Quiz 5 April 30th

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• Quiz 5 April 30th
• Included labs 14, 15 and 16
• Notebook checking April 30th
• 13, 14, 15 and 16.
• Final exam
• May 5

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Most probable number MPN
3
Detection

• Detection of contamination can be difficult
• Pathogens might be present in small number
• Also very large volume of water to analyze
• Solution ? use of indicator organisms

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Indicator organisms

• Not diseases causing agents
• Readily monitored in a quick and easy manner
• Present in number high enough to be detected
• Present only when contamination from a fecal
  source has taken place
• The most commonly used are coliforms bacteria

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Criteria for an Ideal Indicator Organism

• Should be useful for all types of water (drinking
  water, wastewater, recreational water, sea water)
• Should be present whenever enteric pathogens are
  present, and absent when pathogens are absent
• Should survive longer in the environment than the
  toughest enteric pathogen
• Should be a member of the normal intestinal
  microflora of warm-blooded animals

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Coliforms

• Facultative anaerobe
• Gram negative rod
• Do not form endospore
• Ferment lactose with acid and gas production
• Within 48 hours
• At 37oC

No gas
Gas
No acid
Acid
http//www.marietta.edu/spilatrs/biol202/labresul
ts/lactose_broth.jpg
http//biology.clc.uc.edu/fankhauser/Labs/Microbio
logy/Gram_Stain/Gram_stain_images/index_gram_stain
_images.html
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Fermentation

• If acid produced? media turns yellow (pH
  indicator)
• If gas production, bubble get trapped in the
  small tube (Durham tube)

http//faculty.mc3.edu/jearl/ML/glucose.jpg
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MPN Most Probable Number

• Test to detect coliforms
• Presumptive test ? lactose broth
• Confirmative test ? EMB plate
• Confirmative test is necessary because not all
  fermenters are coliforms
• Statistic test (hence the name most probable)
• Compare results to statistic tables

9
Most probable number

• Inoculate 3 double strength lactose broth with
  10ml of pond water
• Inoculate 3 single strength lactose broth with 1
  ml of pond water
• Inoculate 3 single strength lactose broth with
  0.1 ml of pond water

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Most probable number
http//smccd.net/accounts/case/biol240/images/MPN1
.gif
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Most probable number

• After incubation ? confirmative test on positive
  lactose tube
• Streaking a loopful of the positive broth on an
  EMB plate
• Find by looking at statistical table

http//www2.austin.cc.tx.us/microbugz/images/emb_e
coli.jpg
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MPN statistical table

• Tube that turn yellow and gas production? ,
  presumptive evidence for coliformes.
• Tube not yellow ? -
• (Tube without gas ?-

3 1 1
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MPN statistical table
75 is the MPN of coliforms per 100 mL of water
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MPN statistical table
MPN for drinking water 0 coliforms per 100
ml MPN for Municipal waters 4 coliforms per 100
ml MPN for fishing or swimming 70-200 coliforms
per 100 ml
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Objective

• Inoculate your tubes for MPN water
• All inoculated with the same pond water
• Ds ? 10ml
• Ss ? 1 ml
• Ss ? 0.1 ml
• Next lab Pick a sample from the presumptive tube
  which is positive (media turned yellow gas in
  the Durham tube), pick the highest dilution
• Streak 1 EMB plate with a loopful of positive
  lactose broth (3 streaks technique). Just do one
  plate even if you have many positive tubes
• Incubate at 37C. You will observe your plate on
  the 3rd lab period