USE DILUTION AOAC 2010 CHAPTER 6

Use dilution - Staphylococcus aureus ATCC 6538- Salmonella Choleraesuis ATCC 10708- Pseudomonas aeruginosa ATCC 15442

Use dilution (PC) PC 20 Phenol 1:20 Use dilution Stainless steel cylinder 60 1 10

-701. NB 10 mL 371 oC 18 -24 h.

NB 10 mLNA Slant 37 1 oC 48 2 h. 2 5 oC 30 2 TSAMSATSACetrimideTSAStaphylococcus aureusSalmonella CholeraesuisPseudomonas aeruginosastreakXLD

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 = +

Pseudomonas aeruginosaStaphylococcus aureusSalmonella Choleraesuis

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.

3. Carrier 1 M NaOH 12 1 - 1% Phenolphthalein DI 2 Carrier Bottle solution RO 121 oC 15

4.exampleAseptic technical BeakerPipetteBottle solution

example solutionexample solution10 mL20.0Water bath 201 oC

4.Pipette 25 mL 152

Plate 1Plate 2Plate 3SET 136.0Dry 361 oC 402

Carrier 1 2 1 set Control positive20.0 Carrier305 S

20.0305 S36.0 361 oC 482 Letheen broth 10 mL

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

- Enumeration & Quantitation of Test organism on CarrierLetheen broth 10 mL1 minSonicator 1 min 5 sec

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

- 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.

6. Use dilution 60 60 1

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

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

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

Enumeration

Set/plate10-610-710-81212121/125027092832526 1/2255221756732211/3245234656912182/1232276666811212/2212246567823252/3249211435421213/1289300556426233/2211233765714183/326824556671215AVG247.0566.1619.05

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

Control positive

Control negative

Control sample Control positive, Control negative, Control sample

Neutralizationpppppp 6

1000 L1000 L1000 L1000 L1000 L 24 48 h.Pour plate100 L10-110-210-310-410-510-610-710-8100 LPour plate

Carrier 10 100