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Microbiology: Week 2 Notes

by: Amanda Biddlecome

Microbiology: Week 2 Notes 81382 - MICR 3050 - 001

Amanda Biddlecome
GPA 4.0

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About this Document

These notes cover chapters 2 and 7.
General Microbiology
Dr. Rudolph
Class Notes
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This 4 page Class Notes was uploaded by Amanda Biddlecome on Sunday September 4, 2016. The Class Notes belongs to 81382 - MICR 3050 - 001 at Clemson University taught by Dr. Rudolph in Fall 2016. Since its upload, it has received 5 views. For similar materials see General Microbiology in Biology at Clemson University.


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Date Created: 09/04/16
Microbiology  3050   Chapter  2:  Study  of  Microbial  Structure:  Microscopy  and  Specimen   Preparation   Amanda  Biddlecome   August  29,  2016     1)  The  Light  Microscopes     -­‐types  in  microbiology       *bright-­‐field,  dark-­‐field,  phase-­‐contrast,  fluorescence,  confocal         ^bright  and  dark  can  be  done  with  the  same  scope     -­‐compound       *multiple  lenses     2)  Lenses  and  Bending  Light     -­‐when  light  hits  the  lens,  it  bends  (refracts)     -­‐refractive  index       *different  for  different  mediums       *measure  of  how  much  the  velocity  of  light  slows  down  (bends)  as  it         enters  the  new  medium       *air=1;  oil=1.25;  water  varies  depending  on  what’s  dissolved  in  it     -­‐direction  and  magnitude  of  the  bend  depends  on  the  refractive  index  of  both     media     3)  Lenses     -­‐collection  of  prisms     -­‐light  bends  toward  the  normal  of  the  lens       *the  line  perpendicular  to  the  lens     -­‐focal  point=where  all  rays  meet  behind  the  lens     -­‐focal  length=distance  between  the  lens  and  the  focal  point     -­‐strength  of  the  lens:  shorter  focal  length=greater  magnitude     4)  Bright-­‐Field  Microscope     -­‐condenser  lens=focus  light  under  the  stage     -­‐image  is  dark  against  brighter  background  light       *any  light  that  can’t  get  through=darker  object       *denser  objects=darker     -­‐objective  lenses       *parfocal=get  in  focus  at  lower  power  and  should  still  be  in  focus  as         you    move  up  in  strength         ^use  coarse  adjustments  ONLY  at  4  and  10       *parcentric=stays  centered  as  you  increase  strength     -­‐total  magnification=magnification  of  ocular  lens  (ours  is  10X)  X     magnification  of  objective  lens     -­‐upper  limit  of  magnification  for  bright-­‐field=1500X     -­‐upper  limit  of  resolution  for  bright-­‐field=0.2μm   5)  Microscope  Resolution     -­‐ability  of  the  microscope  to  distinguish  between  two  small  objects  that  are     close  together     -­‐wavelength  affects  that  distance  (d)       *shorter=greater  resolution  and  smaller  d       *blue  light=short  wavelength     -­‐d=(0.5λ)/NA=(0.5λ)/(nsinθ)       *NA=Numerical  Aperture:  how  much  light  it  captures       *working  distance=distance  between  specimen  and  lens         ^longer  objective=greater  NA       *n=refractive  index         ^oil  gives  better  clarity,  not  better  magnification       6)  Dark-­‐Field  Microscope     -­‐reverse  of  bright-­‐field  microscope     -­‐bright  image  on  a  dark  background     -­‐image  is  shown  by  the  light  reflected  or  refracted  by  the  specimen     -­‐most  bacteria  don’t  have  pigments,  so  we  have  to  stain  them  for  a  bright-­‐   field,  but  that  kills  them     -­‐use  dark-­‐field  to  observe  living  microbes  that  aren’t  stained       *see  motility     -­‐can  see  internal  structures  in  eukaryotes     -­‐put  dark  field  stop  on  a  bright-­‐field  microscope  to  block  light,  except  for  the     edges     7)  Preparation  and  Staining  of  Specimens     -­‐we  stain  to  be  able  to  see  them:  contrast  with  the  background     -­‐helps  see  morphological  features       *bacteria  we  look  at  overall  shape     -­‐preserve  the  shape  so  it  doesn’t  change  before  our  eyes       *look  normal  and  not  distorted     Chapter  7.5     1)  Culture  Media     -­‐they  are  nutrient  preparations  devised  to  support  growth  and  reproduction     of  microorganisms       *have  macronutrients       *have  micronuetrients:  inorganics,  trace  metals/minerals,  Zn,  Mg,  Cu         ^usually  don’t  have  to  worry  about  these  because  they  are  in           water       *growth  factors=organics  that  they  don’t  make  on  their  own:  vitamins     -­‐can  be  liquid  or  solid       *without  agar=liquid  broth       *solid  media  are  usually  solidified  with  agar     2)  Defined/Synthetic  Media     -­‐all  components  and  their  concentrations  are  known         *know  EVERYTHING  including  formulas     3)  Complex  Media     -­‐very  complex     -­‐some  ingredients  have  unknown  compositions  and/or  concentrations       *ingredients  could  be  different  everytime     -­‐as  soon  as  you  add  agar  to  something,  it  becomes  complex,  even  if  it  started     as  defined     3)  Complex  Media  Components     -­‐peptones       *partial     -­‐extracts       *aqueous  extracts,  usually  beef  or  yeast     -­‐agar       *sulfated  polysaccharide  used  to  solidify  liquid  media;  most           microorganisms  can’t  degrade  it  and  it  stays  solid  at  37 C   o   -­‐ALL  of  these  are  complex     4)  Functional  Types  of  Media     -­‐general  purpose  media  (supportive)       *helps  many  microorganisms  grow       *trying  to  grow  many  different  microorganisms     -­‐enriched  media       *try  to  grow  many  types  of  microorganisms       *add  something  really  complex  to  help  the  picky  ones  eat  and  grow         ^blood       *fortify  it       *chocolate  agar=boiled  blood     -­‐minimal  media       *contains  minimal  necessities  for  growth  of  wild  type         ^wild  type=heartiest  type  of  the  strain       *has  inorganic  salts,  simple  carbon  sources,  and  water       *looking  to  see  if  it’s  a  nutritional  mutant         ^know  it  is  if  it  can’t  grow  on  minimal  media     -­‐selective  media       *takes  enrichment  to  extreme       *try  to  help  things  you  want  to  see  grow  and  kill  other  microorganism       *MacConkey  agar:  bile  salts  and  crystal  violet:  kills  gram-­‐positive  and         selects  for  gram-­‐negative     -­‐differential  media       *distinguish  between  different  groups  of  microorganisms  by           biological  characteristics         ^blood  agars:  hemolytic  versus  nonhemolytic  bacteria  (alpha,           beta,  and  gamma)       *MacConkey  agar:  lactose  fermenters  make  lots  of  acids  and  turn         colonies  pink/medium  pink  and  lactose  nonfermenters     5)  Isolation  of  Pure  Cultures     -­‐pure  culture=population  of  cells  coming  from  a  single  cell       *all  are  the  same  strain       *pick  up  one  colony  and  it  should  be  pure       *individual  cells  need  to  be  well-­‐separated  to  tell  if  they’re  pure       *use  streak  plate,  spread  plate,  and  pour  plate     6)  Streak  Plate     -­‐use  inoculating  loop     -­‐each  cell  can  reproduce  to  form  a  separate  colony     7)  Aseptic  Transfer     -­‐hold  loop  down     -­‐flame  enough  of  the  loop  to  kill  all  bacteria     -­‐flame  mouth  of  the  tube  right  after  you  open  it,  never  put  the  lid  down  on     the  bench,  and  flame  it  again  right  before  you  close  it     8)  Spread  Plate  and  Pour  Plate     -­‐spread  plate       *dilute  out  the  culture,  put  it  on  agar  surface,  spread  it  out  with  a         hockey  stick     -­‐pour  plate       *mix  diluted  sample  with  liquid  agar  and  pour  it  into  the  plate     -­‐both  give  isolated  colonies  but  you  can  also  count  isolated  colonies       *enumerate  bacteria  in  the  sample       *count  30-­‐300  colonies  because  less  than  30  isn’t  an  accurate           representation  and  more  than  300  is  impossible  to  count     -­‐1:10  dilution       *1mL  of  sample  into  9mL  of  water       *dividing  original  concentration  by  10  each  time     -­‐1:100  dilution       *1mL  of  sample  into  99mL  of  sample     -­‐to  get  original  concentration  from  dilution:  multiply  counted  colonies  by     reciprocal  of  dilution    


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