H202 SOP 07.11.13

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CONFIDENTIALAnalytical 360 Standard Operating Procedure

Procedure No: 1

Title:

Hydrogen Peroxide Use In Cannabis CultivationVersion No:Issue date:

17-09-13

Page: 1 of5

Edward Stremlow Randall Oliver 07/09/13

Prepared by Approved By Review Date

1.0 PURPOSEThis document describes how to perform the collection and microbial analysis offlower samples at the Analytical 360 lab. Analytical 360 will analyze microbiallevels (total aerobic plate count and yeast & mold plate count) and absence ofSalmonella and E. coli, in cannabis samples which have been treated with H202immediately after cultivation. Samples will also be analyzed prior to, and duringeach of the drying and curing stages of the flower; to determine effectiveness inreducing pathogens in H202 washed cannabis as compared to untreated flowers.

Aerobic plate count and yeast & mold plate count. The USP microbial limit test isperformed at Analytical 360 and is used to estimate the total number of viablemicro-organisms present and for the absence of specific pathogens in productsof all kinds. The microbial limit test is based on each viable microbe presentforming a colony. The number of the colony-forming units, CFU on the plate pergram 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 adisinfectant in a variety of industries. In recent years, many new agricultural

applications for hydrogen peroxide have emerged as an effective measure forcombating pathogens in livestock, fish aquaculture, and is also used as asterilization agent which is sprayed on crops. Studies have also shown H202treatment in water helps promote root and vegetative growth, improving cropyields, and reduce fungal diseases.

The purpose of this study is to determine whether H202 treatment is a viablemeans of reducing fungal diseases and improve crop viability after cultivation.The scope of this project is not intended to analyze possible beneficialapplications of H202 during the growing stage, and is only intended to analyzeeffectiveness after cultivation. Cannabinoid profiling will also be analyzed to

determine whether H202 treatment will compromise quality or potency.

2.0 SCOPEThis SOP applies to all cultivators donating samples and lab techniciansperforming microbial analysis of flower samples.

3.0 RESPONSIBILITYDonation, Collection and Delivery of Samples - Cultivators

Microbial Analysis -This procedure must be performed by Analytical 360 LabTechnician.

Organizing Cultivators Cannabis Action Coalition

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Procedure No: 1

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4.0 MATERIALS

Cultivators Sterol, sealed Bag for transportation of samples, Cannabis Samples

Cannabis Action Coalition 35% Food Grade Hydrogen Peroxide, Water, Two 5-10 gallon Holding/Wash tubs, Peroxide test strips.

Analytical 360 - TSA plate (trypticase soy agar), Sab Dex plate (Sabourauddextrose 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 EQUIPMENTCannabis Action Coalition Holding/Washing Tubs, Peroxide test strips.

Analytical 360 - incubator (See SOP## for incubator operating instructions)Biohazard boxScale (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, orseveral 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 16 th between 10AM 5PM.

Cannabis Action Coalition:

5. Coordinate with a maximum of 6 cultivators for the sample collection and deliveryof 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.

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ANALYTICAL 360:1. Collect all samples required for initial analysis and equipment necessary2. Put on lab coat3. Wash hands4. 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 wipe6. 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 number8. Remove implements from IPA9. Close all IPA containers and place in designated area10.Make sure the IPA has dried from disinfected surfaces11. Start the torch and set on low12.Flame the tips of implements (scoop, tweezers)13.Make 2 foil boats14.Weigh foil boat A and tare scale15.Using tweezers, transfer herb from container to foil boat

16.Weigh 0.1 g herb17.Using IPA in beaker, rinse mortar & pestle A18.Place IPA in designated area19.Flame the mortar & pestle20.Dump herb from foil boat into mortar21.Grind herb in mortar & pestle22.Open the two control plates and set them at the back of your work area23.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 TSAplate.

24.Quickly open TSA plate, upend mortar, and dispense solution into TSA plate.

25.Quickly cover plate with its lid26.While the plate sits on the table, shake the plate a little back and forth to

distribute herb across the agar27.Weigh foil boat B and tare scale28.Using tweezers, transfer herb from customer's container to foil boat29.Weigh 0.1 g herb30.Rinse mortar & pestle B with IPA31.Flame the mortar & pestle32.Dump herb from foil boat B into mortar33.Grind herb in mortar & pestle34.Using spatula scrape herb away from the mortar sides and distribute evenly on

the bottom35.Perform dilutions in base and prepare samples for Sab Dex.36.Quickly open Sab Dex plate, upend mortar, and dispense contents into plate37.Quickly cover plate with its lid

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38.While the plate sits on the table, shake the plate a little back and forth todistribute solution across the agar

39.Replace the lids on the control plates.40.Check the incubator temperature it should be 98F

See SOP ##-6.0 for incubator operation instructions41.Place TSA sample plate and control plate lid down in oven42.Check growth after 1 day. If numerous colonies are present, count them now

Otherwise, incubate TSA plates in 98F oven 2 days43.Check the biohazard box temperature it should be 72F44.Place Sab Dex sample plate and control plate in biohazard box45.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 ofSalmonella, 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 DOCUMENTATIONLab 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]

2.0 SAFETYIPA is flammable refer to material safety data sheet and adhere to proper fireprecautions

Cultured agar plates contain potentially pathogenic microbes use caution andpersonal protective equipment.

3.0 TRAINING REQUIREMENTS

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Lab Technicians must pass training in basic lab operations (TR##), proper use ofthe required equipment (TR##), safe use of flammable materials (TR##, SOP##),microbial analysis training (TR##).

4.0 ATTACHMENTSMicrobial Log

ANL # Time In Date Time Out Date Pos/Neg CFU Comments

TSA

Sab Dex

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 incubators3 thermometerspipettes: 1000 ul, 20 ulpipette tipsbuffer solnNaOH (0.1 M)Eppendorf disposable micropipettes (2.0 ml)3M Petri-Film plates ($100/50)spreaderssterilizer (70% etOH)Petri-Film plate reader - counts CFU in 4 sec

H202 SOP 07.11.13

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