cleaved by addition of water)
The overall reaction is: ATP Þ ADP + P + Energy (7.4 kcal/mole ATP)
The energy released from this exergonic reaction is used to drive forward energy absorbing (endergonic) reactions in cells
Prokaryotes: bacteria & archaea
- no nuclear membrane enclosing DNA
- DNA is not associated with histones
- no true membrane-bound organelles
- cell walls usually contain peptidoglycan (archaea cell walls do not contain peptidoglycan)
- usually divide by binary fission
Eukaryotes: protists, fungi, plants & animals
- nuclear membrane enclosing DNA; DNA organized in multiple chromosomes
- DNA is associated with histones & other proteins
- membrane-bound organelles (mitochondria, Golgi complex, plasma membrane)
- cell walls, when present, are simple in structure (contain polysaccharides such as chitin or cellulose)
- divide by mitosis (nuclear division) followed by cytoplasmic division (cytokinesis)
Bacterial Cells:
Size: 0.2-2 µm in diameter; 2-8 µm in length
3 common shapes: coccus (spherical); bacillus (rod-shaped); spiral
Cocci:
Remain attached after division:
- Diplococci: divide in 1 plane & remain in pairs
- Streptococci: divide in 1 plane & remain in chains
- Tetrads: divide in 2 planes & remain in clusters of 4
- Sarcinae: divide in 3 planes & remain in groups of 8
- Staphylococci: divide in multiple planes & remain in grapelike clusters
Bacilli:
Usually in the form of single rods
- Diplobacilli: appear in pairs after division
- Streptobacilli: appear in chains
- Coccobacilli: oval bacilli that look much like cocci
Spiral:
Have 1 or more twists
- Vibrios: curved rod-like shape
- Spirilla: helical (corkscrew) shape
- Spirochetes: helical & flexible
Star-shaped: genus Stella
Triangular-shaped
Flat Rectangular: halophilic archaea
Bacteria are generally monomorphic but some environmental conditions (salt, pH) can alter their shape
- some bacteria are genetically pleomorphic (can take multiple shapes)
- the bacterial glycocalyx is composed of polysaccharide, polypeptide, or both
- an organized, firmly attached glycocalyx is a capsule
- an unorganized, loosely attached glycocalyx is a slime layer
- a glycocalyx composed of sugars is an extracellular polysaccharide (EPS)
Flagella: long filamentous structures that propel cells (give the cells motility – the ability to move)
- monotrichous: a single flagellum at one pole
- amphitrichous: flagella at both poles of cell
- lophotrichous: flagella at one pole of cell
- peritrichous: flagella surrounding cell
3 parts of flagellum:
- filament: the long outermost region composed of the protein flagellin
- hook: attached to filament at outer surface of cell wall
- basal body: anchors flagellum to the cell wall & plasma membrane
Bacterial flagella propel the bacterium by rotation from the basal body. Bacteria move in long straight ³runs² or ³swims² and random directional changes called ³tumbles²
- Taxis: movement toward or away from a stimulus
- Chemotaxis: movement associated with a chemical stimulus; a positive stimulus is called an attractant (bacteria ³run² toward attractants) & a negative stimulus is called a repellant (bacteria ³tumble² & then ³run² away from repellants)
- Phototaxis: movement associated with a light stimulus
Axial filaments (endoflagella): bundles of fibrils beneath an outer sheath that spiral around cell provide motility
- found in spirochetes
- can be few to several hundred at poles of cell or surrounding cell
Pili (sex pili): join bacterial cells prior to exchange of DNA
Cell wall: protects cell from adverse environmental changes, maintains shape of cell, anchors flagellum if present & prevents cell from rupture in a hypoosmotic medium
- composed of layers of peptidoglycan repeating disaccharide units composed of N-acetylglucosamine (NAG) & N-acetylmuramic acid (NAM) & linked in rows by polypeptides with tetrapeptide side chains (linked by peptide cross bridges)
- amino acids in tetrapeptide side chains are alternating D & L forms (this is unique, since amino acids in proteins are generally only L forms)
- penicillin interferes with peptide cross bridge linking
- lysozyme catalyzes hydrolysis of the bonds between NAG & NAM
Gram-positive cell wall: many layers of peptidoglycan & teichoic acids (lipoteichoic acid & wall teichoic acid)
- stains with crystal violet (the primary stain) in Gram stain procedure
Gram-negative cell wall: few layers of peptidoglycan in periplasm (between plasma membrane & outer membrane)
- outer membrane consists of lipopolysaccharides (LPS) containing O polysaccharides & lipid A, lipoproteins, & phospholipids
- porins (channel proteins) present in outer membrane
- stains with safranin (the counterstain) in Gram stain procedure
Cell Walls & Gram Stain:
- Gram Stain based on differences in gram-positive & gram-negative cell walls
- Crystal violet, the primary stain, stains both gram-positive & gram-negative cells (dye enters cytoplasm)
- Iodine (the mordant) forms large crystals with the dye that are too large to escape cell
- In gram-positive cells, alcohol (the decolorizer) dehydrates the peptidoglycan in the cell wall, making it more difficult for dye to escape
- In gram-negative cells, alcohol dissolves the outer membrane & leaves holes in thin peptidoglycan layer, allowing dye complex to escape
- The colorless gram-negative cells are then stained with safranin (the counterstain); gram-positive cells remain purple
Atypical cell walls
- Mycoplasmas (smallest bacteria, from genus Mycoplasma) have no cell wall
- Archaea may lack cell walls or have cell walls composed of pseudomurein (not peptidoglycan)
Plasma membrane: selectively permeable membrane; regulates passage of molecules & ions inside & outside cell
- composed of a phospholipid bilayer with partially or fully embedded proteins
- contains integral membrane proteins & peripheral membrane proteins
- bacterial plasma membranes contain metabolic enzymes, and the plasma membrane of photosynthetic bacteria contain enzymes & pigments for photosynthesis in infoldings called thylakoids (chromatophores)
- production of ATP by ATP synthase occurs in bacteria on the plasma membrane
- some alcohols & quaternary ammonia compounds used as disinfectants damage the bacteria plasma membrane
- polymyxins are antibiotics that disrupt the phospholipids of the bacterial plasma membrane (contents of cell leak out)
Movement of Molecules across membrane:
Passive Processes: no energy requirement
- Simple Diffusion: the net movement of molecules or ions (O2, CO2) from a region of higher concentration to a region of lower concentration
- movement continues until equilibrium (equal concentrations of the molecule or ion inside & outside cell) is reached
- Facilitated Diffusion: some molecules (example: glucose) are transported across the membrane by a membrane bound transporter protein
- in bacteria, substances too large to enter the cell by simple or facilitated diffusion (example: nucleic acids) are first broken down by extracellular enzymes, and then taken into the cell by transporter proteins
Osmosis: the diffusion of solvent molecules (usually water in organisms) across a selectively permeable membrane
- osmotic pressure is the pressure required to stop the osmosis of water
Osmotic solutions: isotonic, hypotonic & hypertonic
- isotonic (isoosmotic) solution: the net solute concentration of the solution equals that inside the cell
- hypotonic (hypoosmotic) solution: the net solute concentration of the solution is less that inside the cell
- hypertonic (hyperosmotic) solution: the net solute concentration of the solution is greater that inside the cell
Active Processes: require energy in the form of ATP
- Active Transport: molecules are usually moved from outside the cell inside the cell, often against the concentration gradient (from a region of lower concentration to a region of higher concentration)
- requires transporter proteins & energy from ATP hydrolysis
- generally the transported molecule is not modified during transport
- in group translocation (a form of active transport in bacteria), the molecule is chemically modified during transport (for example, glucose can be phosphorylated during group translocation) such that once inside it cannot leave the cell
- cleavage of a high energy phosphate bond (example: phosphoenolpyruvate) can provide the energy & the phosphate required for group translocation
Cytoplasm: substance inside cell (~80% water) containing proteins, carbohydrates, lipids, inorganic ions, DNA in the nucleoid, ribosomes & inclusions
Nucleoid: the nuclear area (not a true nucleus); contains a single chromosome
- no nuclear envelope in prokaryotes
- attached to plasma membrane
- may also contain one or more plasmids (extrachromosomal circular DNA molecules that replicate independently of the chromosome)
Ribosomes: site of protein synthesis
- prokaryotic ribosome is a 70S ribosome composed of 2 subunits (30S & 50S); each subunit consists of a protein & ribosomal RNA
- -several antibiotics interfere with protein synthesis by attaching to & blocking either the 30S subunit (streptomycin, gentamycin) or the 50S subunit (erythromycin, chloramphenicol)
Inclusions: reserve deposits of nutrients
- Metachromatic granules:
- Polysaccharide granules
- Lipid inclusions
- Sulfur granules
- Carboxysomes
- Gas vacuoles
- Magnetosomes
Endospores: specialized resting cells formed by some gram-positive bacteria (Clostridium, Bacillus); can survive extreme conditions (high temperature, radiation, lack of water) for many (hundreds to millions of) years
- during sporulation (sporogenesis) , a spore septum formed by the plasma membrane encloses cytoplasm & a newly replicated chromosome the spore septum fully encloses the cytoplasm & chromosome & becomes a forespore several layers of peptidoglycan are added between the 2 membranes & a thick protein spore coat is added to the outside membrane
- most of the water is removed from the forespore cytoplasm, which now contains dipicolinic acid & calcium ions (aid in restoring normal metabolism when germinated)
- the vegetative cell ruptures to release the endospore
- damage to the spore coat & rehydration result in germination of the endospore; metabolism resumes
Eukaryotic Cells:
- True nucleus with nuclear membrane
- Organelles: small, membrane-bounded bodies with a specific structure & function (e.g.: mitochondria, chloroplasts, lysosomes) in cytosol (semifluid medium between nucleus and plasma membrane)
Outer boundary:
Cilia and Flagella: composed of microtubules (9 + 2 pattern); anchored to plasma membrane by basal body; used in movement
- Cilia present in some unicellular protists (Paramecium) and cells of respiratory tract in animals
- Flagella present in some unicellular protists (Euglena) and sperm cells
- unlike the rotating prokaryotic flagella, the eukaryotic flagellum moves back & forth in a wavelike motion
Cell Wall: many eukaryotic cells have a cell wall
- unlike the bacterial cell wall, no peptidoglycan is present in a eukaryotic cell wall
- plants & algae have a porous cell wall composed of cellulose fibrils; functions in support & protection
- fungi have a cell wall containing chitin (a NAG polymer)
- the yeast cell wall contains the polysaccharides glucan & mannan
Glycocalyx: if some eukaryotic cells (animal cells), the plasma membrane is covered by a carbohydrate layer that strengthens the cell & enhances attachment to substrates or other cells
Plasma membrane: outer boundary of cells (except plant cells – also cell wall)
- Phospholipid Bilayer: semipermeable and selectively permeable
- Functions in regulation of passage of molecules into and out of the cell
- Capable of endocytosis large molecules or particles are engulfed by a portion of the plasma membrane & taken into the cell in a vesicle
Cytosol:
Cytoskeleton: composed of microtubules, intermediate filaments, and actin filaments
- Functions in maintaining shape of cell and movement of subcellular structures
- Microtubules: composed of tubulin dimers coiled into tubelike structures
- Concentrated & arranged as rings of nine doublets or triplets in centrioles, cilia, and flagella
- Microtubules involved in movement associate with motor proteins kinesin and dynein
- Intermediate Filaments and actin filaments have structural roles throughout the cell
- Actin filaments combine with myosin in muscle cells to enable muscle movement
Nucleus: stores genetic information in all eukaryotic cells
- DNA is organized into distinct chromosomes
- Chromosomes are packaged with proteins to form chromatin
- Chromatin exists in a semifluid medium called nucleoplasm
- Dark regions within the nucleus are nucleoli (1 or more per cell)
- Within each nucleolus, ribosomal RNA is produced and joins with ribosomal proteins to form ribosomes
- The nucleus is bounded by a porous membrane, the nuclear envelope, which regulates passage of molecules into & out of the nucleus through nuclear pores
- The structure of the nucleus is maintained by the nuclear matrix, which contains a protein network called the nuclear lamina, which also provides chromatin attachment sites to maintain organization
Ribosomes: site of protein synthesis in the cell
- free in cytoplasm (polyribosomes) or associated with rough endoplasmic reticulum
- 2 subunits (large & small); mRNA is threaded through subunits during translation (protein synthesis)
Endomembrane System: includes Golgi apparatus, endoplasmic reticulum, vesicles, and nuclear membrane
Endoplasmic Reticulum: (ER)
- Rough ER: associated with ribosomes; proteins translated on ribosomes associated with the rough ER will be transported and/or secreted outside cell
- begins processing & modification of these proteins
- Smooth ER: synthesizes phospholipids in all cells; various other cell type-specific functions
- synthesizes steroid hormones in testes, and detoxifies drugs in liver cells
Golgi Apparatus:
Completes modification of proteins from rough ER (transported to Golgi in vesicles (small membrane-bounded organelles for transport))
- modification of proteins & lipids (addition of carbohydrate chains (glycosylation))
- also transports organic molecules in vesicles; some become lysosomes
Lysosomes: vesicles with digestive enzymes to break down macromolecules & cell debris
Microbodies: smaller version of lysosomes with specific enzyme activities
- Peroxisomes are microbodies that contain enzymes for oxidizing certain organic molecules with the release of hydrogen peroxide (toxic, but breaks down into water & oxygen)
Vacuoles: larger membrane-bounded organelles
- function in storage (mainly in plant cells)
- some plant cell vacuoles store water (central vacuole) for support; some store pigments
Energy-related organelles:
Chloroplasts: plant cells and some unicellular protists
- site of photosynthesis in plant cells (use of solar energy to produce carbohydrates for food)
- contains a fluid-filled space (stroma) within which is a system of interconnected flattened membranes (thylakoids)
- several photosynthetic pigments (e.g.: chlorophyll) are within the thylakoid membranes of grana (stacks of thylakoids)
Mitochondria: all eukaryotic cells
- site of cellular respiration (ATP production from carbohydrates)
- also have folded membrane system (folds are cristae, inner fluid-filled space is the matrix)
- extensive membrane systems are important in both chloroplasts and mitochondria for ATP production
Centrosomes: region near the nucleus that consists of the pericentriolar area (proteins that organize the mitotic spindle) & 2 centrioles
- Centrioles have a 9+0 array of microtubules; aid in formation of cilia & flagella
Chapter 5: Microbial Metabolism
Metabolism: all the chemical reactions in a cell
- Catabolism: the breakdown of complex organic compounds into simpler ones; releases energy
- Anabolism: the building of organic molecules from simpler ones
- Metabolic pathways: sequences of chemical reactions in a cell
Enzymes: increase the rate of a chemical reaction by lowering its activation energy without increasing the temperature or pressure within a cell
- turnover number: the maximum # of substrate molecules an enzyme converts to product per second
- names of enzymes generally end in –ase
- most consist of a protein apoenzyme & a nonprotein cofactor; an organic cofactor is called a coenzyme the whole enzyme (apoenzyme & cofactor) is the holoenzyme
- often assist each step of a metabolic pathway
- each enzyme reacts with a specific substrate to form a specific product
- enzymes are not changed by chemical reaction (usually)
- enzymes lower the energy of activation for a reaction (energy required to activate the reactants)
- the part of an enzyme molecule where the substrate binds is called the active site
Synthetic reaction: two or more reactants combine to produce a larger product (combination of reactants)
Degradative reaction: Larger product is broken down into two or more smaller products
The rate of a chemical reaction is influenced by:
- temperature: moderate is best; high temperatures often denature (inactivate) enzyme
- pH: optimized for reaction conditions
- amount of substrate: usually increased substrate concentration increases rate
Enzyme Inhibition: (slow rate)
Competitive Inhibition: competition of substrate analog for active site of enzyme
Noncompetitive Inhibition: binding of a molecule to a different site of enzyme (allosteric site) induces change of shape at active site – substrate can no longer bind
Feedback Inhibition: product of reaction binds to enzyme (allosteric site), and stops reaction
Ribozymes: RNA enzymes; splice RNA molecules
Oxidation-Reduction reaction (redox reaction): an electron donor is oxidized while an electron acceptor is reduced
Oxidation: loss of electrons or H atoms
Reduction: gain of electrons or H atoms
- most biological oxidations are called dehydrogenation reactions (result in the loss of hydrogen atoms)
- example: cellular respiration glucose oxidized to CO2 & oxygen reduced to water
Exergonic reactions: release energy
Endergonic reactions: require (absorb) energy
In cells, the energy released by ATP hydrolysis (an exergonic reaction) is used to fuel endergonic reactions such as metabolism & muscle contraction
ATP generation: 3 mechanisms for phosphorylation (addition of phosphate) of ADP to yield ATP:
- Substrate-level phosphorylation: a high-energy phosphate is directly transferred from a phosphorylated compound to ADP
- Oxidative phosphorylation: electron transport of electrons derived from NADH & FADH2; chemiosmosis (movement of (hydrogen) ions across a membrane to release energy is used to synthesize ADP from ATP using the enzyme ATP synthase
- Photophosphorylation: photosynthetic cells convert light energy to chemical energy in NADPH & ATP (synthesized by electron transport & chemiosmosis/ATP synthase)... this energy may be used to synthesize organic molecules (glucose)
Cellular respiration includes both Aerobic Respiration and Anaerobic Respiration
- includes all the various metabolic pathways that break down carbohydrates and other molecules resulting in the production of ATP
- aerobic respiration: the complete breakdown of glucose to carbon dioxide and water
o oxidation-reduction reaction; glucose is oxidized & oxygen is reduced
- fermentation: glycolysis followed by the reduction of pyruvate to either lactate or alcohol and carbon dioxide
- ATP synthesis is an endergonic reaction; energy released by metabolism of glucose is used to produce ATP
Glycolysis: the breakdown of glucose (6C) to 2 pyruvate (3C) molecules
- net gain of 2 ATP molecules (4 produced, 2 used)
- 2 NADH also produced (these NADH need to be shuttled into mitochondrion in eukaryotes; in some cell types their electrons are transferred to FADH2)
- no oxygen is required; takes place in cytoplasm of cell (outside mitochondria)
- total (net) output: 2 ATP, 2 NADH
Alternatives to Glycolysis:
- pentose phosphate pathway (hexose monophosphate shunt): used to metabolize 5-carbon sugars (pentoses)
o produces intermediate pentoses used in synthesis of nucleic acids, glucose & some amino acids
o yields NADPH from NADP+ and net 1 ATP from 1 glucose
- entner-doudoroff pathway: produces 2 NADPH & 1 ATP for each glucose
o found in some gram-negative bacteria (Rhizobium, Pseudomonas, Agrobacterium
Aerobic Respiration:
The mitochondrion is an organelle with a double membrane and a fluid matrix in the middle, with an intermembrane space between the inner and outer membranes
- the membranes fold to form cristae, to increase surface area for transport, transfer of hydrogen ions and ATP
- ATP is produced in the matrix, and leaves the matrix through a channel protein
- Glycolysis occurs in the cytoplasm of the cell
- The Krebs cycle takes place in the matrix of the mitochondrion
- The electron transport system is located in the cristae of the mitochondrion, with electron carriers and an ATP synthase complex in the inner membrane
Decarboxylation of Pyruvate (Transition reaction): pyruvate (3C) is oxidized to an acetyl group (2C) with release of carbon dioxide
- acetyl group combined with coenzyme A (derived from pantothenic acid, vitamin B5) to form acetyl-coA
- output per pyruvate: 1 NADH, 1 CO2
- total output: 2 NADH, 2 CO2
Krebs cycle: acetyl-coA is input into a series of reactions that form a cycle, releasing carbon dioxide and producing ATP
- output per acetyl-coA: 1 ATP, 3 NADH, 1 FADH2, 2 CO2 (from 2 Cs in acetyl-coA)
- total output: 2 ATP, 6 NADH, 2 FADH2, 4 CO2
Totals of ATP, NADH & FADH2 & CO2 so far from complete glucose catabolism:
- ATP, NADH & FADH2 produced by decarboxylation of pyruvate & Krebs cycle are doubled since 2 pyruvates enter transition reaction & 2 acetyl-coA molecules enter Krebs cycle from 1 glucose breakdown
- So: 4 ATP, 10 NADH, 2 FADH2 & 6CO2
- electron transport uses electrons from NADH & FADH2 & chemiosmosis to generate 32-34 ATP molecules
Electron Transport System: accepts electrons from NADH (produced from NAD+ in glycolysis and Krebs cycle) and FADH2 (produced from FAD during Krebs cycle), and is transferred to acceptors until it reaches the final acceptor in the chain, oxygen
- 3 types of electron carriers: flavoproteins, cytochromes & ubiquinones (coenzyme Q)
- 17 ATP can be produced per pyruvate from glycolysis, for a total 34 ATP produced (from total of 10 NADH and 2 FADH2 produced by complete breakdown of glucose)
- each NADH yields 3 ATP molecules
- each FADH2 yields 2 ATP molecules
ATP is produced by ATP synthase on the inner membrane of the mitochondrion (plasma membrane in prokaryotes)
- chemiosmosis: electrochemical gradient produced by the flow of H+ ions through membrane pores created by electron acceptor complexes drives the production of ATP
- the H+ ions are released from reduced NADH and FADH2, following the electrons donated to the membrane carriers
The various metabolic reactions of cellular respiration result in a pool of metabolites – metabolic intermediates – that can be used in anabolic (synthesis) or catabolic (degradative or breakdown) reactions
Anaerobic Respiration: ATP yield less than aerobic respiration (only part of Krebs cycle is used & not all carriers in electron transport used)
- final electron acceptor is an inorganic substance; not oxygen
o nitrate (NO3-) in Pseudomonas & Bacillus; reduced to nitrite, nitrous oxide or nitrogen gas
o sulfate (SO4-2) in Desulfovibrio; reduced to hydrogen sulfide
o carbonate (CO3-2) in other bacteria reduced to methane (CH4)
Fermentation: used by yeast and bacteria to produce lactate (lactic acid fermentation) or ethyl alcohol (alcohol fermentation)
- only 2 ATP are produced (low energy yield)
- final electron acceptor is organic acid or alcohol
- NAD+ is regenerated so glycolysis can continue
- ability to ferment a certain sugar can be used as a biochemical test in identification of bacteria... bacteria ferment sugar to acid in media, and pH indicator in media changes color as acid produced (bacterial colonies or media itself changes color)
Lipid & Protein Catabolism:
- lipases break down lipids to fatty acids & glycerol both are metabolized to acetyl coA molecules & enter Krebs cycle
- peptidases break down proteins to amino acids; amino acids must be deaminated (may also be decarboxylated or dehydrogenated) & converted to organic acid (pyruvate to acetyl-coA) prior to entering metabolic pathways
Photosynthesis: conversion of solar energy to carbohydrate molecules using carbon dioxide and water
- overall reaction: solar energy + CO2 + H2O Þ carbohydrate + O2
Photosynthesis uses the visible light portion of the electromagnetic spectrum
Photosynthesis uses pigments (chlorophylls, carotenoids, etc.) that absorb specific wavelength(s) of light
- Plants appear green (mostly) because the dominant pigments of plants (and photosynthesis) are chlorophylls
- chlorophylls transmit green light (absorb most other wavelengths of visible light)
Photosynthesis occurs in chloroplasts in eukaryotes & on thylakoids in prokaryotes
Chloroplasts composed of 2 main parts:
- stroma: large central compartment
- thylakoid: membrane system within stroma of chloroplast
- grana: stacks of thylakoids
Photosynthesis consists of 2 major reactions:
Light-dependent reactions: capture solar energy in chlorophyll in 2 photosystems
- occur in thylakoid membrane where pigments are located
- in each photosystem, the light-gathering antenna (chlorophylls & accessory pigments) absorb solar energy and transfer it to a reaction center chlorophyll a molecule, which then sends energized electrons to an electron acceptor molecule
- chemiosmosis: ATP production occurs in the stroma, tied to an electrochemical gradient produced by flow of hydrogen ions from the thylakoid space into the stroma
- utilizes a membrane ATP synthase complex
- Cyclic photophosphorylation: utilized by some photosynthetic bacteria to produce only ATP
- electrons released by chlorophyll flow back to chlorophyll
- Noncyclic photophosphorylation: noncyclic electron flow; ATP is produced, and electrons from chlorophyll used to reduce NADP+ to NADPH
- to return electrons to chlorophyll, water is oxidized (split) to yield H+, e-, and O2
- ATP & NADPH can be used in Calvin-Benson cycle to synthesize carbohydrates
- most plants uses this pathway
Calvin-Benson Cycle (light-independent reactions):
Carbon dioxide fixation: attachment of carbon dioxide to a 5 carbon sugar (ribulose disphosphate or RuDP) by RuBP carboxylase to form 2 molecules of 3-phosphoglycerate (3C), which is then converted into 2 molecules of glyceraldehyde 3-phosphate (PGAL or phosphoglyceraldehyde)
- these reactions use ATP and NADPH formed by the light-dependent reactions
- 2 glyceraldehyde 3-phosphate molecules are needed to synthesize 1 glucose
- RuDP is regenerated for the next cycle: the cycle must turn 6 times to yield 1 molecule of glucose, since 5 out of every 6 of the glyceraldehyde 3-phosphate molecules produced are used to regenerate 3 RuDP molecules
Photoautotrophs: use light as energy source & CO2 as carbon source
Photoheterotrophs: use light as energy source & organic compounds as carbon source
Chemoautotrophs: use reduced inorganic compounds as energy source & CO2 as carbon source
Chemoheterotrophs: most bacteria & animals; require organic nutrients as energy source
Chapter 6: Microbial Growth
Microbial Growth Requirements
Growth Temperature:
Psychrophiles: cold-loving microorganisms; capable of growth at 0C
- first group: optimum growth temperature of 15C; cannot grow above 25C; found in deep ocean & polar regions
- second group: optimum growth temperature of 20-30C; cannot grow above 40C; microorganisms in this group are responsible for refrigerated food spoilage
Mesophiles: moderate temperature-loving microorganisms
- optimum growth temperature of 25-40C (37C (physiological temperature) is often considered optimal); most common type of microorganism includes most of the common spoilage & disease-causing microbes
Thermophiles: heat-loving microorganisms
- optimum growth temperature of 50-60C; many cannot grow below 45C; found in sunlit soil & hot springs
- endospores formed by these microbes resist extreme heat
- Extreme thermophiles: the hyperthermophiles of the Archaea may have an optimum growth temperature of 80C or higher; found in hot springs associated with volcanoes (sulfur usually important for their metabolism)
pH: most bacteria grow optimally at pH 6.5-7.5; most molds & yeasts have an optimum pH range of 5-6
- acidophiles (acid-loving microbes) can survive at pH 1
Osmotic pressure:
- bacteria are 80-90% water hypertonic solutions remove water from the cell – results in plasmolysis (shrinks plasma membrane) which slows bacterial growth; salts are often used as food preservatives
- extreme halophiles (Archaea) require high salt conditions (up to 30% salt) for growth – they¹re obligate halophiles; found in Dead Sea & Great Salt Lake
- facultative halophiles can grow in up to 2% salt (some up to 15% salt)
Chemical Requirements:
Carbon: chemoheterotrophs require organic macromolecules (proteins, lipids, carbohydrates); chemoautotrophs and photoautotrophs require CO2
Nitrogen: required for proteins & nucleic acids (ATP)
- sources include ammonium ions (NH4), nitrates (NO3)
- some autotrophic bacteria use gaseous nitrogen (N2) as a source for nitrogen fixation
Sulfur: required for proteins & some vitamins; sources include sulfate (SO4) & hydrogen sulfide (H2S)
Phosphorus: required for nucleic acids; source is ATP & phosphate ion (PO4)
Potassium
Magnesium
Calcium
Trace elements: Iron, Copper, Molybdenum, Zinc
- used in enzymatic reactions & in the synthesis of some cofactors
- usually present in tap water
Oxygen:
Obligate aerobes: require oxygen for growth
Facultative anaerobes: can use oxygen, but not required
Obligate anaerobes: cannot use oxygen to make energy; oxygen often toxic
Aerotolerant anaerobes: cannot use oxygen to make energy; oxygen can usually be tolerated
Microaerophiles: need oxygen for growth, but less than atmospheric levels
Toxic oxygen forms:
Singlet oxygen
Superoxide free radicals (O2-); neutralized by superoxide dismutase (produced by all organisms attempting to grow in atmospheric oxygen (aerobes, facultative anaerobes growing aerobically, aerotolerant anaerobes))
Peroxide anion (O22-) from hydrogen peroxide; hydrogen peroxide may be neutralized by catalase or peroxidase
Hydroxyl radical (OH-)
Organic growth factors: some bacteria may require vitamins, amino acids, purines or pyrimidines for metabolism
Culture Medium: used to grow bacteria & other microbes
- growing microbes on a culture medium is a culture
- the polysaccharide agar (from algae) is used as a solidifying agent for solid cultures
Chemically defined media: chemical composition is known can be customized to the specific requirements (optimum conditions) for a given microbe
Complex media: contains most essential nutrients, but chemical composition varies
- nutrient broth (liquid) or nutrient agar (solid); yeast extracts used as source of organic growth factors & other nutrients
Reducing Media: ingredients such as sodium thioglycolate combine with oxygen in media; heating removes absorbed oxygen
- anaerobes plated on solid media can be grown in anaerobic chambers or anaerobic jars hydrogen & carbon dioxide are released by a chemical reaction (involving sodium bicarbonate, sodium borohydride & water); the hydrogen reacts with oxygen in the jar to form water, & the carbon dioxide acts as a carbon source
- carbon dioxide incubators & candle jars can be used to control CO2 levels
- capnophiles: microbes that grow better at high CO2 concentrations
Selective media: suppress growth of unwanted microbes
- example: Bismuth sulfate used to grow the gram-negative Salmonella typhi it inhibits growth of gram-positive & most gram-negative bacteria
Differential media: allows identification of colonies of the desired microbe by a specific appearance or reaction product when growing in the media. (example: Streptococcus pyogenes can be distinguished on blood agar by their ability to lyse red blood cells)
Enrichment culture: media with conditions that favor the growth of one microbe over others allows isolation of the desired microbe with minimal starting material
Pure cultures:
Colony: group of cells (visible as a small spherical mass on solid media) resulting from a single spore or vegetative cell, or from a group of the same microbe attached in chains or clumps.
- individual bacterial colonies can be isolated using the streak plate method for plating the bacteria
Preservation of pure cultures:
Deep-freezing: microbes are frozen quickly in suspension (using media with glycerol or another viscous liquid)
Freeze-drying (lyophilization): the frozen suspension may be dehydrated under vacuum for long-term storage
Bacterial division is by binary fission (equal division of 1 cell into 2; most common type) or budding (formation of a small bud on the parent cell that enlarges to form a new cell)
Generation time: the time required for a cell to divide
- generally 1-3 hours, but could be shorter or substantially longer depending on the microbe
Bacterial growth can be represented logarithmically
- since bacteria grow so rapidly to extremely large cell populations, arithmetic growth curves are often not very informative
- the log10 of the number of cells can be used in a bacterial growth curve to track the cell growth by generation number
- the bacterial growth curve consists of 4 basic phases:
- lag phase: a period of little or no cell division while the cells are replicating DNA & synthesizing organic molecules necessary for cell division
- log (exponential growth) phase: a period of exponential growth generation time remains constant & minimal
- stationary phase: number of new cells is in equilibrium with cell deaths
- death (logarithmic decline) phase: number of deaths exceeds the number of new cells formed for each generation
Direct measurement of microbial growth:
Plate count: colony-forming units (CFUs) are counted (colonies may have arisen from individual or clumped cells)
- To obtain an appropriate number of CFUs (plates with overcrowded CFUs may give inaccurate results due to limited resources), a serial dilution (a series of 1:10 dilutions) may be performed
- Pour plate method: a bacterial suspension is mixed with nutrient media with agar & then poured onto plates; cells may grow in or on top of the agar
- Spread plate method: a bacterial suspension is poured onto a preformed nutrient media agar plate & spread into the plate; may be more
Filtration: small numbers of bacteria in liquid media (for example, water) are filtered through a thin membrane filter the bacteria do not pass through the filter, & the filter with the bacteria is transferred to a nutrient medium for growth & counting
Most probable number (MPN) method: uses serial dilutions to estimate the number of cells in a liquid sample; when no growth is observed in a dilution in the series, it is assumed no bacteria were present
Direct microscopic count: count the number of cells in a defined volume of liquid media under a microscope
- uses slides with square fields (Breed count method) or grids (Petroff-Hauser cell counter) to make several counts & average the resulting cell counts
- Coulter counters automate the process
- difficult to use with motile microbes
- counts may be inaccurate due to inclusion of dead cells
- no incubation time is required
Indirect methods:
Turbidity: as a beam of light is passed though a bacterial culture, the optical density of the bacterial culture (absorbance of light) or transmitted light is measured in a spectrophotometer; the resulting value is compared to a known standard to estimate bacterial numbers in the culture
- requires large numbers of cells (incubation time)
Metabolic activity: the amount of a metabolic product (CO2, acid) is assumed to be in proportion to the number of cells present in a sample
Dry weight: the microbe is filtered or centrifuged from growth medium, lyophilized & weighed; comparison to a set of standards gives an estimate of numbers of cells
- most useful for fungi, where most of the above methods would not give accurate counts, but may also be used with bacteria
Chapter 7: The Control of Microbial Growth
Terminology:
Sterilization: destruction of all forms of microbial life on an object (includes endospores)
- most common method is heating (e.g.: autoclaving)
Commercial Sterilization: limited heat treatment; just enough to destroy endospores of Clostridium botulinum (a gram-positive rod that produces a deadly exotoxin responsible for a form of food poisoning known as botulism)
- heat-resistant endospores may survive this treatment although they may cause food spoilage, they generally will not grow at normal food storage temperatures & are generally not pathogenic
Disinfection: destruction of vegetative pathogenic (disease-causing) microbes (pathogens)
- does not destroy endospores
- may use chemicals (disinfectants), ultraviolet (UV) light, boiling water or steam
Antisepsis: disinfection targeted at living tissue
- chemicals used for antisepsis are called antiseptics
Degerming: mechanical removal of most of the microbes from a limited area (e.g.: swabbing skin with alcohol)
Sanitization: lowering of microbial counts to safe public health levels
- aimed at destruction of most pathogenic vegetative microbes
- generally used in restaurant/food industry to treat eating & drinking utensils
- may use high-temperature washing or chemical disinfectant
Biocide (Germicide): agent that kills microbes (may not kill endospores)
- examples: fungicides kill fungi, virucides inactivate viruses (can¹t be killed since they¹re abiotic)
Bacteriostatic: treatment to inhibit the growth & multiplication of bacteria
- also applies to other microbes; uses –static or –stasis suffix
- once the treatment is removed, the microbe may begin to grow again
Sepsis: indicates a state of bacterial contamination
Asepsis: the absence of significant microbial contamination
- aseptic technique is important in hospitals (esp. during surgery) & in food packaging to prevent contamination
Microbial Death Rate:
- bacteria subjected to an antimicrobial chemical generally die at a constant rate
- as with bacterial growth curves, a bacterial death curve can be plotted logarithmically to show a constant death rate
- the effectiveness of a given microbial treatment can be influenced by: the number of microbes, the presence of organic matter in the material to be treated, temperature & acidity of the material to be treated, the time of exposure and microbial characteristics (such as differences in the cell walls of gram-negative & gram-negative bacteria & the spore coat of endospores)
Mechanism of Action of Microbial Control Agents:
Alteration of membrane permeability: some microbial agents cause damage to the lipids or proteins of the plasma membrane that causes cell contents to leak out of the cell & slows/stops growth
Damage to proteins & nucleic acids:
- heat may cause denaturation of proteins by breaking hydrogen bonds or disulfide linkages that hold the 3D structure of the protein (the function of a protein is dependent on its shape)
- heat breaks hydrogen bonds between adjacent bases in a DNA molecule (denatures DNA); UV light causes thymine-dimers that disrupt replication & transcription
Methods of Microbial Control:
Heat: primary effect appears to be denaturation of enzymes
- heat resistance varies among microbes (& their endospores)
- Thermal death point: the lowest temperature that kills all the microbes in a liquid culture in 10 minutes
- Thermal death time: the minimal length of time that kills all the microbes in a liquid culture at a given temperature
- Decimal reduction time: the time required to kill 90% of the microbes in a liquid culture at a given temperature
- Moist heat: appears to kill microbes by coagulation of proteins
- boiling kills vegetative forms of pathogenic bacteria, fungi & their spores, & destroys viruses within 10 minutes
- autoclaves use heat under pressure to sterilize (generally the preferred means of sterilization)
- increase pressure to increase temperature of the steam generally, steam at a pressure of 15 psi (pounds per square inch) at 121C will kill all microbes & their endospores within 15 minutes
- requires that air be completely exhausted from the chamber – trapped air will prevent steam entry
- more time is required for more/larger items & solid items (must be loosely covered to allow steam to enter all open spaces)
- chemicals that change color when subjected to steam for a given length of time are used as indicators of successful sterilization
Pasteurization: mild heating of beverages, sufficient to kill specific microbial spoilage elements without harming the taste
- eliminates microbial pathogens & lowers overall microbial numbers, but leaves some thermoduric (heat-resistant) microbes unlikely to cause spoilage or disease
- high-temperature short-time (HTST) pasteurization: 72C for 15 seconds used in most milk pasteurization
- ultra-high-temperature (UHT) pasteurization: superheated steam raises temperature of milk to 140C for < 1 second used in applications where milk will be left unrefrigerated for days at a time
Dry Heat-Sterilization: kills microbes by oxidation
- direct flaming & incineration are examples
- hot-air sterilization: items ³cooked² in oven at 170C for 2 or more hours
Filtration: used to sterilize heat-sensitive materials (media, proteins)
- traps microbes on a screen with pores smaller than microbes
- high-efficiency particulate air (HEPA) filters: remove air-borne microbes
- membrane filters: pores range from 0.22 µm & 0,45 µm to as little as 0.01 µm (can trap viruses & proteins)
Low temperature: refrigerator temperatures (0-7C) have a bacteriostatic effect on most bacteria (some psychrophiles can grow at these temperatures & cause food spoilage)
- slow-freezing is more effective at killing microbes – ice crystals interfere with growth
Desiccation: removal of water; stops growth of microbes
- growth of microbes can resume when water is added back
Osmotic pressure: high concentration of salts & sugar used to preserve food
- creates hypertonic medium that removes water from the cell & disrupts metabolism
- molds & yeasts more resistant than bacteria to high osmotic pressure (more able to spoil fruits * grains)
Radiation:
Ionizing Radiation: gamma rays (from radioactive elements), X-rays & high-energy electron beams
- short wavelength, high-energy radiation
- ionizes matter; produces reactive hydroxyl radicals – can react with DNA & cause mutations
- used in sterilization of pharmaceuticals & disposable plastics
Nonionizing radiation: UV light
- long wavelength, lower energy radiation
- UV light causes thymine dimers (thymine-thymine bonds) between adjacent thymines in DNA prevents normal replication & transcription (eventually can kill cell)
- UV (germicidal) lamps can kill airborne microbes
Chemical Methods of Microbial Control:
Phenol Coefficient Test: compares the effectiveness of a disinfectant with that of phenol
Use Dilution Test: tests ability of disinfectant to kill or inhibit growth of 3 bacterial species (Salmonella choleraesuis, Staphylococcus aureus, Pseudomonas aeruginosa)
- metal rings are dipped into cultures of bacteria, then incubated in disinfectant & finally transferred to culture the effectiveness of the disinfectant is determined by growth of bacterial cultures
Disk-Diffusion Method: filter paper disk soaked in disinfectant is placed on culture plate containing target microbe a clear zone showing inhibition of growth around the disk indicates the effectiveness of the disinfectant
Disinfectant types:
Phenol & Phenolics: disrupt plasma membrane of microbe
- phenol rarely used skin irritant & strong odor
- phenolics used on environmental surfaces & instruments
- reactive in presence organic material
- Lysol® uses O-phenylphenol as disinfectant
- bisphenols such as Triclosan used in disinfectant hand soaps & skin lotions
Biguanides (Chlorhexidine): disrupt plasma membrane of microbe
- used for skin disinfection (surgical scrubs)
- bactericidal to gram-positive & gram-negative bacteria
Halogens: iodine is strong oxidizing agent; chlorine as hypochlorous acid is strong oxidizing agent exact mechanism of action unknown, but likely damages cellular enzyme systems
- iodine commercially available as Betadine® & Isodine®; used for skin disinfection & wound treatment
- chlorine as a compressed gas in liquid form is used to disinfect water (drinking water, swimming pools, sewage); calcium hypochlorite used to disinfect dairy equipment & restaurant eating utensils, and was the disinfectant used by Semmelweis to control hospital infections; sodium hypochlorite used as disinfectant in household bleach
- chloramines (chlorine & ammonia) also used as disinfectants, antiseptics & sanitizing agents
Alcohols: denatures proteins & solubilizes lipids
- used for degerming, especially prior to injection (ethanol & isopropanol)
- bactericidal & fungicidal; not effective against endospores or nonenveloped viruses
- water required for denaturation: 70% ethanol (70% ethanol, 30% water) more effective than 100% ethanol (70% is recommended optimal concentration, although anywhere from 60% to 90% may be as effective)
Heavy Metals & their compounds: silver & mercury biocidal; denature proteins
Surface-active agents:
- soaps & acid-anionic detergents used for degerming
- acid-anionic detergents also used as microbicides in commercial sanitizers
- cationic detergents (quaternary ammonia compounds) used as antiseptic for skin, instruments & rubber gloves
- inhibits enzymes, denatures proteins, & disrupts plasma membranes
Organic Acids & derivatives: inhibit metabolic reactions, especially in molds
- sorbic acid, benzoic acid, calcium propionate used in food industry
- parabens used in cosmetics & shampoos
Antibiotics: many varieties; may inhibit synthesis of cell wall, plasma membrane, cellular proteins or nucleic acids
- use is restricted; unlike the other antimicrobials discussed here, antibiotics are generally used for specific treatment of disease
- 2 antibiotics are used to inhibit growth of microorganisms in dairy products (nisin & natamycin)
Aldehydes: denature proteins
- Glutaraldehyde (preferential now to formaldehyde) used for disinfection of medical equipment
Gaseous sterilants: denature proteins
- ethylene oxide used as sterilant (especially in applications where heat would be damaging)
Peroxygens (oxidizing agents): oxidize
- hydrogen peroxide poor antiseptic but good disinfectant, peracetic acid effective disinfectant
- disinfection of contaminated surfaces & to clean wounds