Title: Cuticle and Pollen Image Prep
SOP ID #:Click here to enter text.
Revision #:5
Date: December, 2016
Page 1 of 3
Cuticle and 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]
Hazards
Flammable
target organ toxicity, irritation, flammable
target organ toxicity, irritation, flammable
Flammable, peroxide forming, carcinogen, target organ toxicity
Flammable, health hazard
Relevant References for Material Hazards:
All found on printed in Safety Binders and online in lab manager’s 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
Personal Protective Equipment
Title: Cuticle and Pollen Image Prep
SOP ID #:Click here to enter text.
Revision #:5
Date: December, 2016
Page 2 of 3
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 and cuticle material is allowed to rehydrate w/ water and 1 ml Triton X
2) Following day 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.
Section 4: Waste Disposal/Cleanup
Disposed Xylene/Isopropanol mixture should be placed into appropriate waste container
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.
Section 6: Additional Information
Advice:
1.
2.
Allow all disposable tips and pipettes to dry before placing them in trash bag
Allow all tubes and beakers to dry before removing them from the fume hood
Title: Cuticle and Pollen Image Prep
SOP ID #:Click here to enter text.
Revision #:5
Date: December, 2016
Page 3 of 3
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