A-A-5139A
Inoculums Preparation.
Prepare suspension of Staph aureus and Enterobacter cloacae by washing the growth on the TSA slant into 4-5 ml of sterile phosphate buffer. Transfer 1.0 ml into a sterile cuvette. Adjust turbidity of suspension to appropriate optical density and dilute as follow. A minimum of 1000 organism/ml of product is required, not exceeding 5000 organisms per ml of product as upper limit.
|
Organism |
Optical Density at 580 nm |
|
Staph aureus |
0.4 |
|
Enterobacter cloacae |
0.2 |
Prepare the seed of Aspergillus niger by washing 6 SABS slants into 5 ml of phosphate buffer + 0.5% of TWEEN 80 in a sterile 16 x 100 mm test tube. Inoculate 0.1 ml of 10-2 dilution into 20 ml of the product.
Inoculate 0.1 ml of 10-3 to 0.1 ml of 10-2 (depending upon which spectrophotometer is used) dilution each of Staph aureus and Enterobacter cloacae into two 20 ml samples of the aluminum hydroxide gel.
Sample Inoculation.
Prepare seeds one at a time and complete all necessary dilutions prior to preparing next seed.
Verify inoculums by performing back/titer plates for each organisms.
Dilute each organism to 10-7 in PO4 and inoculate 2 plates per dilution with 0.1 ml to 10-5 -10-7 dilutions. Spread 0.1 ml inoculums over entire surface of plates using a sterile 10 ul inoculating loop or use 1 mk for pour plate. Incubate Enterobacter and Staph plates at 30-35oC for 24-48 hours and Aspergillus plates 20-25oC for 24-48 hours. Use TSA plates for Staph and Enterobacter and SABS plates for Aspergillus niger.
Pipet 0.1 ml of appropriate dilution of each organism into 20 ml of Aluminum Hydroxide Gel into labeled container. Mix well. Keep samples at room temperature for test intervals.
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