Study Guide for Midterm and Final Tests (IN LAB)
Study Guide for Midterm and Final Tests (IN LAB) 81382 - MICR 3050 - 001
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Microbiology Lab 3051 Spring 2015 MidTerm Laboratory Exam Study Guide Labs 1 6 Objectives 1 Know the parts of the microscope and their functions Rotata Arm Stage Nosepiece Objective Ad justme nt Mechanical Stage Power Swrtch Light Intensity Diaphragm Control antral Coarse Adjustme nt Condenser Knob Illuminator ine Adjustment Knob Base if 39 tage Adjustment v Knob 2 Know how to use the microscope adjusting for proper viewing getting an image in focus using oil immersion and how to take care of a microscope a Focus on 10X magnification and use course adjustment knob first Once you find the specimen add the oil move to lOOX and only use fine tuner 3 Describe aseptic technique and understand Why it is important a The aseptic technique ensures that no contaminating organisms are introduced into culture materials b Technique Procedures for test tube i Disinfect table top ii Flame loop iii Flame mouth of test tube iv Once loop is cooled insert loop into test tube to gather bacteria V Remove loop ame test tube and replace cap that was in your hand vi Flame mouth of test tube that Will be inoculated vii Insert loop into test tube 1 If broth turn loop several times 2 If slant touch loop to bottom and drag up in a squiggle viii Flame mouth of tube place cap on and ame loop 0 Technique Procedure for agar plates i Disinfect table top ii Flame loop iii Inoculate your loop from sample iv Lift lid of agar plate and streak loop across entire plate v Replace lid ame loop 4 Compare and contrast how to make a bacterial smear from liquid media and solid media a Smear for Liquid Media i Shake culture in liquid media ii Aseptically transfer 2 loopfuls of media to the center of the slide ame loop in between each transfer iii Spread the liquid into an oval shape on the slide iv Flame the loop and allow slide to air dry V Once slide is dry heat fix it using the Bunsen burner b Smear for Solid Media i Flame loop and apply two loops of water ii Aseptically obtain a very small amount of bacteria just enough so that it s visible on the loop iii Place loop in water in center stage iv Spread the liquid into an oval shape v Flame loop and allow slide to air dry vi Once the smear is dry heat fix with Bunsen burner 5 Explain the importance of heat fixing and airdrying specimens on slides a Heat fixing kills the bacteria in the smear firmly adheres the smear to the slide and allows the sample to more readily take up stains i Allow the smear to air dry ii After the smear has airdried hold the slide at one end and pass the entire slide through the flame of a Bunsen burner two to three times with the smearside up 6 Describe simple staining and what bacterial characteristics can be observed using this technique a Simple staining is the use of a single stain to color bacteria i Common stains used are methylene blue basic fuchsin and crystal violet 1 These dyes have color bearing ions called chromophores that are positively charged a This is important because bacteria is negatively charged so it attracts the chromophores from the dye 7 Distinguish between acidic and basic dyes and when to use each a Basic dyes the dyes that hold the chromospheres positively charged dyes b Acidic dyes contain negatively charged chromospheres Acidic dyes do NOT stain bacteria because of the electrostatic repelling forces c Use basic dyes when you have a bacteria that is negative because Basic dyes are d Use acidic dyes when you have a positive charged specimen because Acidic dyes are negative 8 Recognize the different bacterial morphologies a Types i Bacilli rods 1 Coccobacillus 2 Bacilli 3 Fusiform ii Cocci spherical 1 Forms diplococci 2 cocci and tetrads 4 cocci 2 And streptococci linear form of cocci 3 Staphylococci grapelike cocci iii Spirals curved rods 3 Curved bacilli vibrios spirillum sgiochaete d y M 09 Treponema pamdum 09 Vibrio cholerae eg Splrmum volumns I 1 9 Understand the distribution of bacteria in our world 10 Compare and contrast grampositive and gramnegative cells what colors they stain using the Gram stain and how cell wall structure determines how they stain a Gram positive bacteria has thick layer of peptidoglycan that retains the Crystal Violet iodine complex Therefore the gram positive is purple due to the CRI stain i Gram positive remains purple throughout the entire procedure since the crystal violent is added b Gram negative has a much thinner layer of peptidoglycan in their cell wall below the outer membrane so it does not hold the CVI complex loses it stain with the decolorization Therefore it needs the Safrinin counterstain to colorize the specimen so that it can be observed as a pink color i Gram negative is purple while CV and then Iodine is added ii But it becomes clear when ethyl alcohol is added iii Then ends up being pink with safranin 11 Know all of the steps and stains used in a Gram stain Know the purpose of each stain and how bacteria look at each step Know which stain is the primary stain counterstain mordant and decolorizing agent a Procedure i Primary Stain Crystal Violet 20 sec 1 Water wash ii Mordant Gram s Iodine 1 minute iii Decolorize Alcohol 1020 sec TIP Stop once it runs clear 1 Water wash iv Counterstain Safrinin 1 minute 1 Water wash v Wash amp Blot dry TIP Do not rub slide 12 Understand the purpose of a streak plate and how you would do one a The purpose of a streak plate is to isolate bacteria on agar i 13 Define pure culture and pure colony a A pure culture contains only a single kind of organism b A pure colony a colony that is due to a single bacterium i The entire colony is a clone of the original bacteria c A pure culture allows the study of physiology and morphology 14 Know the purpose of a gelatin stab how you conducted this test and what a positive reaction for protease production would look like a A gelatin stab tests the production of proteases enzymes that degrade proteins b The test is positive if liquefaction the gelatin turning liquid occurs i This test is completed by putting culture in ice bath for several minutes and then observing c However if negative it needs to stay in ice bath for 4 or 5 days because some bacteria can produce protease at a very slow rate 15 Describe endospores their function their unique characteristics and which genera produce them a Endospores produced by bacteria when nutrients are low gone Allows bacteria to survive in unfavorable environments heat chemicals acid UV radiation desiccationdrying out b Exospore is the protective protein coat around the endospore that is the barrier of protection c Genera that produces it bacillus and Clostridia 16 Understand the SchaefferFulton endospore staining procedure know the primary stain and counterstain why heat is used as a mordant and what endospore producers look like under the microscope after being stained a Procedure i Make smear ii Cover slide with small piece of paper towel iii Saturate with malachite green primary stain iv Place resting above beaker with boiling water for 5 minutes heat is mordant v Allow slide to cool vi Remove paper towel vii Rinse with water for 30 seconds viii Counterstain with safranin for 20 seconds ix Water rinse X Blot dry 17 Know what genera of bacteria are acidfast and what structure causes them to be acidfast a Genera i Mycobacterium ii Some Nocardia b The structures that make these genera require acidfast is the presence of mycolic acid which prevents penetration of typical stains 18 Describe the acidfast stain primary stain decolorization counterstain and know how acidfast cells and nonacid fast cells appear after staining a Procedure i Smear and heat fix first ii Primary Stain Cover slide with basiefuehsinl carbolfuchsin stain for 5 mins 1 water wash iii acidalcohol decolorize for 1 min 1 stop decolorize by short water wash b c iv methylene counterstain for 30 seconds 1 short water wash V blot dry Acid fast cells are pink Nonacid fast cells are blue 19 Describe bacterial capsules and their functions a A polysaccharide layer that lies outside the cell envelope of bacteria part of the outer envelope of bacteria cells It is not easily washed off and is the cause of various diseases It is well organized Found in both gram negative and gram positive bacteria 20 Describe the use of negative staining to visualize capsules and know how capsules appear after using this method a 6 f g Negative stains are acidic so they have a negative charged chromophore that does not penetrate the cell it is repelled since the cell is negative also This results in a negative staining Examples of negative stains are India ink and nigrosine Procedure i Place a small drop of Maneval s A solution on one side of the slide ii Placing a small amount of the specimen inside the Maneval s A iii Using another slide spread the specimen over the slide in a thin layer iv After it has air dried ood smear with Maneval s B solution for 1 minute v Drain the excess dye vi Allow to dry Then observe not always heat fixed before the negative stain i Without heat fixing the negative stain can allow for determining cell dimensions ii Heat shrinks the cells size and capsule Maneval s A is a negative stain that also has a pH indicator that turns blue after adding Maneval s B Maneval s B is a positive stain Expect to see blue background with pink cells surrounded by capsule 21 Understand the methods we used to see motility and how motility or lack of was determined a b Wet Mount i Make sure the 40X is completely clean ii Put one to two loops on your slide then quickly coverslip it iii Move fast iv Ignore Brownian motion 1 Movement caused by currents under coverslip jiggling v Make sure light is not too bright Hanging Drop i Dot Vaseline on the four corners of the coverslip ii Place two loopfuls of culture onto the coverslip iii Place depression slide on top iv Quickly invert and view look at edge v Move fast 22 Of the bacteria we used for the motility tests be able to name which bacteria are motile and nonmotile a P vulgaris motile b M luteus nonmotile 23 For each of the following tests know the purpose what a positive reaction would look like what media and or reagents were used and how the media andor reagents work to show negative and positive reactions ex methyl red in MRVP turns red when the pH drops to 5 or less indicating mixed acid fermentation Glucose Sugar Fermentation Oxidative Fermentation test Mixed Acid Fermentation MR Butanediol Fermentation VP Can unknown ferment glucose Distinguish between various gram bacteria that may or may not produce mixed acid Distinguish gram that do not carry out mixed acid fermentation but rather ferment glucose to produce limited Positive Reaction looks like Gas present Red color Positive pinkred color if acetoin is present What mediaReaents used Glucose with Durham Tube MRVP medium is a glucose broth Grown in MRVP medium How mediareaents work to show neative and ositive reactions 39 If color is yellow aerobic the acid has been produced fermentation test If a bubble is present in Durham tube gas was produced oxidative test The pH indicator methyl red turns red if the mixedacid fermentation process has occurred by the bacteria Butanediol is not detected directly but an intermediate product acetoin is tested Old culture 35 days shaken I add 18 drops of I Citrate test Oxidase Test Catalase Test Nitrate Reduction amounts of some acids so that the product is more neutral Test if bacteria use citrate as carbon source Tests for prsense of cytochrome oxidase enzyme in ETC Determine if catalase is present catalase turns H202 into oxygen and water Determine if bacteria uses nitrate as terminal electron acceptor pH indicator in medium turns from dark green to a deep blue Turns from yellow to purple in 10 to 30 seconds if change is after 30 seconds it s still negative Bubbles present hydrogen peroxide is added to small amount of bacteria meaning a break down Gas in Durham Tubes After adding reagent A and B sulfanilic acid Medium includes ammonium salts nitrogen source TSA plate Nutrient agar slant Nitrate reduction broth with Durham tube both Baritt s A and B reagent is used Wait 30 minutes at room temp The blue indicate the presence of ammonia 9 meaning the bacteria is using citrate 9 turns blue because ammonia alkaline The oxidase reagent added to cells once glass is broken and bacteria exposed tests if the cell will oxidize in oxygen The test tells if the bacteria uses an artificial electron acceptor not oxygen which would be unstable and oxidize if left exposed to air The enzyme catalase protects from cell damage due to production of H202 Nitrate makes products gas or nitrate Starch Hydrolysis Test Casein Hydrolysis Tryptophan Degradation Indole Test Urea Hydrolysis implying facultative anaerobic Can the microbe use starch as a carbon source and energy for growth Can bacteria degrade casein meaning the presence of proteases Can bacteria degrade tryptophan Can bacteria hydrolyze urea to produce urease and alpha naphthylamine the unknown turns red If neither gas or red happens check with zinc powder it forces any nitrate to nitrite so it turns red if nitrate is present If none of those then negative If starch is degraded then a clear zone will surround streak If casein is degraded a clear zone will surround streak Red ring at surface if black precipitate is also present it means that amino acid cysteine was broken down in H202 production color change is from yellow to HOT PINK Starch agar with Iodine that causes it to turn blueblack Skim milk plate SIM tubes Urea agar slants If Tryptophan is degraded then indole will be produced Therefore when the Kovac s reagent is added a red ring will form If urease is produced ammonia is a byproduct causing an increase in pH exoenzymes break down starches for transport into the cell ie amylases cellulases exoenzymes break down proteins for transport into the cell ie proteases 24 Know the functional type mechanism of action what type of colonies will grow and how they can be distinguished on the following media MacConkey Agar MAC Phenylethyl Alcohol Agar PEA and Blood Agar Media Info MacConkey Agar MAC 0 Selective and differential culture medium 0 Isolates gram negative 0 Bacilli 0 Has pH indicators that turn if microbe is Phenylethyl Alcohol Agar PEA Blood Agar fermenting lactose Selective medium Grows most gram negative It functions to disrupt lipid structure in gram negative membranes and stops protein synthesis 9 growth is stunted or stopped permanently Differential medium Allows for hemolysis destruction of RBCS complete destruction 9 clears area around the colonies partial breakdown 9 greenish brown color on agar no damage to RBCS 9 no change in medium 25 Understand how to construct a bacterial growth curve by measuring the turbidity of a broth culture a Use a spectrophotometer at 600 nm Make a blank to use in between b Measure the sample every 20 minutes for 2 hours 26 Know the four phases of bacterial growth in a batch culture 13 iilililifi39lhu 1i 31515 g r 75153 wife Il fsg 399 395 E313 Log number of viable cells gt Time gt o Lag phase no increase 0 The energy is spent on synthesizing new components and adapting to the new medium instead of dividing 0 Exponential 0 log phase 0 Rate of growth is constant and maximal 0 Population is most uniform o Inoculate during midexponential phase 0 Stationary phase 0 Total number of Viable cells remains constant meaning reproduction stops or the reproduction rate is balanced by death rate 0 This phase is trigger by nutrition limits limited oxygen toxic increase andor critical population density reached 0 Death phase 0 Total number of Viable cells decreasing o Irreversible loss of reproduction o Lysis may occur 27 Be able to classify organisms based on their oxygen requirements obligate aerobes obligate anaerobes facultative anaerobes microaerophiles aerotolerant as shown by growth in uid thioglycollate medium FTM a Obligate aerobe strictonly oxygen b Obligate anaerobes no oxygen c Facultative anaerobes does not require oxygen but growths better with oxygen 1 Microaerophiles requires small amount of oxygen 210 e Aerotolerant grows with or without oxygen 28 Define neutrophile acidophile and alkaliphile a Acidophiles i Between pH 0 55 b Neutrophiles i Between pH 55 and 7 c Alkaliphiles i Between pH 85 115 29 Define AW hypotonic hypertonic plasmolysis halophiles halotolerant osmophiles a Nonhalophile does not require NaCl b Halophile requires NaCl 115 Halotolerant bridges between nonhalophile and halophile Extreme halophiles requires NaCl 1530 Aw water activity i The amount of water available to organism ranges from 01 Hypotonic solute concentration is lower than outside the cell Hypertonic solution concentration is higher than outside the cell Plasmolysis process where cells lose water in hypertonic solution Osmophiles organisms that can grow where sugar concentrations are excessive 30 Understand what happens to a cell when it is placed in a hypotonic or hypertonic environment a If cell is in hypotonic solution mechanosensitive channels allow solutes to leave so that less water comes into the cell prevention of lysing b If cell is in hypertonic solution it allows an increase in solutes concentration so that water can come in c Bacteria prefers hypotonic solution so that more water can go into cell 31 Know which organism that we tested should have been able to grow at the highest salt concentration Describe how UV light is germicidal its effect on DNA and the wavelength at which UV light is the most lethal a H salinarium is the only extreme halophile b UV light damanges DNA by causing the thymine base pairs next to each other to bond together 9 pyrimidine dimers which is disruptive and cannot be copied c The primary lethal effects of UV light are due to the mutagenic properties d UV at 260 nm is most lethal because it this is the wavelength where DNA maximally absorbs UV light 32 Describe how we examined the germicidal effects of UV light on S aureus and B subtilis 33 Be able to define the following terms antiseptic disinfectant steriliant sanitizer bacteriostatic and bacteriocidal a Sterilization killsremoves all VIALBE organisms INCLUDES ENDOSPORES This is an absolute condition b Disinfectant killstopremove of pathogenic organisms i Usually used on inanimate objects ii Alcohol and peroxide are forms that can be used on living tissues iii Bleach is used on inanimate objects usually c Sanitization reduction of microbial population to safe levels d Antisepsis against rotting i Prevents infection of living tissue ii Chemical agents used to kill growth of microbes when applied to tissue e Bacteriostatic an agent that inhibitsstops growth f Bactericide agent that kills pathogens 34 Explain the Filter Paper Disk Method of evaluating the relative effectiveness of various disinfectants andor antiseptics a A sterile disk is dipped halfway into agent and then layed on the nutrient agar pressed down lightly to secure it b After 2448 hours in incubation zone is measured measure the distance between disk edge and growth to determine how successful the agent was c This tests the effectiveness of disinfectants and antiseptics 090 rprqoth i Disinfectants are agents that are applied to inanimate objects to kill microbes Usually more harsh than antiseptics which is why they are damaging to living tissues 1 Some disinfectants can be steriliants killing endospores also ii Antiseptics agent alcohol that stops microbe growth or kills microbes But are gentle enough for living tissue Do not destroy endospores 35 Distinguish between antibiotics and antimicrobials a Antibiotics microbial products or derivates that kill mircobes or stop their growth b Antimicrobials chemical agents that treats a disease by destroying pathogenic microbes or inhibiting their growth within a host 36 Explain the KirbyBauer Method of evaluating the sensitivity of bacteria to certain antibiotics andor antimicrobials a A streak is done on a MuellerHinton medium b Paper disks with specific concentrations of antibiotic andor antimicrobial are deposited on surface c After 18 hours of incubation the zones of inhibition diameters are measured d The measurements are compared to the KirbyB auer chart 37 Understand how you would measure a zone of inhibition what a zone of inhibition is and how you would determine if a bacterium is R I or S to an antimicrobial agent a Zone of inhibition a zone around a disk where no growth occurs i The diameters of the zones are measured and compared to the chart ii A culture is R resistant if it falls in the smallest diameter category because resistance to the disk s agent would mean a smaller zone of inhibition iii A culture is I intermediate if the diameter falls in the middle range iv A culture is S sensitive if the diameter falls in the largest range because the bacteria isn t resistant it will be killedinhibited by the disk s agent causing a large zone of inhibition 38 Describe the effects of alcohol on bacterial growth a Alcohol is an antiseptic used to treat skin before inoculations or drawing of blood b Alcohol functions to denature proteins DNA and dissolves the lipid membrane The lab midterm exam will include the following types of questions multiple choice truefalse matching short answer llin the blank with word bank and practicaltype questions in which you will have to look at something such as a slide plate tube etc and answer a question about it Micro 3051 Final Lab Exam Study Guide 1 Describe how UV light is germicidal its effect on DNA and the wavelength at which UV light is the most lethal When DNA absorbs UV light it causes the formation of pyrimidine dimers These form when a covalent bond is formed between two adjacent thymine or cytosine molecules in a DNA stand Dimers essentially cause the DNA molecule to become deformed so that the DNA polymerase cannot replicate DAN strands past the site of dimer formation nor can genes past this point be transcribed Wavelength UV light is most lethal 260 nm 2 Describe how we examined the germicidal effects of UV light on S aureus and B subtilis One half of each plate will be shielded from the radiation to provide a control comparison Bacillus megaterium an endospore former and Staphylococcus aureus a nonendospore former will be used to provide a comparison of the relative resistance of vegetative cells and endospores 3 Be able to define the following terms a Antiseptic substances such as alcohol or betadine that inhibit microbial growth or kill microorganisms and are gentle enough to be applied to living tissue Disinfectant chemical agents that are applied to inanimate objects such as oors walls and tabletops to kill microorganisms harsher than antiseptics and damage living tissue Steriliant Destroys all microbial life including endospores Sanitizer Agents that reduce microbial numbers to a safe level but do not completely eliminate all microbes used in food industry Bacteriostatic agents that inhibit the growth of bacterial cells but do not kill them Bacteriocidal agents that kill bacterial cells 4 Explain the Filter Paper Disk Method of evaluating the relative effectiveness of various disinfectants andor antiseptics In this procedure a plate of suitable medium is streaked with the test organism Filter paper disks the same size as antibiotic disks are impregnated with the agent by dipping the disk in a germicide or disinfectant and placing the disk on an inoculated plate The plate is incubated for 48 hours The agent will diffuse from the disk into the agar forming a concentration gradient If the substance is inhibitory a clear zone of inhibition will surround the disk where no growth has occurred The size of the zone can be used to quantitatively compare one agent s effectiveness against other chemical substances 5 Distinguish between antibiotics and antimicrobials By definition antimicrobials are compounds that kill or inhibit microorganisms Antibiotics are antimicrobials usually of lower molecular weight produced by microorganisms that inhibit of kill other microorganisms Many times antibiotics are chemically altered to make them more effective in their mode of action 6 Explain the KirbyBauer Method of evaluating the sensitivity of bacteria to certain antibiotics andor antimicrobials This is a standardized test procedure that is reliable relatively simple and yields results in as short a time as possible It is performed by uniformly streaking a standardized inoculum of the test organism on MuellerHinton medium and then paper disks containing specific concentrations of an antimicrobial or antibiotic are deposited on the agar surface The antimicrobial diffuses out from the disk into the agar forming a concentration gradient If the agent inhibits or kills the test organism there will be a zone around the disk where no growth occurs 7 Understand how you would measure a zone of inhibition what a zone of inhibition is and how you would determine if a bacterium is R I or S to an antimicrobial agent After incubation measure the zone diameter with a metric ruler to the nearest whole millimeter The zone of complete inhibition is determined without magnification To determine which antibiotics your organism is sensitive to S or resistant to R or intermediate 1 consult the table It s only going to be one of the three which one is determined by how large the zone of inhibition is 8 Describe the effects of alcohol on bacterial growth Alcohol destroys the bacterial cell wall and stops the cell from behaving properly thus killing it Alcohol kills most bacteria and viruses 9 Explain the basis of the Reductase Test and how it can be used to distinguish between high and low quality milk Milk that contains large numbers of actively growing bacteria will have a lowered oxidationreduction potential due to the exhaustion of dissolved oxygen by microorganisms This lowered oxidationreduction potential can be measured by the addition of the dye methylene blue to a milk sample The test is based on the change in color of methylene blue from blue to clear when the dye is reduced High quality milk methylene blue is not reduced within 6 hours low quality milk methylene blue is reduced within 2 hours 10 Know the meaning and significance of MBRT 11 12 13 14 15 16 0 The time is takes for the methylene blue to become colorless is the methylene blue reduction time MBRT The shorter the MBRT the lower the quality of the milk An MBRT of 6 hours is very good whereas an MBRT of 30 minutes is of very poor quality Understand the role that microbes have in food production and in food spoilage o The presence of microorganisms in food does not necessarily indicate that the food is spoiled or that it has the potential to cause disease Some foods can have high counts because microorganisms are used in their production like fermentation Postproduction treatments such as pasteurization or smoking will significantly reduce the numbers of bacteria present All of page 175 Understand how to conduct a Standard Plate Count including determining a dilution scheme to determine bacterial numbers in a liquid of solid food sample 0 20 g of food blended in 180 ml of water for 110 ratio 1 ml of that is added to 99 ml of water for 11000 ratio 01 ml of original is added to plate for a dilution of 1100 1 ml of sample 2 is added to plate for dilution of 11000 01 ml of sample 2 is added to plate for dilution of 1 10000 Be able to calculate the number of bacteria in a sample based on the number of CFUs on various plates Know what constitutes a countable plate 0 CFUml of bacteria in plate X dilution 110quot eX 50 X 1 50x 103 103 50000 373 for 50 colonies on plate diluted 3 times 0 Countable plate 30300 colonies Explain how the grape juice fermentation ask was setup and know what observations would indicate fermentation had occurred 0 100 ml of grape juice is inoculated with 3 ml of yeast culture Mouth of ask is sealed with rubber balloon before incubation Lead acetate teststrip is taped to inside of ask neck Let bottle sit at room temp for 25 days The balloon should have swilled up and the gas should produce an odor and the color of test strip should have changed showing that fermentation had occurred Define the process of ammonification 0 Decomposition with production of ammonia or ammonium compounds especially by the action of bacteria on nitrogenous organic matter It is also the release of ammonia from biological molecules such as proteins nucleic acids and nitrogenous wastes Describe how to test for the presence of ammonia in soil samples 0 Deposit a drop of Nessler s reagent into two separate depressions of a spot plate add a loopful of the inoculated peptone broth to one depression and a loopful from the sterile uninoculated tube in the other If color is faint yellow there is a small amount of ammonia deep yellow there is more ammonia brown precipitate there is large amount of ammonia 17 Describe coliforms and how they are good indicators of fecal contamination in water 0 Coliforms are gramnegative facultative anaerobic nonendospore forming rods that ferment lactose to produce acid and gas in 48 hours at 35 C 0 E coli is a common fecal contaminate in water that has the same properties as a coliform 18 Explain how the presumptive and confirmed tests are conducted and how they are used to determine the presence of coliforms in a water sample Know the media used for each and what a positive test looks like 0 In the presumptive test 15 tubes of lactose broth are inoculated with measured amounts of water to see if the water contains any lactosefermenting bacteria that produce gas If after incubation gas is seen in any of the lactose broths it is presumed that coliforms are present in the water sample This test is also used to determine the most probable number MPN of coliforms present per 100 ml of water 0 In the confirmed test plates of Levine EMB agar and Endo agar are inoculated from positive gasproducing tubes to see if the organisms that are producing the gas are gramnegative another coliform characteristic On EMB agar coliforms produce small colonies with dark centers On Endo agar coliforms produce reddish colonies The presence of coliformlike colonies confirms the presence of a lactosefermenting gramnegative bacterium 19 Know how to determine the MPN of a water sample MPN100 m1 number of positive tubes x 100 ml sample in negative tubes x ml sample in all tubes 20 Describe the distinguishing characteristics of bacteriophages o Bacteriophages are viruses that infect bacterial cells 0 Bacteriophage quotbacterial eater 0 These phages are obligate intracellular parasites meaning they must invade a host cell in order to replicate and reproduce o Viruses lack their own necessary components so they depend on hosts 0 Viruses are made up of single kind of nucleic acid and is enclosed by protein coat capsid o Viruses exhibit specificity for their hosts 0 T4 phages consist of o Nucleocapsid nucleic acid and protein capsid o Sheath hollow tube that is contractile and made out of proteins 0 Base plate that has tail fibers and spikes attached 0 A single virus virion 21 Explain the procedure for determining a bacteriophage titer o This is accomplished by making a serial dilution of bacteriophage stock culture The dilutions of bacteriophage are then mixed with a soft agar medium and this mixture is layered on top of an agar plate The bacteria grow as tiny colonies within the soft agar layer These colonies appear as a translucent lawn of growth covering the surface of the plate The phages form clear patches in the bacterial lawn called plaques The plaques form because the phages have bust open the bacteria at that site on the plate By counting the number of plaques and multiplying by the serial dilution factor one can determine the titer which is the number of phage particles in the original phage culture 22 Know how to calculate the number of phage particles in a suspension by counting the number of PFUs on plates 0 PFUs can be counted to determine the number of viral particles in suspension 0 A Plaque Forming Unit is a clear zone causes by the lysis of a cell because of a phage infection on the cell 23 Describe the two mutant isolation procedures we used and know what type of mutants we were trying to isolate p 209 0 We used replica plating to isolate the mutant E coli 1 we spread the E coli broth over nutrient agar with a sterile bent glass rod 2 after incubation colonies are picked up with velveteen colony carrier 3 nutrient agar is inoculated by lightly pressing the carrier onto it 4 Streptomycin agar is inoculated with same carrier directly after the nutrient agar 24 Know the purpose of the gradient plate and the three plates used in the replica plating procedure 0 The gradient plate using a plate with and without antibiotic to prove that mutations to streptomycin occur spontaneously in a population of cells on any plate 0 Three plate types 0 Petri plate of culture of E coli from previous lab where the growth starts 0 Use velveteen cloth to print growth on Nutrient agar 0 Then onto Streptomycin agar o The velveteen cloth has tiny threats that each act as tiny inoculating loops 25 Describe the general characteristics of Staphylococcus aureus as part of the microflora of the human body and its significance in disease Nonmotile nonspore forming and able to grow in high salt concentrations Found in nasal membranes hair follicles skin and the perineum in healthy individuals Can cause skin infections wound infections bone tissue infections scalded skin syndrome toxic shock syndrome and food poisoning S aureus is has coagulase production so it can cause serum to form a clot to form around the staphylococcal infection to protect it from host defenses 26 Describe how we isolated S aureus from our noses and how we can identify it using Blood Agar MSA and CHROMagarTM Isolated S aureus by swabbing noses and growing culture on plates It can be identified in blood agar because S aureus produces hemolysin alpha toxin that degrades DNA so it causes a clear zone on blood agar S aureus ferments mannitol to produce acid so when grown on MSA plate the pH indicator within the media indicates S aureus because it changes from red to yellow meaning acid is being produced 27 Define transformation Transformation occurs when a bacterial cell receives small amounts of DNA from the environment 28 Know the four basic steps to isolate DNA from bacterial cells PWP Centrifuge bacterial cells to concentrate them Use detergent to break down the cell wall and cell membrane so that the DNA is released Use heat and a protease to denature and digest proteins within the cell Use cold alcohol to precipitate the DNA from the solution allowing it to be spooled onto a glass or metal rod 29 Describe the heat shock method we used to artificially transform E coli with pGLO We used a heat bath Cooled with ice bath 30 Know the selectable characteristics of pGLO that allowed us to select for transformants pGLO is a plasmid that contains an ori the bla gene and the gene that codes for production of green fluorescent protein GFP 31 Know the role of araC in pGLO and why arabinose was added to one of the plates araC role 0 regulates the arabinose promotor I If arabinose is not present araC prevents RNA polymerase from binding to the arabinose promotor o In the pGLO plasmid araC is used to regulate transcription of the GFP gene 0 pGLO role 0 it s a vector used to introduce the GFP gene into E coli through transformation the vector is taken into e coli from the environment with help of competent cells which function to uptake as much DNA as possible 32 Be able to predict the outcomes of a hypothetical transformation o If transformation takes place hypothetically the broth will grow on the selective antibiotic agent medium proving that transformation has taken place because resistance allowed the cells to grow on the antibiotic medium 33 Describe the effects of lysozyme on bacterial cells 0 Lysozymes are enzymes that damage bacterial cell walls because they cause hydrolysis of 14 beta linkages between amino sugar molecules in peptidoglycan o Lysozymes are found in secretions such as tears saliva human milk and mucus 0 Also found in egg white 0 Gram negative bacteria are usually more resistant to lysozyme than gram positive because the outer layer in gram negative prevents the lysozyme from reaching the peptidoglycan 0 But S aureus is resistant although it is gram positive because of its presence of teichoic acids in their cell wall 34 Be able to define and calculate o Morbidity illness due to a specific disease no cases per period susceptible population size at midpoint of period 0 Mortality number of deaths Within a specified period among people having a Morbidity particular disease no disease related deaths per period M t it or a l y no people with the disease 0 Incidence compares the number of new cases of a disease during a specified period to the size of the susceptible population during that period no new cases x susceptible population Size at midpoint of period Incidence 35 Define a Communicable infectious diseases that are transmissible to other persons b Epidemiology the study of how when Where What and who are involved in the spread and distribution of disease in human populations c Endemic an infection disease that exhibits a steady frequency over a long period of time in a particular region d Epidemic if the number of newly reported cases in a given period of time in a specific area is excessive e Pandemic if the disease spreads to one of more continents 36De ne 0 antigen 0 quotAntibody generator foreign substance which serves as a target for receptors of an adaptive immune response 0 The immune response antibodies secreted to fight off antigens o Antiserum 0 Blood serum containing polyclonal antibodies and is used to pass on passive immunity to many diseases 0 serological typing 0 A technique based upon antibodyantigen reactions by which pathogenic bacteria are identified It is particularly useful for strains of pathogens difficult to differentiate by morphological methods 0 The scientific study of serum and other bodily fluids In practice the term usually refers to the diagnostic identification of antibodies in the serum1 Such antibodies are typically formed in response to an infection against a given microorganism2 against other foreign proteins in response for example to a mismatched blood transfusion or to one39s own proteins in instances of autoimmune disease 0 Serological tests may be performed for diagnostic purposes when an infection is suspected in rheumatic illnesses and in many other situations such as checking an individual39s blood type1 o Agglutination o Occurs if an antigen is mixed with its corresponding antibody called isoagglutinin This is commonly used term in blood grouping This occurs in biology in three main examples The clumping of cells such as bacteria or red blood cells in the presence of an antibody or complement 37 Explain how we used serological typing to identify S aureus Serologlcal typing of Staphylococcus aureus 1 Mix the latex reagent by shaking expel any latex from the dropper for complete mixing 2 Dispense 1 drop of Test Latex contains Staph antibody onto one of the circles on the reaction card and add 1 drop of Control Latex onto another circle 3 Using a loop pick up and smear 5 suspect colonies either a suspect Staph from your body OR a known Staph aureus supplied from the lab onto the Test Latexcontaining circle and mix this into the Test Latex reagent Spread to cover the circle Be sure you have a homogenous bacterial suspension Clumps will make the reaction difficult to identity and possibly give a false reaction 4 Repeat step 3 for the Control Latex using the same bacterial culture 5 Pick up and hand rock the card for up to 20 sec and observe for agglutination under normal lighting conditions 6 Rock the slide back and forth for no longer than one minute and look tor blue agglutination Clunping is a positive reaction for the identi cation of S aureus Id 38 Distinguish between a positive and negative test for an agglutination reaction 0 Positive control and negative control reagents are used to test two complete colonies o In those two circles and another one drop of latex reagent is added 0 Negative reaction smooth cloudy suspension free of particles 0 Positive reaction clumps of aggregated yellow latex beads with cloudy background
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