lab practical

  1. What is dark field microscopy?
    The dark field is when the specimen is lit up and the background is dark
  2. How does drak field work
    The light passes through the specimien and becomes defracted. Light curves around the condenser.
  3. When would you use dark field
    Dark fields would be used on organisms that are hard to stain and when looking at internal structures larger than eukaryotic organisms.
  4. What role do the annular stop and the phase plate and ring play in phase contrast microscopy
    The annular has a circular like cut out and allows the light to pass through the specimens. The phase plate is similar to the annulus but is placed above the objective lenses.
  5. What happens to the phase of diffracted light in comparison to undiffracted light in a phase-contrast microscope
    The phase contrast bends light and makes the organism a little darker than the surrounding light. Giving it a contrast.
  6. whaT IS DARK pahse contrast
    Dark-phase makes the organism darker than the surrounding light.
  7. When would you use dark phase?
    Useful in studying living cells and determining structures like endospores and inclusion bodies.
  8. What was the negative stain that we used in calss?
    The example of negative stain that we used in class is the India ink.
  9. How do you prep a slide from a liquid sample?
    Just a loop full will do. Smear on the slide making sure it is spread out. Let air dry and the heat fix
  10. How do you prep a slide from a "dry" sample?
    Place a tiny drop of water on the slide (usually with the loop) and the smear the sample in the drop and spread around the slide. Let air dry and then heat fix.
  11. what is the general characteristics of negative stains?
    Cells are color less and the background is stained.
  12. why dont you heat fix the cells?
    Since you are spreading the sample and stain, you want the cells to spread out. Thus you dont want them stuck to the slide.
  13. positive stains?
    these are stains that are able to penetrate the cell wall and stain the cells.
  14. What are some basis stains we used in class?
    Simple stain with crystal violet or methylene blue
  15. what is the purpose of the heat fix?
    thius is to adhere the cell to the slide.
  16. What is a differential stain? What is the role of a counterstain in differential stains?
    A differential stain is like gram stain, used to differntiate between two kinds of cells. The counter stain is there to show a contrast between the two types of cells. used to stain cells that didnt hold onto the stain.
  17. What are the steps to the gram stain?
    Heat fix, flood slide with cystal violet for 30s, rinse w/ dh2o 5s, flood with grams iodine 1m, rinse w/ dh2o 5s, decolorise for 5s, rinse w/dh2o 5s, stain with safran 60-80m, and rinse w/ dh2o. Blot dry
  18. What is happening to the gram - cell.
    violet stains the cells, iodine has no effect, outer membrane weakened and wall looses dye from decolorzer, and red dye (Safran) stains the colorless cell.
  19. what is happening to gram + cell.
    Violet stains the cell, iodine traps stain in wall, decolorize doesnt remove stain, and safran is masked by the violet.
  20. How do gram - cells look?
  21. how do gram + cells look?
  22. What is the ideal number of colonies on a plate in order to do a CFU?
  23. What are CFUs?
    each seperate colony represnts a seperate cell, thus the number of colonies should give you the number of cells.
  24. how does turbidity effect the absorbance and help determine the growth?
    light is pased through the sample. The more turbid the sample the more light is refracted and less light can reach the sensor.
  25. why is it reffered to optical desity and not absorbance?
    because it has nothing to do with how much light is being absobed. the more turbid the more light is being refracted.
  26. how to calculate # cells from direct cell count?
    number of cells counted / number of squares times 5000000
  27. Why is heat used for endospore staining?
    It drives the stain into the endospore.
  28. what is the primary and secondary stains for endospore stainsing.
    Malachite Green and Safranin
  29. In the endospore stain what do the vegatative celss and spore look like?
    Vegie cells will be pink and green in the center and the spore will be green.
  30. what is the differenc between selective and differential media?
    Slective media grows only a certain kind of organism while differentail medias allow you to differntiate one organism from another.
  31. Mannitol salt agaris selective why?
    Mannitol salt agar is going to be selective because it has a high salt concentration.
  32. Eosin Methylene Blue Agar is differencial why?
    This is the dark red that we saw. the cells like E.coli fermenting lactise will shoiw up a blue -black color and a metacllic shine.
  33. what is the purpose of the duram tubes and the phenol red?
    The tubes will show gas and the red is to show acid production from fermentation.
  34. What was the purpose of using three differnt sugars in the fermentation tests?
    to see what kind of sugars they can use. Sucrose, glucose, lactose. Simple and complex
  35. What are the three sugars in the TSI slants?
    Glucose, Lactose, and Sucrose. 0.1% G and 1%S majority Lac
  36. What is the purpose of the iron sulkfate in the TSI medium?
    To see if it can produce sulfide. It will turn black.
  37. In a TSI tube what does it mean if the slant has a color change at the bottom, top, both, and/or gas?
    If only the butt, then only glucose was used, slant and butt are yellow then all sugars are used, and if there is gas, then gas was produced.
  38. What is the indicator for the starch hydrolysis test?
    A starch agar plate is used and Gram's iodine is poured onto it. A clearing around the streak will show it there is starch
  39. what is in the starch agar?
    there is beef extract, soluble starch, and agar
  40. Is the starch agar selective or differential?
    It is differntial because iut can support other organisms, but will show the ones that can use starch,
  41. What is the function of lipases? Where would the enzymes likely act?
    It is an enzyme that breaks down triglyerides. It is outside
  42. What type of medium was used for testing for lipase activity?
    Spirti blue agar
  43. What are the four main test that comprise the IMViC set of tests?
    Indol reaction, Methyl red test, Voges-Posaure test, and citration test
  44. Although the individual IMViC tests can be useful in identifying a variety of microbes, members of which specific group of bacteria can be differentiated using the suite of IMViC tests?
  45. What ius the indol test proxy for? what does it suggest?
    That the trytophan has been used up and tryptophanase is present
  46. SIM medium shows an indol test how and what does that mean?
    By having Kovac's regaent driopped onto it. shows that tryophanse is present and will be a red color.
  47. What will happen if you let the SIM test incubate too long?
    All the indol would be metaoblized.
  48. What is the other test that SIMs is used for?
  49. What is the methyl red test meant to distinguish and how can you tell its positive?
    If there is acid present. The top ring will be red and yellow for negative.
  50. What compound is the voges proskauer test detecting adn what does it mean?
    Acetone, and that glucose is being fermented.
  51. how do you verify that the VG worked?
    After 15 mins there will be a red color in the medium
  52. would an organism that was not able to utilize citrate be able to grow on that medium?
    NO, citrate is the mnain carbon source.
  53. How can you tell that Citrate test is positive and what does it mean?
    It will turn blue beacuse pyruvic acid is present.
  54. What is the casein, what does a positive test look like adn what dioes it mean?
    A protein in milk, There will be a clearing around the colony, and it means there is proteasespresent breaking it douwn.
  55. What is gelatin?
    A soluble mixture of polypeptides and amino aicds
  56. What does hydrolysis mean of gelatin?
    Means that the enzyme gelatinase is present and is breaking down the gelatin.
  57. What is used for the catalase test and what does a positive mean?
    3%H2O2 is used and it means that bacteria have enzymes that oprotect them from O2
  58. Haw can you determine a positive vs negative for Dnase test?
    There will be a clearing around the colony.
  59. Is Nitrate testing a dissimilatory or assimilatory reduction ?
  60. What are some possible products of the nitrate reduction test and how do we destinguish them?
    Presence of gas means that the is N2 or NH4 + test. Nirtate reagen A and B added, if a color change then Nitrite was made and used. NO2- You can use zinc powder to verify that there is nitrate in the tube.
  61. What ar5e the three methods of cell counting we used in class?
    Viable cell count, Direct ceel count, and turbidity testing Spectrophotometric.
  62. what are the adv. and disadv. of Viable cell count?
    each colony is a cell and can represent each cell in the sample. For it to be accurate needs to be repeaded multiple times.
  63. What are the adv. and disadv of Direct cell count?
    eay to do and quick. Cells move a lot and hard to count.
  64. What are the adv. and disadv. of the spectrophotometric test?
    Fastest , but is limited to suspensions of at leat 10^7
Card Set
lab practical
Micro lab