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