Microscopy and Microbial Diversity Intended Learning Outcomes After the completion of the chapter, students will be able: • enumerate the different types of microscopes and their uses; • explain the three-domain classification of microorganisms • enumerate distinct characteristics of archaea, bacteria, fungi, protozoa, algae, etc.; • differentiate the acellular agents from bacteria; • compare some morphological features of prokaryotes and eukaryotes Lecture Topics 1 Microscopy 4 Prokaryotic Microbes 2 Classification Schemes 5 Eukaryotic Microbes 3 Acellular Agents Microscopy Staining Procedure Dyes can be used to stain cells. • organic compounds, which have affinity for specific cellular materials • most are positively charged, known as basic dyes, that bind strongly to negatively charged cell components • examples: methylene blue, crystal violet, and safranin Gram Staining A procedure that render different kinds of cells with different colors. It is used to see the purity of the cultures and to observe the gram-reaction, shape, and size of cells Gram Stain Reaction • Gram-positive: appear purple-violet • Gram-negative: appear pinkish red Staining Procedure Phase-Contrast and Dark-Field Microscopy Saccharomyces cerevisiae visualized by different types of light microscopy Fluorescence Microscopy Cyanobacteria, fluorescence microscopy Cyanobacteria, bright-field microscopy E. coli, fluorescence photomicrograph Differential Interference Contrast and Atomic Force Microscopy Yeast cells, differential interference contrast Bacterial cells, atomic force microscopy Confocal Scanning Laser Microscopy Microbial biofilm community Filamentous cyanobacterium Electron Microscopy Electron microscopes use electrons instead of visible light (photons) to image the cells and cell structures Fitted with cameras called electron micrograph Types of Electron Microscopy Transmission Electron Microscopy • Examine cells and structures at very high magnification and resolution up to molecular level • Wavelength of electrons are shorter than wavelength of light, and wavelength affects the resolution Scanning Electron Microscopy • used to observe the external features or surface of an organism without doing thin sections • Specimen is coated with a thin film of a heavy metal such as gold Prokaryotic and Eukaryotic Cells Prokaryotic Cells • Have a simpler internal structure in which organelles are absent • Can couple transcription directly to translation • energy conservation reaction happens in their cytoplasmic membrane • have nucleoid region where small circular DNA is placed Prokaryotic and Eukaryotic Cells Eukaryotic Cells • House their DNA inside the nucleus; typically, larger and complex • DNA replication and transcription occur in the nucleus while translation occurs in cytoplasm • include protists (algae and protozoa), fungi, slime molds, and lichens • have membrane-enclosed organelles Evolutionary Tree of Life Evolution • Is the process of descent with modification that generates new varieties and eventually new species of organisms • occurs in any self-replicating system in which variation is the result of mutation and selection is based on differential fitness. Phylogeny • Phylogenetic relationship between cells can be deduced by comparing the genetic information (nucleotide or amino acid sequence); ribosomal RNA (rRNA) is an excellent tool Ribosomal RNA (rRNA) gene sequencing and phylogeny Ernst Haeckel’s Tree of Life Whittaker’s Five-Kingdom System of Classification Three Domains of Life The phylogenetic tree of life as defined by comparative rRNA gene sequencing Evolutionary facts revealed by the phylogenetic tree of life All prokaryotes are not phylogenetically closely related. Archaea are actually more closely related to Eukarya than to Bacteria. Microbial Diversity Can be seen in many ways: • Phylogeny • Cell size and morphology (shape) • Physiology • Motility • Mechanism of cell division and pathogenecity • Developmental biology • Adaptation to environmental extremes Metabolic diversity Chemoorganotrophs These organisms utilize organic compounds as their energy source. Chemolithotrophs These organisms utilize inorganic compounds as their energy source Phototrophs These organisms utilize light as their energy source All cells require carbon in large amounts and can be acquired from two sources: Organic compounds and carbon dioxide Heterotrophs Use organic compounds as carbon source Autotrophs Use carbon dioxide as carbon source • Chemoorganotrophs are heterotrophs • Most chemolithotrophs and phototrophs are autotrophs Extremophiles Organisms inhabiting extreme environments. Acellular Agents Viruses Intracellular parasite that consist nucleic acid core surrounded by protein coat (capsid); Viroids Composed of small, single strand of RNA in a loop form; parasitic only to plant Prions Misfolded form of normal proteins that are transmittable; infectious to animals including humans SARS-CoV-2 causing COVID-19 (Knowlton, 2020) Domain Bacteria Contains an enormous variety and best-known prokaryotes, such as: Proteobacteria Gram-Positive Bacteria Cyanobacteria Other major phyla of Bacteria Proteobacteria • Make up the largest phylum of Bacteria • Many are chemoorganotrophic bacteria (e.g. Escherichia coli) • Several are phototrophic and chemolithotrophic species that use H2, S, N in their metabolism Chromatium Achromatium Proteobacteria • Includes nitrogen fixers (e.g. Azotobacter) and toxic organic compounds metabolizers (e.g. Pseudomonas) • Includes a number of key pathogens, including Salmonella (gastrointestinal diseases), Rickettsia (typhus), Neisseria (gonorrhea) • The key respiratory organelle (mitochondrion) of eukaryotes has evolutionary roots within Proteobacteria Gram-Positive Bacteria • Contains many organisms that are united by their common phylogeny and cell wall structure • Included endospore-forming Bacillus Bacillus w/ endospore Streptococcus Cyanobacteria • Phylogenetic relatives of G-positive bacteria • first oxygen phototrophs to evolve on earth • cells of some cyanobacteria join to form filaments • morphological forms: ◦ unicellular ◦ colonial ◦ heterocystous (contain heterocysts for nitrogen fixation) Oscillatoria Spirulina Other Major Phyla of Bacteria Gram-Negative Bacteria Planctomycetes Cells have distinct stalk that allows the organism to attach to a solid substratum in aquatic environment Spirochetes Planctomyces Helically shaped cells; causative agents of notable syphilis and Lyme disease Spirochaeta zuelzerae Other Major Phyla of Bacteria Phototrophic and Autotrophic Bacteria Green sulfur bacteria E.g. Chlorobium, undergo anoxygenic photosynthesis and use sulfur as electron donor Green non-sulfur bacteria E.g. Chloroflexus, a filamentous phototroph that inhabits hot springs and associate with cyanobacteria to from microbial mats. Anoxygenic photosynthesis; doesn’t use sulfur as e- donor Chlorobium Chloroflexus Other Major Phyla of Bacteria Chlamydia • Intracellular parasites that harbor respiratory and sexually transmitter pathogens of humans • E.g. Rickettsia, Mycobacterium tuberculosis Deinoccous-Thermus Mycobacterium tuberculosis • Unusual cells walls and innately resistant to high levels of radiation • E.g. Deinococcus radiodurans Deinococcus radiodurans Other Major Phyla of Bacteria Aquifex and Thermotoga • Both include organisms that grow in hot springs that are near the boiling point • Extremophiles Hyperthermophile Aquifex uses H2 as its energy source and can grow in temperatures up to 95 °C. Hyperthermophile Aquifex Domain Archaea • Most cultured Archaea are extremophiles, with species capable of growth at the highest temperatures, salinities, and extremes of pH known for any microorganisms • Comprises chemotrophic and chemolithotrophic organisms Phylum Euryarchaeota Phylum Crenarchaeota Phylogenetic tree of some representative Archaea Euryarchaeota Four groups of organisms: Methanogens Extreme Halophiles Thermoacidophiles Hyperthermophiles Phylogenetic tree of some representative Archaea Euryarchaeota Methanogens • Strict anaerobes and cannot tolerate even very low levels of oxygen • Metabolism is unique in that energy is conserved during the production of methane (natural gas) • Important in anaerobic degradation of organic matter • E.g. Methanobacterium Methanobacterium thermoautotrophicum Euryarchaeota Extreme Halophiles • Most require oxygen • Unified by their requirement for very large amounts of salt (NaCl) for metabolism and reproduction • Inhabit salt lakes, salterns (salt evaporation ponds), and other very salty environments • e.g. Halobacterium Halobacterium sp. Euryarchaeota Thermoacidophiles • Organisms that grow best at high temperatures plus acidic pH • E.g. Thermoplasma - lacks cell wall and grows best at 60 to 70 °C and pH 2 Thermoplasma acidophilum • E.g. Picrophilus - most acidophilic of all known prokaryotes, which grow at 60 °C and pH 0.7 Picrophilus torridus Euryarchaeota Hyperthermophiles • Organisms whose growth temperature optimum lies above 80 °C • Autotrophs (carbon source is CO2) • These organisms show a variety of Methanopyrus kandleri and Pyrococcus furiosus physiological activities including: ◦ methanogenesis (Methanopyrus) ◦ sulfate reduction (Archaeoglobus) ◦ iron oxidation (Ferroglobus) ◦ sulfur reduction (Pyrococcus) Archaeoglobus fulgidus Ferroglobus placidus Crenarachaeota • Either chemolithotrophs or chemoorganotrophs • Organisms grow in acidic and hot environments such as hot springs and hydrothermal vents • Anaerobes (because of the Solfolobus high temperature, their habitats are typically anoxic) Pyrolobus • Many of them use H2 as energy source Desulfurococcus Domain Eukarya • They phylogeny of Eukarya based on rRNA sequencing shows plants and animals to be farthest out on the branches of the tree; such latebranching groups are said to be the "most-derived" • Some early-branching are simple prokaryotes Phylogenetic tree of some representative Eukarya Domain Eukarya The major groups are the following: Protists (Algae and Protozoa) Fungi Slime Molds Lichens Protists: Algae • Phototrophic, which contains chloroplasts and can live in environments containing only few minerals (e.g. K, P, Mg, N, S), water, CO2 and light • Primary producers and inhabit both soil and water Polysiphonia, a marine red alga • They have cell walls • Types: Red, Green, Yellow-green, Goldenbrown, Brown Algae, Diatoms, and Euglenoids Volvox, a colonial green alga CHARACTERISTICS OF ALGAE 1. Relatively simple eukaryotic photoautotrophs that lack the tissues (roots, stem, and leaves) of plants. 2. Algae can be either unicellular or multicellular organisms. 3. Most algae are found in the ocean. Their locations depend on the availability of appropriate nutrients, wavelengths of light, and surfaces on which they can grow. 4. Reproduction in algae occurs in both asexual and sexual forms. Asexual reproduction occurs by spore formation. ● Algal cells consist of a cytoplasm, cell wall, cell membrane, nucleus, plastid, ribosomes, mitochondria, and Golgi bodies. ● Pellicle – thickened cell membrane ● Stigma – also known as an eyespot, light sensing organelle. ● Chlorophyl - a green pigment found in almost all plant algae and cyanobacteria. They have five kinds of photosynthetic pigments (chlorophyll a, b, c, d, and f) and have many accessory pigments that are blue, red, brown, and gold. The body of a multicellular alga is called a thallus. Holdfasts anchor the alga to a rock. Diatoms Tiny, unicellular algae with complex cell walls that consist of pectin and a layer of silica. They live in both freshwater and seawater. Over 200 genera of living diatoms are known, the distinctive patterns of the walls are a useful tool in diatom identification. Dinoflagellates Dinoflagellates are unicellular algae collectively called plankton, or free-floating organisms. Their rigid structure is due to cellulose embedded in the plasma membrane. Some dinoflagellates produce neurotoxins. Brown algae or kelp, are macroscopic; some reach lengths of 50 m. Most brown algae are found in coastal waters. Brown algae have a phenomenal growth rate. Green algae have cellulose cell walls, contain chlorophyll a and b, and store starch, as plants do. Prominent examples of green algae include Spirogyra, Ulothrix, Volvox, etc. Red Algae also called Rhodophyta, are red in color due to the presence of a pigment called chlorophyll A, phycocyanin, and phycoerythrin. However, they lack chlorophyll b or beta-carotene. Algae are sometimes grown to make algae biofuels, which make up the third generation of biofuels. Many types of algae can be used and processed to become biofuel. Biofuel is a fuel made from living things, or the waste of a living thing, also known as biomass. The algae oils can be converted to biodiesel and the remaining material can be used to create bioethanol. Protists: Protozoa • Typically, motile • Widespread in nature in aquatic habitats or as pathogens of humans and other Amoeba Paramecium Plasmodium falciparum Giardia lamblia animals • They lack cell walls • Groups: Amebas, Flagellates, Ciliates, Apicomplexa 1.Protozoa are unicellular, eukaryotic chemoheterotrophs. 2.Found in soil and water and as normal microbiota in animals. 3.They live as predators or parasites and feed on organic materials such as bacteria, detritus, and other microbes. 4.Protists that have characteristics in common with animal cells also have mitochondria, which provide energy for the cell. 5.Protists that are like plant cells have a cell wall and chloroplasts. Chloroplasts make photosynthesis possible in these cells. 6.Protista reproduces by asexual means. The sexual method of reproduction is extremely rare and occurs only during times of stress. Ciliated protozoans Ciliates move about by means of large numbers of cilia on their surfaces. Cilia exhibit an oarlike motion. Amoeboid protozoans Amebae move by extending blunt, lobe-like projections of the cytoplasm called pseudopodia. Flagellated protozoans Flagellates move by means of whiplike flagella. The basal body anchors each flagellum within the cytoplasm. Fungi • Lack photosynthetic pigments • Either unicellular (yeasts) or filamentous (molds) • Major agents of decomposition in nature and recycle much of the organic matter produced in soils and other ecosystems. Yeast Mushroom Mold AMF • They have cell walls • groups: Yeasts, molds, mushrooms, Arbuscular mycorrhizal fungi (AMF) Examples of fungi: Many species of fungus produce the familiar mushroom (a) which is a reproductive structure. This (b) coral fungus displays brightly-colored fruiting bodies. This electron micrograph shows (c) the spore-bearing structures of Aspergillus, a type of toxic fungi found mostly in soil and plants. (Image source: https://bit.ly/3qy8fJS) UNICELLULAR FUNGI: YEAST − Nonfilamentous, unicellular fungi that are typically spherical or oval. − Frequently found as a white powdery coating on fruits and leaves. − They multiply by budding a daughter cell off from the original parent cell called blastopores or blastocondia. One yeast cell can in time produce up to 24 daughter cells by budding. − The budding yeasts reproduce asexually by budding off a smaller daughter cell; the resulting cells may sometimes stick together as a short chain or pseudo hyphae. MULTICELLULAR FILAMENTOUS FUNGI: MOLDS − The thallus (body) of a mold or fleshy fungus consists of long filaments of cells joined together; these filaments are called hyphae (singular: hypha) − Hyphae that have walls between the cells are called septate hyphae; hyphae that lack walls and cell membranes between the cells are called non septate or coenocytic hyphae. − Many hyphae join to form a network-like structure called mycelium. Mycelia are of three kinds: 1. Vegetative mycelium are those that penetrate the surface of the medium and absorb nutrients. 2. Aerial mycelium are those that grow above the agar surface 3. Fertile mycelium are aerial hyphae that bear reproductive structures such as conidia or sporangia. Reproduction in Fungi A. Fragmentation - In this process, the mycelium breaks into two or more similar fragments either accidentally or due to some external force. Each fragment grows into a new mycelium. B. Budding - The parent cell produces one or more projections called buds, which later develop necessary structures and detach to grow into new individuals. Budding is common in unicellular forms like yeast. C. Fission – The parent cell splits into two equal halves, each of which develops into a new individual. D. Sclerotium - The hyphae become interwoven to form a compact mass and get surrounded by a rind that varies in form and has a dark-colored covering. E. Rhizomorphs - In some higher fungi, several hyphae may become interwoven to form ropelike structures called rhizomorphs. Under favorable conditions, they resume growth to give rise to new mycelia. SPORES Sexual spores are produced by the fusion of gametes and are formed in different varieties depending on how they are formed. Most fungi are classified taxonomically according to the type of spores that they produce of the type of structure on which the spores are produced. A. Ascomycota (Sac Fungus) They can be coprophilous, decomposers, parasitic or saprophytic. The sexual spores are called ascospores. Asexual reproduction occurs by conidiospores. Ex. Edible mushrooms, truffles, Penicillium B. Basidiomycota (Club Fungus) Sexual reproduction occurs by basidiospores. Asexual reproduction occurs by conidia, budding or fragmentation. This phylum includes fungi that produce mushrooms. Ex. Club fungi, puffballs, mushrooms C. Zygomycota (Conjugated Fungus) Saprophytic molds that have coenocytic hyphae. These are formed by the fusion of two different cells. The sexual spores are known as zygospores, while the asexual spores are known as sporangiospores. Ex. Rhizopus stolonifera (black bread mold) Asexual spores are produced by an individual fungus through mitosis and subsequent cell division; there is no fusion of the nuclei of cells. This takes place with the help of spores called conidia or zoospores, or sporangiospores. If the reproductive structure if formed within a sac-like structure called sporangium, then the asexual form is referred to as sporangiospore (commonly known as spore) If the reproductive structure arises from a fungal component called conidiophore, the spore will be identified as conidia. Fungal spores or conidia are very resistant to temperature, acids, bases, and other chemicals and are carried by wind. Many people are allergic to fungal spores. Slime Molds • They are motile and lack cell walls • During life cycle, motile cells agregate to form a multicellular structure called a fruiting body from which spores are produced that yield new motile cells • Types: Plasmodial and Cellular slime molds A. Acellular Slime Molds (Plasmodial) − A plasmodial slime mold exists as a mass of protoplasm. with many nuclei. This mass of protoplasm is called a plasmodium. − They can grow up to 1 ft in diameter. − They are found creeping as a slimy mass over leaf litter, moist and decaying logs. It feeds on dead and decaying organic matter and microorganisms. − When the food is scarce and moisture is less, they reproduce asexually. − The entire plasmodium moves as a giant ameoba; it engulfs organic debris and bacteria (phagocytosis) B. Cellular Slime Molds − The feeding stage is a single-celled amoeboid, which lives as a solitary organism. − They have a close resemblance to amoebas. − Individual cells feed on microorganisms and other food matter while creeping on decaying logs or freely swimming in freshwater. − When the food is less, they form aggregate but retain their individuality due to the presence of a thin plasma membrane and reproduce asexually by spore formation Lichens • Leaf-like structures often found grown on the surface of rocks and trees • Are an example of a microbial mutualism, a partnership in which two organisms live together for mutual benefit • Consist of a fungus and a phototrophic partner, either an alga (a eukaryote) or a cyanobacterium (a prokaryote) The lichen medulla is composed of fungal hyphae surrounding the algal layer. The protective cortex is a layer of fungal hyphae that covers the surface and sometimes the bottom of the lichen. Photosynthesis occurs in the algal zone. Lichens use hyphal bundles called rhizines to attach to the substrate. The lower cortex also provides protection. Crustose lichens Foliose lichens Fruticose lichens Helminths 1. They may lack a digestive system. 2. Their nervous system is reduced. 3. Their means of locomotion is occasionally reduced or completely lacking. 4. The reproductive system is often complex. A. Nematodes (Roundworms) Members of the phylum Nematoda, the roundworms, are cylindrical and tapered at each end. Roundworms have a complete digestive system, consisting of a mouth, an intestine, and an anus. They can lead to infection in the intestines or elsewhere in the body. B. Platyhelminths (Flatworms) Members of the phylum Platyhelminthes, the flatworms, are dorsoventrally flattened. The flukes and tapeworms are medically important parasites. Reference Madigan, MT., Martinko, JM., Stahl, DA., and Clark, DP. 2012. Brock Biology of Microorganisms 13th Edition. Benjamin Cummings
