Negative Stain Grid Preparation
Category: Sample preparation
Sub-category: Room temperature work
Certification for use of negative stains and the negative stain work area.
Safety
You must complete all required safety training at the center you are visiting before hands on training. Coordinate this with the user office or training coordinator.
Background
Relevant Training Modules (to review until you can pass the knowledge test):
- Review the basics of room temperature methods in EM in Part 4 of Getting Started in cryoEM
- Review negative stain fundamentals for single particle analysis in CryoEM 101: Chapter 1 part 5
Videos of staining procedures
The following resources will show you negative staining protocols:
- Jove article showing to methods of staining: https://dx.doi.org/10.3791/57199
- The “side blot method” which is shown in the video included in this reference, at minute 1:08: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912373/
Additional protocols and useful references:
- A trusted reference for negative staining: https://pmc.ncbi.nlm.nih.gov/articles/PMC389902/
- A study of commonly used detergent micelles by negative stain: https://pubmed.ncbi.nlm.nih.gov/29852220/
- Using non-radioactive stains: https://pubmed.ncbi.nlm.nih.gov/39601624/
SOP for Negative Stain Grid Preparation (for review before demonstration)
The pass/fail knowledge assessment will test understanding of the following concepts:
- Safety precautions for working with stains
- What types of grids are used and why
- The purpose of the main steps in the staining protocol
- Common modifications of the protocol for troubleshooting
Here is the current quiz version:
A workflow demonstration should be scheduled with center staff. The demonstration will cover all points of the SOP. You must have passed the knowledge quiz and reviewed the SOP before the demonstration.
Each center has additional specific Safety, Setup, and Shutdown procedures. Below, you will find those protocols for each center.
- PNCC Site Specific Procedures – Negative Stain
- NCCAT Site Specific Procedures – Negative Stain
- S2C2 Site Specific Procedures – Negative Stain
When you’re ready for your training, schedule your supervised practice with the user office or appointed user coordinator. This can be scheduled for the same session as the demonstration. During supervised practice, center staff will be present for the full duration of the session to provide feedback and answer questions with the goal that you can pass the practical test for independence with no assistance. The number of supervised practice sessions will be tailored to the user, and if necessary, more than one session can be scheduled before taking the practical test.
Center staff will assist in arranging time for access to the necessary equipment and testing for independence. The practical test can not be scheduled during the same session as supervised practice.
A Negative Staining independence checklist will be used by the certifying staff to test for proper use of the instrument with a pass/fail outcome. All steps on the checklist must be performed to pass. You may use any instructions normally posted as a guide during the session.
Recertification period
- Sample preparation merit badges for room temperature work are valid for 12 months.
- Recertification (to maintain active status) requires passing only the practical test again with one center staff member. If supervised training is needed to pass the practical test, this can be arranged.
