Lab 29

Water labs

Objectives

• To learn about the specific procedure for declaring water safe to drink or swim

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

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

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

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

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?

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

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