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BIOL 241 - Introduction to Applied Microbiology Lab Notes

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BIOL 241
Vivian Dayeh

BIOL  241L  –  Introduction  to  the  Microbial  World     Experiment  1:  Direct  Microscopic  Count  of  Microorganisms  in  Milk   − Microbial  population  of  milk  is  typically  composed  of  Gram -­‐positive,  non-­‐motile,   microaerophilic  or  anaerobic  rods  and  cocci   − Lactobacillus,  Micrococcus  and  Steptococcus  are  typically  represented   − Microbial  content  can  be  determined  by  microscopic  examination  of  a  stained  film  of  a  milk   sample  using  a  direct  count  method   − Advantages:   o Results  are  quickly  available   o Can  count  clumps  and  individual  bacteria   o Minimal  equipment  required   o Allows  you  to  determine  morphology   o Slides  can  be  kept   − Disadvantages:   o Counts  can’t  be  made  accurately  on  low -­‐count  milk   o Dead  cells  present  in  pasteurized  milk  are  counted   o Continuous  microscopic  examinations   are  tiring   o Cells  may  be  difficult  to  discern   o Debris  may  be  confused  with  bacterial  cells   − Milk  samples  vortexed  to  disperse  organisms  and  break  up  clumps  of  bacteria   − SlideBrite  is  used  to  remove  fat  globules   − Slide  stained  with  Methylene  Blue,  decolourized  w ith  ethanol     Calibration  of  Microscope:  Microscope  Factor   − Microscope  Factor  (MF) :  relationship  of  the  field  to  a  mL   − Measure  the  diameter  of  the  microscope  field   –  each  space  =  0.01mm   − Use  πr  to  calculate  the  area  in  mm   − Convert  this  area  to  cm  by  dividing  by  100   − Divide  1  cm  by  the  area  of  the  field  to  calculate  the  number  of  fields/cm   − MF  =  fields/cm  /  0.005  mL/cm     Experiment  2:  Standard  Plate  Count  of  Milk   − Standard  Plate  Count  (SPC)  is  an  agar-­‐plate  method  for  estimating  bacterial  populatio ns   o Official  method  for  determining  sanitary  quality  of  milk   o Uses  serial  dilutions  and  the  spread  plate  technique,  or  the  pour -­‐plate  technique   − Each  colony  is  assumed  to  have  arisen  from  an  individual  bacterial  cell   − Total  number  of  colonies  formed  is  equival ent  to  the  number  of  viable  microorganisms   − Serial  dilutions  reduce  the  population  to  ensure  that  a  sample  will  be  obtained  that  will   produce  separate  and  distinct  colonies   − Advantages:   o Counts  only  viable  microorganisms   o Accurate  for  use  with  low -­‐count  milk   − Disadvantages:   o Detects  only  organisms  capable  of  growing  under  specific  conditions   o Colonies  may  have  arisen  from  single  cells  or  a  clump  of  cells   − Should  be  considered  as  providing  only  an  estimate  of  the  total  population   − Quebec  Colony  Counter  used  to  count  colonies   o CFU/mL  =  number  of  colonies  x  dilution  factor     Experiment  3:  Microbial  Analysis  of  Cheese   − Cheese  is  the  result  of  microbial  metabolic  activity   − Streptococcus  and  Lactobacillus  produce  lactic  acid  that  is  responsible  for  acidification  of   milk  and  the  cheese  flavours   − Other  microbial  enzymes  cause  the  curd  (protein  molecules)  to  separate  from  the  whey   (liquid  portion)   o Curd  is  further  processed  to  become  cheese  product   − Three  groups  of  cheese:   o Soft,  acid-­‐curd  cheese  –  cottage  cheese,  cream  cheese   o Hard,  rennet-­‐curd  cheese  –  Swiss  cheese,  cheddar  cheese   o Semisoft,  rennet-­‐curd  cheese  -­‐-­‐  Camembert   − Clostridium  and/or  coliform  bacteria  (Escherichia  coli,  Enterobacter  aerogenes )  produce  gas   that  can  cause  defects  in  cheddar  cheese   − Spoilage  may  result  from  “dairy  mold”  in  cured  cheeses   − Geotrichum,  Cladosporium  and  Penicillium  may  grow  on  the  surface  or  in  crevices  of  cheese   and  give  it  an  off-­‐flavour   − EMB  agar  –  differentiates  coliforms  like   E.  coli   − Malt  Extract  agar  plates   –  pH  =  5.4,  used  to  detect  mold  growth,  high  sugar  content   − APT  agar  –  selects  for  lactic-­‐acid  bacteria     Experiment  4:  Food  Illnesses  Caused  by   Staphylococci  and  Salmonellae   − Staphylococcus  aureus  can  enter  food  and  excrete  a  heat  stable  exotoxin  that  may  lead  to   food  poisoning   − Salmonella  infections  involve  liberation  of  endotoxins  from  LPS  in  the  outer  membrane   − Small  numbers  of  Salmonella  are  found  in  food  products  so  enrichment  steps  are  necessary   o Selenite-­‐Cystine,  Tetrathionate  Brilliant  Green  enrichment  broths  and  Brilliant   Green  Sulfa  and  MacConkey  agars  used   − Large  numbers  of  Staphylococci  are  required  to  produce  food  poisoning   − Usually  in  food  poisoning  outbreaks,  only  one  organism  is  the  etiologic  agent   − Staphylococcus  110  agar  plates  –  select  for  Staphylococcus;  pathogenic  strains:  orange  to   pale  yellow  (Staphylococcus  aureus);  non-­‐pathogenic  strains:  white  ( Staphylococcus   epidermidis)   − Brain  Heart  Infusion  Broth   –  enriches  Staphylococcus   − MacConkey  agar  plate   –  purifies  Salmonella  to  get  a  clonal  population;  differentiates  between   lactose  fermenters  and  non-­‐lactose  fermenters   o Salmonella  does  not  ferment  lactose,  so  it  will  be  transparent  to  pink  in  colour   − Triple  Sugar  Iron  (TSI)  slant   –  isolates  Salmonella  based  on  the  ability  to  ferment  glucose,   sucrose,  dextrose;  produces 2  H S  on  slant   o Can’t  use  lactose  or  sucrose;  slant  should  become  more  red  (negative)   o Utilizes  dextrose  to  produce  yellow  butt  with/without  gas  formation  (black  is  very   positive)   o Blackening  of  butt  extending  into  slan2  rom  H Sproduction  (positive)   o Gas  pockets  appear  in  medium  (positive)   − Coagulase:  enzyme  that  produces  a  clot   o Relationship  between  strains  capable  of  producing  this  enzyme  and  producing   exotoxins     Experiment  5:  Determination   of  Coliforms  in  Water  by  the  Most -­‐Probable-­‐Number  Method   (MPN  Test)   − Sanitary  quality  of  water  is  primarily  determined  by  the  kinds  of  microorganisms  present   − Coliform  bacteria  are  used  as  indicators  since  sewage  always  contains  them  in  large  number   and  they  can  be  detected  quickly/easily   o Most  are  non-­‐pathogenic  so  they  indicate  the  presence  of  fecal  matter,  not  harmful   microorganisms   − Presumptive  test:  inoculate  known  amount  of  water  sample  into  multiple  lauryl  tryptose   broth  tubes   o If  gas  is  produced,  the  water  is  assumed  to  be  contaminated  with  fecal  matter  and  is   unsafe  to  drink   − Confirmed  test:  loopful  from  positive  presumptive  test  tube  inoculated  into  brilliant  green   lactose  bile  broth,  which  is  more  selective  for  coliform  detection   o Positive  if  gas  produced   − Alternative  confirmed  test:  streak  culture  from  positive  presumptive  test  tube  onto  Eosin   Methylene  Blue  (EMB)  Agar  or  Endo  agar   o Typical  coliforms  are  green  metallic  sheen  ( E.  coli)  or  pink  colonies  with  dark  center   (E.  aerogenes)   o If  they  form,  indicates  that  gas  was  likely  produced  by  coliforms   o E.  coli  is  the  more  reliable  sewage  indicator  though,  as  it  is  not  normally  present  in   soil   − Completed  test:  typical  coliform  colony  inoculated  into  lauryl  tryptose  broth  and  a  Gram   stain  is  conducted  after  incubation   o If  organisms  selected  are  gram  negative  and  non -­‐spore-­‐forming  bacilli  and  they   produced  acid  and  gas  from  lactose,  it  is  a  positive  test;  indicates  that  the  organisms   are  coliforms   − Most  probable  number  of  coliforms  present  can  be  estimated  by  use  of  the  MPN  index     Experiment  6:  Determination  of  Coliform  Numbers  in  Water  by  the  Membrane  Filter   Technique   − Endo  medium  promotes  the  growth  of  coliforms  and  discourages  the  growth  of  most  other   species  of  bacteria   o Otherwise,  these  organisms  would  overgrow  the  test  filter  and  mask  the  presence  of   any  coliform  colonies   − Coliforms  can  break  down  lactose  to  produce  simpler  substances,  including  aldehydes   o Endo  medium  contains  lactose  and  other  nutrients,  as  well  as  bas ic  fuchsin,  which   reacts  with  the  aldehydes  to  produce  a  shiny  green  complex   − Total  coliforms:  organisms  which  ferment  lactose,  produce  acid  and  gas  within  48  h  at   O 35 C,  or  which  form  colonies  with  a  green  sheen  on  membrane  filters  using  Endo  broth   when  incubated  under  those  conditions   − Fecal  coliforms:  coliform  organisms  able  to  grow  and  ferment  lactose  with  the  production   of  acid  and  gas  or  to  produce  blue  colonies  on  membrane  filters  using  m -­‐FC  broth  when   incubated  at  44.5 C  for  24  h.   o More  indicative  of  fe cal  pollution  by  man  and  other  warm -­‐blooded  animals  than   total  coliform  counts   − Membrane  filter  technique  advantages:   o Greater  sensitivity  because  larger  volumes  of  water  can  be  tested   o Higher  degree  of  reproducibility  of  results   o Shorter  time  to  obtain  results,  since  it  doesn’t  require  confirmed/completed  tests   − Disadvantages:   o Water  must  be  free  from  extraneous  matter  (ex.  cyanobacteria)  since  it  can  plug  up   the  filter  quickly   − Millipore  filter  must  be  screwed  on   − Pal  filter  (used  in  the  lab)  is  magnetic     Experiment  7:  Comparison  of  Colony  Appearance  of  Coliforms  and  Other  Bacteria  Grown  on   EMB  and  Endo  Agars   − Differential  media  as  E.  coli  develops  distinctive  colonies  on  them   − Some  other  bacteria  grow  readily  on  the  media  but  produce  colonies  that  look  different  from   typical  E.  coli  colonies   − Other  microorganisms  may  have  their  growth  inhibited           Expected  Results:   Model  Organism   EMB  Agar   Endo  Agar   Nutrient  Agar   Escherichia  coli   ++   +++   ++   green,  metallic   dark  pink,  green   metallic   Salmonella   +   ++   +   typhimurium   colourless   pale  pink   Pseudomonas   +   +   +   aeruginosa   colourless   pale  pink   fluorescent,  green   Citrobacter  freundii   ++   +++   +   green,  metallic   dark  pink,  metallic   Bacillus  subtilis   -­‐   -­‐   +   irregular  margin   Staphylococcus  aureus   -­‐   -­‐   +   yellow     Experiment  8:  The  IMViC  Test   − Developed  to  separate  various  types  of  coliforms,  specifically   Escherichia  coli  and   Enterobacter  aerogenes   − I  =  indole  test   o Tryptone  broth  is  rich  in   tryptophan,  which  can  be  hydrolyzed  by  organisms  that   produce  the  enzyme  tryptophanase  to  produce   indole,  pyruvic  acid,  and  ammonia   o Bacteria  utilize  pyruvic  acid  and  ammonia,  but  can’t  use  indole   o Kovak’s  reagent  forms  a  red  complex  with  the  indole  ring  in  a  positive  test;   negative,  it  remains  yellow   − M  =  methyl  red  test   o E.  coli  ferments  glucose  to  acidic  end  products,  creating  a  pH  below  4.5   o Other  bacteria  utilize  the  peptone,  forming  ammonia  and  producing  a  basic   environment   o Methyl  red  is  a  pH  indicator  that  is  red  below  4.5  (positive)  and  yellow  above  that   (negative)   − Vi  =  Voges-­‐Proskauer  reaction     o Detects  production  of  acetylmethylcarbinol  ( acetoin)  from  sugar  fermentation   o Positive  test  indicated  by  development  of  cherry  red  colour   o Negative  is  indicated  by   brown  colour   − C  =  citrate  test   o Sodium  citrate  is  the  carbon  source   o If  bacteria  can  utilize  that,  the  pH  becomes  basic  and  changes  the   bromothymol   blue  indicator  from  green  to  blue   o Green  is  a  negative  test     Expected  Results   Organism   Indole   Methyl  Red     Voges-­‐Proskauer   Citrate   Escherichia  coli   +   +   -­‐   -­‐   red   red   brown   green   Enterobacter   -­‐   -­‐   +   +   aerogenes   yellow   yellow   red   blue   Citrobacter   -­‐   +   -­‐   +   freundii   yellow   red   brown   blue     Experiment  9:  Determination  of  Chlorine  Demand   − Chlorine  is  used  to  purify   water  as  effective  concentrations  kill  microorganisms  but  are  not   toxic  to  higher  life  forms   − Solutions  containing  chlorine  depend  on  liberation  of  free  chlorine  and  oxidation  of  enzymes   and  proteins  by  nascent  oxygen  for  effectiveness   o Hypochlorite  à  hypochl
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