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Method: Multiple tube fermentation technique (MPN) A. I. Presumptive test: Day 1 Culture medium: LTB 1. Label 9 test tubes of LTB 1-3(3)---LTB 10mL 4-6(3)---LTB 1mL 7-9(3)---LTB 0.1mL 2. Shake the bottle w/ the H2O , inoculate all 9 LTB tubes using a sterile pipet. 3. Mix all tubes at least 20x. 4. incubate at 37C for 24 hours. 5. Observe for the presence of gas in the inverted vials. 6. submit all (+) tubes to confirmed test. II. Confirmed Test: Day 2 Culture media: BGLB & EC 1. Shake each (+) tubes in the PT. 2. Using a sterile wire loop, transfer 1 loopful of culture to BGLB tubes. (sc all (+) LTB tubes to BGLB) 3. Choose 1(+) LTB (10mL, sc to EC medium 4. Incubate BGLB at 37C and EC at 44C for 24 hours 5. Observe for the presence of gas. *if (-) reincubate for another 24 hours 6. Record all (+) BGLB tubes and determine the MPN. Example: MPN Dtermination (Most Probable Number of Coliforms) -determine in the confirmed test (BGLB) after 48 hours of incubation BGLB 10mL BGLB 10mL BGLB 10mL BGLB 1mL BGLB 1mL BGLB 1mL BGLB 0.1mL BGLB 0.1mL (+) turbid w/ gas=3
(+) turbid with gas=3
(-) no gas=0
BGLB 0.1mL MPN=3-3-0=240 col./100mL H2O III. Completed Test Culture media: EMB, LTB & NA slant *remember: EMB: differential mildly selective differentiates lactose fermenters from non-lactose fermenters 1. Choose 1(+) BGLB (10mL)=Streak an EMB plate (4Q streaking) 2. Incubate at 37C for 24 hours. a. After 24 hrs incubation. from EMB plate -look for typical colonies (pick 1 or more) -look for atypical colonies (pick 2 or more and SC to LTB and NA slant) b. Incubate at 37C for 24 hrs. c. Observe for gas production in LTB tube d. Make a smear---do Gram stain from NA slant