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75 Cards in this Set
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What is microbiology?
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• The biology of very small, mostly unicellular, organisms that are too small to be viewed with the naked eye
o The foundation of all the biological sciences because microorganisms are central to the very functioning of the biosphere o Microorganisms are excellent models for understanding the functions of the cells of all organisms, including “higher forms of life,” such as humans |
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what are examples of model organisms in microbiology?
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• Escherichia coli
o Commonly used as a model organisms to study prokaryotic cell functions • Saccharomyces cerevisae (yeast) o Often used to study eukaryotic cell functions • Chlamydomonas reinhardtii o Model for studies on plant cell functions • Microorganisms aren’t used to study developmental processes unique to multicellular organisms o Only used for studies of functions that are common to all cells o Chlamydomonas cannot be used to model flower development of higher plants • Drosophila malanogaster and Arabidopsis thaliana are very small, but are not microorganisms o They are multicellular and therefore great models for stidies on the development of multicellular organisms even humans |
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what are six features associated with living organisms?
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• Metabolism
o Uptake of nutrients from the environment, their transformation within the cell, and elimination of wastes into the environment • The cell is an open system • Reproduction/ growth o Chemicals from the environment care turned into new cells under the direction of preexisting cells • Differentiation o The formation of a new cell structure, such as a spore, usual as part of a cellular life cycle • Communication o Cells communicate or interact primarily by means of chemicals that are released or taken up • Movement o Living organisms are often capable of self-propulsion • Evolution o Cell contain genes and evolve to display new biological properties o Phylogenetic trees show the evolutionary relationships between cells |
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how are cells coding devices and machines?
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• Cells are postulated to have decended from a universal ancestor or primordial cells, which existed more than 3.8 billion years ago
o This is thought because all cells are constructed in similar ways and evolutionarily related o Very little is known about the process that lead to the emergence of the cellular form of life • Pro-cellular evolution • Cells are coding devices that store and process information o The information is the blueprint of their own characteristics and function o The information is stored, replicated, and passed on to offspring during reproduction through DNA • Cells are considered machines that carry out chemical transformations o Enzymes are the catalysts of this chemical machine • They greatly accelerate the rate of chemical reactions • The product of the interactions of cells as coding devices and cells as machines is growth o Coding function • DNA undergoes replication and gene expression through the transcription of RNA, which is translated into proteins This results in reproduction or growth The proteins may be enzymes that are used in metabolic catalysis (one form of machine function) • Energy for reactions require ADP and ATP (second form of machine function) • Metabolism results after generations of precursors of macromolecules like sugar, amino acids, fatty acids, purines, pyrimidines, etc (third machine function) |
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how can Single microbial cells have an Independent Existence and State Clonal Population of Cells?
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• Purple bacteria were among the first phototrophs on Earth
• Cyanobacteria were the first oxygen-evolving phototrophs on Earth o Oxygenated the atmosphere, allowing for the evolution of oxygen-dependent “higher” life forms |
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What are the natural environments of microorganisms?
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• Populations
o Assemblages of many cells of the same species living in associations with other cells • Communities o Assembly of populations of different species • Habitat o Location in an environment where a population of cells lives • Can be anywhere! • Microbial ecology o The study of microorganisms in their natural habitat • Ecosystem o The dynamic interactions of microbial communities with the chemical and physical components of a habitat and with each other • Affected by nutrient utilization, waste excretions, predation, poison production, etc |
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How much microbial biomass is there?
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• Estimates of the total number of microbial cells on Earth is on the order of 5 X 10^30
• The total amount of carbon present in this very large number or very small cells equals that of all plants on Earth o Most prokaryotic cells reside underground in the oceanic and terrestrial subsurfaces • At least 60% of all photosynthesis is carried out by microorganisms o Mostly marine microorganisms • SAR11 o Pelagibacter ubique o Discovered in 1990 by Stephen Giovanni by “environmental” rRNA sequencing o Is a small bacterium estimated to account for 20% of all oceanic bacteria |
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What is the impact of microorganisms on humans?
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• Microorganisms can be both beneficial and harmful to humans
o Tend to emphasize pathogens • Infectious disease agents o Many more microorganisms in nature are beneficial than harmful o Knowledge has vastly improve out ability to prevent and treat diseases caused by pathogens • Influenza, pneumonia, TB, gastroenteritis, and diphtheria do not cause as great of harm as they did in 1900 • Microorganisms are important in the agricultural industy o Legumes have nodules on their roots that convert atmospheric nitrogen into fixed nitrogen that plants use for growth • Live in close association with bacteria • The activities of bacteria reduce the need for costly and polluting plant fertilizers o Microorganisms are used in carbon cycling, sulfur cycling, meat and milk production, animal and plant diseases, too • Microorganisms are important in the processing of foods o Cheeses, yogurts, buttermilk, sauerkraut, pickles, sausages, baked goods, alcoholic beverages, and many more o Culinary microbes add tang to food and deacidify wine • Microorganisms play an important role in the safety of food o Food fit for human consumption can support the growth of many microorganisms and must be properly prepared, preserved, and monitered to avoid the transmission of disease or poisoning of the consumer by production of powerful toxins • Clostridium botulinum • Escherichia coli O157:H7 strain • Biotechnology is the use of microorganisms in industrial biosynthesis by microorganisms that have been genetically modified to synthesize products of high commercial value o Production of pharmaceuticals from genetically modified organisms (like insulin and other human proteins) can be used as gene therapy for certain diseases to correct the genetic lesion • Microorganisms are used in energy production o Methane (natural gas), energy stored in organisms (biomass), and ethanol • Microorganisms are used to consume spilled oil, solvents, pesticides, and other environmental toxic pollutants • Microorganisms can be used to treat human and animal diseases through the use of probiotics • Microorganisms are important in the formation or ores and mineral deposits |
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who were influential people in the early years of microbiology?
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• Robert Hooke
o Described fruiting bodies of fungi in 1665 and presented drawings of what he observed in Micrographia o Was the first person to describe microorganisms • Antoni van Leeuwenhoek o Made simple microscopes because he wanted to examine fibers in cloth o Make a new microscope for each specimen o Examined water and infusion and saw tiny wee animalicules, later classified as fungi, algae, single-celled protozoa, and bacteria o Presented observations in a serried of letters to the Royal Society of London o The father of Protozoology and the discoverer of bacteria • Spontaneous Generation of Microbial Life o Aristotle proposed spontaneous generation o Francisco Redi generated experiments that doubts Aristotle’s theory • Maggots grew on open piece of meat • No maggots grew on corked or gauzed meat • Turberville Needham’s Experiments o Scientists believed that microbes could arise spontaneously, but animals could not o Microbes grow when exposed to air • Spallanzani’s Experiments o Lazaro Spallanzani repeated Needham’s experiments with a different protocol • Boiled and immediately sealed broth No microbes grew o Concluded that • Needham failed to heat vials sufficiently to kill all microbes or didn’t seal vials tightly enough • Microorganisms exist in air and can contaminate experiments • Spontaneous generation of microorganisms does not occur o Critics said that the sealed vials did not allow enough air for organisms to survive and that prolonged heating destroyed the “life force” |
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What questions were asked during the golden age of microbiology?
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• Searched for answers to four questions
o Is the spontaneous generation of microbial life possible? o What causes fermentation? o What causes disease? o How can we prevent infection and disease |
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What were Louis Pasteur's experiments?
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o Reported the results of experiments he had designed to answer Spallanzani’s critics
• Definitively disproved spontaneous generation • Non-sterile infusion is poured into a flask and the neck of flask is drawn out into an S shaped open tube • The liquid is “sterilized” by heating and the air is forced out the open end • The infusion is heated and then sits for days while remaining sterile indefinitely • Air is able to move in and our and the dust from the air settles in the bend • If the sterile infusion has been sitting for a long time and then the flask is tilted, the solution comes in contact with the dust in the neck and microbes appear in the infusion • Pasteur’s Conclusions o Microbes in the air were the “parents” of the microorganisms that appeared in Needham’s flasks of boiled brother • Now we would assert that Needham did not use “proper sterile techniques” o Never will spontaneous generation recover from the mortal blow of this simple experiment |
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how was Pasteur a top notch scientist?
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o The father of Microbiology
o Notable discoveries • Sterilization The killing of microorganisms on objects or in liquids by heat or chemical treatment • Pasteurization Preservation of foods by keeping them at a temperature above 60 C and below the boiling point for at least 60 mins • Facultative anaerobes Organisms that can live with and without oxygen Yeast and acid producing bacteria • Founder of industrial microbiology Biotechnology Microbes are intentionally used to manufacture products • Development of vaccines Against anthrax, cholera, and rabies • Discovered fermentation Of pasteurized grape juice by yeast produces alcohol (wine) Of pasteurized grape juice by bacteria produces acid (vinegar) • Postulated the germ-theory of disease Proved by Robert Koch |
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What is Ferdinand Cohn known for?
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o Contemporary of Louis Pasteur and a botanist interested in microscopy
o Studied unicellular algae and pigmented bacteria and believed they were plants o Described the filamentous, sulfur oxidizing bacterium Baggiatoa mirabilis and recognized that granules inside the cells of this organism consist of elemental sulfur produced by the oxidation of hydrogen sulfide o Discovered bacterial endospores and described the lifecycle of Bacilli • Vegetative cell to endospore to vegetative cell o Discovered that endospores could not be killed by boiling and explained why boiling was an unreliable means of sterilization o Credited with helping to devise simple methods that are effective in preventing contamination of sterile cultures like plugging vials and flasks with cotton o The Father of Bacteriology o Director of a research institute in Berlin and a strong advocate of Robert Koch’s work, the founder of Medical Microbiology |
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What is Robert Koch known for?
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• Robert Koch
o Developed a set of postulates to prove that a specific microorganism causes a specific disease o Studied Anthrax o Examined blood of infected animals and found a large rod-shaped bacterium was always present in animals that were succumbing to the disease, but never in healthy animals o Could no tell if the bacterium was the cause or the result of the disease o Ferdinand Cohn became aware of Koch’s studies and gave him the means to pursue his studies on pathogens o In Berlin, they developed • Simple staining techniques • Photomicrography Methods for photographing bacteria from cultures and in diseased tissues under a microscope • Techniques for estimating number or live bacteria in liquids by determining colony forming units (CFU/ml) by serial dilutions • The use of steam to sterilize media and equipment • Aseptic techniques Flame-sterilization of wire loops, stem sterilization of pipettes, etc • The use of glass Petri dishes filled with media solidified by agar for the preparation of pure cultures • Koch understood that each colony represented a clone of cell that had arisen from the growth of a single cell or spore that was deposited on the surface of the medium and could be used to start a pure sulture of the corresponding organism |
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What were Koch's postulates?
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o Koch’s postulates
• The suspected pathogenic organism should be present in all cases of the disease and absent from healthy animals Observed blood tissue under the microscope • The suspected organism should be grown in pure culture Streak agar plate with sample from either diseased or healthy animals • Cells from a pure culture of the suspected organism should cause disease in a healthy animal Inoculate healthy animal with cells of suspected pathogen Remove blood or tissue sample and observe by microscopy • The organism should be reisolated and shown to be the same as the original |
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How was Koch a top notch scientist?
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• Proved that anthrax is caused by Bacillus anthracis and set out to find the agent of tuberculosis
• Developed a staining procedure (alkaline methylene blue followed by Bismarck brown, forerunner of acid fast stain) which revealed blue rod shaped bacterium in tb tissue • Caused tb in Guinea pigs by inoculating them with tissue samples from diseased humans • Grew tb in a medium containing coagulated blood serum • Used Guinea pigs to prove that pure cultures of tb curltured from diseased humans and different types of animals elicit tb after being introduced into healthy animals • Demonstrated that animal testing is critical for the id of disease agents that are deleterious or lethal to humans • Received the Nobel Prize for Physiology or Medicine • Recognized as the Father of Medical and Laboratory Microbiology |
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What did Martinus Beijerinck and Sergei Winogradsky do?
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o Two giants of the age were Martinus Beijerinck and Sergei Winogradsky
• Studied bacteria in soil and water and developed the enrichment culture techniques for the isolation of representatives of various physiological groups The enrichment culture technique involved the inoculation of synthetic media that contained or lacked chemicals that were deemed to be important or unimportant for the growth of some types of cells The omission of ammonia and nitrate from a medium potentially would allow the growth of organisms that could “fix” nitrogen (N2) from the air, and inhibit the growth of those that derive nitrogen for the synthesis of amino acids, purines and pyrimidines from ammonia and or nitrate • Physiological groups of microorganisms o Enrichment techniques allowed Beijernck and Winogradsky to identify aerobic nitrogen-fixing bacteria, sulfur-reducing and sulfur oxidizing bacteria o Winogradsky recognized the metabolic significance of biogeochemical processes • From his studies on sulfur-oxidizing bacteria, he developed the concept of chemolithotrophy Defined as energy conservation linked to the oxidation of inorganic compounds o From his studies on nitrification, Winogradsky concluded that the nitrifying bacteria obtained their carbon from CO2 and concluded that they were autotrophs (chemoautotrophs not photoautotrophs) meaning they used CO2 as their sole source of carbon • Winogradsky’s most important concepts o Nitrogen fixation • N2 + 6H > 2 NH3 Uses ATP Product used for proteins and nucleic acids • Chemolithotrophy Using ADP and P to make ATP • H2S > S0 > SO4(2-) • NH3 >NO2(-) > NO3 (-) • Chemoautotrophy Uses CO2 • A waste compound produced by one organism may be an important metabolic substrate of another! |
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what happened during the emergency of virology?
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o Ivanowski and Beijernck observed that extracts from tobacco plants that showed the symptoms of a mosaic disease contain an infection agent that passes through filters that normally retain bacteria
• Beijernck recognized that the pathogen was not a bacterium and called it a filterable virus (TMV) Such agents are called viruses today o In 1900 Walter Reed proved that yellow fever in humans is called by a virus o The first bacterial virus (bacteriophage) was discovered in 1915 or 1917 by Twort or d’Herelle |
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What happened during the invention of vaccinations and the emergence of immunology?
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• Vaccination and the Emergence of Immunology
o Jenner incoluated a boy with pus from a cowpox on a milkmaid then with smallpox • Showed that the cowpox virus protects against smallpox • Developed the concepts of vaccination and immunity o Immunology is often defined narrowly as the study of the ability of higher-animals to resist infection and overlaps partially with Serology o In a more liberal interpretation of the discipline, “immunology” also includes the consideration of • Mechanisms by which microbes escape detection by the immune system • The role of microorganisms in eliciting immune deficiencies and autoimmune disease • Invertebrate immune responses • Infection and injury-induced local and systemic defense responses in plants |
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what happened during the emergence of serology?
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• The emergence of Serology
o The study of blood serum (blood fluid left after blood cells and clotting factors have been removed from whole blood o Emil von Behring and Kitasato demonstrated the existence of chemicals (antibodies) and cells (macrophases and lymphocytes) in the blood that fight infection (pathogen or toxin is inactivated in vitro by serum from an immunized animal) o Agglutination or inactivation of microorganisms by sera from immunized humans and animals soon were used to classify microorganisms into serovars • Sera from non-immunized humans are used to determine blood types (ABO, MN, Rh factor, etc.) |
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What happened during the modern age of microbiology?
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o The astute observations and simple experiments of medical practitioners, scientists, monks, and people in all kinds of trades instigated the practices and concepts that became the foundation for the amazing developments of the Modern Age of Microbiology
o Would be unfair to only credit microbiologists because much would have been impossible without chemistry, physics, mathematics, and computer science o All biologists are driven by finding answers to questions such as: • 1. What are the Basic Chemical Reactions of Life? Biochemistry and Molecular Biology. • 2. How Do Genes Work? Molecular Genetics, Recombinant DNA Technology, Gene Therapy, Genomics, Enzymology, Proteomics. • 3. What Role Do Microorganisms Play in the Environment? Environmental Microbiology, Ecology, Bioremediation. • 4. How Do We Defend Against Disease? Hygiene, Epidemiology, Food safety, water and sewage treatment, Serology, Immunology, Chemotherapy. • 5. How can we use Microorganisms? Industrial Microbiology, Agriculture, Bioremediation, Food Processing, Bioengineering, Mining. o Some subdisciplines of applied microbiology include medical microbiology, immunology, agricultural microbiology, industrial microbiology, aquatic microbiology, marine microbiology, and microbial ecology. o In the middle to latter part of the twentieth century, basic and applied microbiology worked hand in hand to usher in the current era of molecular microbiology. |
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what are the general principles of microscopy?
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• The general principles of microscopy are
o Wavelength of radiation (light or electrons) • Determines resolution • Can cause excitation and fluorescence of molecules o Resolution is a functions of the physical properties of the radiation (light electrons) o Magnification can be unlimited, but high magnification does not improve resolution o Contract is achieved by • Staining or shading of the specimens (many methods) • Manipulation of light or electron beam • Electromagnetic spectrum o As resolution increases, energy increases, and wavelength decreases • Size comparisons o Atoms, molecules, viruses, small bacteria, unicellular algae, bacteria, cyanobacteria, colonial algae, protozoa, fungi, multicellular organisms |
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what are the parts of a light microscope?
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• Light microscope
o Ocular lens • Remagnifies the image formed by the objective lens o Body • Transmits the image from the objective lens to the ocular lens using prisms o Arm o Objective lenses • Primary lenses that magnify the specimen o Stage • Holds the microscope slide in position o Condenser • Focuses light through specimen o Diaphragm • Controls the amount of light entering the condenser o Illuminator • Light source o Course focusing knob • Move the stage up and down to focus the image o Fine focusing knob o Base • Why oil? o With oil, unrefracted light rays enter lens o Without oil refracted light rays lost to lens o An oil that has the same refractive index as glad reduces light scattering |
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how can you improve and adjust contrast for light microscopy?
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• Improving and Adjusting Contrast for Light Microscopy
o Cells that do not contain pigments usually are hardly visible in a bright field microscope because they lack contrast o Contrast (but not resolution) can be increased in two ways • Staining Can differentiate between groups of organisms Cells usually are killed Procedures often cause distortions in the specimen • Manipulation of the light that is used to illuminate the specimen Live cells can be observed Structures that can not be stained can be observed (eg flagella) Motility of microorganisms can be studied • Staining cells for microscopic observation o Spread culture in thin film over slide o Dry in air o Pass slide through flame to fix o Flood slide with stain; rinse and dry o Place drop of oil on slide and examine with 100x objective o Tissues may require embedding and slicing |
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how is differential staining done?
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• Differential Stains
o Gram Stain • Step 1 Flood the heat-fixed smear with crystal violet for 1 min All cells purple • Step 2 Add iodine solution for 3 min Mordant intensified stain All cells remain purple • Step 3 Decolorize with alcohol briefly for about 20 seconds Gram-positive cells are purple; gram-negative cells are colorless • Step 4 Counterstain with safranin for 1-2 min Gram-positive (G+) cells are purple; gram-negative (G-) cells are pink to red • Gram stained cells of a mixture of bacteria o Gram-postitive cocci (purple) mixed with Gram-negative rogs (pink/red) o What is the objective of a Gram stain? • It is the first step in the classification of bacteria, distinguishing between two major groups: Gram+ and Gram- o Why do Gram-positive bacteria retain crystal violet • Crystal violet (CV) combines with iodine (I) can forms an insoluble complex, CV-I, that is trapped in the thick peptidoglycan layer of Gram-positive cells and can not be washed out by alcohol |
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what are differential staining fundamental concepts?
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• Differential Staining Fundamental Concepts
o Bacteria are slightly negatively charged at pH 7, thus attract cations o Strains consist of positive and negative ions o In a basic dye, the chromophore is a cation • Crystal violet, methylene blue, malachite green, safranin o In an acidic dye, the chromophore is an anion • Eosin, acid fuchsin, nigrosin o Staining the background instead of the cell is called negative staining |
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what happens to cells that undergo differential staining
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o Cells that retain the basic stain, carbolfuchsin (basic fuchsin), in the presence of acid-alcohol are called acid-fast
o Non-acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually conterstained (with a different color basic stain) to see them o Only cells that have a “waxy” coat (mycolic acid, present only in the Mycobacteria) retain the acid-fast stain • Some Nocardia straings also stain; their cells and cell walls are extremely high in lipid content (both are technically Gram+) o Important Mycobacteria and Nocardia • Mycobacterium tuberculosis • M. leprae • Nocardia asteroids (causes hard-to-treat pulmonary ad skin infections) |
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what is acid fast staining?
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• Acid-fast staining
o Basic fuchsin (Rosaniline) o Cationic amino groups form ionic bonds with anionic carboxyl groups of mycolic acids • Mycobacteria derive their name from the fact that they have mycolic acids in their cell walls o Some mycolic acids of Mycobacterium tuberculosis are • A-mycolic acid, methoxy-mycolic acid, keto-mycolic acid |
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what are the schematic parts of a mycobacterial cell wall?
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• Schematic of a mycobacterial cell wall
o Trehalose dimycolate • Made up of Trehalose and Mycolic acid o Mycolylarabinogalactan • Mycolic acid, Arabinofuranose, Galactofuranose, Diglycosylphosphoryl bridge, peptidoglycan, protein, connected to the plasma membrane o For Mycobacteria, the chains are 60-90 carbons in length o For Corynebacteria, chain of 28-40 carbons are common o For Nocardia, chains are 40-56 carbons in length |
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what are the different types of contrast for light microscopy?
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o Bright field (grey everything)
• Has condenser lens, specimen, objective lens, ocular lens o Dark field (black background an light light specimen) • Has an opaque disk, specimen, unreflected light, only light reflected by the specimen is captures by the objective lens o Phase contrast (grey background and halo around specimen) • Has an annular diaphragm, condenser lens, specimen, refracted or diffracted light (altered by specimen), objective lens, undiffracted light (unaltered by specimen) Diffraction plate, and ocular lens |
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what is differential interference contrast (DIC) microscopy?
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• Differential interference contrast (DIC) microscopy
o Good for observing living cells o Accentuates diffraction of the light that passes through a specimen: uses two beams of light o Has an eye or camera, polarizing filter, upper wollaston prism, objective lens, sample, condenser lens, lower wallaston filter (prism), polarizing filter, light source |
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what is fluorescence microscopy?
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• Fluorescence microscopy
o Directs a UV-light beam at the specimen, causing the specimen to radiate energy (photons) of longer, visible wavelength (fluorescence) o UV light increases resolution and contrast o Come cells and molecules are naturally fluorescent, while other must be stained with compounds that fluoresce when irradiated with UV light o Used in immunofluorescence to identify pathogens and to determine the location of proteins and other antigens in tissues and within cells o Used in FISH (fluorescent in situ hybridization) to identify organisms in complex tissue and environmental samples, and the expression of genes within cells • A One-Step Gram Stain Uses Two Fluorescent “Dyes” o One version of the one-step “Gram” stain uses a mixture of two nucleic acid-binding dyes: hexidium iodine (HI, red orange) and SYTO 13 (green) o Cells have to be observed with a fluorescence microscope o HI penetrates Gram-positive but not Gram-negative organisms, whereas SYTO 12 penetrates both o When the dyes are used together, Gram-negative organisms are rendered green fluorescent by SYTO 13; conversely, Gram-positive organisms are rendered red/ orange fluorescent by HI, which simultaneously quenches SYTO 13 green fluorescence • Fluorescence microscopy o DNA stained with a dye that fluoresces red (oglionucleotide probe, FISH) o Spore protein stained with fluorescein (green) o RNA for beta- galactosidase is stained with a blue fluorescent dye (FISH) |
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what is immunofluorescence?
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• Immunofluorescence
o A microbe is injected into an animal and serum is taken out o The antibodies are combined with fluorescein isothiocyanate (fluorescent dye) o The bacterial cell is bound with antibodies combined with dye o Fluorescent dye-tagged monoclonal antibodies that are specific for many different pathogens are commercially available • A second Antibody Trick o A trick commonly used to avoid the pain of binding a fluorescent dye to all the different antibodies (Ab) involved the production of large batches of a “second” antibody (goat, sheep) that reacts with all the “first” antibodies (produced in mice or rabbits) |
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what is the principle of fluorescent in situ hybridization (FISH)?
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o Synthetic oligonucleotide probe has a sequence complementary to a sequence in DNA or RNA
o One strand of the DNA has fluorophore (fluorescein) attached and the other has a quencher (Dabecyl) attached |
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what is confocal scanning laster microscopes (CSLM)?
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• Confocal Scanning Laser Microscopes (CSLM)
o Uses a UV-laster directed through a pinhole that precisely focuses the light a very narrow vertical plane in the specimen o This system is coupled to a scanning devise that makes many “optical slices” through the sample o A computer then generates a 3-D image from these slices of data o The CSLM is coupled to a light microscope • An image taken by the light microscope can be superimposed to the image generated by the laser scan o Excellent devise for determining the intracellular location of structures marked with fluorescent dyes |
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what is electron microscopy?
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• Electron microscopy
o The principle of transmission electron microscopy is the same as for light microscopy: it uses a beam of electrons focused by electromagnets • Wavelength Determined by voltage • Magnification 1000 to 10^6 fold • Resolution Limited more by technology than wavelength o Has an electron gun, condenser lens (magnet), specimen, secondary electrons, objective lens (magnet), projector lens (magnet) and final image on fluorescent screen o Types of electron microscopes • Transmission electron microscopes (TEM) Require thin sections of materials Dehydrated samples Permeation of sample with electron-dense strains (uranyl acetate) • Scanning electron microscopes (SEM) Can detect only surfaces: good for shapes Samples must be coated with metal (gold or platinum) • Live cells can not be examined by TEM or SEM because objects must be viewed in a vacuum Modern instruments combine TEM SEM capacity o Scanning EM and specimen coating chamber • Very narrow beam moves bak and forth over object and data is transmitted to a detector and converted into an image |
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What is electroncryotomography?
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• Electroncryotomography
o Relatively new technique o Can be performed on “live” cells o Cells in a liquid are plunge frozen in liquid nitrogen o Layers of ice with embedded cells are sliced off with a microtome and kept frozen in the object chamber (vacuum) o The slices are then bombarded with electrons at different angles in a transmission cryoelectron microscope o Images are computergenerated from deflected (dislodged) electrons captured on a screen o Technique reveals very fine structures including the existence microfilaments and compartments in prokaryotic cells (cytoskeleton) |
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what is scanning tunneling and atomic force microscopy?
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• Scanning Tunneling and Atomic Force Microscopy
o Scanning tunneling and atomic force microscopy can give extremely good resolution: in some cases, it is possible to vaguely locate atoms within molecules o No special preparation of the specimens is required: live cells and native macromolecules can be scanned o Scanning tunneling microscopes (STM) and atomic force microscopes (AFM) use a stylus consisting of a pointed tungsten probe that is sharpened to a single atom |
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what happens in scanning tunneling microscopy (STM)?
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• Scanning tunneling microscopy (STM)
o Tunneling microscopes pass the tiny tungsten probe at a short distance back and forth over the object o The generate data be measuring electron flow to and from the tip of the probe o By converting the rate of electron flow into distances, a computer generates a (topographical) image of the specimen • Electron flow is inversely proportional to distance of the probe from the object |
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what happens in atomic force microscopy (AFM)?
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• Atomic force microscopy (AFM)
o In atomic force microscopes, the tiny tungsten stylus is placed very close to the surface of the speciment o Weak repulsive forces between the stylus and the specimen make the stylus move up and down as it is passed over the object o This up and down movement is detected by a laser bean pointed at the tip of the probe o A 3-D image is produced by a computer |
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What are the elements of cell structure?
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• Elements of Cell Structure
o All microbial cells share certain basic structures, such as • A cytoplasmic membrane, through which nutrients and other substances needed by the cell enter, and waste materials and other cell products exit • Cytoplasm, which is a complex mixture of substances and structures bounded by the cell membrane Major components dissolved or suspended in the cytoplasm of all are • Macromolecules o Mostly proteins and nucleic acids • Many organic small molecules o Precursors of macromolecules • Inorganic ions • Ribosomes o Little machine that consist of RNA and many protins, and are dedicated to the assembly of proteins as they read and translate information contain in messenger RNAs • Porous cell wall (usually) o Located outside the cell membrane and protects cells from osmotic and physical damaged o Usually a strong, inelastic sheath of polysaccharide fibers and/or proteins o Plant cells and most microorganisms have a cell wall, but most animal cells do not • Cytoskeleton o Present in all cells, acts as a scaffolding that reinforces cell structure |
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what are the different types of cells?
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• Types of cells
o By the structural characteristics of their cells, organimsms are classified as prokaryotes (Bacteria, Archaea) and eukaryotes (protista, which includes all unicellular eukaryotes, and higher plants and animals) o Prokaryotic cells have a simpler internal structure than eukaryotic cells, mostly lacking distinct membrane-enclosed organelles o Prokaryotic cells • Sizes and shapes vary trememdously • Have cell wall, cytoplasmic membrane, cytoplasm, ribosomes, plasmid, nucleoid • Belong to two phylogenetically different domains, distinguished by characteristic rRNA sequences: Bacteria and Archaea o Eukaryotic cells • Generally are much larger than prokaryotic cells and have a complex internal structure, including membrane-bound organelles that compartmentalize functions One nucleus or several nuclei • Gene replication and transcription Mitochondria • Energy metabolism in all eukaryotes Chloroplasts • Photosynthesis, plants only Golgi bodies • Protein sorting and packaging for secretion Endoplasmic reticulum • Transport, multiple functions Lysosomes • Digestion of macromolecules Peroxisomes • Enzymatic reactions that produce hydrogen perioxide (H2O2) • Can vary tremendously in size and shape • Include algae, fungi, and protozoa • Have rRNA features distinct from those of the bacteria and archaea, thus they are grouped into a single biological domain, the eukarya |
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what are the characteristics of viruses?
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• Viruses
o Are not cells, but depend on cells for their replication o Have limited metabolic capacity of their own o Lack ribosomes and depend totally on ells for the synthesis of their proteins o Generally considered to be “microorganisms” o Only when they infect cells to viruses acquire the key attribute of being living systems- reproduction o Are static particles consisting of a nucleic acid (DNA or RNA “chromosome”) which is packaged into an envelope consisting either of one or more proteins or a membrane derived from the cell membrane o Infect the cells of all organisms in the domains bacteria, archaea, and eukarya o Many viruses cause disease in the organisms they infect o Many viruses have profound effects on the properties of cells, including genetic alterations that can either deleterious or beneficial o Vary in size, shape, and complexity o Small that the smallest cells |
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What is Acanthamoeba polyphaga Mimivirus (APMV)?
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o Free-living amoeboid protozoan and opportunistic pathogen that can cause keratitis and encephalitis
• Linear dsDNA virus • 90% coding capacity • 10% junk DNA • ~911 protein coding genes • Has additional genes, including aminoacyl tRNA snthetases, sugar, lipid, amino acid metabolism o The genome of their virus consists of 1.2x10^6 bp and is larger than the genomes of small parasitic bacteria o Sputnik is a virophage that infects APMV (Mimivirus) • Sputnik can not infect Acanthamoeba castellanii on its own: no new virus is produced • Sputnik can infect and multiple only in APMV-infected A. castellanii cells • Sputnik reduced the yield of infective APMV particles of 70% and decreases lysis of castellanii cells threefold • The genome of Sputnik is a 18.3-kb circular DNA and has only 21 predicted protein coding genes |
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What are viroids?
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• Small infectious, single-stranded, circular RNAs
• Have been found only in plants • Do not produce proteins • Replicate in the nucleus or in chloroplasts • Cause a variety of plant diseases (PST disease, chrysanthemum stunt disease, cucumber pale fruite disease ASB disease o Most likely by interference in the translation of host-cell mRNAs (RNAi- RNAinhibition) • Have a sequence that will complex with cellular RNase • Ribosome cannot translate the gene o Replicated in the chloroplast or the nucleus o Go to other cells and then get into the vascular system the plant and are distributed throughout the plant and invade new cells • Travel around via insects |
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What are prions?
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• Infectious proteins that exist in two forms in mammals and yeast
o Harmless form (PrP), which is found on the plasma membrane of mammalian cells, especially brain cells o Harmful form (PrPSc), which is an incorrectly folded form of PrP • Causes the conversion, refolding, of PrP to PrPSc Referred to as seeding • PrPSc molecules stick together inside the cells, forming insoluble fibrils that are associated with degeneration of brain tissue (spongiform encephalomyopathy) Plack formation o No species specific, and can be transmitted through protein supplements in feed, consumption of raw meats or brains, skinning and butchering of affected animals |
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what are chracteristics of genes and genomes?
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• Genes govern the metabolism of cells
o Therefore the growth and reproduction • A cell’s complement of genes is its genome • DNA is arranged in cells to form chromosomes • Most prokaryotes have a single circular chromosome, which is folded by proteins into a compact nucleoid (no nuclear membrane) o histones o Some prokaryotes, have linear chromosomes and some have more than one chromosome • Plasmids are circular or linear extrachromosomal genetic elements (DNA), nonessential for growth o Often are attributed exclusively to prokaryotes o Common in fungi and plants • No plasmids have been found in higher animals • Through non-essential for growth, plasmids sometimes contain genes that confer special properties to cells, such as antibiotic resistance, toluene degradation, pathogenicity (render cells pathogenic), and toxin production o Conjugation allows them to move around and the plasmid transmits itself • In eukaryotes, the genome usually consists of several chromosomes that are sequestered in the membrane-bound nucleus o Each chromosome consists of a long linear DNA molecule which is wound around proteins (histones) and forms an array of nucleosomes • Many eukaryotic microorganisms are haploid (have a single set of chromosomes, yeast has 16), while the cells of most multicellular organisms are diploid (have a pair of each chromosome) HOW DO THEY UNDERGO MEIOSIS?!? • Mitochondria and chloroplasts in eukaryotic cells also contain DNA o Organelle chromosomes are small and resemble prokaryotic chromosomes • Most genes code for proteins • On average, E. coli cell contains 1900 different proteins and a total of about 2.4X10^6 protein molecules o Total of about 400 genes, but not expressed • Some proteins are very abundant, other are rare o Call have mechanisms for controlling the expression of different genes • Arabidopsis and rice have more genes than homo sapiens |
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what is evolution and phylogeny?
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Evolution and Phylogeny
• Evolution is the sum of the genetic changes (mutation) in a line of descent over time heading to new species or varieties • Mutation o Heritable change in the nucleotide sequence of an organism, which happens in the genome of an individual cell and then is transmitted to furture generations of “progeny” • The mutant genotype which may or may not be manifested as an easily-obserable phenotype • The evolutionary relationships between life forms are the subject of the science of phylogeny • Although the evolutionary relationships among organisms can be assessed in different ways o Comparison of morphological and/or physiological traits, lipid composition of membranes o The direct assessment of genetic similarity/dissimilarity at the DNA level has become the most phylogenetic, molecular phylogeny |
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what are characteristics of ribosomes?
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• Carl Woese demonstrated that comparisons of the nucleotide sequences of rRNA genes is a very reliable way to determine the phylogenetic relationships among large numbers of different organisms (rRNA phylogeny)
• The sequences of 16S rRNA of the small ribosomal subunitis are most commonly used to construct phylogenetic trees for the prokaryotes o The equivalent rRNA in eukaryotes is 18S • Large subunit rRNAs o Prokaryoties: 23S o Eukaryotes: 28S • rRNA phylogeny is the bases for the grouping of microorganisms • The Tree of Life o All cells have ribosomes, and the analysis of the nucleotide sequences of ribosomal RNA genes showed that they are highly conserved • Such genes tolerate few mutations (related to the requirement of their functional integrity for the survival of cells) o Comparative ribosomal RNA (rRNA) sequencing, inititated by Carl Woese, as defined three domains of life: Bacteria, Archaea, and Eukarya • The rRNAs of the organisms in each domain of life share characteristics “signature sequences” o The ribosomes of each of the three domains of life, Bacteria, Archaea, and Eukarya, have unique characteristics: different sequences and ribosomes respond differently to antibiotics that inhibit protein synthesis |
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what are major features that group bacteria?
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o Ester linked membrane lipids, chlorophyll photosynthesis, muramic acid in cell wall, no histone proteins in chromosome structure, 1 RNA polymerases (4 subunits), -10, -30 sequences for promoter structure, 1 to over 50 transcription factors, initiator tRNA is fMet, ribosomes not sensitive to diptheria toxin but yes to Cap, Str, Kan
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what are major features that group archaea?
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o ether linked membrane lipids, no chlorophyll photosynthesis, no muramic acid in cell wall, yes histone proteins in chromosome structure, 1 RNA polymerases (8-12 subunits), -TATA box sequences for promoter structure, TBP, BRE, TFB transcription factors, initiator tRNA is Met, ribosomes sensitive to diptheria toxin but not to Cap, Str, Kan
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what are major features that group eukarya?
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o Ester linked membrane lipids, chlorophyll photosynthesis, no muramic acid in cell wall, yes histone proteins in chromosome structure, 3 RNA polymerases (12-14 subunits each), TATA box sequences for promoter structure, many, different for each RNAP transcription factors, initiator tRNA is Met, ribosomes sensitive to diptheria toxin but not to Cap, Str, Kan
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why are there mixed origins of eukaryotic genomes?
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• Mixed origin of Eukaryotic genomes
o In addition to the genome in the chromosomes of the nucleus, mitochondria and chloroplasts of eukaryotes contain their own genomes (DNA arranged in circular fashion, as in Bacteria) and ribosomes. o On the basis of rRNA sequence data, these organelles have been shown to been derived from ancestors of specific lineages of Bacteria (see next slide). o Mitochondria and chloroplasts were thus once free-living cells that established stable residency in cells of Eukarya eons ago. The process by which this stable arrangement developed is known as endosymbiosis. o Many of the genes of the original endosymbionts that gave rise to mitochondria and chloroplasts have since been moved into the nucleus: the nuclear genomes of eukaryotes are composed of a set of genes derived eukaryotic ancestors and genes derived from bacteria. |
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What are the criteria for assessing diversity?
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• Criteria for Assessing Diversity
o Microbial diversity is overwhelming and can be observed in many ways • Cell morphology: size, shape, prokaryotic, eukaryotic, natural pigmentation, staining (e. g. Gram positive and Gram negative). • Cell physiology: metabolic strategies • Cell motility: swimming, gliding, sessile, flying. • Mechanism of cell division: fission, budding, endospore formation, mitosis, meiosis. • Pathogenicity: non-pathogenic, opportunistic pathogen, pathogen. • Habitat: “Common” soil, water, animals and plants “Extreme” glaciers, polar seas, hot springs, thermal vents, brine, deep ocean, low and high pH. • Genetic diversity: rRNA signatures, evolutionary divergence. |
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how are organisms classified by carbon sources?
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o Classification of organisms by carbon sources:
• Autotrophs use carbon dioxide (CO2) as their carbon source. • Heterotrophs use organic compounds as sources of carbon (carbohydrates, lipids, organic acids, etc.). |
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how are organisms classified by energy sources?
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o Classification of organisms by energy sources:
• Chemolithotrophs obtain their energy from the oxidation of inorganic compounds. Most are autotrophs. • Chemoorganotrophs obtain their energy from the oxidation of organic compounds (frequently carbohydrates). By definition, they are heterotrophs. • Phototrophs contain pigments that allow them to use light as an energy source. Almost all are autotrophs. |
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what are characteristics of proteobacteria?
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• The phylum Proteobacteria represents the largest lineage of Bacteria, and includes:
Many chemoorganotrophic species, such as Escherichia coli, the Gram-negative, model organism of bacterial physiology. Several chemolithotrophs and phototrophic chemolithotrophs particularly those using sulfur in their metabolism, such as Chromatium and Achromatium. Many important pathogens, including Salmonella (gastroenteritis, typhoid fever), Neisseria (gonorrhea), and Rickettsia (typhus, Rocky Mountain spotted fever). Many of the bacteria that live in water, soil, animals and plants, including species that can be casual (opportunistic) pathogens or degrade toxic organic compounds, particularly Pseudomonas. Organisms that can fix nitrogen, including Azotobacter. |
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what are characteristics of gram-positive bacteria?
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• Gram-positive Bacteria have in common a thick peptidoglycan cell-wall structure and lack an outer membrane. Included are:
Endospore-forming Bacillus and Clostridium species Antibiotic and spore producing Streptomyces. Milk-spoiling Lactobacillus Many non-pathogenic and a few pathogenic Streptococcus species that produce chains of spherical cells and frequently occur in milk products. The Mycoplasmas, cell-wall lacking obligate parasites (many are pathogenic, e.g. M. genitalium). |
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what are characteristics of cyanobacteria?
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o Cyanobacteria
• The Cyanobacteria are phylogenetic relatives of Gram-positive bacteria and are oxygenic (O2 producing) phototrophs (have light-harvesting pigments- bacteriochlorophylls) • Cyanobacteria were critical in the transition of Earth from an anoxic environment (and anoxic life) to an oxygen-containing environment allowing for the evolution of “aerobic” life • Cyanobacteria can be unicellular, filamentous, colonial and heterocystous (heterocysts are special cells that carry out nitrogen fixation). |
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what are characteristics of green sulfur and non sulfur bacteria?
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• Two additional distinct phylogenetic groups of Bacteria also are phototrophic autotrophs, but they are anoxygenic.
The green sulfur bacteria, represented by Chlorobium. The green non-sulfur bacteria, represented by Chloroflexus. • Though distinguished by rRNA characteristics, the two groups have similar photosynthetic pigments (bacteriochlorophyll c-e (purple bacteria have bacteriochlorophyll a or b). • Chloroflexus often is the predominant species in communities inhabiting hot springs and shallow marine environments (primitive Earth environments) and is an important link in the evolution of photosynthesis. |
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what are characteristics of phyla of bacteria with unique shapes?
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o The Planctomyces are stalked and often form rosettes that are attached to substrates.
o Spirochetes are helical and include some well-known pathogens: Treponema pallidum (syphilis), Borrelia burgdorferi (Lyme disease) |
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what are characteristics of chlamidyia?
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o Chlamydia are obligatory intracellular parasites and most are pathogens.
o Because of their intracellular life style, they escape detection by the immune system. o They are the agents of disorders such as trachoma, urethritis, pneumonia, spontaneous abortion. |
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what are characteristics of deinococcus?
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o Bacteria in the Deinococcus lineage have legendary innate capacities to survive exposures to very high doses of radiation and other mutagens.
o Deinococcus radiodurans has enzymatic systems that can repair massive damage to its DNA, including extensive fragmentation of its DNA. |
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what were “Early” Lineages of Bacteria?
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o Thermatoga and Aquifex are two of several lineages of bacteria that branch of “early” from the trunk of the phylogenetic tree
o Thermatoga and Aquifex are hyperthermophiles that grow in environments (hot springs) near the boiling point of water. o The early-branching points of these lineages indicates that life arose when planet Earth was still much hotter than it is now. • Interestingly, the early-branch lineages of the Archaea, Methanopyrus and Pyrolobus, also are hyperthermophiles. |
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how are anonymous species identified even when they cannot be cultured?
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o Sequencing of PCR-amplified, cloned rRNA genes obtained from DNA isolated from samples of cells retrieved from different habitats have shown that many unique phyla of yet to be cultured prokaryotes exist in nature
o Phyla and species that have been detected by rRNA analysis without any members having been cultured are called environmental (Env). • This kind of analysis indicates that < 10% of the Bacteria and Archaea that exist on this planet have been “cultured” |
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how are organisms classified by habitat preference?
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• Classification by Habitat Preference
o Extremophiles • Thrive under environmental conditions in which high organisms cannot survive • Temperature, pH, pressure, salt |
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what are characteristics of archaea?
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• All Archaea are chemotrophic, although Halobacterium has a light-harvesting pigment, a bacteriorhodopsin/retinal complex, that drives ATP synthesis by generation of a proton-motive force.
o Some are chemoorganotrophs. o Most are chemolithotrophs, with hydrogen gas (H2) being widely used as the energy source. • Most Archaea are extremophiles some extreme extremophiles are: o Hyperthermophiles that grow at temperatures as high as 121 C. (abundant in Yellowstone Natl. Park). o Psychrophiles that grow at temperatures as low as 0 o Acidophiles that grow at pH as low as 0 o Alkaliphiles that grow at pH as high as 12 o Barophiles that grow at pressures >1000 atm o Halophiles that grow at dissolved salt concentrations >32% saturation o Note: Many of the extremophile Archaea thrive at more than one extreme; e.g., thermal vents in deep oceans, 121 at >100 atm • There are two phylogenetic (rRNA) lineages of Archaea, the Euryarchaeota and the Crenarchaeota |
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what are characteristics of euryachaeota?
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• The Euryarchaeota consist of three physiological types of organisms:
The methanogens: • produce methane (natural gas, CH4) by biodegradation of organic matter. Most, if not all, natural-gas deposits in the Earth have been produced by these organisms. • are strictly anaerobic (killed by oxygen), hence they generate CH4 rather than CO2 as a waste product. The extreme halophiles (very high salt environments): • are relatives of the methanogens, but • are strictly aerobic (require oxygen) Thermoacidophiles (Thermoplasma and Picrophilus) • lack cell walls (have only archaean cell membranes). • prefer moderately high temperatures • prefer extremely low pH • Picrophilus is the most acidophilic organism of all known prokaryotes. |
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what are characteristics of crenarchaeota?
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• The Crenarchaeota
o The Crenarchaeota are ubiquitous in all environments: marine, fresh water and soil. o The group also includes many thermophiles and psychrophiles. o Most of the non-extremophile Crenarchaeota survive well under aerobic conditions, but are chemolithotrophs that use ammonia (NH3) as their energy source. o Chemoorganotrophs, important in carbon “fixation” (biomass) o Many have shapes similar to Bacteria. o Unlike other Euryarchaeota, the Crenarchaeota lack histones. o A typical organisms in this group is Sulfolobus, first found in geothermally-heated sulfur springs in Italy. o Sulfolobus can function as a chemolithotrophic (H2S as energy source) heterotroph or autotroph and is capable of aerobic (oxygen is the final electron acceptor) and anaerobic respiration (sulfuric acid is the final electron acceptor). |
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what are eukaryotic microorganisms diversity?
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o Collectively, “unicellular” microbial eukaryotes are known as the Protista.
o Microbial eukaryotes are a diverse group that includes • Algae • Protozoa • Fungi • Slime molds o Cells of algae and fungi have cell walls, whereas the protozoa and slime molds, do not. o Many are haploid (for example some yeasts), others are diploid (slime mold Physarum). o Some algae and fungi have developed mutualistic associations called lichens. o Early branching eukaryotes • Were believed to lack mitochondria, but, it was discovered in 2005 that Giardia, a water-borne pathogen, has “remnants” of mitochondria, indicating that the early-branching organisms may have lost some of the features of “typical” mitochondria |
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what are characteristics of algae?
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o Unicellular or multicellular eukaryotes.
o Photosynthetic (have chloroplasts and convert CO2 + H2O to “food”, usually in the form of a “sugar”) o Most reproduce asexually and sexually. Simple reproductive structures. o Categorized on the basis of pigmentation, storage products, and composition of cell wall. • Unicellular algae are common in freshwater ponds, streams and lakes. • Large multicellular algae, called seaweeds and kelps, are common in oceans. |
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what are characteristics of protozoa?
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o Single-celled eukaryotes.
o Similar to animals in their nutritional needs and cellular structure. A few have chloroplasts (Dunaliella), but rRNA phylogeny places them with other protozoans. o No cell wall, but many have a though outer cuticle o Typically live freely in water or as pathogens in humans and animals. o Most reproduce asexually;; some reproduce sexually. o Most are capable of locomotion by: • Pseudopodia ␣ cell extensions that flow in direction of travel (Amoeba). • Cilia - numerous, short, hair-like protrusions that propel organisms through environment (Ciliates). • Flagella - extensions of a cell that are fewer, longer, and more whip-like than cilia (Flagellates). |
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what are characteristics of fungi?
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o Eukaryotic: have membrane-bound nuclei and mitochondria.
o Saprobes: obtain food from other organisms, dead or alive. Are major agents of biodegradation. o Have cell walls that contains some chitin. Included are: • Molds - multicellular; have hyphae;; hyphae usually are coenocytic; reproduce by growth of hyphae, asexual spores; some also reproduce sexually and generate sexual spores by meiosis. Many have spectacular fruiting bodies known as mushrooms • Yeasts - unicellular; reproduce asexually by budding (budding yeasts) or fission (fission yeasts); some reproduce sexually and produce sexual spores by meiosis. |
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what are characteristics of slime molds?
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o Slime molds resemble protozoa because they lack cell walls.
o Molecular phylogeny, however, does not group slime molds with the protozoa or with the fungi. o Slime-mold cells show social behavior and aggregate to form fruiting bodies (organs dedicated to cell differentiation into spores). o Slime molds are the earliest known protists that show cellular cooperation to form organ-like structures (development and differentiation). o Slime molds can be o Acellular, which grow as a mass of protoplasm, called a plasmodium, and expand by amoeboid movement (e.g., Physarum) o Cellular, which grow by division of vegetative cells that aggregate and migrate as a mass, called a slug, when they become starved (e. g. Dictyostelium). |
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what are characteristics of lichens?
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o Lichens often are found growing on rocks, trees and other surfaces.
o Lichens are examples of mutualism, the coexistence of two organisms that benefit each other. o Lichens consist of a fungus and a phototrophic partner, an alga or a cynobacterium, that has chlorophyll and carotenoid pigments. o The phototroph is the primary producer and the fungus provides an anchor and protection from the elements. |
