CONFIDENTIAL

Analytical 360 Standard Operating Procedure
Title:

Procedure No: Version No: Issue date: Page:

1 1 7-09-13 1 of 5

Hydrogen Peroxide Use In Cannabis Cultivation
Edward Stremlow
Prepared by

Randall Oliver
Approved By

07/09/13
Review Date

1.0

PURPOSE This document describes how to perform the collection and microbial analysis of flower samples at the Analytical 360 lab. Analytical 360 will analyze microbial levels (total aerobic plate count and yeast & mold plate count) and absence of Salmonella and E. coli, in cannabis samples which have been treated with H202 immediately after cultivation. Samples will also be analyzed prior to, and during each of the drying and curing stages of the flower; to determine effectiveness in reducing pathogens in H202 washed cannabis as compared to untreated flowers. “Aerobic plate count and yeast & mold plate count. The USP microbial limit test is performed at Analytical 360 and is used to estimate the total number of viable micro-organisms present and for the absence of specific pathogens in products of all kinds. The microbial limit test is based on each viable microbe present forming a colony. The number of the colony-forming units, CFU on the plate per gram determines the acceptability of the product. Microbial Counts (USP 2012) provide meaningful information regarding the microbial acceptability of products.” Hydrogen Peroxide is used for oxidation, bleaching, sterilization and as a disinfectant in a variety of industries. In recent years, many new agricultural applications for hydrogen peroxide have emerged as an effective measure for combating pathogens in livestock, fish aquaculture, and is also used as a sterilization agent which is sprayed on crops. Studies have also shown H202 treatment in water helps promote root and vegetative growth, improving crop yields, and reduce fungal diseases. The purpose of this study is to determine whether H202 treatment is a viable means of reducing fungal diseases and improve crop viability after cultivation. The scope of this project is not intended to analyze possible beneficial applications of H202 during the growing stage, and is only intended to analyze effectiveness after cultivation. Cannabinoid profiling will also be analyzed to determine whether H202 treatment will compromise quality or potency.

2.0

SCOPE This SOP applies to all cultivators donating samples and lab technicians performing microbial analysis of flower samples. RESPONSIBILITY Donation, Collection and Delivery of Samples - Cultivators Microbial Analysis -This procedure must be performed by Analytical 360 Lab Technician. Organizing Cultivators – Cannabis Action Coalition

3.0

CONFIDENTIAL
Analytical 360 Standard Operating Procedure
Title:

Procedure No: Version No: Issue date: Page:

1 1 7-09-13 2 of 5

Hydrogen Peroxide Use In Cannabis Cultivation
Edward Stremlow
Prepared by

Randall Oliver
Approved By

07/09/13
Review Date

4.0

MATERIALS Cultivators – Sterol, sealed Bag for transportation of samples, Cannabis Samples Cannabis Action Coalition – 35% Food Grade Hydrogen Peroxide, Water, Two 510 gallon Holding/Wash tubs, Peroxide test strips. Analytical 360 - TSA plate (trypticase soy agar), Sab Dex plate (Sabouraud dextrose agar), 2 mortar & pestles, 2 foil pieces approx. 3” x 3”, scoop, tweezers, 70% isopropyl alcohol (IPA), beaker, HEPA mask, gloves, permanent marker

5.0

EQUIPMENT Cannabis Action Coalition – Holding/Washing Tubs, Peroxide test strips. Analytical 360 - incubator (See SOP## for incubator operating instructions) Biohazard box Scale (See SOP## for scale operating instructions) Torch

6.0

PROCEDURES: Cultivators:

1. Remove 3 branches or stems from each plant, containing either 1 large flower, or several small to medium size flowers. Leave all foliage on branch. 2. Place samples in a large sterol, and sealable bag to prepare for transportation. Take caution to limit or avoid contamination. 3. Samples collected from different plants need to be separated in separate bags. 4. Deliver to Analytical 360, Tuesday, July 16th between 10AM – 5PM. Cannabis Action Coalition: 5. Coordinate with a maximum of 6 cultivators for the sample collection and delivery of 6 different cannabis strains. 6. Oversee the collection of supplies and assist with H202 treatment and cleanup. 7. Prepare 2, 5-10 gallon washing tubs. 1 tub treated with H202 for soak, and 1 tub with untreated water for rinse. 8. Mix 1/2 pint of 35% food grade H202 per 10 gallons of water. 9. Prepare untreated water for rinse. 10. Soak plants for 3-5 minutes in treated H202 and rinse following treatment. 11. Hang Plants for drying and prepare for transportation, for drying and curing offsite.

CONFIDENTIAL
Analytical 360 Standard Operating Procedure
Title:

Procedure No: Version No: Issue date: Page:

1 1 7-09-13 3 of 5

Hydrogen Peroxide Use In Cannabis Cultivation
Edward Stremlow
Prepared by

Randall Oliver
Approved By

07/09/13
Review Date

ANALYTICAL 360: Collect all samples required for initial analysis and equipment necessary Put on lab coat Wash hands Put on gloves Change gloves anytime you touch something that has not been disinfected. 5. Disinfect the torch, table surface, and other equipment with an IPA wipe 6. Pour isopropyl alcohol into the beaker 2 cm deep and submerge implement tips (scoop, tweezers) 7. With a permanent marker, label the bottom of the TSA plate and Sab Dex plate with sample's ANL number 8. Remove implements from IPA 9. Close all IPA containers and place in designated area 10. Make sure the IPA has dried from disinfected surfaces 11. Start the torch and set on low 12. Flame the tips of implements (scoop, tweezers) 13. Make 2 foil boats 14. Weigh foil boat A and tare scale 15. Using tweezers, transfer herb from container to foil boat 16. Weigh 0.1 g herb 17. Using IPA in beaker, rinse mortar & pestle A 18. Place IPA in designated area 19. Flame the mortar & pestle 20. Dump herb from foil boat into mortar 21. Grind herb in mortar & pestle 22. Open the two control plates and set them at the back of your work area 23. Using scoop, scrape herb away from the mortar sides and distribute evenly on the bottom of the mortar. Perform dilutions in base and prepare samples for TSA plate. 24. Quickly open TSA plate, upend mortar, and dispense solution into TSA plate. 25. Quickly cover plate with its lid 26. While the plate sits on the table, shake the plate a little back and forth to distribute herb across the agar 27. Weigh foil boat B and tare scale 28. Using tweezers, transfer herb from customer's container to foil boat 29. Weigh 0.1 g herb 30. Rinse mortar & pestle B with IPA 31. Flame the mortar & pestle 32. Dump herb from foil boat B into mortar 33. Grind herb in mortar & pestle 34. Using spatula scrape herb away from the mortar sides and distribute evenly on the bottom 35. Perform dilutions in base and prepare samples for Sab Dex. 36. Quickly open Sab Dex plate, upend mortar, and dispense contents into plate 37. Quickly cover plate with its lid 1. 2. 3. 4.

CONFIDENTIAL
Analytical 360 Standard Operating Procedure
Title:

Procedure No: Version No: Issue date: Page:

1 1 7-09-13 4 of 5

Hydrogen Peroxide Use In Cannabis Cultivation
Edward Stremlow
Prepared by

Randall Oliver
Approved By

07/09/13
Review Date

38. While the plate sits on the table, shake the plate a little back and forth to distribute solution across the agar 39. Replace the lids on the control plates. 40. Check the incubator temperature – it should be 98°F See SOP ##-6.0 for incubator operation instructions 41. Place TSA sample plate and control plate lid down in oven 42. Check growth after 1 day. If numerous colonies are present, count them now Otherwise, incubate TSA plates in 98°F oven 2 days 43. Check the biohazard box temperature – it should be 72°F 44. Place Sab Dex sample plate and control plate in biohazard box 45. Check growth after 1 day. If numerous colonies are present, count them now Otherwise, leave Sab Dex plates at room temp in biohazard box for 4 days Assessing colony forming units (CFU):  Set plate on grid and count the total number of colonies  Count number of colonies on plate  Determine CFU/gram: number of plate colonies x 10 = CFU/gram  Record CFU/gram for TSA and Sab Dex plates in the log  Refer to the table of Flower Microbial Limits below  If CFU is greater than allowable limits, mark positive in Positive/Negative column  If no CFU are visible, mark negative in Positive/Negative column 1. If any growth is seen on the TSA plate, determine presence/absence of Salmonella, E. coli by referring to Salmonella/E. coli ID method Flower Microbial Limits (USP 2012)  TSA plate: total aerobic 1,000,000 CFU, absence of Salmonella spp, E. coli in 10 g  Sab Dex plate: total combined yeast & mold 1,000 CFU 1.0 DOCUMENTATION Lab Technician Training Requirements (TR## – Binder 1) Flammable Materials Safety Training (TR## – Binder 1) Equipment Operation Manuals: Incubator (SOP## – Binder 2), Scale (SOP## – Binder 2), Torch (SOP## – Binder 2) [required training for equipment is in its SOP] SAFETY IPA is flammable – refer to material safety data sheet and adhere to proper fire precautions Cultured agar plates contain potentially pathogenic microbes – use caution and personal protective equipment. TRAINING REQUIREMENTS

2.0

3.0

CONFIDENTIAL
Analytical 360 Standard Operating Procedure
Title:

Procedure No: Version No: Issue date: Page:

1 1 7-09-13 5 of 5

Hydrogen Peroxide Use In Cannabis Cultivation
Edward Stremlow
Prepared by

Randall Oliver
Approved By

07/09/13
Review Date

Lab Technicians must pass training in basic lab operations (TR##), proper use of the required equipment (TR##), safe use of flammable materials (TR##, SOP##), microbial analysis training (TR##). 4.0 ATTACHMENTS Microbial Log ANL # Time In TSA Sab Dex

Date

Time Out

Date

Pos/Neg

CFU

Comments

3M Petri-Film: AOAC International Official Methods of Analysis Petri-Film Method Supplies: Plates: Aerobic count, Yeast & Mold count, Enterobacteriaceae, and E. coli/Coliform, Listeria 2 mini incubators 3 thermometers pipettes: 1000 ul, 20 ul pipette tips buffer soln NaOH (0.1 M) Eppendorf disposable micropipettes (2.0 ml) 3M Petri-Film plates ($100/50) spreaders sterilizer (70% etOH) Petri-Film plate reader - counts CFU in 4 sec

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