Introductory Microbiology COMMON CHARACTERISTICS OF LIVING THINGS (ORGANISMS): All biological systems [living things] have the following characteristics in common: 1. The ability to ingest or assimilate nutrients (food substances) and metabolize them for energy and growth. 2. The ability to excrete waste products. 3. The ability to react to their environment sometimes called irritability. 4. The ability to reproduce its own kind. 5. There is susceptibility to mutation. Microbiology is a science that deals with the study of living organisms that cannot be seen by the naked eye. These can be seen with the aid of microscopes, which magnify objects. Microbiology: Study of structures and activities of microorganisms. Form, Structure, Reproduction, Physiology, Metabolism and Identification. Distribution in Nature Relationship to each other and to other living things. Beneficial and detrimental effects on humans, animals, plants. Physical and chemical changes they make in the environment Microorganisms are unicellular or consist of same kind of cells held together. Higher Organisms have a hierarchy of organization: Cells > Tissues > Organs > Organ systems > Organism. An Overview of microbiological Basics 1 Cell is the smallest unit of living things that can maintain its structure and reproduce itself in its suitable environment. Cell is the true basic unit of life. Viruses: Viruses are not cellular organisms. Viruses are obligate intracellular parasites of living host cells. Viruses are particles that represent the border line of life. They can reproduce themselves by being parasites on cells of living things, destroying the host cells. There are viruses which use animal, plant or human cells as their hosts being specific to one kind of host. Haeckel’s three kingdom classification of microorganisms: Plants [Protista] Animals Haeckel (1866), a German zoologist suggested a third kingdom Protista to include those organisms that are not typically plants and animals. Bacteria, cyanobacteria, algae, fungi and protozoa are cellular organisms placed under protista. Bacteria and cyanobacteria were lower protists while algae, fungi and protozoa were higher protists. The cells of living organisms are either procaryotic or eucaryotic in nature and there is not any intermediate condition. The size, shape, morphology and the internal cellular organizations are different in these two groups. Protista: Procaryotic: Eubacteria, Archaebacteria, Cyanobacteria Eucaryotic: Fungi (Molds and Yeasts), Algae and Protozoa Procaryotic cells do not have a membrane enclosing a nucleus, and are smaller in size and have simpler internal constitution in comparison with the eucaryotes. Procaryotes are organisms with primitive type of nucleus lacking a well-defined membrane, a less complex nuclear division than mitosis. The nuclear material is a DNA molecule in prokaryotes compared to chromosomes of higher organisms. Cell wall is made of peptidoglycan (murein or mucopeptide), a component that is absent in eucaryotic cell walls. 2 Eukaryotes are organisms with cells having true nuclei enclosed in a nuclear membrane and are structurally more complex them prokaryotes. A varying degree of localization of cellular functions in distinct membrane bound intracellular organelles like nuclei, mitochondria chloroplasts etc. are occurring in eucaryote cells. Eucaryotes have membrane bound organelles. Their DNA is complex and typically associated with structural and regulatory proteins and it is contained within a membrane bound nucleus. The cells are about ten times larger than those of prokaryotes. Some eucaryotes (e.g. plants) have cell walls but are not made up of peptidoglycan molecules. Replication in eucaryotes involves mitosis and meiosis. Meiosis occurs in sex cells like sperm and egg. The eucaryote cell member is a fluid phospholipid bilayer containing sterols and carbohydrates. The membranes can endocytose, phagocytose, pinocytose and exocytose. In Meiosis a diploid parent cell creates four haploid daughter cells. The DNA has usually undergone some crossing over so the chromosomes are not only halved but are also changed through rearrangement. Mitosis: Cells have two main stages in the life cycle. Interphase: the cells grow and duplicate their DNA, in the second stage the cell’s nucleus divides. In mitosis nuclear division starts after the cell has duplicated its DNA. The result is two exact copies of the DNA. Whittaker (1969) proposed five kingdoms based on three levels of cellular organization and three principal modes of nutrition, photosynthesis, absorption and ingestion. The prokaryotes lacking ingestive mode of nutrition are included in the kingdom. Monera. In the kingdom Protista unicellular eukaryotic microorganisms representing all the three modes of nutrition are included. The multicellular green plants and higher algae were placed in the kingdom Plantae while multinucleate higher fungi in the kingdom Fungi and the multicellular animals in the kingdom Animalea. (1) Binomial nomenclature (a) Organisms are named using binomial nomenclature (viruses are exceptions) (b) Binomial nomenclature employs the names of the two lower level taxa, genus and species, to name a species (c) Conventions when using binomial nomenclature include: (i) Genus comes before species (e.g., Escherichia coli) (ii) Genus name is always capitalized (e.g., Escherichia) (iii) Species name is never capitalized (e.g., coli) (iv) Both names are always either italicized or underlined (e.g., Escherichia coli) 3 (v) The genus name may be used alone, but not the species name (i.e., saying or writing "Escherichia," alone is legitimate while saying or writing "coli" is not) (vi) The genus name may be abbreviated but · It must be used first without abbreviation · If abbreviated it must be used with the species name (no E. all by itself) · It must be abbreviated unambiguously · If abbreviating as the first letter of the genus is unambiguous, then abbreviating as the first letter is what one does (e.g., Escherichia abbreviated as E. but only if no other genera considered also starts with E) · Genus abbreviations are only used in conjunction with the species name (i.e., E. coli) Introduction to Bacteria: The characteristic compound found in all true bacterial cell walls is peptidoglycan. Coccus Chain = Streptoccus Cluster = Staphylococcus Bacillus Chain = Streptobacillus Coccobacillus Vibrio = curved Spirillum Spirochete Square Star 4 Shape Gram Flagella, Fimbriae, staining Morphological pili Traits Colonial Morphology Capsule and slime layers Gram-positive cell walls Gram-negative cell walls Thick peptidoglycan Thin peptidoglycan 90% peptidoglycan 5-10% peptidoglycan Teichoic acids No teichoic acids 1 layer 3 layers Outer membrane has lipids, Not many polysaccharides polysaccharides In acid-fast cells, No acid- fast cells (mycolic contains mycolic acid acid) 5 The Gram staining process includes the use of: a primary stain (crystal violet) a mordant (helper) iodine solution, a decolorizer (95% ethanol), a counterstain (safranin). 6 Classification on the bases of source of carbon, electron and energy CARBON SOURCES: Autotrophs CO2 sole or principal biosynthetic carbon source. Reduced, preformed, organic molecules Heterotrophs from other organisms ENERGY SOURCES: Phototrophs Light Oxidation of organic or Inorganic Chemotrophs compounds ELECTRON SOURCES: Lithotrophs Reduced inorganic molecules Organotrophs Organic molecules 7 Archaebacteria have distnictive chemistry. Originally thought to exist only in extreme environments, archaea are now found to be ubiquitous in soil and water, and even in the human digestive tract. Included are the thermophilic Crenarchaeota, such as Sulfolobus and Pyrodictium, as well as mesophilic crenarchaeotes and even sponge endosymbionts. They cover the salt-loving Haloarchaea, the methanogens, and the elusive Nanoarchaea, whose tiny size pushes the limits of viability. 8 History of development of Microbiology as a science: Many scientists contributed to the science of microbiology. Louis Pasteur (1822-1895) Louis Pasteur was a French chemist and a crystallographer. His contribution to microbiology is so great that he is considered to be the “Father of Microbiology”. Contribution to science: As a chemist He was working with tartaric acid crystals. He could pick up the dextro and levo rotatory crystals by seeing the morphology of the crystals. Later he was called to solve some of the problems in fermentation industry and turned his attention to biological process of fermentation. Contribution to wine industry 1. He discovered that alcohol production from grape juice was due to Yeast. 2. He found out that large amounts of lactic acid production was due to the presence or contamination of rod shaped bacteria. 3. He observed that the process of alcohol production i.e. FERMENTATION took place in the absence of air(high dissolved oxygen in the medium) . 4. He coined the terms aerobic to describe those organisms requiring air and anaerobic to describe those organisms which do not require air for their growth. Contribution to modern microbiology Pasteur disproved the theory of spontaneous generation. The theory proposed that living organisms originated spontaneously, particularly from decaying organic matter. He disproved it. Pasteur’s swan neck flask Pasteur poured meat infusions into flasks and then drew the top of each flask into a long curved neck that would admit air but not dust. He found that if the infusions were heated, they remained sterile (free from any growth) until they were exposed to dust. He opened them on a dusty road and resealed them and demonstrated the growth of microorganisms in all the flasks. The unopened flasks were sterile. Thus he disproved the theory of spontaneous generation. Louis Pasteur defined pasteurization to prevent spoilage of food by bacteria, develop vaccines and disproved the scientific dogma of “Spontaneous Generation”. He defined “Germ Theory” and demonstrated that germs were responsible for disease. 9 Edward Jenner 1796 It was an ancient observation that persons, who had suffered from a specific disease such as small pox or mumps, resisted the infection on subsequent exposures. They rarely contracted it second time. Such acquired resistance is specific. Edward Jenner a country doctor in England noted a pustular disease on the hooves of horses called the grease. This was carried by farm workers to the nipples of cows (cow pox). This was again carried by milk maids. They got inflamed spots on the hands and wrists. The people who got this cow pox were protected from small pox. He reported that 16 farm workers who had recovered from cow pox were resistant to small pox infection. He took the material from the cow pox and inoculated into the cut of an 8 year old boy on 14 May 1796. Two months later Jenner inoculated the same boy with material taken from small pox patients. This was a dangerous but accepted procedure of that time and the procedure was called variolation. The boy was protected against small pox. His exposure to the mild disease cow pox had made him immune to the disease small pox. In this manner Jenner began the science of Immunology, the study of the body’s response to foreign substances. Robert Koch (1843-1910) Robert Koch was a German physician. 1. For the first time he showed the evidence that a specific germ (Anthrax bacillus) was the cause of a specific disease (splenic fever in sheep) 2. He established that a specific germ can cause a specific disease and introduced scientific approach in Microbiology. 3. He discovered Bacillus anthracis (Anthrax bacillus), Mycobacterium tuberculosis, and Vibrio cholerae. 4. He modified Ziehl-Neelsen acid fast staining procedure which was introduced by Ehrlich. 5. He devised the solid medium to grow the microorganism to get single colonies. 6. He introduced Koch’s thread method to find out the efficacy of disinfectants 7. He established certain rules that must be followed to establish a cause and effect relationship between a microorganism and a disease. They are known as Koch’s Postulates 8. He also described the Koch’s Phenomenon The need for Koch’s postulates: In those days there were no perfect techniques to identify the organisms. Solid media and staining techniques were not available. So the etiological role of organisms was not known. To prove the etiology there were not strict criteria. So there was a need to establish criteria. Koch’s Postulates 1. The organism should be regularly seen in the lesions of the disease. 2. It should be isolated in pure culture on artificial media. 10 3. Inoculation of this culture should produce a similar disease in experimental animals. 4. The organism must be recovered from the lesions in these animals. Postulate 1 The organism should be found in lesions of the disease. All the causative agents of the disease are seen in the particular diseases. If we take pneumococci as example, they are seen in all the pneumonia cases. Postulate 2 It should be isolated and grown in solid media. Pneumococci are grown in solid media and are isolated from the diseases. Some organisms do not grow on solid media or the solid media are not developed yet. Example: Mycobacterium leprae and Treponema pallidum Postulate 3 The organisms should produce the exact disease in experimental animals Almost all the pathogenic organisms produce the same disease in experimental animals. Usually rats, mice, rabbits or guinea pigs are used as experimental animals. Pneumococci produce pneumonia in animals. Salmonella species do not produce typhoid fever in rat, mice or rabbit. So chimpanzee is taken as experimental animal and it produces fever in chimpanzee. Postulate 4 It should be isolated from the diseased animal also Pneumococci are isolated from the experimental animals also. Modern addition to Koch’s Postulates Today we recognize additional criteria of causal relation between a microorganism and a disease. The important one is the demonstration of abnormally high concentration of specific circulating antibodies to the organism in the infected host Or, the presence of abnormally high degree of specific immunity or hypersensitivity to the infecting agent in a recently recovered host. Limitations Some organisms have not yet been grown in artificial culture media Example: Mycobacterium leprae and Treponema pallidum. Usefulness of Koch’s Postulates 1. It is useful in determining pathogenic organisms 11 2. To differentiate the pathogenic and nonpathogenic microorganism 3. For the classification of organisms 4. To detect the susceptibility, resistance of the laboratory animals. Conclusions Koch has done a valuable work in the field of Microbiology and has made postulates, which have merits, demerits and limitations with modern omission and addition. Other ‘applied microbiologists’: Ignaz Semmelweis was the first to recognize the need for good hygiene during medical procedures. The first to identify nosocomial infections. 1827-1912 Joseph Lister developed antiseptic methods for use in surgery and medicine. 1854-1915 Paul Ehrlich developed chemotherapy to cure infectious diseases and discovers antibiotics to treat sleeping sickness and syphilis. 1881-1951 Alexander Fleming discovered penicillin and lysozyme. 1864-1920 Dmitri Ivansvski discovered the first virus which is known as the tobacco mosaic virus (TMV) 12 13 14 EXPLOIT THE USEFUL MICROBES. / COMBAT THE HARMFUL ONES. YOU CAN’T BE TOTALLY FREE OF THEM. Industrial Microbiology Medicinals, food supplements, alcoholic beverages, enzymes and organic acids _ these are some of the substances produced on a commercial basis by using microorganisms. The beneficial chemical activities of microorganisms like bacteria, yeasts, molds and algae are exploited to obtain valuable products from these organisms after they have been grown in a relatively inexpensive medium. Industrial Microbiology and Food processing: Yeast is used make breads, baked goods, alcohol, yogurt and other foods and drink items. Today’s yeast are specially engineered to work in large scale industrial applications. Specialized bacteria and molds are used to make cheeses of different types. Biofertilizers include bacteria such as Rhizobia that fix nitrogen. Food additives increase nutritional value, retard spoilage, change consistency and enhance flavor. These may be natural compounds such as guar gum and xanthan gum or flavor enhancers and vitamins. Industrial Microbiology and Medicine: Biosensors are monitors used in the detection of specific targets in the environment, human body or other organisms. Antibiotic production is a capacity that many microbes have naturally. Microbes have been developed as a drug delivery system. Lactic acid bacteria (LAB) has been exploited to make and deliver vaccines and other bioactive materials. 15 Microbes have been developed that degrade oil so that they it may be more easily extracted. Industrial Microbiology and Economics: In the cosmetic industry the botulism toxin derived from Clostridium botulinum is utilized. Biopesticides have been developed for the control of insect, nematodes and other pathogens that effect plants. Synthetic energy fuels such as ethanol, methane, hydrogen and hydrocarbons are produced by microbes. Gasohol which is a 9:1 blend of gasoline and ethanol is a popular fuel alternative. The ethanol is produced as a by product of yeast fermentation. Microbes have been used in mining. An example of this is the recovery of metals is facilitated by bacteria by helping to solubilize it making it more easily extracted. Microorganisms have been used to clean up the environment in a process called bioremediation. In bioremediation a microbe is introduced into an environment where its natural metabolism results in the detoxification or break down of hazardous chemicals or pollutants. Specialized Microbes: Rhizobia are bacteria that fix nitrogen and make it available for plant nutrition and growth. They form nodules on the roots of legumes. Azolla is a fee floating water plant that fixes nitrogen in association with cyanobacteria. It acts as a renewable biofertilizer. Azotobacter are nitrogen fixing bacteria that do not form nodules on plant roots or associate with legumes. They are free living and in addition to fixing nitrogen they can produce antibiotics and beneficial growth substances. 16 Azospirillum fix nitrogen inside plant roots. They produce beneficial compounds for plant growth and can survive in wetland conditions as well as soils. Mycorrhiza are fungi that form symbiotic relationships with plant roots. Vesicular arbuscular mycorrhiza (VAM) is the most important member of this group. VAM colonies take up nutrients and water which is available for the plant and they act as root extensions. 17 18 19 20 21 22 Eukaryotes Basic structure The basic eukaryotic cell contains the following: 1. plasma membrane 2. glycocalyx (components external to the plasma membrane) 3. cytoplasm (semi fluid) 4. cytoskeleton - microfilaments and microtubules that suspend organelles, give shape, and allow motion 5. presence of characteristic membrane enclosed subcellular organelles Characteristic biomembranes and organelles Plasma Membrane A lipid/protein/carbohydrate complex, providing a barrier and containing transport and signaling systems. Nucleus Double membrane surrounding the chromosomes and the nucleolus. Pores allow specific communication with the cytoplasm. The nucleolus is a site for synthesis of RNA making up the ribosome. 23 Mitochondria Surrounded by a double membrane with a series of folds called cristae. Functions in energy production through metabolism. Contains its own DNA, and is believed to have originated as a captured bacterium. Chloroplasts (plastids) Surrounded by a double membrane, containing stacked thylakoid membranes. Responsible for photosynthesis, the trapping of light energy for the synthesis of sugars. Contains DNA, and like mitochondria is believed to have originated as a captured bacterium. Rough endoplasmic reticulum (RER) A network of interconnected membranes forming channels within the cell. Covered with ribosomes (causing the "rough" appearance) which are in the process of synthesizing proteins for secretion or localization in membranes. Ribosomes Protein and RNA complex responsible for protein synthesis Smooth endoplasmic reticulum (SER) A network of interconnected membranes forming channels within the cell. A site for synthesis and metabolism of lipids. Also contains enzymes for detoxifying chemicals including drugs and pesticides. Golgi apparatus A series of stacked membranes. Vesicles (small membrane surrounded bags) carry materials from the RER to the Golgi apparatus. Vesicles move between the stacks while the proteins are "processed" to a mature form. Vesicles then carry newly formed membrane and secreted proteins to their final destinations including secretion or membrane localization. 24 Lysosymes A membrane bound organelle that is responsible for degrading proteins and membranes in the cell, and also helps degrade materials ingested by the cell. Vacuoles Membrane surrounded "bags" that contain water and storage materials in plants. Peroxisomes or Microbodies Produce and degrade hydrogen peroxide, a toxic compound that can be produced during metabolism Cell wall Plants have a rigid cell wall in addition to their cell membranes. 1. Yeasts · Unicellular fungi, nonfilamentous, typically oval or spherical cells. Reproduce by mitosis: · Fission yeasts: Divide evenly to produce two new cells (Schizosaccharomyces). · Budding yeasts: Divide unevenly by budding (Saccharomyces). Budding yeasts can form pseudohypha, a short chain of undetached cells. Candida albicans invade tissues through pseudohyphae. · Yeasts are facultative anaerobes, which allow them to grow in a variety of environments. o When oxygen is available, they carry out aerobic respiration. o When oxygen is not available, they ferment carbohydrates to produce ethanol and carbon dioxide. CHARACTERISTICS OF FUNGI Molds and Fleshy Fungi 25 Multicellular, filamentous fungi. Identified by physical appearance, colony characteristics, and reproductive spores. Thallus: Body of a mold or fleshy fungus. Consists of many hyphae. Hyphae (Sing: Hypha): Long filaments of cells joined together. Septate hyphae: Cells are divided by cross-walls (septa). Coenocytic (Aseptate) hyphae: Long, continuous cells that are not divided by septa. Hyphae grow by elongating at the tips. Each part of a hypha is capable of growth. Vegetative Hypha: Portion that obtains nutrients. Reproductive or Aerial Hypha: Portion connected with reproduction. Mycelium: Large, visible, filamentous mass made up of many hyphae. 26 27 28 29 30 31 32
"Microbiology-basics for biochem engnrs"