Microbiology for Nurses 3053 PDF

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Microbiology for Nurses 3053 Humans and The Microbial World What is Microbiology?  A study of microbes or micro-organisms (m.o)  Organisms so small that they cannot be seen with a naked eye  Microorganisms Includes: - Bacteria - Virus - Fungi - Protozoa - Microscopic Algae  Less than 1% known m.o causes disease  Have a close association with humans - In us - On us - Everywhere around us Importance of Microbiology  Activities of m.o are responsible for survival of all other organisms, including humans  Provide nitrogen: Certain bacteria convert the nitrogen in the air into a chemical form that plants can use  Provide oxygen: Micro-organisms (m.o) are responsible for replenishing the supply of oxygen  Marine and fresh water m.o form the basis of the food chain  Soil microbes help break down wastes and incorporate nitrogen gas from the air into organic compounds  Certain microbes play a role in photosynthesis; food and oxygen is generated  Industrial application in the synthesis of various chemicals  Commercially valuable products can be synthesized by bacteria cheaper and faster  Used in the food industry in production of - Dairy products - Baking industry - Brewing industry (alcoholic beverages) - Pickles, olives, sour croute - Probiotic products

 Used by sewage treatment plants. Combination of physical and chemical processes with action of beneficial microbes  Used to clean up pollutants and toxic wastes from industrial processes  Can be used to degrade various environmental pollutants e.g. PCB, DDT, trichloroethylene (a processes called Bioremediation)  Ability to degrade cellulose important so that leaves, fallen trees do not pile in environment (leaves can be broken down)  Humans/animals depend on microbes in their intestines for digestion and synthesis of vitamins that their bodies require  Cellulose is degraded by m.o in the digestive tract of cattle, sheep, etc. The digestion products are used for energy  Certain bacteria are used for insect pest control. Avoids the danger of chemical insecticides polluting the soil and getting incorporated in to the food chain  Ability to synthesize antibiotics e.g. Penicillin from molds  Major tools of Genetic Engineering  Production of medically important products - Interferon - Insulin (people with diabetes) - Human growth hormone (disease with lack of HGH) - Blood clotting factors (hemophiliac) - Enzymes that dissolve blood clots  Production of vaccines using recombination DNA techniques. Safer vaccines now possible  Important outcome of recombinant DNA techniques is Gene Therapy. Used in various disease conditions. Person is missing gene, gene therapy - virus is used to carry disease to person.  Genetic engineering applied to agriculture. Genetically altered strains of bacteria developed to obtain pest-resistant plants, protect fruits against frost damage  Micro-organisms serve as model organisms - Display same metabolic and genetic properties as higher forms of life - Easy to study - Multiply rapidly on simple inexpensive media The Microbial World  Living things classified into 3 major groups, called domains 1. Bacteria 2. Archaea 3. Eucarya

Features of Bacteria  Single-cell prokaryotes (no membrane bound nucleus, no intracellular organelles)  Specific shape: Cylindrical (Bacillus), Spherical (Coccus) or Spiral (Spirillum) – few are spirial, most are cylindrical or spherical  Rigid cell-wall. Contains unique peptidoglycan – very unique to bacteria, since they have this we use this to our advantage  Multiply by binary fission – when one bacteria divides into two (daughter cells)  Many move by flagella Features of Archaea  Single-celled prokaryotes (no membrane-bound nucleus or intracellular organelles)  Same shape, size and appearance as Bacteria  Rigid cell-wall; however, no peptidoglycan  Multiply by binary fission  Move mainly by flagella  Grow in extreme environments eg. High salt concentrations, high temperatures Features of Eucarya  Single-celled or multi-cellular eukaryotes (true membrane-bound nucleus and internal cell organelles)  Far more complex than simple prokaryotes  Have mitochondria; some have chloroplasts  Cells in our body are eukaryotic  Have a cytoskeleton; gives cells their shape  Egs. Algae, Fungi and Protozoa Algae  Some single-celled; others multi-cellular  Different shapes and sizes  All contain chloroplasts; some have chlorophyll  Cell walls are rigid; distinct chemical composition compared to bacteria  Move by flagella; more complex than prokaryotes  Derive energy from sunlight Fungi  Diverse group  Some single-celled yeasts  Others large, multi-celluar eg. Molds, mushrooms  Derive energy from organic materials Protozoa

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Microscopic, single-celled organisms Complex, larger than prokaryotes No rigid cell wall but many do have a specific shape Require organic compounds as food Most are motile (have pseudopods, flagella or cilia)

Viruses, Viroids and Prions  Not living  Termed as infectious agents  Obligate intracellular agents (grow inside the cell) – not capable of growing anywhere else besides inside the cell Viruses:  Consist of nucleic acid (RNA or DNA) surrounded by a protein coat  Variety of shapes  Multiply inside living host cell  Borrow the multiplication machinery and nutrients from their host to reproduce  Inactive when outside the host  Obligate intracellular parasites  Can infect Bacteria, Archaea and Eukarya  Agents of infection – cannot grow, reproduce or anything on their own. They are totally dependant on the host  Non-living Viroids:  Simpler than viruses  Smaller  Nucleic acid (RNA); no protective coat  Reproduce only inside host cells  Cause plant diseases  May cause human diseases  Relatively new, do not know much about it  Non-living Prions:  Consist only of protein  No nucleic acid  Responsible for neuro-degenerative disease in humans and animals  Also do not know a whole lot about them  Non-living  DIAGRAM 1 PAGE 13

Nomenclature  System of binomial nomenclature  Dual scientific name  Genus name followed by species name  Genus capitalized; species in lower case  Latin origin; italicized or underlines  Eg Neisseria gonorrhoeae or Neisseria gonorrhoeae  If repeated N. gonorrhoeae or N. gonorrhoeae  Sometimes referred to informally by name that resemble the genus name but are not italicized  E.g. Species of Staphylococcus are often referred to as staphylococci Microscopy and Cell Structures  Technology of making very small things visible to the human eye  Human eye cannot perceive objects smaller than 100 micro metres  2 general types of microscopes: - Light Microscopes - Electron Microscopes Light Microscopes  Use visible light waves  Magnification up to 1000X  Easy to operate  Used in teaching and general labs Electron Microscopes  Use electron beam  Magnification above 100.000X  Large, expensive, complex  Used in specialized labs with trained personnel  DIAGRAM 2 PAGE 42 Bright-Field Microscopes  Most common type of light microscope; easiest to use  Light typically passes through a specimen and then through a series of magnifying lenses  2 magnifying lenses  Objective lens: primary magnifying lens positioned close to the object  Ocular lens: secondary magnifying lens positioned close to the eye  Total magnification = Magnification of objective lens X magnification of ocular lens

 Selection of objective lenses of different powers: 4X, 10X, 40X and 100X  Choice of different magnifications is possible with the same instrument  Lenses are mounted on a nose-piece; moved into position as needed  Ocular lens 10X  Total magnification 40X, 100X, 400X, and 1000X Condenser Lens  Does not affect magnification  Positioned between light source and specimen  Helps focus the illumination on to the specimen  Usefulness of a microscope depends on its resolution  Resolution: ability to clearly separate or resolve two objects that are very close together  Resolving Power: minimum distance existing between two objects when they can be observed as separate  Maximum resolving power of LM is 0.2 micro meters  To obtain maximum resolution when using high power objectives, oil is used to displace the air between the lens and specimen  Avoids the bending of light rays when light passes from glass to air  Oil has nearly the same refractive index as glass  Light can then pass without refraction giving a sharper image  DIAGRAM 3 & 4 PAGE 42  Microscopes are designed so that when magnification is increased or decreased by changing from one objective to another, the specimen will remain very nearly in focus  Such microscopes are said to be parfocal  The amount of contrast affects how easily cells can be seen  The more contrast, the easier the cells can be seen  Bacteria are transparent against a bright colorless background  Problem is overcome by staining the bacteria with dyes  However, staining process kills microbes (bacteria), so living cells cannot be observed Special Light Microscopes  Special light microscopes are useful in that they increase contrast between m.o. and the surroundings. Overcomes difficulties of observing unstained bacteria  Particularly valuable if we want to examine characteristics of living organisms eg. Motility  Staining kills bacteria The Phase-contrast microscope  Amplifies the difference between refractive index of cell and the surrounding medium  Light passing through cells is refracted differently than when it passes through surroundings

 Darker appearance of denser material  Increased contrast with the help of special optical devices The Differential Interference Contrast Microscope  Depends on differences in R.I. as light passes through different materials  Device separates light into 2 light beams that pass through specimen and recombine  Light waves are out of phase when they recombine  3-D appearance of the specimen The Dark-Field Microscope  Organisms stand out as bright objects against a dark background  Special mechanism directs light toward the specimen at an angle  Only light scattered by the specimen enters the objective lens  Useful for Treponema pallidum The Fluorescence Microscope  Used to observe cells or other materials that are naturally fluorescent or have been stained with fluorescent dyes  Projects ultraviolet light through a specimen  Captures only the light emitted by the fluorescent molecules  Fluorescent cells stand out as illuminated objects against a dark background The Confocal Scanning Laser Microscope  Detailed view of the interior of an intact cell  Lenses focus a laser beam to illuminate a given point  Mirrors scan the laser beam across the specimen  Successive regions illuminated to scan the entire specimen  Computer constructs 3-D image; displayed on a screen Electron Microscopes  Here we use electromagnetic lenses, electrons and fluorescent screen to produce the image  Electrons have a wavelength 1000X shorter than visible light; R.P. increases 1000X  More details can be observed; 100 000X magnification  Lenses and specimen must all be in a vacuum; molecules composing air would interfere with the path of electrons  Expensive, bulky, require complex sample preparation  Not useful to observe the activities of living cells Transmission Electron Microscope  Fine details of cell structure  Beam of electrons directed at the specimen  Varied scattering depending on density  Darker region corresponds to denser portions of the specimen  Elaborate specimen preparation: thin sectioning, freeze-fracturing or freeze-etching Scanning Electron Microscope

 Observe surface details of cells  Beam of electrons scans over surface of specimen coated with a thin film of metal  Electrons released are reflected back into the viewing chamber  3-D effect obtained Atomic Force Microscope  Detailed images of surfaces  Resolving power greater than electron microscopes  No special preparation needed; can view samples either in air or submerged in liquid  Sharp probe moves across the surface ‘feeling’ the bumps and valleys of the atoms  Laser measures its motion  Computer produces topographic map of the sample’s surface Preparation of Specimens for Light Microscopy 1. Wet preparation: in the living state suspended in a fluid 2. Smear preparation: in a dried state where microbes are no longer alive Wet Preparations  M.o. can be studied in the living state to determine shape, size, motility or group arrangements  Useful for larger microbes  Internal structures can be seen  Dyes may be used eg. Methylene blue, iodine  All particles exhibit ‘brownian movement’  Continuous movement of molecules of the liquid, which constantly bombard the suspended particles Smear Preparation  Thin film of material containing m.o. is spread on to the surface of the slide  Air dried  Heat-fixation: passed 3-4 times through an open flame  Heat fixation serves 3 purposes: - Kills the organisms - Causes the organisms to adhere to the slide - Alters the organisms so that they accept stains more readily - Important step in smear preparation, important that it is done properly - DIAGRAM 5 PAGE 46 Principles of Staining  A stain or a dye is a molecule that can bind to cellular structure and give colour  Most commonly used dyes are positively charged or cationic or basic dyes eg. Methylene blue, crystal violet, safranin and malachite green

 Attracted to negatively charged cell components like cell membrane  Two main types of stains: 1. Simple stain: uses a single dye, reveals basic cell shape and arrangement. Eg. Methylene blue, Safranin, Carbolfuchsin, Crystal Violet 2. Differential Stain: makes use of 2 or more dyes, used to distinguish one group of bacteria from another. Egs. Gram stain, Acid-fast stain Gram Stain  Most frequently used fidderential stain  Devised by Gram in 1884  Bacteria separated in to 2 major groups - Gram + - Gram –  Reveals a fundamental difference in the nature of cell walls of bacteria  DIAGRAM 6 PAGE 47  Decolorizing the smear too long may cause G+ cells to lose the CV-I complex. Such cells will appear pink after counter staining  As bacterial cells age, they lose their ability to retain CV-I complex due to changes to the cell wall. G+ cells will appear pink  Fresh cultures (less than 24 hrs. old. 18-24 hours is the best) are more reliable Acid-Fast Stain  Used to stain members of the genus Mycobacterium (cause tuberculosis and leprosy)  High concentration of lipids (mycolic acid) in the cell wall prevents uptake of Gram stain  Used to identify mycobacteria in clinical specimens  Primary stain is carbolfuchism, a red dye  Heated to facilitate staining; rinsed  Treated with Acid-alcohol (decolorizer); removes dye from most bacteria except those that are acid-fast  Treated with Methylene Blue (counter stain)  Acid-fast cells: bright reddish-pink  Non Acid-fast cells: blue Special Stains to Observe Cell Structures  Dyes can be used to stain specific structures inside or outside the cell  Staining procedure varies, depending on chemical composition and properties of that structure Capsule Stain  The capsule is a viscous layer that envelops the cell

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It is sometimes correlated with organism’s ability to cause disease Stains poorly Wet mount is prepared using India ink Negative staining is used; color the background The capsule stands out as a halo around the organism DIAGRAM 7 PAGE 48

Endospore Stain  Members of certain G+ genera (Bacillus and Clostridium) form a dormant cell called endospore  Resistant to destruction and staining  A special spore stain has to be used  Primary stain: malachite green  Gentle heat to facilitate uptake of dye  Rinsed with water  Counter stain with safranin  Endospores appear green  Vegetative cells (non-sporing) appear pink  DIAGRAM 8 PAGE 49 Flagella Stain  Appendages for mobility  Too thin to be seen with a light microscope  Use a mordant that will allow the staining agent to adhere and coat the flagella  Increases the diameter making them visible  Presence and distribution of flagella is used to identify bacteria  Difficult and time-consuming  DIAGRAM 9 PAGE 49 Fluorescent Dyes  Sometimes fluorescence can be used to observe total cells, a subset of cells or cells with certain proteins on their surface  Acridine Orange binds to DNA. Useful to determine total number of m.o. in a specimen  Dye CTC made fluorescent by cellular proteins involved in respiration. Fluoresces when bound to live cells. Helps distinguish between live/dead cells  Calcofluor White binds to component of cell walls of fungi; cells fluoresce bright blue  Auramine and Rhodamine bind to mycolic acid in cell wall or Mycobacterium; bright yellow or orange fluorescence occurs Immunofluorescence  Technique used to tag specific proteins with a fluorescent compound

 Used to detect specific organisms in a sample containing a mixture of cells by tagging a protein unique to a microbe  Uses an antibody to deliver the fluorescent tag  DIAGRAM 10 PAGE 49  Microscopy and staining techniques can offer valuable information about m.o.  Not enough to permit identification of most microbes  Positive identification requires various biochemical tests and determination of genetic characteristics Morphology of the Prokaryotic Cell Morphology  Shapes: - Spherical, called coccus (plural cocci) - Cylindrical, called bacillus or rod (plural bacilli) - Short rod: coccobacillus - Short, curved rod: vibro (plural vibrios) - Curved rod forming spiral: Spirillum (plural spirilla) - Long, helical cell with a flexible cell wall: Spirochete - Varied shape: pleomorphic - DIAGRAM 11 PAGE 50 Groupings  Cells adhering after cell division form a characteristic arrangement  Pairs (diplococcic) eg. Neisseria gonorrhoeae  Long chains eg. Member of genus Streptococcus  Cubical packets eg. Members of genus Sarcina  Clusters eg. Members of genus Staphlycoccus  DIAGRAM 12 PAGE 51 Multicellular Associations:  Eg. Myxobacteria, move as a pack  Cells release enzymes, degrades organic materials  Cells aggregate to form fruiting body when nutrients and water are depleted  Eg. Bacteria living on surfaces in associations called biofilms Dynamics of Prokaryotic Growth Principles of Prokaryotic Growth  Prokaryotes multiply by binary fission  One cell divides into 2, 4, 8, 26, 32……

 Increase in cell numbers is exponential  Microbial growth refers to increase in the number of cells in a population; not increase in relative size  DIAGRAM 13 PAGE 83  Time taken for a population to double in number is called generation time  Varies with the species  Equation to express the relationship: Nt=N0X2n  Nt= number of cells at a given time in a population  N0= original number of cells in the population  n= number of divisions undergone during the time  DIAGRAM 14 PAGE 83 Obtaining a Pure Culture  Prokaryotes are generally isolated and grown in pure culture  A pure culture is a population of organisms descended from a single cell and separated from all other species  Important basic procedure in microbiology  Used extensively in laboratory diagnosis of infectious diseases  Pure cultures are obtained using special techniques  Sterile conditions for all containers, media and instruments; aseptic technique is used  These are procedures that minimize the chance of other organisms being accidentally introduced Culture Medium  The medium that bacterial cells are grown in or on  A nutrient material prepared for the growth of m.o. in a laboratory  Mixture of nutrients dissolved in water  Liquid broth or solidified gel-like form  Provides all the essential nutrients, proper concentration of salts and ions, proper pH and moisture  Contains products of partial protein breakdown eg. Peptides, amino acids  Provided as partially cooked meats or meat extracts  Need energy; provided as carbohydrates such as glucose, lactose or starch  Minerals and vitamins in purified form or in natural foods eg. In meat digests, yeast extracts  Culture Media is in 2 forms - Liquid broth form - Solid form  Broth: dissolving nutrients in water  Solid media: a solidifying agent, agar is added Agar  ...