BIOL 142 Lecture 4 Chapter 4: Microbial Diversity Part 1: Acellular & Prokaryotic Microbes & General Staining Techniques 62 slides 1 Acellular Microbe Types a virion is a complete, infectious viral particle a virus with all its parts present. synonymous with the terms virus & infectious particle. a prion is a protein with infectious properties. it has no DNA and no RNA. a viroid is an infectious RNA molecule with no protective protein coat. Viruses, Prions, and Viroids do not meet the definition of a living organism they are NOT ALIVE!!! 2 Virus / Virion / Infectious Particle viruses are incredibly small: most viruses range in size from 10 300 nm. viruses were not able to be seen until the electron microscope was invented in 1931. the first photographs of viruses were obtained in 1941. No type of known organism has been found to be completely safe from viral infections: humans animals plants fungi protozoa algae bacteria 3
Virus / Virion / Infectious Particle acellular no cell nucleus no organelles no cytoplasmic membrane no cytoplasm Viruses lack the genes and enzymes necessary for energy production. Viruses contain only one form of nucleic acid: DNA or RNA some recent discoveries may soon modify this rule. Some rare discoveries of DNA viruses with RNA coded protein coats have been found in harsh locations like hot springs. 4 Virus / Virion / Infectious Particle Viruses do NOT divide by binary fission, mitosis, or meiosis. Viruses are obligate intracellular parasites Viruses can only replicate inside a living host cell. replication uses the cellular machinery of the host cell (such as ribosomes) to make all virus components. replication usually kills the host cell and results in hundreds to thousands of new viral copies. Viruses can not carry out any metabolic pathway on their own a host cell is required for metabolism of any kind. Viruses can have an extracellular state / form & intracellular state / form. Viruses do not grow (no increase in size, start as adults). Viruses do not respond to the environment. 5 Characteristics of a Virus Extracellular State: the genetic material (DNA or RNA) plus: the protein coat (capsid) some viruses also have a phospholipid envelope. function is to surround the nucleic acid and to provide it some protection and a site of recognition for host cells. Intracellular State: capsid (and any envelope) is removed. virus exists as a nucleic acid (DNA or RNA) inside the host cell. 6
Components of a Virus 7 Components of a Virus Capsid: determines the shape of a virus. Icosahedral (polyhedral), Helical & Complex. protects it. encloses the nucleic acid. plays a key role in the attachment of some viruses to the host cell. composed of subunits called capsomeres. each capsomere can be the same protein repeated over and over to make the capsid, or many proteins may make the capsid. the arrangement and the number of proteins in the capsomeres can be useful in identification and classification of the virus. 8 Components of a Virus Envelope: typical bilayer phospholipid membrane outside the capsid. acquired from the host cell as it buds through one or several membranes. some viruses have no envelope are are called: naked non-enveloped the composition of the envelope is derived from: the viral nucleic acid instructions. the substances found in the parasitized cell s membranes. Spikes: glycoproteins that project from some envelopes that serve to attach viruses to specific receptor sites on susceptible host cell surfaces. can result in hemagglutination. 9
Components of a Virus Envelope: can help a virus evade detection from the host s immune system. can help with fusion to make infection of new host cells easier or possible. the envelope is easily damaged, and once it occurs the virus has NO way to repair it. it has no organelles nor genetic instructions to assist in its repair. very sensitive to temperature changes, ph, lipid solvents, chemical disinfectants... all of which will easily destroy the envelope. 10 Bacteriophage 11 Enveloped Virus with a Helical Capsid 12
Virus Classification Viruses are difficult to classify into typical taxonomic categories such as Kingdom, Phylum, Class, and Order. The Family is the highest taxonomic category that is used i.e., there are no viral kingdoms. Naming is also a bit different. For Example, the rabies virus is taxonomically ranked as follows: Family: Rhabdoviridae Genus: Lyssavirus Species: rabies virus NOTE that the English Common Name is used to designate a virus and NOT the Latin Binomial name!!! 13 Nucleic Acid Classification The presence of RNA or DNA is the main physical characteristic which divides viruses into 2 groups. RNA viruses single-stranded (ssrna) most are single-stranded. Positive (+) Stranded RNA looks and acts like mrna and can be translated by the host s ribosomes. Negative (-) Stranded RNA acts as a template during transcription and uses the RNA Polymerase it carries to make it into a positivesense RNA strand. double-stranded (dsrna) RNA viruses must carry their enzymes or have genes to make the enzymes as part of their genome. DNA viruses single-stranded (ssdna) double-stranded (dsdna) 14 Lytic Replication Cycle in Bacteriophages 15
Pattern of Viron Abundance in Lytic Cycle Notice that the Y-axis is for Infective Virions. This means that the virus is OUTSIDE the host cell and is completely assembled and ready to infect another host cell. Few viruses go into a host cell and many new ones burst out of the host cell. 16 Lysogeny Viral Replication Lysogeny: modified replication cycle. infected host cells grow & reproduce normally for generations before they lyse. while the host cell replicates its genome and divides, it is also replicating the genome of the virus. known as Lysogenic phages or Temperate phages. Lysogenic conversion results when phages carry genes that alter the phenotype of a bacterium. this can result in the bacteriophage obtaining new genetic information. 17 Lysogenic Replication Cycle in Bacteriophages: phage lambda & E. coli 1. Attachment 2. Entry / Penetration Lytic cycle Lysogeny / Temperate Phages 6. Biosynthesis 8. Release 7. Assembly 18
3 Mechanisms of Entry of Animal Viruses: 1. Direct Penetration Receptors are very specific and will limit a specific viruses to being able to only enter specific cells of a specific species species. This is how one virus can cause a cold in a human, but not another species. 19 3 Mechanisms of Entry of Animal Viruses: 2. Membrane Fusion 20 3 Mechanisms of Entry of Animal Viruses: 2. Endocytosis 21
Replication of Animal Viruses Assembly & Release of Animal Viruses: Most DNA viruses assembled in the nucleus. Most RNA viruses assembled in the cytoplasm. the number of viruses produced depends on: the type of virus the size & initial health of the host cell. typically about 50 1000 new virions are made. Enveloped viruses cause persistent infections. Naked viruses are released by: exocytosis cell lysis 22 The process of Budding in an Enveloped Virus 23 Inclusion Bodies Inclusion Bodies: remnants or collections of viruses in infected cells. useful as a diagnostic tool. can be found in the cytoplasm or in the nucleus. Examples: Negri Bodies: Rabies virus, found in the cytoplasm. Inclusion Bodies: HIV virus, found in the cytoplasm. Guarnieri Bodies: Smallpox virus, found in the cytoplasm. Inclusion Bodies: Herpes virus, found in the nucleus. Inclusion Bodies: Polio virus, found in the nucleus. 24
Nucleic Acid Classification Positive-Stranded (+) RNA Translation Structural Proteins & Enzymes for the new virus. Negative-Stranded (-) RNA Transcription with virus RNA dependent RNA Polymerase Positive-Stranded (+) RNA Translation Structural Proteins & Enzymes for the new virus. RNA Reverse Transcription with enzyme Reverse Transcriptase DNA Transcription mrna Translation Structural Proteins & Enzymes for the new virus. 25 RNA Viruses General Properties most are single-stranded. Exception: Retroviridae are double-stranded most are enveloped. Exceptions: non-enveloped RNA viruses: Picornaviridae, Caliciviridae, Reoviridae most have helical capsid symmetry. Exceptions: icosahedral symmetry RNA viruses: Picornaviridae, Caliciviridae, Reoviridae Togaviridae, Flaviviridae Rhabdoviridae (helical symmetry but shaped like a bullet). most will replicate in the cytoplasm. Exceptions: nuclear replication: Retroviridae, Orthomyxoviridae. 26 DNA Viruses General Properties Most are Double-Stranded: Exception: Single-Stranded DNA: Parvoviridae Most show Icosahedral Symmetry: Exception: unique complex box-like protein structure: Poxviridae Most will replicate in the Nucleus. Exception: cytoplasmic replication: Poxviridae 3 have Envelopes: Hepadenaviridae, Herpesviridae, Poxviridae 3 are non-enveloped: Parvoviridae, Adenoviridae, Papovaviridae 27
Characteristics of Prions Prions: Cellular PrP protein: made by all mammals (best understood is from raw brains). normal structure is the alpha-helix called cellular PrP. Prion PrP (a proteinaceous infectious agent): Disease causing form with beta-pleated sheets called prion PrP. Prion PrP changes shape of cellular PrP so it becomes another prion PrP. All cause various forms of spongiform encephalopathy. Mad Cow Disease Kuru (ritualistic cannibalism in Papua, New Guinea) Creutzfeldt Jakob disease (C J disease) Gerstmann Sträussler Scheinker (GSS disease) Fatal Familial Insomnia Scrapie Chronic Wasting Disease 28 Characteristics of Prions 29 General Characteristics of Prokaryotes Prokaryotes: Most diverse group of cellular microbes. Habitats are virtually everywhere: Antarctic Glaciers Thermal Hot Springs Distilled Water Supersaturated Brine Disinfectant Solutions Basalt Rocks (thousands of meters below the Earth s crust). Colons External surfaces Inside the cytoplasm of cells Despite this diverse list, less than 1% cause disease! 30
General Characteristics of Prokaryotes Typical Prokaryotic Morphologies (shapes): 31 General Characteristics of Prokaryotes Reproduction of Prokaryotic Cells: all reproduce asexually by 3 main methods: binary fission (this is the most commonly used method). snapping division. budding. 32 Asexual Reproduction via Binary Fission 33
Asexual Reproduction via Snapping Division Snapping Division is a variant of Binary Fission. only the inner portion of the cell wall forms a cross wall. as the daughter cells grows, the tension increases till the outer portion of the cell wall snaps leaving the new daughter cells connected by a hinge of old cell wall material. 34 Asexual Reproduction via Budding & Spores Budding: the parental cell retains its identity during and after the budding process. a outgrowth of the original parent cell (a bud) which receives a copy of the genetic material then enlarges. Spores: the parental cell retains its identity during and after the spore production process. each spore is a clone of the original parent cell. 35 note that the daughter cell that results from budding is much smaller than the parent cell, which survives to produce more buds. Budding 36
Arrangement of Cocci Cells 1 plane of cell division: just 1 Diplococci a chain Streptococci 37 Arrangement of Cocci Cells 2 planes of cell division 3 planes of cell division Random planes of cell division 38 Arrangement of Bacilli Cells 1 plane of cell division: just 1 Diplobacilli perpendicular to long axis of bacteria 39
Arrangement of Bacilli Cells 1 plane of cell division: a chain Streptobacilli snapping division results in palisade Example: Corynebacterium diphtheria causative agent of diphtheria. 40 Endospores Endospores: a defensive strategy against hostile or unfavorable conditions. is a stable resting stage that forms from a vegetative cell, which barely metabolizes (thus it can t last forever) and will germinate when conditions improve. is NOT a reproductive structure. they are NOT the same as the reproductive spores of other species: actinomycetes, algae, and fungi. the process of sporulation requires about 8 to 10 hours to complete. many endospore bacteria produce deadly toxins: anthrax, tetanus and gangrene. 41 Endospores Bacillus cereus causes food poisoning. Clostridium botulinum causes botulism. 42
Modern Prokaryotic Classification Original classification methods looked at: physical features (cell wall structure, shape). metabolism capabilities (presence of enzymes). growth location (in gut, on skin, on ocean floor). Modern classification methods now focus on: genetic relatedness of rrna sequences. resulted in 3 Domains of Living organisms: Archaea (a prokaryotic group of life) Bacteria (a prokaryotic group of life) Eukarya (a eukaryotic group of life) Over 99% of prokaryotes have never been isolated or cultured and are only known from their rrna sequences from samples obtained in nature. 43 Prokaryotic Taxonomy based on rrna sequencing data 44 Survey of Archaea Shared features of all Archaea: lacks peptidoglycan in the cell wall. cell membrane lipids have branched hydrocarbon chains. AUG codon codes for methionine this is the same coding as seen in eukaryotes. the Domain Bacteria code AUG as N formylmethionine. Three Phyla: Crenarchaeota Euryarchaeota Korarchaeota known only from environmental rrna samples. no species has ever been seen!!! 45
Survey of Archaea Reproduction: binary fission, budding, and fragmentation Shapes: most are cocci bacilli spiral forms pleomorphic NOT known to cause any diseases of humans and other life forms. Most live in moderate environmental conditions. Some are extremophiles: thermophiles, hyperthermophiles, halophiles, methanogens. 46 Principles of Staining Principles of Staining: staining increases the contrast and resolution by coloring specimens with stains (dyes). smear of microorganisms (a very thin film) is made on a glass slide prior to staining. microbiologic stains contain a chromophore: at least one of the two ions (the cation or the anion) in a dye is colored, this colored ion is the chromophore. chromophores bind to chemicals via covalent, ionic or hydrogen bonds. Example: Methylene Blue - Chloride Methylene Blue is a cation & the chromophore. It will seek out negatively charged cell parts and stain them. Chloride is an anion and it is colorless. 47 Principles of Staining Principles of Staining: Anionic Chromophores: also called Acidic Dyes stain Alkaline structures and work best at low ph Examples: Eosin, Picric Acid Cationic Chromophores: also called Basic Dyes stain Acidic structures and work best at high ph. Examples: Methylene Blue, Crystal Violet, Safranin, Malachite Green we use basic dyes in microbiology more due to the negative charge found on most cells and their structures. 48
Techniques of Light Microscopy Wet Mounts: a drop of medium is placed on a slide with the organisms swimming in it. usually adding something like Protoslow slows them for easier viewing. a special version of the wet mount is the hanging drop and is often used with Dark-Field Microscopy. coverslip has a ring of vaseline to prevent the drop from evaporating. good to see living microbes move. Treponema pallidum -spiral bacterium -cause of Syphilis 49 Techniques of Light Microscopy Smears: microbes are picked up with a loop / swab and smeared onto a slide. can view live or dead microbes. they will be killed if you fix them to the slide. takes practice to not make them too thick or too thin and to avoid disrupting the cells if normally found in specific arrangements. Heat Fixation: air dried completely then slide is passed 3 or 4 quick times over bunsen burner if it is not dried completely it will boil the microbe and destroy it!!! microbes with capsules are usually just air dried to avoid destruction. this kills the organisms and makes them stick to the slide. it alters the microbes so they are more able to accept stains / dyes. if you heat-fix too little, they will wash off when adding the dyes. if you heat-fix too much, you will incinerate the microbes!!! 50 Preparing a Specimen for Staining using Heat Fixation 51
Types of Staining Techniques Simple Stain: uses a single dye to reveal basic cell shapes and arrangements. Examples: Methylene Blue, Crystal Violet, Safranin, Carbolfuchsin. Differential Stain: uses 2 or more dyes to distinguish between 2 kinds of organisms or between two different parts of an organism. Examples: Gram Stain Ziehl-Neelsen Acid-Fast Stain Schaeffer-Fulton Endospore Stain Histological Stain Special Stain: Negative (capsule) stain Flagellar stain 52 Simple Stain of S. aureus and E.coli with Crystal Violet 53 1 The Gram Stain 2 3 4 54
The Gram Stain 4 Groups of organism can be distinguished with the Gram Stain. Gram-Positive organisms whose cell wall retains the crystal violet. Gram-Negative organisms whose cell wall does NOT retain the crystal violet (remember these were counterstained with Safranin). Gram-Nonreactive organisms which stain poorly or not at all. Gram-Variable organisms that stain but do so unevenly. The gram stain takes practice to get it right... many things can go wrong... even using pure cultures that are a bit older than a day can mess up the coloration process. 55 Ziehl-Neelsen Acid-Fast Stain modified stain that was developed by Paul Ehrlich in 1882. used to stain and detect: Mycobacterium tuberculosis: the cause of Tuberculosis. Mycobacterium leprae: the cause of Leprosy. humans are the only species that are infected with these two disease causing microbes. both are thin rods with lots of lipids in their cell walls. Procedure: a smear of the organism is put on a slide. the red stain Carbolfuchsin is added and the slide heated to aid penetration of this dye. Rinse slide. the slide is now decolorized with Acid Alcohol (3% HCl and 95% ethanol). Rinse slide. Loeffler s Methylene Blue counterstain is added which will any other bacteria that were decolorized. Acid-Fast microbes hold FAST to the Red Carbolfuchsin stain! 56 Ziehl-Neelsen Acid-Fast Stain Mycobacterium leprae 57
Negative Staining Used when a specimen, or part of it, like the capsule resist taking up the stain. the Capsule is a defensive structure made up of a layer of polysaccharides which can repel stains. the background if filled with stain versus staining the actual microbe: India Ink Nigrosin Streptococcus pneumoniae 58 Flagellar Staining difficult to see with a light microscope as they are very thin. can be coated to make visible: dyes silver Pseudomonas 59 Endospore Staining some bacteria produce endospores that can be resistant to virtually everything and last for years. this makes them very difficult to stain. Schaeffer-Fulton Spore Stain: heat-fixed smears are covered with malachite green then gently heated till they steamed for 5 minutes to allow the dye to penetrate the endospore s wall. the slide is then rinsed with water for 30 seconds to remove the green dye from all parts of the cell except the endospores (which retain it). counterstain with Safranin to color the non-spore regions. Bacillus megaterium 60
Schaeffer-Fulton Endospore Stain of Bacillus anthracis 61 Colony Morphology a single bacterial cell that lands on the surface of a solid culture medium will form a mound / pile which has a specific size, color, shape, edge appearance, and elevation. 62