BIO 211 lab - quiz #5 notes PDF

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lab portion of microbiology. notes for quiz 5 content ...

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1 Quiz 5: Exercises 18, 19, 27, 29AB, 30 continued, and BSL2 Safety Exercise 18—Gram Positive Cocci: Staphylococci  Describe the Gram reaction and recognize the arrangement of staphylococci cells on a Gram stain slide gram positive (purple) cocci (spheres), divide in various directions forming grape like clusters

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List the types of media that the staphylococci grow on and describe their appearance 1. M-staphylococcus broth

2. Mannitol salt agar plates

3. SM 110 plates

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4. sheep blood agar plates (BAP)

5. Coagulase plasma (rabbit) test tubes

6. DNA agar plate

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Perform and describe the catalase, coagulase, and DNAse tests Catalase = used to differentiate genera, in this lab used to determine if genus Staphylococcus or Streptococcus Staph. Catalase positive Strep. Catalase negative

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Coagulase = enzyme that binds plasma fibrinogen causing plasma to clot, can distinguish S. aureus from other staph., S. aureus produces the coagulase enzyme therefore it will clot the rabbit plasma S. aureus positive (plasma clotted) S. epi negative (plasma liquid)

DNAse test = aids in further identification of S. aureus which produces Depxyribonuclease (enzyme that can alter DNA), DNAse agar with toluidine blue: when S. aureus inoculated on this the nucleus cleaves the DNA which changes color of agar from blue to pink, S. epi can degrade the dye without cleaving the DNA leaving a clear zone S. aureus positive (pink halo) S. epi negative (clear halo)

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Define nosocomial infections and give some examples of some Infections acquired while in the hospital Ex: UTI’s, surgical site infections, bloodstream infections, pneumonia List some of the normal flora found on skin and in the nasal area S. epi skin and nose S. aureus skin and nose

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Corynebacteria skin and nose Define carrier, opportunistic pathogen and pathogen Carrier = hosts (humans) that carry pathogenic organisms that produce no symptoms, but are capable of transmission Opportunistic pathogen = an organism that exists harmlessly as part of the normal human body environment and does not become a health threat until the body’s immune system fails Pathogen = aka infectious agent is a biological agent that causes disease or illness to its host Describe the types of hemolysis Alpha – incomplete/partial lysis of the RBCs Greenish, cloudy zone, darkening of the agar around the colony Beta – complete lysis of the red cells Transparent (clear) zone of the agar around colony Gamma – no lysis of the red cells No change in the blood agar around the colony

Identify colonies of staphylococci that are beta hemolytic and nonhemolytic and give examples of each Beta hemolytic – S. aureus Non-hemolytic – S. epi  Compare and contrast the term pathogen and opportunistic pathogen using the staphylococci as examples Harmful strep that cause disease are pathogens and opportunistic pathogens are those like normal flora that are helpful to the body and don’t cause sickness Exercise 19—Gram Positive Cocci: Streptococci  Describe the Gram reaction and recognize the arrangement of streptococci cells on a Gram stained slide Gram positive (purple) cocci (spheres), division occurs in one linear direction forming chain of round cells

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Perform the proper technique of obtaining a throat culture on yourself Sterile cotton swab, use mirror to swab self, swab area on both sides of the uvula (tonsil area)

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List some of the normal flora found in the throat and mouth Mouth : S. epi, S. mitis, S. aureus Recognize positive and negative results for catalase, 6.5% NaCl and bile esculin tests Catalase Positive = bubbles Negative = no change/bubbles

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6.5% NaCl Positive = (organism has growth in the salt broth) cloudy medium Negative=(organism has no growth in salt)no change in medium, clear

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Bile esculin tests Positive = (hydrolyzes esculin in the presence of bile) turns more than half the medium dark Negative = (does not hydrolyze esculin in the presence of bile) no dark brown is formed

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o know the purpose of each test in the identification of streptococcus/bacteria looked for in each test Bacitracin susceptibility – (A disc) used for the presumptive identification of group A beta hemolytic strep. Optochin susceptibility – (P disc) differentiates between S. pneumonia and other alpha hemolytic strep Bile esculin hydrolysis – differentiates b/t Group D strep and other strep, E. faecalis (a Group D strep) can grow in presence of 40% bile and can hydrolyze esculin to esculetin 6.5% salt tolerance – another test for E. faecalis, Group D enterococci are able to grow in 6.5% NaCl broth Immuno serological identification – help detect antibodies Recognize the types of hemolysis on a blood agar plate

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Name the types of hemolysis and give examples of streptococci for each Alpha – S. pneumoniae (and group of oral strep) Beta – S. pyogenes (Group A) Gamma – E. faecalis (Group D) Perform the identification tests using Bacitracin, SXT and Optochin discs Bacitracin – antibiotic interfering w/ the synthesis of peptidoglycan (major part of bacterial cell walls), inoculum transferred using cotton swab to plate then disk containing bacitracin is places on plate surface and incubated, growth up to the disc indicates the bacteria is resistant to the antibiotic, clear zone around disc is the zone of inhibition which indicates the bacteria is susceptible to the antibiotic SXT - ? use the discs Optochin discs – used to differentiate b/t S. pneumoniae and other alpha hemolytic strep, use P disc

List some diseases that Group A streptococci cause in humans Pneumonia, streptococcal toxic shock syndrome List the different groups of streptococci and give examples of each Group A strep – S. pyogenes Group B strep – S. agalactiae Group D enterococci – E. faecalis List examples of streptococci that cause dental plaque S. mutans Know how agglutination tests help in identifying the beta hemolytic group of streptococci Latex agglutination tests for rapid identification of group A strep directly from throat swabs

8 Exercise 27—Spoilage of Meat  Perform standard plate counts on food samples Instructor blends 10 grams meat with 90 mL buffered water, 4 test tubes, 4 plates (1:1,000 1:10,000 1:100,000 1:1,000,000), transfer 1mL from the meat suspension to tube #1, perform a ten-fold serial dilution by transferring 1mL from each tube to the next all the way through tube 4, plate 0.1mL from tube #1 to plate 1, put on turn table and dip rod in alcohol then flame and let cool then distribute the inoculum on plate, repeat for all 4 tubes, incubate half the plates at room temp and the other half in at 4 degrees Celsius

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List the types of microorganisms that grow at refrigerator temperatures Psychrophiles and some low temp mesophiles o MEMORIZE one organisms that grows at refrigerator temperatures that is pathogenic to humans Listeria Define: o Psychrophile Etremophilic bacteria that are cold loving, optimal temp for growth at about 15 degrees Celsius, range -8-18 degrees Celsius (polar regions) o Psychrotroph Cold tolerant bacteria that have the ability to grow at low temps but have optimal growth at 22 degrees Celsius, range 0 to 30-40 degrees Celsius (soil, plants) o Mesophile Organisms that grow best in moderate temp, neither too hot nor too cold and have an optimum temp of 25-40 degrees Celsius, range 10-48 degrees Celsius (animals, plants, soil) o Thermophile Organisms that thrive at relatively high temps with an optimum temp of 50-60 degrees Celsius, range 40-70 degrees Celsius (hot springs, compost piles)

9 o Hyperthermophile Organisms that thrive at extremely hot temps with an optimum temp of 80-105 degrees Celsius, range 65-113 degrees Celsius (volcanic hot springs , deep sea hyro-thermal vents)  List some examples of each of the growth temperature groups Psychrophiles – genera Pseudomonas Psychrotrophs – M. cryophilus Mesophiles – E. coli Thermophiles – B. stearothermophilus Hyperthermophiles – P. furiosus  Discuss what temperature human pathogens grow at and why 37 degrees Celsius b/c that is human body temp therefore they can grow easily in that temp  List some examples of food borne pathogens E. coli, Salmonella, S. aureus Exercise 29—Microbiology of Milk  Describe raw and pasteurized milk Raw milk = milk from animals that has not been pasteurized to kill harmful bacteria, can carry dangerous bacteria (Salmonella, E. coli) Pasteurized milk = uses process called pasteurization which kills harmful bacteria by heating milk to a specific temp (60 degrees Celsius) for a period of time (20 mins) and holding it at 10 degrees Celsius after pasteurization, kills harmful organism responsible for diseases  Determine the amount of bacteria from a raw and pasteurized milk from a standard plate count Certified pasteurized milk can’t exceed 500 bacteria/mL Certified raw milk can’t exceed 10,000 bacteria/mL Grade A pasteurized milk can’t exceed 20,000 bacteria/mL Grade A raw milk can’t exceed 100,000bacteria/mL Exercise 30—Isolation of an Antibiotic Producer from Soil  Know steps to perform dilution plating using the spread plate technique Ten fold serial dilution, plate tubes 4, 5, and 6 using spread plate technique w/ rod and alcohol

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List some of the bacteria found in soil that produce antibiotics Penicillium S. aureus Describe why the soil was diluted

10 So bacteria will be in suspension in water and will transfer to inoculating loop easier w/ less solid soil chunks...