Coliforms

1. Coliforms Laboratory 4 James Douglass

2. 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

3. Where are coliforms found? • Ubiquitous • Fecal AND non-fecal origins • Water • Soil • Intestinal tract

4. Food quality and acceptability • “Indicator Organism” • Personal hygiene and sanitation • Presence of coliforms does not mean a food is hazardous

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

6. 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

7. 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

8. 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

9. 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

10. 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)

11. 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

12. 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

13. 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

14. Coliforms on EMB

15. Not a coliform!