Download Lab 29

Lab 29
Water labs
Objectives
• To learn about the specific procedure for
declaring water safe to drink or swim
Coliform Bacteria
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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