TITLE: Step by Step from Environmental Samples to Preservation Vials
AUTHORS: phangguanjie

[DESCRIPTION]
This protocol shows how to generate a MICROBES ISOLATION FROM ENVIRONMENTAL SAMPLES into LAB COLLECTION. Please follow the instructions and do not modify the protocols by yourself. If any changes are needed please inform the lab manager or admin.

[STEPS]
SECTION: Isolate Primary Culture from Environmental Samples
1. Once you received your environmental samples. 

Please catalog them in your Environmental Samples spreadsheet.
   


SECTION: Upload your datasheet to Specify database
3. **The account setup is skipped**

Login to the Specify database. Choose the first collection (HuangLab-Insect & Environmental & MacroFungi).
  
 

SECTION: Isolate Primary Culture from Environmental Samples
2. Fill in the columns of the spreadsheet step by step (As shown in the following steps 2.1-2.15)
*In the template, some template data are there for your reference, please delete them in your copy and fill in only your data.
* Remember to scroll to the right and fill in as much as information you can.
    

SECTION: Isolate Primary Culture from Environmental Samples
2.1. Leave the Catalog Number (Column A) blank. The Catalog Number will be generated automatically from Specify. Copy and paste it directly after the upload.
   

SECTION: Isolate Primary Culture from Environmental Samples
2.2. Accession Number (Column B) is for your own reference, you can name your sample as you want, there is no specific format for this.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.3. Field Vial (Column C) is the iNaturalist code. You only need to fill this column when you got the samples from citizen scientists.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.4. Leave the Vial Extracted From (Column D) blank.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.5. For Column E, F, G, G:
If your samples are SOIL SAMPLES, please follow this example.
  
If your samples are ENVIRONMENTAL SAMPLES, please follow this example.
   

SECTION: Isolate Primary Culture from Environmental Samples
2.6. Fill in the Verbatim Date (Column I) with the date you COLLECTED the samples. 
Fill in the Cataloged Date (Column J) with the date you UPLOADED the data to Specify.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.7. Collector First Name (Column K) and Collector Last Name (Column L)
Fill in the name of the collector.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.8. Fill in these columns when the sample is identified with a scientific name, remember to fill Column E and F (Genus and Species names) as well. If not, leave it blank. 
Fill in the date you did the determination (identification).
   

SECTION: Isolate Primary Culture from Environmental Samples
2.9. Fill in these columns with the geographical information of your samples. 
Leave it blank, when you don't have the information or the materials is outsourced lacking this info.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.10. The values of these columns were set using a formula. You only need to choose the Prep Type (Column W), which is the way you preserve your samples.
*Inform admin when other Prep Type is needed.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.11. Fill in the name (Column AA and AB) of who uploaded the data to Specify.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.12. You can leave any informative notes of your samples. If not, leave it blank.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.13. Done!! Download the spreadsheet as .csv format.
  

SECTION: Isolate Primary Culture from Environmental Samples
2.14. Delete the row with existed Catalog Number if any. 
**Leave the not-uploaded rows only.
   

2.15. Rename the file with the date you are going to upload the datasheet to Specify.
  

SECTION: Upload your datasheet to Specify database
3.1. Go to the Workbench tab. 
  

SECTION: Upload your datasheet to Specify database
3.2. Click Import Data.
  

SECTION: Upload your datasheet to Specify database
3.3. Choose the file you wanted to import (upload), and click Open.
  

SECTION: Upload your datasheet to Specify database
3.4. Let everything as default. Then, click OK.
  

SECTION: Upload your datasheet to Specify database
3.5. Select the second option: Use an existing Data Set mapping.
  

SECTION: Upload your datasheet to Specify database
3.6. Check for any invalid cells (red highlighted cells). 
Remember to tick Auto-fill Catalog Number (marked with red box).
   

SECTION: Upload your datasheet to Specify database
3.7. Click Upload (pointed with red arrow).
   

SECTION: Upload your datasheet to Specify database
3.8. Click Upload again (pointed with red arrow).
    

SECTION: Upload your datasheet to Specify database
3.10. Click Yes to finish the upload progress.
  

SECTION: Upload your datasheet to Specify database
3.9. Then Click Commit (marked with red arrow).
   

SECTION: Upload your datasheet to Specify database
3.11. Now you can go to Query -> catNumber determination...(under the Saved Queries in left panel) to check if the upload succeeded.
Click Search to start querying.
   

SECTION: Upload your datasheet to Specify database
3.12. Ta-Da!! The upload was succesful.
   
**Remember to delete the Data Sets & Record Sets before closing the Specify.
   

SECTION: Isolate the microbes from your environmental samples
4. Prepare the equipment and materials first for sample isolation. Put them on another bench first.
   

SECTION: Upload your datasheet to Specify database
3.13. Paste the Catalog Number of uploaded data back to your spreadsheet.
SECTION: Isolate the microbes from your environmental samples
4.3. Before isolation, key the information about your isolation (how many plates you are going to isolate, what kind of media you will use, etc) into the 1 Fungi Isolation Plates (Direct edit) 菌株分離培養基編號 (直接編輯) spreadsheet first
  
SECTION: Isolate the microbes from your environmental samples
4.1. Sterilize the microbes isolation area (air and bench) using a 75% ethanol sprayer.
**Make sure the mist is emitted.**
                                                                         
                                                  
    
SECTION: Isolate the microbes from your environmental samples
4.2. Now, move the equipment and materials for microbes isolation back to the sterilized area.
SECTION: Isolate the microbes from your environmental samples
4.4. Write the plate information on the downside of the plate following the example photo shown below.
  
SECTION: Isolate the microbes from your environmental samples
4.5. Make sure that you sterilize every tool before using them for each isolation:
1. Dip the scalpel, tweezer, or spreader into 95% ethanol cooling bottle, and flame it immediately. 
2. Repeat the step(dip and flame) THREE times for sterilization of equipment.
SECTION: Isolate the microbes from your environmental samples
4.6. Now, you can start to isolate microbes from your environmental samples. We called them primary cultures.
**Isolation method varied depend on the type of sample, check published papers or consult with  seniors if needed**
SECTION: Isolate the microbes from your environmental samples
4.7. After finishing all the isolation, seal the plates with grafting tape for 2 rounds.
   

SECTION: Isolate the microbes from your environmental samples
4.9. Check the growth condition of the primary cultures every day. Make sure they are not overgrown.
Once they are matured, you can start to subculture them.
SECTION: Isolate the microbes from your environmental samples
4.8. Pack all the plates using a PP bag (no. 8, 10, or 12 depending on how many plates you have). Incubate the plates in your slot in the incubator for 7~21 days (depending on the growth rate of the microbes).

no. 8: 8 plates
no. 10: 24 or 32 plates
no. 12: 48 or 56 plates
  

SECTION: Record and upload the data of primary cultures
5. 1. Examine the microbes colonies under a dissecting microscope. 
2. Choose your desired colonies, which are your targeted colonies.
3. Mark the targeted colonies with their later corresponding secondary plate number. 
4. If there is more than one morphotype targeted colony in a certain plate, you need to record them into a distinct plate number, e.g., TWO morphotypes will have TWO distinct secondary plate numbers from the same primary plate number.
  

SECTION: Record and upload the data of primary cultures
5.1. Record all the data of primary cultures to the second worksheet: Secondary Isolate PDA (in 1 Fungi Isolation Plates (Direct edit) 菌株分離培養基編號 (直接編輯)). 
Fill in all the columns following the photos and instructions shown below.
 
  

SECTION: Record and upload the data of primary cultures
5.3. Before scanning plate, you need to sterilize the environment and scanner glass using paper towels with 75% ethanol.
                    

SECTION: Record and upload the data of primary cultures
5.5. Keep the order of the plates, remove the plates cover, and put the plates upside down onto the scanner glass.
**Do not change the order before and after scanning**
  

SECTION: Record and upload the data of primary cultures
5.4. Wipe the scanner glass using paper towels with dishwashing detergent (this prevents water condensation on the glass).
                    

SECTION: Record and upload the data of primary cultures
5.2. Record or take a photo of the order of your plates that are ready for scanning. 6 plates as a group, so you can refer back during the photo editing.
   

SECTION: Record and upload the data of primary cultures
5.6. Search and open EPSON Scan software. Choose Professional mode. Go to the File Save Settings (red arrow), and make sure the image format is .jpg (follow all the settings as the photo below shown).
   

SECTION: Record and upload the data of primary cultures
5.7. Then, click OK. (It will start scanning like the photo shown below).
   

SECTION: Record and upload the data of primary cultures
5.8. Move the files from your Pictures folder to the D:\PlateSegmentation\images
  

SECTION: Record and upload the data of primary cultures
5.9. Search and open Anaconda Navigator software.
  
You will see the interface as the photo shown below.
  

SECTION: Record and upload the data of primary cultures
5.10. Select onxx under Environments tab (as the photo shown below). 
  
Left click the play button. Select Open Terminal.
   

SECTION: Record and upload the data of primary cultures
5.11. Follow the commands, and you will have the similar output as the photos shown below.
Type D: , then press enter
 
Type cd PlateSegmentation , then press enter
  
Type python ONXX_predict.py , then press enter
 
Press enter again and again. You will see a _ flashing.
                    
Then, a new command line comes out, which represent the program execution was complete.
   

SECTION: Record and upload the data of primary cultures
5.12. The output files are in the D:\PlateSegmentation\output. 
  
Rename the output files with their corresponding plate number.
 
Upload them to the HuangLab Collection/Plate picture/20XX
 
SECTION: Subculture the primary cultures
6. Follow   to   for the subculture of primary cultures. Now, we called them secondary cultures. 
You may need to keep the primary cultures for a while before the secondary plate are all secured.
SECTION: Ready for preservation
7. After 7-21 days (depending on the growth rate of your targeted microbes), the secondary cultures are ready to be preserved. If the secondary cultures were contaminated, please redo the subculture from the primary cultures.
SECTION: Ready for preservation
7.1. Before prepare preservation vials, the For Specify cateloging COPY to MODIFY!!! worksheet need to be filled.
 
   
There is 2 part for all the locality information, you need to replace the each information: spreadsheet code (red arrow pointed), worksheet name (purple arrow pointed) with your own spreadsheet information.
  
If the Cataloged Date is filled, the Column U to Column Z is auto-filled with the written formula. Do not change it unless it needed. The Column AA is synchronized with the Column O of the Secondary Isolate PDA worksheet.
    

SECTION: Ready for preservation
7.2. Once the spreadsheet is filled, download the data with .csv format.
Open Specify, go to HuangLab-Microbes collection.
  

SECTION: Ready for preservation
7.3. Follow   to   for preservation vials data upload.

SECTION: Ready for preservation
7.4. For each Catalog Number, you must prepare 2 water vials and 2 glycerol vials. You also need a 2 ml microtube with lysis mixture and beads (for details, check DNA extraction protocols).

Each tube needed a sticker with its corresponding Catalog Number on top of the cap. The label should be written with a black color marker pen or a technical pen alternative. 

Please follow the example in the photo shown below.
 
The lysis mixture microtubes that are ready for use will look like this.
  

SECTION: Ready for preservation
7.5. Operate UV sterilization of the laminar flow for 15 minutes. After UV sterilization, you need to ethanol-sterilize the materials that are going into the laminar flow. 

Light up the alcohol lamp and flame the material.

**The materials you need are the same as   , but you only need a scalpel instead of a tweezer and a spreader.**

SECTION: Culture preservation
8. Preserve the plates with one water and one glycerol vial for one colony. The rest is for DNA extraction. Make sure you flame-sterilize the scalpel every time before slicing the agar.

**The details of culture preservation are not provided here.**
SECTION: Culture preservation
8.1. Store the preservation vials in their corresponding freezing box. 

For water vials, you can keep them directly.

For glycerol vials, you need to keep them in a freezing container and then freeze them inside a -20ºC fridge for 1 day before keeping them in the -80ºC fridge collection.
**The freezing boxes of water vials are kept in the steel cabinet in front of Prof. Huang's office; the freezing boxes of glycerol vials are kept in the -80ºC fridge outside N927**