Title: Cuticle and Pollen Image Prep
SOP ID #:Pollen02
Revision #:6
Date: December, 2017
Page 1 of 4
Pollen Image Prep
Standard Operating Procedure
Lab: Punyasena
Department: Plant Biology
Written By: M. A. Urban
Section 1: Overview
Type of SOP:
☒Process
☒Hazardous Material
☐Hazardous Class of Chemicals ☐Equipment
Synopsis:
This SOP describes the preparation of cuticle and pollen for imaging with SR-SIM, Airyscan and
Nanozoomer
Section 2: Risk Assessment Summary (Hazards and control measures)
Materials:
Material (name, CAS #, other ID)
Isopropyl alcohol [63-67
Xylenes [1330-20-7]
Eukitt [94555-21-2]
Periodic Acid [10450-60-9]
Schiff’s reagent [507-09-5]
Triton X [9002-93-1]
Hazards
Flammable
target organ toxicity, irritation, flammable
target organ toxicity, irritation, flammable
Flammable, peroxide forming, carcinogen, target organ toxicity
Flammable, health hazard
Irritation
Relevant References for Material Hazards:
All found in printed in Safety Binders and online in Punyasena Lab Safety Documentation Box folder.
Equipment hazards:
Equipment is considered low hazard.
Hazardous Conditions:
Conditions are considered low hazard.
Technique Hazards:
Transferring xylene via syringe.
Rinsing excess xylene from tubes.
Mounting material to slides with Eukitt
Title: Cuticle and Pollen Image Prep
SOP ID #:Pollen02
Revision #:6
Date: December, 2017
Page 2 of 4
Personal Protective Equipment
The following are in addition to proper dress for work in a laboratory:
Nitrile disposable gloves (minimum)
Flame resistant lab coat
Engineering Controls
This procedure is done in a chemical fume hood.
Section 3: Procedures
1) Pollen material is allowed to rehydrate w/ water and 1 ml Triton X
2) The material is placed in Pluriselect sieves and incubated in periodic acid for 30 min
3) Material is then given three washes with ddH20
4) Schiff’s reagent is added to sieves and allowed to incubate until a color change occurs in the material
5) Material is washed with ddH20 until runs clear
6) Material is then given a stepwise dehydration from water to 100% Xylene. (70% isopropanol, 80%
isopropanol; 100% isopropanol; 2:1 iso/xylene; 1:1 iso/xylene; 1:2 iso/xylene; 100% xylene). Each
incubation lasts 5 minutes.
7) Material is transferred into centrifuge tubes and 1 drop of Eukitt mounting media is placed in tubes.
8) Incubate samples overnight
9) using a pipette, drain the tubes of xylene and add appropriate amount of Eukitt; mix with wooden stick
10) Apply small droplet of Eukitt/sample mixture to slides
11) place slide cover on top of droplet
12) Depress the slide cover slightly with your finger
13) Allow to dry overnight in fume hood.
Special clean up procedures:
1) All liquid waste should be stored in an appropriately labeled waste container
2) All solid waste should be stored in an appropriately labeled 1 gallon Ziploc bag
3) Sieves should be soaked overnight in water to allow xylene to evaporate off
4) Sieves should then be placed in a clean sonic bath. Turn the bath on for the maximum time. Afterwards
remove the sieves, rinse and place in drying rack.
Section 4: Waste Disposal/Cleanup
Disposed liquid Xylene/Isopropanol mixture should be placed into appropriate waste container. Solid waste
(transfer pipettes) should be placed in a sealed 1 gallon ziplock bag. When requesting a waste pick up these
chemicals should be noted in the form: Xylene, Isopropanol, Perioidic Acid, Schiff’s Reagent.
Section 5: Emergency Response
An eyewash and sink are nearby
Review the location of nearest safety shower before starting the procedure.
In case of a fire, identify two routes to exit the room.
Title: Cuticle and Pollen Image Prep
SOP ID #:Pollen02
Revision #:6
Date: December, 2017
Page 3 of 4
Section 6: Additional Information
Advice:
1.
2.
Allow all disposable tips and pipettes to dry before placing them in ziploc bag
Allow all tubes and beakers to dry before removing them from the fume hood. Xylene is highly
volatile and will produce a powerful odor if left out of hood.
Title: Cuticle and Pollen Image Prep
SOP ID #:Pollen02
Revision #:6
Date: December, 2017
Page 4 of 4
Training Documentation
Signing this document means that you have read and understand all aspects of this Standard Operating Procedure.
The supervisor is the person that acknowledges you took the training and understand the procedure. They can be a
lab manager or researcher assigned by the PI to oversee this particular SOP.
Name (Printed)
Name (Signed)
Supervisor
Date