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Coliforms. Laboratory 4. James Douglass. 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:

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slide1

Coliforms

Laboratory 4

James Douglass

slide2

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

slide3

Where are coliforms found?

  • Ubiquitous
  • Fecal AND non-fecal origins
  • Water
  • Soil
  • Intestinal tract
slide4

Food quality and acceptability

  • “Indicator Organism”
  • Personal hygiene and sanitation
  • Presence of coliforms does not mean a food is hazardous
slide5

Detection method

  • Presumptive (e.g., LST broth, VRB)
  • Confirmatory (e.g., BGLB broth)
  • Completed (e.g., EMB for probable E. coli)
slide6

Food Sample (mushrooms, sprouts or tofu*)

11g into 99 ml of peptone water (10-1)

Homogenize

1

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

2

Do not vortex

Inoculate presumptive colonies and tubes to BGLB

Score tubes for coliform count

3

Inoculate coliforms to EMB

4

Inspect EMB plates

slide7

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

slide8

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

slide9

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
slide10

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)
slide11

E. coli /Coliform petrifilm

  • Contains:
  •    VRB gel
  •    Two dyes-neutral red & indicator of glucuronidase activity
  • E. coli =>  BLUE with gas bubble
  •                         Coliforms =>  RED with gas bubble
slide12

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
slide13

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