LECTURE NOTES WEEK 3
LECTURE NOTES WEEK 3 BIOL 2460-001
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This 6 page Class Notes was uploaded by Mercy on Saturday September 19, 2015. The Class Notes belongs to BIOL 2460-001 at University of Texas at Arlington taught by Dr. Michelle Badon in Summer 2015. Since its upload, it has received 88 views. For similar materials see Nursing Microbiology in Biology at University of Texas at Arlington.
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Date Created: 09/19/15
Lecture Notes week 3 Tuesday September 15 2015 0824 Pay attention to words in bold and italics Tools of Laboratory Ecoli 0104H4 There were reports of the outbreak of enterohemorrhagic Ecoli EHEC in Germany Generally strains of Ecoli cause relatively mild cases of diarrhea also known as traveler39s diarrhea or a mild form of dysentery both of which resolve with minimal treatment When the reporting of illness subsided in midJune there were a total of 855 cases of hemolytic uremic syndrome HUS Thirtyfive deaths were attributed to HUS while 18 were due to EHEC gastroenteritis making this the largest outbreak of foodborne disease ever recorded in Germany 0 What are the steps that should be taken to identify the specific cause of this outbreak 0 How is a case of EHEC different than other foodborne infections caused by E coli Cases of EH EC are usually associated with the notorious strain E coli 0157H7 but this outbreak was caused by the lesserknown strain E coli 0104H4 Both strains of E coli produce the Shiga toxin that causes bloody diarrhea and HUS but the 01042H4 strain had previously not been found in animals and only rarely in humans Scientists and doctors analyzed stool samples of patients showing symptoms of EH EC and HUS in an attempt to isolate the Shigatoxin producing pathogen Genomic analysis of specimens revealed that the 01042H4 strain rather than the 0157H7 strain was causing illness in the patients Moreover the scientists confirmed that it was resistant to at least 14 different antibiotics 0 What type of microbial media might have been used to identify E coli 0104H4 Some type of solid surface to encourage microbial growth was needed Robert Koch was able to isolate Bacillus anthracis on thin slices of potatoes but other species of bacteria didn39t thrive in this medium Koch had some success with using gelatin but when left out in the warm laboratory gelatin quickly liquefied ruining the isolation experiment Additionally it was discovered that microbes produced an enzyme called gelatinase that cleaved the bonds between protein molecules also liquefying the medium How was the problem solved Walter Hesse and his wife Fanny started working as lab assistants for Robert Koch in 1881 One day Walter noticed that Fanny39s puddings in his lunch weren39t melting in the warm laboratory unlike the gelatin in the Petri dishes When asked about this Fanny told him that she39d learned the trick to perfect puddings was using a product made from seaweed called agaragar Knowing the trouble Walter was having with finding solid microbial media she suggested that they combine the agaragar with the nutrient broth already used in the lab Suddenly Robert Koch39s lab had the perfect microbial media for isolating bacterial colonies The agar mixed with nutrient broth stayed firm at incubator temperatures and was impervious to bacterial enzymes After this seemingly simple modification Koch and his laboratory assistants were able to isolate the microbial cause of tuberculosis and numerous other pathogens Methods of culturing Microganisms M0 Sterile complete absence of viable microbes Aseptic prevention of infection or contamination pure culture growth of a single species of microbes 1 Inoculation Put or introduce microbes right there for growth Culture microbes by introducing a sample in a sterile medium which will sustain the growth of MO Samples can be taken from clinical species like blood feces urine sputum disease tissues and microbiological analysis like soil water sewage foods air 2 Incubation Give it the right temperature for it to grow Microbes are placed in a temperaturecontrolled chamber to encourage growth Put in the cold room to slow the growth Usual temperature in the lab is 20 to 40 C 3 Isolation separating one species of microbes from another Axenic means a culture that is free of other living thing except for the one being studied Subculture make a second level culture from a well isolated colony A tiny bit of cell from a previously cultured bacteria is transferred into a separate medium and is incubated Pure culture population of cells coming from a single culture characterized as an individual species In order to obtain and maintain a pure culture aseptic techniques sterile environment is necessary All glassware media and instrument that comes in contact with the bacteria must be sterile Mixed culture two or more identified species of MOs Contaminated culture Once pure or mixed but now contains unwanted microbes of uncertain identity Methods of isolating bacteria Streak plate common tech to isolate pure colonies Purpose is to reduce the number of cells being spread with each successive series of streak diluting the conc of cells Spread plate a tech where a mixture of cells is spread out on an agar surface so that every cell grow into a completely separate colony i Colony a macroscopically visible growth or cluster of M0 on a solid medium where each colony represents a pure colony About million cells are needed for a colony to be visible with the naked eye Enumeration of bacterial colonies viable plate count contains 30300 colonies Pour plate original sample is diluted several times to reduce microbial population to obtain separate colonies when plating i Small volume of several diluted samples are mixed with agar ii mixtures are poured immediately in sterile culture Petri dishes iii After agar solidifies each cell is fixed in place and forms an individual colony 4 Inspection Check evaluate and examine cultures macroscopically color texture and size and microscopically cell shape size and motility colony morphology and growth Form and margin can be determined by looking down at the top of the colony and elevation can be viewed from the side as the plate is held at eye level Most rapid cell growth occurs at the colony edge At the colony edge oxygen and nutrients are plentiful Slower growth takes place at the colony center Oxygen does not diffuse readily into the center toxic metabolic products cannot be eliminated quickly and growth in the colony center is slowed or stopped 5 Identification identify what MO grew MO are identified in terms of their Macroscopic or immunological morphology Microscopic morphology biochemical reactions Genetic characteristics Types of culture media Media 500 different types of media are used to culture and identify MOs Use test tubes flasks and Petri plates for culture media o For inoculation use inoculating loop inoculating needle pipettes and swabs Some microbes and all viruses require cell cultures or host animal Solid semisolid gelatin or liquid preparation used to grow transport and store MOs Medium must contain necessary nutrients required for the growth of MOs Medium is used to select and grow specific MOs to help identify a particular species Media can be classified in the physical state chemical composition and functional type Synthetic or defined media Media whose compositions are chemically defined They contain pure organic and inorganic compounds Complex media or non synthetic TSA TSVMedia that contain at least one ingredient that is not chemically definable It is rich and complete to meet the nutritional requirements of many MOs Used because nutritional requirements are unknown ie used forfastidious picky MOs It contains components like peptones meat extracts and yeast extract Peptones protein hydrolysates prepared by partial digestion of meat casein soy gelatin and other protein sources serving as C energy and Nitrogen sources Broth can be solidified with the addition of agar Agar is a polysaccharide It is an extract from red algae Gelidium t melt at temp above 95 C can solidify at temp below 45 C It is also translucent Types of Media General purpose media Non synthetic that support the growth of many MOs Examples are TSA nutrient agar or broth Enriched media Blood or other special growth factors vitamins AA nutrients are added to general purpose media to encourage the growth of picky fastidious MOs Examples are blood agar used for growing bacteria from human throat eg Streptococcus pyogens chocolate agar Nisseria sp Specific for identifying gonorrhea Selective Media A medium that is specific and favors the growth of a particular MO Contains one or more agents that inhibit the growth of a certain microbe or microbes but encourages or selecting the growth of others Example Mannitol Salt Agar MSA contains a high conc of NaCl that is inhibitory to most human pathogens Staphylococcus grow well here Bile salts favors the growth of gram by inhibiting the growth of gram MacConkey agar is used for the detection of E coli Differential Media t distinguishes between different groups of bacteria It incorporates different ingredients that cause certain organisms to develop a different appearance from other microbes growing on the same medium Example Blood agar is both a differential and enriched medium t distinguished between hemolytic and nonhemolytic bacteria It allows you to determine if a bacterial colony has produced the enzyme hemolysin and whether the resulting hemolysis that is gamma beta or alpha Microbes exhibiting gammahemolysis have no change in the blood agar due to the lack of hemolysin production Microbes exhibiting betahemolysis have a clearing of the blood agar due to complete red blood cell lysis Microbe exhibiting alphahemolysis have a greening of the blood agar due to incomplete red blood cell lysis CHROMagar orientation a state of the art medium developed for cultivating and identifying the most common urinary pathogen at least 7 different species Ecoi Klebsiella Proteus Enterococcus S saprophyticus and S aureus MICROSCOPY The light microscope is the single most important research tool that microbiologists have ever had It is an optical instrument which operates on the principle that light energy will pass through and around a suitably thin object and with the aid of lenses form a magnified impression on the visual sensory layer of the eye c When light energy passes from one medium to another ie AIR and GLASS the light rays are bent at the point of interface This process is called refraction The measure of how greatly a substance slows the velocity of light is called the refractive index The brightfield microscope Called the ordinary microscope because it forms a dark image against a brighter background It is common multipurpose micro for live and preserved stained specimens Provides fair cellular detail Parafocal image should remain in focus when objectives are changed Path of light The obj lens forms an enlarged real image within the microscope and the eyepiece lens further magnifies this primarily image Darkfield microscopy is one such tech that is often used to observe living unstained cells and organisms Phase contrast microscopy exploits the differences between refractive index of the cells and the surrounding medium resulting in a darker appearance of the denser material It is used for live organisms and excellent for internal cellular detail The Interference Microscope Brightly colored and highly contrasting 3D images of live specimens Fluorescence Microscopy Specimens are treated with dye molecules called fluorochromes which brightly fluoresce when exposed to light of a specific wavelength The color that the cell will appear depends on the type of dye used Confocal Microscopy Is used to construct a 3D image of a thick structure such as a community of microorganisms and provides a detail sectional view of the interior of an intact cell It eliminates a murky fuzzy and crowded image A focused laser beam strikes a point in the specimen and light from the illuminated spot is focused by an objective lens onto a plane The laser is scanned over a plane in the specimen beam scanning or the stage is moved stage scanning and a detector measures the illumination from each point to produce an image of the optical section Factors that determine the quality of an optical image Magnification To enlarge it is the apparent increase in size affected by a convex lens A compound microscope uses two sets of lenses with different focal lengths to facilitate magn Total mag mag of obj lens x mag of ocular lens usually 10x Resolution is the ability to separate points to observe fine details Clearly separate One way to increase the resolution of an image is to increase the amount of light by using Immersion oil Immersion oil with a density closer to glass can be used to reduce the refraction of light rays compared to air and allows more light to enter the objective This improves resolution Contrast reflects the number of visible shades in a specimen Microbes are composed of water nucleic acids proteins and lipids transparent Most appear colorless against a colorless background when observed using bright field microscopy Therefore in order to see them we must devise a way to increase the contrast In bright field microscopy we have direct staining of the M05 and indirect staining of the background In order to stain a specimen it must first be fixed to the slide which results in the death of a specimen Electron Microscopy A physical relationship exists between resolution and light Are used to achieve up to more than 100000 greater magnification and more than 1000 times greater resolution than light microscope Do not use lenses to focus the electron beams instead they use magnetic fields Used only for preserved materials Types of Electron Microscopy Transmission Electron Microscope TEM projects a beam of electrons through a specimen Scanning Electron Microscope SEM observing surface details but not internal structures of cell Preparation and staining of specimens MOs must be fixed and stained to increase visibility specific morphological features and preserve them for future study Fixation Process by which internal and external structures of the cells and M05 are preserved and fixed in position This process inactivates enzymes that might disrupt cell morphology and toughens cell structure so that they do not change during staining and observation A MOs usually is killed and attached firmly to the microscope slide during fixation Two types of fixation Heat fixed by gently flame heating a bacteria preserving the overall morph but not structures within cells Chemical fixation is used to protect fine cellular substructures and the morphology of larger more delicate mos Common fixative mixtures ethanol acetic acid mercuric chloride and formaldehyde Biological stains One way to increase contrast of an image is to stain colorless microorganisms Stains are very pure dyes Stains are typically salts composed of positive and negative ions eg Na Cl Only one of the ions carries the chromogen or colored part of the molecule Many dyes used to stain MO have two common features 1 They have chromophore groups groups with conjugated double bonds that gives dyes its color 2 They can bind with cells by ionic covalent or hydrophobic bonding Positive vs Negative staining Positive stain also known as Basic stain Examples methylene blue is a chloride salt MBCl Basic fuchsin crystal violet safranin malachite green Basic dyes bind to negatively charged molecules like nucleic acids proteins and cell wall components and the surface of the cells themselves Because the surfaces of bacterial cells are negatively charged basic dyes are most often used Negative stain also known as Acidic stain The dye does not stick to the specimen the cells themselves but settles around its outer boundary Stains the glass slide to produce a dark background around the cells The cells themselves do not stain because these dyes are negatively charged and are repelled by the negatively charged surface of the cells If the negative ion anionic contains the chromogen the stain is an acidic stain The smear is not heat fixed Examples Nigrosin bueblack India nk a black suspension of carbon particles Simple vs Differential staining Simple stains requires only a single dye Differential stains use 2 differently colored dyes called primary dye and the counterstain Special stains those that were developed for a single purpose Staining a Bacterial smear too thick can cause dye to accumulate between cells a too thin too hard to see ideal smear is one cell layer thick The gram stain The most important differential stain is the gram stain which was devised by Hans Christian Gram in 1884 Almost all bacteria can be divided into two groups Grampositive and Gramnegative by this stain Results of the Gram stain are correlated with cell wall composition The Gram stain is usually the first step taken to identify an unknown bacterium Grampositive which stain purple and Gramnegative which stain pink red Acid Fast staining To stain mycobacteria you must use a combination of heat and phenol to drive the basic fuchsin stain into the cell wall Once the stain has penetrated it is not easily removed or decolorized with acid alcohol Thus the cells are said to be ACID FAST Acid fast red
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