Microbiology Lab-1

  1. what is needed to see microbes?
    compound microscope (2 lens between eye and object)
  2. microscope used in lab?
    brightfield compound microscope
  3. types of objective lenses on microscope?
    • scanning (4x)
    • low power (10x)
    • high dry (40x)
    • oil immersion (100x)
  4. determining total magnification?
    multiply magnification of ocular (10x) by magnification of objective lens.
  5. which lens has highest magnification?
    oil immersion
  6. define resolution/resolving power?
    ability of lens to reveal fine detail or two point distincly separated
  7. when is resolving power the greatest?
    greatest when 2 objects are seen as distinct even though they are close together. the smaller the distance the better resolving power.
  8. when is light refracted and why?
    refracted when emerged from slide b/c of change in density as light passes from glass to air
  9. oil immersion and light refraction?
    light rays continue without refraction b/c immersion oil has same refractive index ax glass. as light rays pass through lens they are bent to converge at focal point where image is formed.
  10. what is spherical aberration?
    periphery may be fuzzy when you bring center of microscope field into focus d/t curvature of lens which results in multiple focal points.
  11. rules for microscope use?
    • carry microscope with both hands
    • observe slide with both eyes open
    • focus slowly/carefully
    • adjust light. more needed with higher resolution
    • focus under low power first
    • focus slide under 40x before going to oil immersion
    • keep stage clean
    • use only lens paper for cleaning
  12. occular lens?
    remagnifies image formed by objective lens. (eyepeice)
  13. body tube
    transmits image from objective lens to ocular lens
  14. objective lens
    primary lens to magnify specimen
  15. stage
    holds microscope slide in position
  16. condensor
    focus light through specimen
  17. diaphragm
    controls amount of light entering condensor
  18. illuminator
    light source
  19. to make sure color/shades are accurate what can you adjust on microscope?
    make sure numeric measurement on each lens is same as iris diaphragm (understage)
  20. what is brownian movement and what is the cause?
    particles and microorganisms all vibrate at same rate and maintain relative positions d/t molecules in liquid striking an object, causing it to shake or bounce.
  21. what is true motility?
    motile organisms move from one place to another in a directed manner, sometimes as if to spin and roll.
  22. procedure for wet mount?
    • stir contents of container
    • using pipette transfer small drop to slide
    • lay coverslip over drop
    • place onto sgtage and observe with low power (10x)
    • observe slide under high power lens (40x). bacteria should now be visible
  23. purpose of addding a stain to specimen?
    slow down microbes so easily seen
  24. who first described morphology of bacteria (shape) and what were the shapes?
    Anton Van Leeuwenhoek in 1600 described rod, coccus, spiral
  25. three patterns of coccus/spherical?
    • diplococci- pairs
    • streptococci- row of 4+
    • staphylococci- irregular clumps resemble grapes
  26. describe rod shaped bacteria
    aka bacillus , occassionaly found in diplobacilli and streptobacilli but not cocci.
  27. coiled/spiral shaped bacteria
    • spirochetes- flexible, waving forms with several coils
    • spirilla- rigid with one or several curves. if they are short without complete coils then called vibrios.
  28. pleomorphism?
    variations in shape and size of bacteria
  29. ways to handle microbes safetly using aseptic technique
    • read procedure prior
    • organize lab space
    • dont rush
    • use test tube rack
    • hold loop secure in dominant hand
    • dont hold test tube by cap
    • sterilize loop using bunsen burner until red at 45 degree
    • pass tops of tube through flame 1-2 x before recap
    • incubate petri dish agar side up and lid side down
    • hold all lids/caps in hand not on table
    • clean space before and after lab
  30. what is agar?
    • extract from marine red algae
    • remains solid during microbial growth
    • few microbes degrade it
    • liquifies at 95-100 celcius remains liquid until cool at 45 celcius
  31. why use petri plates for culture?
    provide lare surface area for exam of colonies.
  32. what is a colony?
    visible growth on agar surface that started out as 1 single bacterium and replicated self many times.
  33. what is colony forming unit?
    orginal bacteria that turn into colony during incubation
  34. steps to isolate bacteria by streak plate ?
    • first pass with culture sample
    • second pass with sterile loop
    • third pass with sterile loop
    • leave space between first and third pass
  35. pure culture?
    colony not touching upon any other colony
  36. how do you achieve isolation for pure culture?
    streak plate used as dilution technique.
  37. define streak plate
    dilute out organism in mixed culute until cells are seperated from each other enough that single bactyeria become isolated in discrete location on agar surface. plate then incubated and cells undergo division resultint in visible growth upon surface of agar.
  38. what is chromagen? what happens when stain added?
    colored molecules positively charged and attracted to negative charged bacterial cell. this results in cell becomeing colored against a white background.
  39. what colors used in simple stain?
    • methylene blue
    • crystal violet
    • safranin (red)
  40. what is heat fixing and why use it?
    kills bacteria adn ensures that smear will stay attached to slide. it distorts specimen to small extent so dont overheat.
  41. steps to smear and simple stain
    • use loop, place loopful sterile water on slide
    • use same loop, transfer small amt bacteria from single colony into water
    • let dry completely (or will wash off)
    • heat fix slide (pass thru flame 2-3 x)
    • add few drop methylene blue/crystal violet/safranin to slide
    • leave for 1 min
    • use deionize water to rinse off slide
    • coverslip to slide
    • observe on 10x then 40x
  42. what is negative stain
    • stain background NOT bacteria
    • bacteria appears clear against dark background
  43. in negative stain, why isnt bacteria stained?
    ionic repulsion- bacteria and acid stain both have negative charge
  44. when is negative stain used?
    when other stain techniques dont show cell size/morphology.
  45. why isnt heat/strong chemical used in negative stain?
    bacteria less distorted so visualize easier
  46. steps to negative stain
    • small drop of nigrosin at end of slide (dont spread let dry)
    • flame loop and add small loop of culture onto drop of nigrosin
    • reflame loop
    • using edge of another slide, spread drop to produce smear varying from dark at one end to gray
    • let smear air dry - DO NOT HEAT FIX
  47. why use gram stain?
    identify and classify bacteria
  48. what is gram stain
    differntial stain allowing classification of bacteria as either positive or negative
  49. history of gram stain
    1884 Hans Christian Gram discovered when he attempted to stain cells and found some lost color when excess stain was washed off
  50. steps to gram stain
    • pick up loopful of sample using sterile loop
    • smear sample all around slide and let air dry (dont blow on it)
    • flame loop again
    • heat fix slide
    • apply primary stain to stain all bacteria purple (crystal violet- alkaline dye) 60 sec
    • wash off with deionize water
    • apply mordant (iodine) 60 seconds
    • wash off with deionize water
    • apply decolorizing agent (alcohol) only small amt until no large amt of purple wash out
    • IMMEDIATE wash with deionize water
    • apply secondary stain/counterstain (safranin) 60 sec
    • wash with deionize water and blot dry
  51. in gram stain, what is used as primary stain and why is it used?
    crystal violet - stains all bacteria purple (alkaline dye)
  52. in gram stain, what is mordant and why use it?
    mordant- iodine (combines with crystal violet in cell to from crystal-iodine complex)
  53. in gram stain, what is decolorizing agent and why use it?
    decolorizing agent- alcohol (primary stain washed out/decolorized of some bacteria while others remain purple)
  54. in gram stain, what is secondary stain/counter stain and why use it?
    secondary stain/counter stain- safranin (alkaline dye stains decolorized bacteria red)
  55. in gram stain, which bacteria are red and which are purple?
    • red= gram negative
    • purple= gram positive
  56. in gram stain, why do some bacteria stain different?
    • chemical and physical properties in cell walls.
    • crystal violet picked up by all bacteria, iodine react with crystal violet in cytoplasm to form CV-1
    • gram negative= decolorize agent dissolves outer lipopolysaccharide layer and CV-1 washes out through thin layer of peptidoglycan
  57. why do you want loop to cool before adding to bacteria? what happens if too hot?
    cool loop for 30 sec so bacteria pick up wont be killed. if too hot will splatter medium and move bacteria into air
  58. what is acid fast stain and who discovered?
    • differential stain discovered by Paul Ehrlich 1882
    • TB retain primary stain even after washing with acid alcohol.
    • used as diagnostic tool for mycobacterium /nocardia as they contain pathogenic species
  59. what does alcohol due to bacteria? which bacteria are not affected/acid fast?
    • most bacteria decolorize by alcohol.
    • acid fast: mycobactrium, nocardia, goronia, tsukamurella
  60. how acid fast works?
    result of relative solubility of carolfuchsin and impermeability of cell wall.
  61. in acid fast stain, describe fuchsin and carbolic acid? relation to lipids and alcohol
    fuchsin more soluble in carbolic acid (phenol) then in water and carbolic acid becomes soluble easier in lipid then alcohol. so carbolfuchsin has higher affinity for lipid then alcohol and will remain with cell wall when washed with alcohol.
  62. steps to acid fast stain
    • heat fix a smear
    • cover smear with carbolfuchsin (leave for 5 min)
    • wash slide with deionize water
    • without drying, wash smear with alcohol until no red color runs off slide
    • wash smear with deionize water
    • counterstain with brilliant green(1 min)
    • wash with deionize water and blot dry
  63. consistancy of acid fast bacteria?
  64. capsule stain and why use it?
    negative stain that stains negative space around bacteria for visual of capsule or slime layer
  65. what chemicals used for capsule stain and what they do?
    • negative charged congo red chemical stains negative space
    • alkaline Manevals reagent chemical stain bacterial cells
  66. specimen used for capsule stain? (in lab)
    klebsiella pneumoniae- mild pathogenic
  67. steps to capsule stain?
    • small drop of congo red at end of slide- let air dry/and dont spread
    • flame loop and add small amt of culture to drop of congo red
    • reflame loop
    • using end of another slide, spread drop to produce smear
    • let air dry- DO NOT HEAT FIX
    • after dry, cover with maneval reagent (1 min)
    • wash with deionize water and blot dry
  68. define endospore
    stress resistant form of bacterium allows for survival in poor environment conditions
  69. what endospore is resistant to and why resistant?
    resistant to heat and chemical d/t production of outer layer of tough keratin
  70. what we use to create endospore?
    steam to stress bacteria into forming endospores
  71. endospore color?
    visible as small green structure
  72. endospore location
    • central- middle of cell
    • terminal- end of cell
    • subterminal- between middle and end of cell
  73. endospore shapes?
    • spherical
    • elliptical
  74. steps to endospore stain
    • prepare heat fix smear
    • put paper towel on top of smear and place over steming beaker under fume hood
    • soak paper towel with malachite green (leave 7-10 min)
    • paper towel must remain soaked and add more green prn
    • remove slide, place paper towel in trash
    • rinse with deionize water
    • counterstain with safranin (1 min)
    • rinse with deionize water and blot dry
  75. why endospores form?
    when nutrients deplete, gram positive bacteria form these resting cells internal to bacterial cell membrane
  76. what is sporulation and what steps?
    • formation of endospore:
    • spore septum isolate new replicated DNA and small part of cytoplasm
    • plasma membrane surround DNA
    • spore septum surround isolated portion to form forespore
    • peptidoglycan layer form between membranes
    • spore coat forms
    • endospore is freed from cell
Card Set
Microbiology Lab-1
handwashing, microscope, microbes, smears, stains