Published on August 21, 2013
THE METHODS FOR STUDYING MICROORGANISMS
Specimen collection: Nearly any object, common ones are body fluids and tissues, foods, water or soil. Specimens are removed by some form of sampling device. This may be a swab, syringe, or a inoculation needle and inoculation loop that holds, maintains and preserves the microbes in the sample.
The Five basic techniques to grow, examine and characterize microorganisms INOCULATION INCUBATION ISOLATION INSPECTION IDENTIFICATION
Inoculation(producing a culture) To cultivate or culture, one introduces a tiny sample(the inoculum)into a container of a nutrient medium which provides an environment in which they multiply Selection of media with specialized functions can improve later steps of isolation and identification.
Incubation To adjust the proper growth conditions of a sample Promotes multiplication of the microbes over a period of hours, days and even weeks. Produces a culture- the visible growth of the microbe in the medium
Isolation The end result of inoculation and incubation in macroscopic form The isolated microbes may take the form of separate colonies (discrete mounds of cells) on solid media, or turbidity in broths
Methods for isolating bacteria Streak method -a small droplet of culture or sample is spread over the surface of the medium according to a pattern that gradually thins out the sample and separates the cells spatially over several sections of the plate
Loop Dilution/ pour plate -the sample is inoculated serially into a series of cooled but still liquid agar tubes so as to dilute the number of cells in each successive tube in the series. Inoculated tubes are then plated out into sterile plates and are allowed to solidify. The end result is that the number of cells per volume is so decreased that cells have ample space to grow into separate colonies
Spread plate technique -A small volume of liquid diluted sample is pipetted onto the surface of the medium and spread around evenly by a sterile tool (like a hockey stick).Like the streak method, cells are pushed into separate areas on the surface so that they can form individual colonies
Media: Providing Nutrients in the lab 3 Primary levels of media classification: 1. Physical form 2. Chemical composition 3. Functional type
Physical state (medium’s normal consistency Chemical composition ( type of chemicals a medium contains) Functional type (purpose of medium) 1. Liquid 2. Semisolid 3. Solid(can be converted into liquid 4. Solid (cannot be liquefied) 1. Synthetic (chemically defined) 2. Nonsynthetic (not chemically defined) 1. General purpose 2. Enriched type 3. Selective 4. Differential 5. Anaerobic growth 6. Specimen transport 7. Assay 8. enumeration
Physical states of media Liquid media- are water based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted. These media, termed broths, milks or infusions, are made by dissolving various solutes in distilled water. Growth occurs throughout the container and can then present a dispersed, cloudy or particulate appearance.
Semisolid media- exhibit a clotlike consistency because they contain an amount of solidifying agent(agar or gelatin) that thickens them but does not produce a firm substrate. Semisolid media are used to to determine the motility of bacteria and to localize a reaction at a specific site.(0.3-0.5% agar) ex. SIM(hydrogen sulfide production and indole reaction)
Solid media- provide a firm surface on which cells can form discrete colonies and are advantageous in isolating and culturing bacteria and fungi. 2 forms 1. Liquefiable- contain a solidifying agent that changes its physical properties in response to temperature. Agar a complex polysaccharide isolated from the red alga Gelidium. It is solid at room temperature, and it melts at the boiling temperature of water (100). Ex. Nutient agar(beef extract and peptone, as well as 1.5% agar by weight) 2. Nonliquefiable –they do not melt. They include materials such as rice grains (to grow fungi), cooked meat media(anaerobes), and potato slices
Chemical content of Media Synthetic- contain pure organic and inorganic compounds Nonsynthetic/compex media-contain at least one ingredients that are not chemically definable- extracts of animals, plants or yeasts, ground up cells, tissues and secretions, milk and peptone . Blood agar, nutrient broth and MacConkey agar.
Media to suit Every Function 1. General purpose media- are designed to grow as broad spectrum of microbes as possible. As a rule, they are nonsynthetic and contain a mixture of nutrients that could support pathogens and nonpathogens alike. Ex. BHI, TSA 2. Enriched medium- conains complex organic substances such as blood serum, hemoglobin or special growth factors that certain species must have inorder to grow. Bacteria that require growth factors and complex nutrients are fastidius. Ex. Blood agar to grow fastidius streptococci and other pathogens. Thayer Martin medium- for neisseria
3. Selective medium- contains one or mre agents that inhibit one or more agents that inhibit the growth of a certain microbe or microbes but not others and thereby selects or encourages microbe nd allow it grow.MSA contains a concentration of NaCl that is quite inhibitory to most human pathogens except staphyloccoccus.
Selective media, agents and functions Medium Selective agent functions Mueller tellurite Enterococcus faecalis broth Phenylethanol agar Tomato juice agar MacConkey agar Salmonella/shigella agar Lowenstein-Jensen Sabourauds agar Potassium tellurite Sodium azide tetrazolium Phenylethanol chloride Tomato juice, acid Bile, crystal violet Bile, citrate, brilliant green Malachite green dye Ph 5.6(acid) C. diptheriae Fecal enterococci Staph and Strep Lactobacilli from saliva Gram negative enterics Salmonella and shigella Mycobacteria Fungi, inhibits bacteria
Differential media Grow several types of microorganisms and are designed to display visible differences among those microorganisms. Differentiation shows up as variations in colony size or color, in media color changes or in the formation of gas bubbles and precipitates
medium Substances that facilitate differentiation Differentiates between Blood agar Intact red blood cells Manitol,phenol and 7.5% Types of hemolysis Species of stap. NaCl Hektoen enteric agar Spirit blue agar Urea broth Sulfur indole motility Triple sugar iron gar Nacl Bromthymol blue, acid fushin,sucrose, salicin, thiosulfate, ferric ammonium citrate, and bile Spirit blue dye and oil Urea, phenol red, thiosulfate, iron Triple sugars, iron and phenol red dye Also inhibits the salt- sensitive species Salmonell, shigella and othe lactose fermenters and nonfermenters Dyes and bile also inhibit gram positive bacteria Bacteria that use fats from those that do not Bacteria that hydrolyze urea to ammonia H2S gas producers from nonproducers Fermentation of sugars, H2S production
medium Substances that facilitate differentiation Differentiates between XLD agar Birdseed agar Lysine, xylose, iron, thiosulfate,phenol red Seeds from thistle plant Enterobacter, escherichia, proteus, providencia, salmonella and shigella Cryptococcus neoformans and other fungi
Miscellaneous media A reducing medium contains substance(cystine or thioglycollic acid) that absorbs oxygen or slows the penetration of oxygen in a medium, thus reducing its availability. Reducing media are important for growing anaerobic bacteria or determining oxygen requirements.
Transport media Are used to maintain and preserve specimens that have to be held for a period of time for clinical analysis or to sustain delicate species that die rpidly if not held under stable conditions.
Assay media To test effectiveness of antimicrobial drugs and by drug manufacturers to assess the effect of disinfectants, antiseptics, cosmetics and preservatives on the growth of microorganisms
Enumeration media Used by industrial and environmental microbiologists to count the numbers of organisms in milk, water, food, soil and other samples
A culture may exist in one of the following forms: 1. Pure 2. Mixed 3. contaminated
Inspection Cultures are observed macroscopically for obvious growth characteristics(color, texture,size) that coild be useful in analyzing specimen contents. Slides are made to assess microscopic details such as shapes, size, and motility. Staining techniques may be used to gather specific information on microscopic morphology
Identification A major purpose of the 5 I’s is to determine the type of microbe, usually to the level of species. Specialized tests include biochemical tests to determine metabolic activities specific to the microbe.
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