Lab Practical 1

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Lab Practical 1
2013-03-14 19:42:19
BI 301

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  1. A ____ consists of one or more species of microorganisms growing in a nutrient growth medium often at specific temp
  2. A culture consisting of ONLY 1 species of microorganism is called a what?
    Pure Culture
  3. The composition of nutrients that all organisms require to grow and survive is called a ___ medium.
  4. A liquid solution in which microbes, especially microbes & protozoa, will grow is known as a ___ ___.
    Nutrient Broth
  5. A broth supplemented with a solidifying agent that produces a relatively solid medium on which bacteria and fungi easily grow is called what?
  6. Agar is a polymer of what extracted from what?
    Galactose extracted from cell walls of red algae
  7. Agar liquifies at ___C and solidifies at ___oC.
    100 and 40
  8. Describe a "circular" form of a colony.
    Circular w/smooth edges like and "O"
  9. What type of microscope utilizes visible light to magnify and resolve objects?
    Compound light microscope
  10. Which staining technique uses 2 contrasting colored stains to allow bacteria to be separated into 1 of 2 grps and also visualize structures such as endospores and capsules?
    Differential Staining
  11. What are the 3 purposes of simple staining?
    • Measure cell size
    • Determine cell shape
    • Determine cell arrangements
  12. What are the 3 main purposes of differential staining?
    • Cell size, shape and arrangements
    • Separate Gram-pos & Gram-neg
    • Visualize cell structures
  13. Define working distance as it relates to microscopy.
    Proximity of the slide to the bottom of the objective lens
  14. What aspect of microscopy pertains to the light-bending ability of glass, oil and air media thru which light must pass during image formation?
    Refractive Index
  15. What are 3 types of basic stains?
    • Methylene Blue
    • Crystal Violet
    • Safranin
  16. Basic stains carry a ___ electrical charge.
  17. The ___ stain technique allows us to see unstained bacteria on a stained background and determine their morphology.
  18. ____ stains such as nigrosin, congo red & india ink carry a neg charge & are therefore what?
    • Acidic 
    • Repelled by negatively charged bacteria
  19. What are the steps in preparing a bacterial smear?
    • Asceptically place bacteria on glass slide
    • Air dry and heat fix
    • Stain appropriately
    • Rinse, dry and view
  20. Define a bacterial smear.
    Thin layer of bacteria placed on a slide for staining
  21. If agar slants are used when making a bacterial smear then a what should be added to the slide as well?
    A loopful of water
  22. What are the 3 things accomplished by heat fixing?
    • Kills any bacteria that may still be alive
    • Facilitates stain penetration
    • Fixes cells to the slide so they do not wash off when stained
  23. Simple staining is an easy way to determine what 3 things about bacterial cells?
    • Size 
    • Shape 
    • Arrangement
  24. Name 3 basic stains we discussed?
    • Methylene Blue
    • Crystal Violet
    • Safranin
  25. The ___ ___ technique permits one to see unstained bacteria on a stained background & determine their morphology.
    Negative stain
  26. Nigrosin, congo red & India ink are ___ stains that carry a ___ charge and are therefore repelled by ____ charged bacteria.
    • Acidic
    • Negative
    • Negatively
  27. What form of bacteria found in dental plaque are best stained in a negative manner and why?
    Spirochetes b/c they do not stain well with other techniques
  28. The use of excessive stain when utilizing the negative stain technique causes what problem?
    It makes the organisms difficult to locate
  29. Negative stain must not be allowed to become contaminated because. . . . 
    It will support bacterial growth
  30. What is the quick procedure for the negative stain technique?
    • Add loopful of bacteria at end of slide 
    • Place sm drop of acidic dye at end w/bacteria
    • Pull dye/bacteria mix across face of slide w/ second slide
    • Air dry and observe
  31. What are 2 advantages to the negative stain technique?
    • It does not distort bacterial cells
    • Some cells such as spirochetes dont stain well w/other techniques
  32. T or F. Negatively stained slides are air dried and heat fixed.
    False - Air dried ONLY
  33. When the negative stain technique is used what are we looking for in the microscope?
    White or clear cells on a colored background
  34. When labeling a diagram of a slide what should we note?
    • Size, shape, arrangement
    • Full binomial name
    • Total magnification 
    • Dye used
  35. What is a second method for preparing a negative stain?
    • Prepare air dried smear
    • Pass a felt tipped marking pen several times over the smear
    • Ink will stick to slide & bacteria will appear white against colored background
  36. Why do we not heat fix bacterial smears iN negative staining?
    B/c it will disrupt the bacterial cells
  37. What is aseptic transfer?
    Sterile, microbe free mvmt or transfer of microorganisms fr one medium to another.
  38. Why do we use aseptic technique?
    In order to maintain pure cultures & create subcultures
  39. What is the red pigment produced by S. marcescens called?
  40. What are 3 advantages of the streak plate technique over the pour plate technique?
    • No time constraints due to agar cooling
    • Less likelihood of contamination
    • Easier access to bacterial colonies
  41. Why do we incubate plates in the inverted position?
    So when condensation forms on the lid it doesn't fall into the cultures
  42. In 1884 ___ ___, a Danish physician, discovered certain bacteria aft being stained w/crystal violet & iodine, would lose their color on subsequent tx w/alcohol while other bacteria would retain the color.
    Christian Gram
  43. When Gram staining bacteria that retain crystal violet stain are known as ___-___ & those that do not are ___-___.
    • Gram positive
    • Gram negative
  44. Gram staining is classified as a ____ staining technique.
  45. What 2 types of bacteria do not respond to Gram staining?
    Spirochetes & Mycobacteria
  46. ___ ___ & ____ form a complex that exits easily fr Gram neg bacteria but not fr Gram pos when ___ is applied.
    • Crystal violet
    • Iodine
    • Alcohol
  47. What is it in the cell walls of Gram positive bacteria that trap the crystal violet/iodine complex?
  48. What is it that makes Gram pos cell walls retain the crystal violet/iodine complex and causes Gram neg walls not too?
    Gram pos bacteria have larger amts of peptidoglycan in their cell walls that traps the complex whereas Gram neg walls have less & fail to retain it
  49. The Gram stain technique is a ___ part procedure in which 2 dyes, a ____(iodine) & a ___ agent are used.
    • Four
    • Mordant
    • Decolorizing
  50. Other than a culture what supplies are needed for a Gram stain procedure?
    • Crystal violet stain
    • Grams Iodine
    • 95% ethyl alcohol
    • Safranin stain
  51. What are the steps in a Gram stain?
    • Prepare air dried, heat fixed smear
    • Apply crystal violet & set for 1 min
    • Rinse
    • Flood w/Grams iodine & set for 1 min
    • Rinse
    • Decolorize w/95% ethyl alcohol for 15 sec, x2
    • Rinse
    • Counterstain for 30 sec
    • Rinse
    • Blot dry w/bibulous paper
  52. The iodine in a Gram stain acts as a mordant which means what?
    It increases the affinity of dye to the cell by forming a complex w/the stain
  53. As a general rule how long should decolorization go on for?
    Until the ethyl alcohol running off is no longer purple
  54. What is the possible result of excessive decolorization in Gram staining?
    Gram pos bacteria may lose their color and appear Gram neg
  55. What is the possible result of insufficient decolorization when performing a Gram stain?
    Gram neg bacteria may retain purple color and appear Gram pos 
  56. Prior to counterstaining in the Gram technique what would the slide look like under a microscope?
    Gram pos bacteria would appear purple & Gram neg bacteria would appear transparent
  57. What effect will safranin have on Gram pos bacteria?
  58. After decolorization, flood smear w/____ for ___ secs.
    • Safranin
    • 30
  59. Gram pos bacteria will appear ___ to ___ & Gram neg will appear ____ to ____.
    • Blue to purple
    • Orange to Red
  60. When observing Gram stained slides, be sure to confine your work to areas containing thin smears of bacteria b/c why?
    Thicker areas may resist decolorization due to heavy concentration of bacteria
  61. Endospores are formed by members of several gram-___ bacterial genera such as ____ &____.
    • Positive
    • Bacillus and Clostridium
  62. What are 3 characteristics of endospores?
    • Resist boiling for 2 hrs or more
    • Contain little water
    • Exhibit few chem reactions
  63. What happens to spores when the chem environment are favorable?
    Protective layers break down & vegetative cells emerge to grow & reproduce
  64. Name 4 notable diseases caused by sporeformers.
    • Tetanus
    • Botulism
    • Gas gangrene
    • Anthrax
  65. What name is given to a layer of polysaccharides & proteins secreted by certain bacteria including pathogens? 
  66. What functions does a capsule perform?
    • Buffer between cell & its external environment
    • Protects against dehydration
    • Traps nutrients fr surrounding environment
    • Contributes to est of disease 
    • Helps resist phagocytosis
  67. A thin flowing capsule isnt really a capsule at all. It is called a ___ ___.
    Slime layer
  68. The term ____ refers to both capsule and slime layer
  69. ____ are protein appendages that facilitate motion of bacteria?
  70. Many species of bacterial ___ & ___  & a few species of ___ possess flagella
    • Rods & spirilla
    • Cocci
  71. Why is it that flagella are so long but cannot be seen with a light microscope?
    B/c although they are many times longer than a cell they are extremely thin
  72. What is the purpose of the spore stain technique?
    To contrast vegetative cells fr endospores
  73. Why is it that bacterial endospores cannot be penetrated easily by stain using simple or Gram stain techniques? 
    B/c they contain numerous protective layers
  74. What do we do to assist with stain penetration when staining bacterial endospores?
    Apply heat
  75. What stain do we use as the primary stain for spore stain?
    Malachite Green
  76. When doing a spore stain we should allow the slide to remain over ___ for ___ mins, continually adding stain. 
    Steam for 3 mins
  77. Heat during spore stain does what?
    Forces stain into endospores & vegetative cells  
  78. When rinsing spore stained slides vegetative cells will ___ their color while endospores will what?
    • Lose
    • Remain green
  79. When spore staining we counterstain w/___ for __ min.  This will have what effect?
    • Safranin for 1 min
    • this will stain the vegetative cells but have no effect on the spores
  80. When spores are found within vegetative cells, their position could be ___, ___ or ___.
    Central, subterminal, terminal 
  81. Young cultures often contain spores within ___ ___ while older cultures contain more free ___ & fewer ___ ___.
    • Vegetative cells
    • Spores
    • Vegetative cells
  82. When utilizing the alternate spore stain technique, we cover the smears w/___ ___ ___ for how long?
    • 7 1/2% malachite green
    • 10 minutes
  83. Visualization of the bacterial capsule by staining is a 2-step procedure involving both ___ & ___ staining.
    Negative & simple
  84. Why is it that water or heat should not be used in any step of capsule staining?
    Capsules are easily destroyed by both
  85. Why is it helpful to use milk cultures of organisms when capsule staining? 
    Media containing milk encourage capsule production
  86. What are the quick steps to produce a capsule stain?
    • Prepare neg stain of bacterial smear
    • Stain w/crystal violet for 1 min
    • Rinse w/ SALINE SOLUTION, dry, observe
  87. When viewing capsule stain, what are we looking for?
    Purple cells surrounded by capsules which appear as white halos
  88. What are 2 methods for determining motility?
    • Hanging drop technique
    • Utilizing motility agar
  89. What characteristics of motility should we not when viewing our slide?
    • Patterns of motion
    • Where cells accumulate
    • Relative shapes & sizes
    • Configurations displayed
    • Speed of mvmt
  90. Erratic vibrations of cells in place & lack of directed mvmt demonstrates a phenomena called what? 
    Brownian Mvmt
  91. What causes Brownian mvmt?
    Molecules striking organisms & displacing them briefly
  92. What is motility test agar?
    Semisolid growth medium containing a reduced amt of agar
  93. What is special about semisolid motility agar?
    The semisolid state allows bacteria to move freely throughout the medium
  94. How will motile organisms present in test agar in comparison to non-motile organisms?
    Motile organisms will spread away fr the inoculation line & est broad zone of growth where nonmotile organisms will grow only along the line of inoculation
  95. Name 3 characteristics that are used to help ID an organism.
    • Size, Shape, Arrangement
    • Response to staining
    • Presence of spores, capsules, flagella
  96. Why is it necessary to invert plates when incubating them?
    B/c condensation develops on the lid of the plate & we don't want it to drip on the culture
  97. In ____ colonies, bacteria form many hairlike filaments as they grow outward from the center of the colony.
  98. ____ colonies grow outward in fewer and thicker filaments, like the roots of a miniature tree.
  99. ____ colonies bulge up from the plate in a pronounced semicircle.
  100. ____ colonies look like raised colonies, but they have a small projection in the middle.
  101. ____ colonies resemble raised colonies, but they have a tiny depression like a dimple in the middle.
  102. The ___ describes the edge of a bacterial colony.
  103. A colony where the edge is lumpy and irregular is considered to be what shape?
  104. Some margins are ____, with tiny filamentous strands of bacteria growing out from a central mass.
  105. ____ margins have a wavelike edge, while ____ margins have fingerlike projections growing outward from the edges of the colony.
    • Curled
    • Lobate
  106. Cloudyness in a broth culture is described as being ____.
  107. A broth culture that contains small particles is said to be what?
  108. A broth culture that contains small masses is said to be ____.
  109. Broth cultures that contain large particles are described as ____.
  110. Sediment at the bottom of a tube that clumps together is said to be ____.
  111. Aerobic growth is located where in a thioglycollate medium tube?
    Growth at the top of the tube
  112. Growth at the bottom of a thioglycollate tube indicate what type of growth?
    Anaerobic Growth
  113. Growth throughout a thioglycollate tube indicates what type of growth?
    Facultative Growth
  114. Why is it said that biochemical characteristics serve as fingerprints for a bacterial species?
    B/c the results of a series of biochemical tests can help to ID an unk organism & potentially ID a disease
  115. What is contained in Bergey's Manual of Systematic Bacteriology?
    Std results for the biochemical tests we performed 
  116. ___ is a type of microbial metabolism which an organic intermediary molecule serves as an electron acceptor  
  117. What are the two products of fermentation that our tests looked for?
    • Carbon dioxide gas production
    • Acid production
  118. What is the media used in fermentation testing consist of?  
    Nutrient broth supplemented w/0.5% fermentable carbohydrate
  119. For our carbohydrate test we used phenol red indicator.  This solution is ___ in neutral or basic solutions & ___ in acidic solutions.
    • Red
    • Yellow
  120. What item was added to our fermentation test tubes to indicate the formation of gas?
    Durham tube
  121. ____ is a poly saccharide consisting of thousands of glucose molecules chemically bonded to one another.
  122. Certain bacteria utilize this enzyme to biochemically break bonds between glucose monomers.
  123. What indicator do we use to test for starch digestion and how does it indicate?
    • Iodine
    • It is added to determine whether starch is still present or has been digested
  124. What does starch normally do in the presence of iodine?
    Forms a blue black chemical complex 
  125. Catalase is an enzyme that breaks down ___ ___ to ___ & ___.
    • Hydrogen peroxide 
    • oxygen & water
  126. Why is catalase important in the bacterial metabolism?
    Hydrogen peroxide produced during energy yielding processes must be broken down
  127. What happens if hydrogen peroxide fr energy yielding processes is not broken down by catalase?
    It will accumulate and kill the cells?
  128. Why is hydrogen peroxide generally a poor antiseptic?
    B/c it is rapidly degraded by bacterial & tissue catalase
  129. How can we test for catalase production?
    Growing bacteria on a nutrient medium & the adding hydrogen peroxide
  130. How do we know if catalase is being produced by a bacteria?
    If oxygen bubbles appear & the area effervesces w/the application of 3% hydrogen peroxide
  131. Certain bacteria produce the enzyme ____ which digests DNA into its constituent nucleotides.
  132. How is the DNA test performed?
    • By incubating a bacteria on a med that can contains DNA
    • Add hydrochloric acid to determine whether hydrochloric acid has been produced
  133. What chem is added to a culture grown on what med to determine if DNA has been digested?
    1N HCL added to bacteria grown on medium containing DNA
  134. DNA will normally react w/HCL & form what?
    Very fine precipitate giving the media a cloudy appearance 
  135. If DNA digestion has occurred the area nr the bacterial streak will be ____ & the remainder of the plate will become ___.
    • Clear
    • Cloudy
  136. The enzyme ___ ___ breaks down the amino acid cysteine to form ___ & ___ ___.
    • Cysteine desulfurase
    • Alanine & hydrogen sulfide
  137. When testing for hydrogen sulfide, if Fe ions are present what happens?
    They will react w/hydrogen sulfide to form hydrogen sulfide causing the medium to become black
  138. Hydrogen sulfide has significance in the food industry.  Why?
    B/c it leads to black rot in eggs w/rotten egg odor
  139. What medium is commonly used for the hydrogen sulfide test & what does it contain?
    • Sulfide Indole Motility (SIM) medium 
    • It contains cysteine & Fe ions
  140. What 3 things can be tested in SIM medium?
    • Hydrogen sulfide production 
    • Indole production 
    • Motility
  141. ___ is an end product of protein metabolism in bacteria.
  142. Certain bacteria produce the enzyme ___ which breaks down urea into ___ & ___.
    • Urease
    • Ammonia & carbon dioxide
  143. Urea digestion is important in ___ cycles in the soil b/c urea is a major component of animal ___ & bacteria are responsible for ___ ___ ___ to release nitrogen for reuse.
    • Nitrogen
    • Urine
    • Breaking it down
  144. In the test for urea digestion, bacteria are inoculated to a nutrient broth that contains what 2 things?
    Urea & phenol red indicator 
  145. If urea is digested during incubation, ammonia will ___ the pH of the medium and cause the indicator to become what color?
    • Raise
    • Deep fuchsia or purple
  146. Certain bacteria secrete enzymes called lipases that ___ the ___ in lipids.
    Hydrolyze linkages 
  147. When lipase hydrolyzes linkages in lipids, what is separated?
    Glycerol molecule fr fatty acid molecules
  148. What happens to glycerol & fatty acid molecules that result fr lipids broken down by lipases?
    They are transported into the bacterial cytoplasm & used as building blocks for new cell structures & as energy sources
  149. What is the name given to a type of food spoilage in which foul odors & flavors develop b/c of fatty acids released fr lipids
  150. What 3 foods are particularly susceptible to rancidity? 
    Butter, vegetable oil & fish
  151. What special medium is used to test for lipid digestion?
    Spirit Blue agar
  152. Spirit blue agar is rich in ___ & contains spirit blue which is an indicator for what?
    • Lipids
    • pH
  153. Spirit blue agar is ___ ___ in a neutral environment but becomes ___ ___ in the presence of acids.
    • Pale lavender 
    • Deep blue
  154. If spirit blue agar becomes then the bacteria being tested are what causing what?
    Able to digest lipids causing fatty acids to accumulate & pH to lower