Coliforms

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Coliforms
Laboratory 4
James Douglass
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Coliform characteristics

• Aerobic or facultative anaerobic
• Gram-negative, non-spore forming rod
• Major genera Escherichia, Klebsiella
  Enterobacter
• Able to grow in the presence of bile salts
• FERMENT LACTOSE
•      Lactose ? acid gaseous H2 CO2

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Where are coliforms found?

• Ubiquitous
• Fecal AND non-fecal origins
• Water
• Soil
• Intestinal tract

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Food quality and acceptability

• Indicator Organism
• Personal hygiene and sanitation
• Presence of coliforms does not mean a food is
  hazardous

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Detection method

• Presumptive (e.g., LST broth, VRB)
• Confirmatory (e.g., BGLB broth)
• Completed (e.g., EMB for probable E. coli)
•  

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Food Sample (mushrooms, sprouts or tofu)
11g into 99 ml of peptone water (10-1)
Homogenize
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Prepare decimal dilutions (10-2 , 10-3, 10-4)
Pour plate overlay
MPN (LST Broth)
Plate on E. coli/Col. Petrifilm
Plate on VRB
Do not vortexMPN tubes
Determine presumptive count of VRB and MPN
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Do not vortex
Inoculate presumptive colonies and tubes to BGLB
Score tubes for coliform count
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Inoculate coliforms to EMB
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Inspect EMB plates
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MPN
1 mL
1 mL
1 mL

• Most Probable Number
• Easier for counting

9 mL
9 mL
9 mL
10-1
10-2
10-3
10-4
1 mL
1 mL
1 mL

-
- - -
LST tubes
3 2 0
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MPN Enumeration
10-1 10-2 10-3 0.1g 0.01g
0.001g MPN/g 3 2 0
93
Adjustment for dilutions used
Used 10-2, 10-3, 10-4
Final result 930 MPN/g
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Lauryl Sulfate Tryptose (LST) Broth

• Selective-differential
• Used for coliform detection in water and MPN
  determination
• Contains
•   Lactose  to detect lactose fermentation
•   Sodium lauryl sulfate  a selective agent
• (selects against non-coliforms)
•   Tryptose to aid in growth
•   NaCl  to control osmotic balance
•   Durham tubes  to detect CO2 formation

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Violet Red Bile (VRB) Agar

• Selective-differential
• Contains
•   Bile salts/crystal violet  to select against
• G, non-enteric organisms
•   Lactose/neutral red
• Lactose fermenting microorganisms will be
  PURPLE-RED (acidic) with halo (coliforms)
• Non-lactose fermenting microorganisms will be
  YELLOW/STRAW COLORED (basic)

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E. coli /Coliform petrifilm

•   Contains 
•    VRB gel
•    Two dyes-neutral red indicator of
  glucuronidase activity
•                         E. coli gt  BLUE with gas
  bubble
•                         Coliforms gt  RED with
  gas bubble

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Brilliant Green Lactose Bile(BGLB) Broth

• Selective-differential
• Confirm the presence of coliforms in food, or
  water
• Contains
•  Oxgall (bile)/brilliant green  to inhibit G and
  non-coliform G- organisms
•  Inverted Durham tubes  trap CO2 produced by
  lactose fermentation

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Eosin Methylene Blue (EMB) Agar

• Selective-differential
• Detection and differentiation of coliforms 
• Contains
•   Eosin/methylene blue  under acidic conditions,
  combine to form a  precipitate to differentiate
  lactose fermenters from non-lactose fermenters. 
  They are selective agents, though G and yeasts
  may form pinpoint colonies. 
• Lactose fermenters  DARK COLORATION
• Non-lactose fermenters PALE STRAW COLORATION
• Sucrose-fermenters  PINK COLOR
• E. coli colonies  GREEN SHEEN
•  

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Coliforms on EMB
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Not a coliform!