Yeast-NS1 Project SOP Outline
Offline Optimization and ValidationRoom 1031 • All yeast growth will be minimized to room 1031 of the main wet lab located on the first floor. • Room 1031 has been outfitted with all necessary instrumentation for yeast growth, harvest, dispensing, and incubation. • There has been a sign posted on the door informing other lab members that these instruments are strictly for yeast cultures. • The following instruments are located in room 1031: • 1x floor shake/incubator • 2x stackable incubator • 1x Multidrop Combi • 1x spectrophotometer (for O.D. measurement) • 1x table top centrifuge with adaptors for microplates and conical tubes
2% Virkon is used for decontaminating the surfaces used in yeast work • Virkon – General Description • Efficacy against bacteria (including mycobacteria), viruses, spores and fungi • High level disinfection of laboratory equipment and instruments • A safe, easy to use product that can be used in a large number of applications and is environmentally friendly. • Chemistry and Mode of Action • VIRKON is a balanced stabilized blend of peroxygen compounds, surfactant, organic acids and an inorganic buffer system. • By oxidation of proteins and other components of cell protoplasm, resulting in inhibition of enzyme systems and loss of cell wall integrity. • Safety • VIRKON working solution has an exceptional safety profile. • The working solution has a low oral and dermal toxicity, no toxic vapor phase • Compatibility • VIRKON is compatible with a wide range of materials including stainless steel, plastics and rubber.
Offline Optimization and Validation • Yeast Culture and Plating Protocol: • 100 mls of a yeast will be cultured daily using disposable flasks • 2 x 50 mls of yeast will be harvested daily using disposable conical tubes • The harvested cells will be washed 1x 50 mls • The pellet will then be re-suspended in ~10 mls of medium and diluted accordingly for microplate dispensing • A multidrop cassette has been designated for yeast only dispensing in room 1031 and one has been designated for medium only • The used cassettes will be disposed of in biohazard waste box in Room 1031 • Any medium in which yeast cultures have been cultured in will be treated with 2% Virkon and disposed of using the sealed deposable flasks and conical tubes in biohazard waste box located in room 1031
Offline Optimization and ValidationAssay Protocol • Dispensing 2.5 ul medium to 1536-well plates using a dedicated Multidrop Combi located in the main lab (no yeast in assay plates) • Pining 23 nl compounds into medium-containing assay plates using offline Kalypsys pin-tool located in the main lab (no yeast in assay plates) • Transfer assay plates to room 1031 using a “yeast ONLY” designated sealable carrier and Dispensing 2.5 ul of yeast suspension using the “yeast ONLY” cassette • Incubate the assay plates using the special labeled lids for yeast assays in room 1031 for 24-30 hrs • Transfer assay plates to room 1014 using a “yeast ONLY” designated sealable carrier and Read on the HRE Viewlux with yeast-only holders • After read is complete, the plates will be transferred back to room 1031 using the “yeast ONLY” carrier for sealing in plastic bags and disposing in the biohazard box • The area surrounding the Viewlux and the designated area in room 1014 that carrier will be placed will be decontaminated using 2% Virkon • Room 1031 will be decontaminated using 2% Virkon on a daily basis
Online Validation- Assay Protocol • The online Kalypsys system will be used to dispense 2.5 ul of medium into assay plates (no yeast in assay plates) • The online Kalypsys pin-tool will be used to pin compounds into the assay plates (no yeast in assay plates) • The plates will then be transferred manually to the HRE incubator (capacity is 200 plates / incubator) • The Multidrop Combo on the HRE system will be used to dispense 2.5 ul of yeast suspension into assay plate • The plates will be lidded and stored in HRE incubator for 24-30 hrs • The plates will be read using the HRE Viewlux • After each read, the plate will be sealed using the HRE heatseal and disposed of in a biohazard yeast only waste box that will be sealed on a daily bases during the screen • The Kalypsys metal lid will be collected in a bin located on the HRE and decontaminated using 2% Virkon followed by autoclaving
Online Validation- Special Procedure for Decontamination • The Kalypsys metal lids used during the yeast screening process will be meticulously kept separate from the rest of the lids and will only be released once they have been treated with 2% Virkon and autoclaved • Once again “yeast ONLY” cassettes will be used for dispensing yeast cultures and the cassettes will be disposed of in biohazard waste box designated for yeast in room 1014 after yeast screening is completed • The designated biohazard box in room 1014 will be sealed on a daily bases during screening as to eliminate yeast escaping into room 1014 • Once again all areas that have been in contact with yeast will be decontaminated using 2% Virkon (this includes the HRE system and surrounding areas of HRE system)
Online ScreenFinal Decontamination after HTS • Bioquell Inc. will present their decontamination technology using Hydrogen Peroxide Vapor (HPV) on March 13th 1:00-2:30 pm • This company was used by Dr. Clifton Berry’s BSL-3 lab when they transferred from their Twinbrook facility to Building 33 of main campus and they were highly recommended • I encourage all to attend this seminar that could be used in future for other projects!