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USE DILUTION AOAC 2010 CHAPTER 6
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Use dilution - Staphylococcus aureus ATCC 6538- Salmonella Choleraesuis ATCC 10708- Pseudomonas aeruginosa ATCC 15442
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Use dilution (PC) PC 20 Phenol 1:20 Use dilution Stainless steel cylinder 60 1 10
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-701. NB 10 mL 371 oC 18 -24 h.
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NB 10 mLNA Slant 37 1 oC 48 2 h. 2 5 oC 30 2 TSAMSATSACetrimideTSAStaphylococcus aureusSalmonella CholeraesuisPseudomonas aeruginosastreakXLD
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TSAMSATSACetrimideTSAPseudomonas aeruginosaStaphylococcus aureusSalmonella CholeraesuisMicroscopeMicroscopeMicroscopeXLD
TSI = K / A Gas = -TSI = K /A, A/A, Gas = +/-TSI = N/N Gas = -H2O3 = -H2O3 = -H2O3 = -LIM = Lysine -/+, Motile = -LIM + Lysine -, Motile = +LIM = Lysine -/+, Motile = -Indole = -Indole = -Indole = +
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Pseudomonas aeruginosaStaphylococcus aureusSalmonella Choleraesuis
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2.1 loop (10l.)NB 10 mL 361 oC 22 -26 h.10 loop (100l.)1 loop (10l.)NB 10 mL 361 oC 22 -26 h.( 3 )NB 100 mL 361 oC 48-54 h.
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3. Carrier 1 M NaOH 12 1 - 1% Phenolphthalein DI 2 Carrier Bottle solution RO 121 oC 15
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4.exampleAseptic technical BeakerPipetteBottle solution
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example solutionexample solution10 mL20.0Water bath 201 oC
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4.Pipette 25 mL 152
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Plate 1Plate 2Plate 3SET 136.0Dry 361 oC 402
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Carrier 1 2 1 set Control positive20.0 Carrier305 S
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20.0305 S36.0 361 oC 482 Letheen broth 10 mL
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1000 L1000 L1000 L1000 L1000 L10-110-210-310-410-510-610-710-810-61000L10-610-710-710-810-8Pour plateTest cultureMix 361 oC 24 48 h.- Quantitation of Test organism
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- Enumeration & Quantitation of Test organism on CarrierLetheen broth 10 mL1 minSonicator 1 min 5 sec
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1000 L1000 L1000 L1000 L1000 L10-110-210-310-410-510-610-710-810-6100 L10-610-710-710-810-8Mix 361 oC 24 48 h.Pour plate
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- Control positive, Control negative, Control sampleControl negativeControl positiveexample solutionControl samplePipette 1 mL 361 oC 24 48 h. 361 oC 24 48 h. 361 oC 24 48 h.
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6. Use dilution 60 60 1
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Quantitation of Test organism & Test organism on Carrier 25 250 n =c(1 n1) + (0.1 n2) dc = 25 250 n1 = dilution d = dilution n2 = dilution : Test organism on Carrier 10
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10-6 = 250, 30910-7 = 98, 6510-8 = 15, 24 25 250 = 250 + 98 + 65 = 453n =413(1 1) + (0.1 2) 10-6n = 344.16 106n = 3.44 108 ( Carrier 3.44 10 108 ) CFU/mL
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Enumeration 0 300 CFU/mL =(avg. CFU for 10-x)+(avg. CFU for 10-y)+(avg. CFU for 10-z)
10-x+ 10-y+ 10-z
2CFU/mL =1CFU/mL 10 mL of broth / Carrier CFU/Carrier taking the log10 6 6 Dry carrier 6
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Enumeration
Set/plate10-610-710-81212121/125027092832526 1/2255221756732211/3245234656912182/1232276666811212/2212246567823252/3249211435421213/1289300556426233/2211233765714183/326824556671215AVG247.0566.1619.05
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CFU/mL =(247.05)+(66.16)+(19.05)
10-6+ 10-7+ 10-8
CFU/mL = 332.26
1.11 10-6
CFU/mL = 3.0 108 CFU/Carrier = 3.0 108 10 CFU/Carrier = 3.0 109 CFU/log10 = 9.477 : Enumeration 6
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Control positive
Control negative
Control sample Control positive, Control negative, Control sample
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Neutralizationpppppp 6
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1000 L1000 L1000 L1000 L1000 L 24 48 h.Pour plate100 L10-110-210-310-410-510-610-710-8100 LPour plate
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Carrier 10 100