water bacteriology
DESCRIPTION
Lab ExerciseTRANSCRIPT
Water BacteriologyMethod: Multiple tube fermentation technique (MPN)
A.I. Presumptive test: Day 1
Culture medium: LTB
1. Label 9 test tubes of LTB1-3(3)---LTB 10mL4-6(3)---LTB 1mL7-9(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 2Culture media: BGLB & EC
1. Shake each (+) tubes in the PT.2. Using a sterile wire loop, transfer 1 loopful of culture to BGLB
tubes. (scall (+) LTB tubes to BGLB)
3. Choose 1(+) LTB (10mL, sc to EC medium4. Incubate BGLB at 37C and EC at 44C for 24 hours5. Observe for the presence of gas.
*if (-) reincubate for another 24 hours6. 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 10mLBGLB 10mL (+) turbid w/ gas=3BGLB 10mL
BGLB 1mLBGLB 1mL (+) turbid with gas=3BGLB 1mL
BGLB 0.1mLBGLB 0.1mL (-) no gas=0
BGLB 0.1mL
MPN=3-3-0=240 col./100mL H2O
III. Completed TestCulture media: EMB, LTB & NA slant
*remember: EMB: differential mildly selectivedifferentiates 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 tubed. Make a smear---do Gram stain from NA slant