1. Lab 29
Water labs

2. Objectives
To learn about the specific procedure for declaring water safe to drink or swim

3. Coliform Bacteria Gram negative rods Facultative anaerobes, aerobes
Not spore forming
Ferment lactose AND produce gas
Non-pathogenic versions of coliforms can be indicator organisms
The presence of these organisms indicate fecal contamination in water

4. 3 phases of testing Presumptive test (today)
Selective (inhibits spore formers)
Differential (lactose fermentation)
Turbidity and gas = +
Most Probably Number (MPN)
Correlates a number of + tubes with a concentration range of organisms

5. 3 phases of testing Confirmed Test (session 2)
Selective (detects coliforms and dairy products)
Presence of bile inhibits some bacteria and stimulates others
Differential (Gas production)
Completed Test (Session 3)
LES Endo agar
Looks for presence of E. coli, Enterobacter aerogenes. Salmonella species

6. For today I spiked on of the samples
USE THE CORRECT PIPETTE FOR THE JOB
Work in pairs, select 1 of the 3 possible water sources
Inoculate 5 10mL double strength broths, 5 1mL regular and 5 0.1mL regular presumptive test

7. Session 2 Review MPN, 15 tubes
A positive if durham tube >10% filled
70/100mL water unsafe for shellfish harvesting
1000/100mL water unsafe for swimming
Use the least positive tube to inoculate BGLBB
Brilliant Green Lactose Bile Broth
Confirmation test: do you still have fermentation with bile and additional selection chemicals present?

8. Session 3 BGLBB is positive if >10% durham tube filled with gas
Inoculate LES Endo Agar
Determines if one or more DIFFERENT organisms are present
E. coli? E. aerogenes? Etc.
Lactose Positive colonies will be redish from the dye in the media
E. coli specifically will have a green sheen
Inoculate a TSA Slant for a final gram stain in session 4

9. Session 4
Gram stain sample off TSA slant to confirm the presence of gram negative rods
We don’t have much success gram staining off LES agar because the selective media messes up morphology