this is my study guide. can you please read through it and answer any of the questions and explain whatever it says to explain….I have 2 exams and need all the study info I can get….It came to me in the form of a power point so the pictures when I copied and pasted , it didn’t come out.
SUMMER 2020GEN. MICRO MIDTERM STUDY GUIDE/REVIEW (LABS 1 – 7)
MICROSCOPE USE & DEFINITIONS•
Know the definitions of parfocal, resolution, numerical aperture etc.
Calculate total magnificationOcular lens – 10XRemember importance of field of view and working distance in quickly resolving a specimen
Know the parts of the microscope
CELL MORPHOLOGICAL STAININGGram stain – crystal violet (1min) – rinse – iodine (1 min) – rinse – acetone/alcohol decolorization(10-15 secs) – rinse immediately – counterstain safranin (1min)•Know staining procedures for capsule or negative stain, and endospore stains•Know the theory/mechanism behind each stain•Significance of the different organisms and structures being stained
CELL MORPHOLGY•Know the difference between cell and colony morphology•Know at least two bacteria that stain gram+/-ve and their cell morphologies
ASEPTIC TECHNIQUES•Definition of an aseptic technique•Examples of aseptic techniques and tools used•Aseptic techniques – do’s and don’ts
ISOLATION TECHNIQUES•Quadrant streak – use up as much of agar as possible, flame the loop in between streaks, use four quadrants•Be able to describe the quadrant streak for isolation•Know procedures for spread plate method and pour plate method and difference in tools used
CONTROL OF MICROBIAL GROWTHpH – acidophiles, neutrophiles, alkalophilesTemperature – psychrophile, psychrotrophic, mesophile, thermophiles etc. – give one or two examples and be able to identify them on media or based on range of growthWhat are cardinal temperatures?Aerotolerance – capnophiles, obligate anaerobe, facultative anaerobe, obligate aerobe etc
CONTROL OF MICROBIAL GROWTH•Effect of UV – thymine dimers – inhibit replication•UV-C most detrimental to bacteria•Disadvantages of UV?
GERMICIDES & ANTIBIOTICS•Difference between Antiseptics and Disinfectants?Disinfectants – designed for use on surfacesAntiseptics – designed for use on living tissue•What is the zone of inhibition?•The smaller the zone of inhibition the more resistant the organism to the antimicrobial or the least effective the antimicrobial is to the organism•Effect of lysozyme on Gram –ve vs Gram +ve bacteria•Purpose of the Use-dilution test, controls used?
CLOSED SYSTEM GROWTH – LAB REPORT•Know the four distinct growth phases and what happens in each phase•Define and calculate k and g•Know procedures for use of the spectrophotometer •Direct vs indirect methods of determining cell density/growth for bacteria – eg. Hemocytometer vs viable plate counts vs turbidity measurementsRemember Calculations!!
BACTERIAL GROWTH CURVEtA1A2Log phaseLag phaseStationary phaseDeath phase
SERIAL DILUTIONS•Be able to carry out a serial dilution using micropipettes•Perform 1/10 or 1/100 or 1/1000 dilutions with fixed total volumes•1/10 – 100ul in 900ul of water•1/100 – 10ul in 990ul of water•1/1000 – 1ul in 999ul of water•Calculate dilution factor given different sample volumes and diluent
PLAQUE ASSAY•What are bacteriophages?•Know the different stages of the lytic cycle•Remember only plates with PFU between 30-300 are statistically valid for calculations, same for CFU (bacteria)•Calculate original viral titer from plaques on prepared media plates•Original viral titer = PFU/sample volume x dilution
MEASURING THE EFFECT OF ANTIMICROBIALSKirby Bauer/ Disk Diffusion test•Size of the zone of inhibition reveals whether a bacterial species is resistant or susceptible to the antibiotic.•Larger zone – more effective antimicrobial/weak resistance of bacteria to antimicrobial•Smaller zone – less effective antimicrobial/strong resistance of bacteria to antimicrobial•What is MIC – minimum inhibitory concentration of an antibiotic – least concentration of antibiotic required to prevent visible growth (zone of inhibition)
DNA EXTRACTIONKnow the different steps of DNA extraction and examples of chemicals usedCell lysis – Protein denaturation – DNA precipitation and purificationBe able to calculate DNA concentration and purity given the absorbance values at 260nm and 280 nmPurity range – 1.65-2.0
GEL ELECTROPHORESIS•Understand the concept of gel electrophoresis•Separation of DNA fragments based on size using an electrical charge – negatively charged cathode and positively charged anode•Which direction does DNA run? •Purpose of the ethidium bromide, DNA ladder and loading dye?
POLYMERASE CHAIN REACTION (PCR)•Know the three stages of PCR and what is happening at each step, enzymes, temperatures and reagents involved•Denaturation – Annealing – Extension•Be able to calculate number of DNA fragments amplified after a definite number of cycles•Number of fragments = orig # x 2n . n=number of cycles
RESTRICTION ENZYME DIGESTION•What is a restriction enzyme?•What are palindromic sequences?•What purpose restriction enzymes play in bacterial organisms? •Be able to determine the number and size of fragments obtained from different cuts of a restriction enzyme on a circular or linear plasmid.
BIOINFORMATICS•Define bioinformatics•How is the NCBI database used to search for sequences – DNA/RNA/protein•What is the significance of the e value, % identity?•What other information can you obtain from a query search using NCBI BLAST