Recently in Protocols Category

Last summer I conducted a biweekly survey of aphids and ants in the common garden, an experimental prairie restoration containing Echinacea from various remnant populations. I was interested in the spatial distribution of aphids and ants within and across years. This year I plan to scale down my survey to once a month, beginning next Friday. Here's a detailed protocol:

Common Garden Aphid Survey Protocol 2012.doc

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Yesterday, the Mettler Toledo BALNT software was throwing up an error when it was started, preventing weighing.

Exact error message: BALNT.exe has generated errors and will be closed by Windows. You will need to restart the program. An error log is being created.

This seems to be an error with the config file in the C:\Windows or C:\WINNT directory. To fix this, take the zip file in I:\Departments\Research\EchinaceaVolunteers\Balance\ and extract it to the C:\ drive. This should overwrite the Balance-May2012 directory on the C: drive. Take the BALNT.ini file from that directory and copy it to C:\Windows or C:\WINNT, whichever exists. You should be able to start the balance software and begin collecting points.

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It's been a busy week for everybody. Plants are blooming, pollen is shedding, and everyone is dashing about madly to catch the field season before it passes us by. I have been running around like a chicken with its head cut off trying to get ready for my aphid addition/exclusion experiment. Everyone has been a wonderful help setting up cages in this sweltering heat. My goal for this week is to finish my first round of experimental treatments: exclusion on Thursday and addition on Friday. Before then, I need to finish setting up nets and teach everyone how to wrangle aphids. Here is a protocol I wrote up to assist the teaching process and the data sheets I mention in the protocol. These are works in progress, so any feedback is appreciated.




Happy wrangling,


View image

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I wrote this up last week, but neglected to post it on the flog. Here is a detailed protocol for the 2011 recruitment searches. See Wagenius et al. 2011* for a description of the seedling recruitment study.


*Wagenius, S., A.B. Dykstra, C.E. Ridley, and R.G. Shaw. 2011. Seedling recruitment in the long-lived perennial, Echinacea angustifolia: A 10-year experiment. Restoration Ecology.

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Today we surveyed a patch of the common garden for aphid infestation. I chose my survey area based on observations I made Wednesday morning, during my initial search for aphids. I wanted my square to include at least one heavily-infested plant with ant domatia (dirt structures that ants build to cultivate aphids). Otherwise my choice of positions and rows was random.

Here is a description of today's survey. This weekend I will go over the data and make a map of aphid infestation. Because it's still early in the season for aphids, I hope to repeat this survey one or more times this summer to look for spatial and temporal patterns of infestation. Thank you to the Echinacea team members for your diligent data gathering.

Aphid survey protocol 1June2001.doc

I'm happy to say the survey went smoothly. Everyone seemed to have an easy time recognizing aphid life stages and ant domatia. My only goof-up was accidentally assigning the same row to two people, leaving us one row short, but thankfully we caught it in time to finish up before a thunderstorm hit. Next time I will be more careful about my row assignments.

I would like to repeat this survey several times throughout the summer--maybe once every two weeks. Here are some thoughts I have based on my observations in the common garden:

1. We have observed that plants with heavy infestations early in the season tend to have wrinkly, stunted leaves--possibly due to aphid overwintering. I suspect that these plants may serve as aphid source populations that spread to surrounding host plants. It will be interesting to see whether aphid infestation is more prevalent among plants nearby heavy aphid infestations (i.e. plants with all three aphid life stages, wrinkly leaves, and ant domatia).

2. I noticed that on plants with small infestations, ants seemed to be carrying away gravid females. Perhaps ants play a role in mitigating population-wide aphid infestation by concentrating aphids on a few heavily-infested plants. This survey won't tell me much about the role of ants, but I am curious to see whether some plants lose their aphids throughout the season.

Thanks again everyone for helping me gather my first data set!

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Today we started the seedling search at Steven's Approach (SAP). The wind was strong and air temperature was chilly. We searched 3 circles; in one of the circles we found 6 seedlings! We drew a map and filled in a matrix, as we have done in previous years. We also tried out the new coordinate frame.
I (Amy) have revised the protocol. Please read it and feel free to suggest ways it can be improved.
Seedling Search Protocol 2011.doc

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Here's the protocol for creating demoMerge.csv
Protocol for creating DemoMerge.doc

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Here's the protocol for re-finds in the remnants. Please look it over, and critique!
Protocol for seedling refinds 2010.docx

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2010 GPS Surveying Draft Protocol.pdf

Here's the GPS Surveying Protocol. Post some comments with thoughts, ideas, and changes.

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2010 Stipa Draft Protocol.pdf. The Google Docs wasn't really working out... Comment with your thoughts and changes.

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In case we need help :)

Petri dish
mesh bags
twist ties
a partner!

1. Sync visor, get randomized plant (row and position) from Hillary.
2. Collect aphids from several plants in Common Garden. Find leaves with fat, dark aphids (aka mature). Gently disturb aphids with brush tip. When aphids start to scurry (remove stylets), brush them gently into the petri dish. Do not mash them by rubbing paintbrush against the plastic (attempting to dislodge them).
3. When approximately 20 mature aphids have been collected (not including tiny green ones), find assigned plant.
4. Check transfer plant for ants and aphids and record presence in form.
5. Find a suitable leaf (close to ground and small enough to fit in bag) that is free of any ants and aphids (if there are no empty leaves, squish present aphids).
6. Prepare bag over most of the leaf (opening bag and pulling over leaf). Then lift the bag up enough to stick the aphids in.
7. Transfer two random aphids to the top of the leaf (one big aphid and one slightly smaller). It works best if one partner holds the leaf and bag and the other transfers the aphids. Then the bag holder pulls the bag down and the transferrer twists the tie.
8. Pull bag completely over leaf and gently twist tie it closed. Avoid strangling the leaf or squishing or dislodging the aphids.
9. If aphids take a death plunge or fall off make sure they are not in the bag and transfer additional aphids as needed.

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Today we will start measuring Echinacea in the Common Garden. Here is the link to the protocol: CGmeasureprotocol2010.htm

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View image

So these are the Stipa that have been collected so far. I've labeled a couple of the places on the map.

I was having trouble projecting the data exported from GPS Pathfinder Office (trimble) and noticed that no coordinate system was defined (same issue with the DOQ maps Stuart gave me; the GeoTIFFs didn't have a spatial definition). The GPS data should be North American 1983 in the Geographic Coordinate System folder (right click on the data in ArcCatalog, hit the XY Coordinate System tab and hit the Select button). The DOQ maps ought to be using the NAD_1983_UTM_Zone_15N from the Projected Coordinate System folder. Dumping all of these files into ArcMap (and a little fiddling) gave me this nice map.

Next plan of attack is to make it work in GPS Pathfinder Office, as it's much less complicated than ArcGIS.

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Josh, Gretel, Hillary, and Ian are also trained on the TopScan to collect the GPS data. The ideal is that the radio signal is at 100% and the designation of the location is described as Fixed - (Float will do and Auto works if you are unable to connect to a radio signal)
Josh and I plan to collect from Hegg Lake and the road adjacent on Monday. In the quest to collect from 300 parent plants, we are likely onto roadsides - where we are trying to stay at least a meter off the road and using plants about 5m apart from each other. Generally, as the black color appears and as the capsule opens around the pointy head, the seeds are ripe and will pop off as you gently pull up the stem containing the seeds.
I plan to be around Sun afternoon and Mon. to finish the collection before starting to cross some plants.

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Here are the data on the three pollen types and the protocol for measuring.
I used the same plant/pollen from each plant and measured at least 30 different pollen grains from each. I didn't use any pollen if its pole faced forward - only if it was sideways.

Bad news - the Ech. ang. and Heli. heli. are very close. Good news - maybe the pollinators and plants can't tell them apart either.


PS - I am in SE Minn and the Monarda Sunflower and Miss. Goldenrod are in full bloom all over.

Protocol for slide image recording and measuring.docx

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So, I've been working hard creating slides from all the styles everyone helped to collect (thanks!), and this is the protocol I've been using:

1. Place the blank slide on a clean surface
2. Pull out the correct vial, open carefully (sometimes the styles are on the lid of the vial), use clean tweezers to remove the contents. Place vial contents onto the slide.
3. Remove any anther parts from the slide; organize the stigmas so they are separate/easily differentiated from each other.
4. Place one drop of glycerin on top of the stigmas. If there are any bubbles, try to move them away from the stigmas.
5. Place a cover slip carefully over the glycerin and allow it to settle.
6. Use mounting medium to seal the cover slip to the slide. Allow the slide to dry on a flat surface for 24-48 hours.
E. Put completed slides into slide boxes.

Of the approximately 380 slides I started with, I have 150 left to do, so I'm more than 1/2 way there. Once I've created all these slides, I'll start taking photos and uploading pictures.

Anyway, just felt like it was about time I updated. Gonna go make slides now... ^_^

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We have made a few changes to the protocol for tomorrow's FINAL outing for pollinator observation and collection:
1) DO NOT capture syrphid flies!
2) If you observe a small common syrphid fly (Family: Syrphidae, Sphaerophoria sp.) on your Echinacea head, make a note that one SCSF was observed. Here are some photos of syrphid flies:

Here's a video that might help:


As for pollinator collection and analysis...

We've gone out collecting three times now (tomorrow is the fourth and final day), and we have about 140 Echinacea insect visitors in vials in the freezer. Here is my protocol for making slides and labeling specimens:
1) Remove vial from the freezer and allow insect to thaw
2) Cut a very small (2mm cubed) piece of agar and situate it in the middle of a slide.
3) Remove the insect from the tube and dab and sweep every surface of the body across the agar cube. This is intended to simulate the amount of pollen that might be transferred to Echinacea styles during a visit.
4) Set the insect aside on Styrofoam.
5) Sweep the inside of the vial with the agar cube for any loose pollen.
6) Place a cover slip atop the agar cube and put the slide on the edge of a hotplate on LOW heat. Watch the slide carefully and remove it when the agar is soft but not melted (once melting starts, it will quickly boil and create bubbles in the slide).
7) Pin the insect (for identification if it is an unknown species, or quick-n-casual for a known species) and pin it with a new one letter, three number code.
8) Label the slide with the vial code and the specimen code.

I photograph the slides on the same day as I make them so that the agar doesn't dry out before the photo. To sample the slides, I have generated random pairs of numbers from 1 to 22 (inclusive). The cover slips are 22 x 22 mm and I use the ruler on the microscope to scroll to the randomly selected "plots" from one corner of the cover slip. From there, I take the photo at 40x. I take ten photos for each slide, and sometimes take more than one photo per plot if I capturing all of the pollen grains requires multiple focuses.

I will post some photos of this process soon, so hang in there!
As always, feel free to leave questions or comments.


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Please review this protocol for measuring plants in the common garden.

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Ech jenkins FNC protocol revised.doc
Ech Guide to Co-Fl Sp.xls

I have a photo guide but I can't upload it b/c the file is too large.

If you plan on helping with FNC, please read the documents above. It's important that everyone counts inflorescences the same way. Thanks!

Also, for tomorrow, there are a couple of notes I thought I'd add:
>Please make a note if you see ants on the head of the plant you are observing.
>Also make a note if it is mostly cloudy.
>Try to get to your site with about 10 min to spare so you can get your supplies ready and orient yourself with the placement of the flags to avoid time spent wandering in search of flags.
>Please try to start your observation as close to 8am as possible. End at 11am. Do not start an observation if you can't finish by 11.
>Remember that you will only be collecting styles at the end of the observation pd from now on. Clean your tweezers with your shirt in between collections.

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So, for those of you who were wondering what Team Echinacea will be doing tomorrow, here is the field protocol for the transect searches that we will be using.

Field Protocol.doc

Any questions, please let us know!

Also, we were searching for Stipa today (a prairie grass that Dr. Ridley is planning to add to the common garden), and this is the setup we used to mark the sites with the GPS:


The antenna allowed us to get about a 9 cm margin of error when using the Trimble. And yes, that is yours truly manning the antenna, ensuring that the carrier lock is not lost. We were all ready to tell the next person who asked us what it was that we were searching for nuclear waste.


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I have returned to take images of pollen as seen below. It will still be a few hours/days/more? to get the images as desired but this is a start. I am predicting some trouble to distinguish between coreopsis, helianthus, and echinacea so be ready to be distinguishing.
Attached are a protocol for pollen slides and the image of echinacea 7.1.09.ech7.1

Protocol for Pollen Slide Prep.doc

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Amanda, Kate, and I have combined our protocols and I've included a revised equipment list.
Echinacea PONS equip list.doc
Ech combined PollComp Protocols.doc

The following people have agreed (I think) to help with this project on the mornings of July 7th and July 9th, as well as July 21st and July 23rd. If you cannot for some reason, let us know tomorrow.

Stuart, Gretel, Caroline, Amy, Daniel, Greg, Megan, Mimi, Amanda, Kate. Thanks everyone!

Also, after some discussion with Allegra about her pollination and painting needs for this week, I think that we need to either A) give up one person and therefore one remnant to help Allegra or B) have the normal 4 observations for remnants with less than 8 plants instead of doing more observations within the 3 hr pd, and have those people help Allegra when they are done early with pollinator observations/style collection or C) a combination of A&B. Thoughts?

Note: I have included the protocol in with pollinator observations and style collections because we are using the same plants for all 3. However, 3 people can do this over the course of 3 pm's. Those who are helping so far are: Kate, Allegra (when not doing pm pollinations)

Friday we practiced catching insects for a half hour but much more practice is needed and those who were not there on friday need to be trained. I think a group training/practicing session before lift-off is imperative.

Today Daniel and Allegra helped me assess the flowering plants situation at some of the remnants we plan on using. We flagged flowering plants at: Riley (5),YOH (3), NRRX (4), LC (8), Steven's appch (3). Some of these sites had plants that seemed likely to flower by Tuesday so we flagged those plants as well.We randomly selected 8 plants for LC since there were 17 that were either flowering or seemed very likely to flower by Tuesday. There was only 1 plant flowering at NWLF so that site has been eliminated. We also noted co-flowering species for each remnant. Tomorrow we need to finish flagging and randomly selecting plants at the 5 other remnants.

Stay tuned for July 4th picnic and kick-ass sandcastle pictures!

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For some time, the powers that be on the Echinacea project have thought, "Wouldn't it be nice to compare all we've learned from coneflower with another important native component of the prairie?" Well, this summer, study of habitat fragmentation and its ecological and evolutionary impacts broadens to include an additional species. And the winner is ... Stipa spartea aka porcupine grass! The time is right now to collect the seed, which will be sown into the common garden later this summer.

Thumbnail image for stipa spartea.jpg

Here's a link to a page from the Bell Museum of Natural History about Stipa spartea.

Below, you will find the sampling protocol that Greg and I used to collect seeds from Staffenson Prairie this afternoon. Stuart may have thoughts to add and the strategy might change a bit as we run into the reality of different Stipa remnant populations. In addition to remnants, we will also be collecting seeds from roadside transects. Expect that challenge to be met later next week.

Protocol for sampling Stipa spartea in prairie remnants.doc

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As promised, you can read the protocol I've developed for collecting pollen styles from Echinacea plants. This protocol will be joined with Mimi and Amanda's to form one complete and very awesome experiment. You can find my protocol here:
Kate's Proposal_1.doc

Input welcome!


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We mowed most of the CG this morning. Putting flags in went smoothly. It helped that we left many flags overwinter. We mowed according to the plan established two years ago. We started removing clippings & pulling flags that marked fl pla from 2008.

I noticed a plant I do not recognize at R46 P~903. Also, in R14 near P870 there is a patch of somethings that is starting to spread. We should determine if it's a weed we should eliminate.

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Here are some clarifications about how mfl and immfl should be filled in for heads in and past the flowering stage. Selecting status "Flowering" means that the last day of flowering is certainly 4 days away or longer. If the last day of flowering is 3 or fewer days away, then select "End of flowering."


Flowering It is not necessary to fill in mfl, ffl, or immfl. (mfl and Immfl are presumed to be well over 11).

End of flowering Fill in mfl and immfl! (Both may 11.)

Last day of flowering Fill in mfl and immfl! (immfl should be zero.)

Done flowering Fill in mfl and immfl! (mfl & immfl should be zero.)

Note 1: When the action is xxxx, then fill in a status (usually: flowering, end of flowering or done).

Note 2: When status is "Flowering," "End of flowering," "Last day of flowering", or "Done flowering" then don't fill in ffl!


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Hi folks! Here's the protocol for measuring plants in the common garden this year. The protocol hasn't changed much from last year, but the description has improved; the protocol is now a html file and there are many nice images from 2007. Thanks to Jameson and Gretel for taking the photos. And thanks to the wonders of digital photography, Pendragon forms, the UMN library's blog, and contributors to this flog. Wahoo! Let the counting of leaves, ants, and aphids begin!

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A protocol for Team Video is in the early stages, and there are many aspects to consider. While it started slow, we have found a relatively quick and efficient system for placing cameras, taking down and storing cameras, and uploading videos. Unfortunately, many additional challenges await.


Our initial protocol for painting bees called for painting bees as they were collecting pollen on the flower heads using a small paintbrush. Before starting painting, we had created "paint bandoliers" that consisted of microfuge tubes filled with different colors of paint and then taped in a line with duct tape to keep them together. We ordered the colors according to the rainbow to make it easier to keep track of the colors. Each color was given a three letter abbreviation. Painting the bees with paint brushes was fairly easy, but the shape and thickness of the dot had the possibility of being very variable. After researching bee painting, in particular queen honeybee marking, it appeared that the ideal dot that would last the longest amount of time is circular and uniformly thin. To obtain this ideal dot, it was suggested that a piece of wire whose diameter was the size of the desired dot be used.

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The frequency of bee sightings has slowed down in the past couple of days, but in the mean time we have been typing up our updated protocols, and begun looking at the data that we've collected. Read on for detailed protocols, the musings of this year's Bee Team, and tips for next year's Bee Team.

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Here are some preliminary instructions on how to observe and record bee/ant presence or absence while we are doing phenology measurements. I thought that while we are looking so closely at each head, we might as well try to garner some information on the Hymenopteran vistors as well. It will not be continuous data, but rather a simple 'yes' or 'no' measurement for each plant (bees) or for each head (ants).


Bee collecting composite pollen, copyright Jon Sullivan


As you begin your phenology measurements only scan for bees once you are within 1m of the plant. If there are any moving, active bees on any head, mark the appropriate box in your phenology form. As soon as you touch a head for phenology measurements, stop recording any bee presence/absence. So, if you are looking at a head and a bee lands on an adjacent head, just ignore it. For bees, we are interested in bee presence for the ENTIRE plant, so as soon as you see one, you are finished entering data.

Remember, wee are only interested in active bees, so don't count sleeping bees, dead bees, bees caught by crab spiders or assassin bugs, etc.


Ant, copyright Alex Wild


Ant data are collected on a per-head basis. As you take each head into your hand and begin your anther count, notice if you see any ants on the top of the inflorescence (either ray OR disk florets). Don't make any special effort at looking underneath the inflorescence. If no ants make an appearance as you are handling the head, mark the head as having no ants, if ANY ants appear whilst you are searching the head, enter the data as such.


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Tomorrow marks the official kick-off day for the Bee Team. We plan to mark several species of generalist bees found in the common garden with paint spots on the thorax. Our painted bees will hopefully provide data that will answer questions about their population size in the common garden, home ranges of individual bees, and flight path distances between echinacea heads. Before we begin marking our bees tomorrow, we need to:
-find and/or make nails with rounded ends with which to paint the bees (nails are the recommended tool, according to Ian's online research, as they transfer thin, even circles) and
-make a pendragon form for the visors with which to collect data (this form should include date, time, bee species, paint color, plant on which the bee was originally observed, and a place for notes)

Equipment needed:
paint bandoliers
nails/other paint applicators
list of random numbers

Tomorrow's protocol:
1. Generate a list of random number 10-56. Distribute evenly divided lists of these numbers to the 2 groups of 2 people participating. Walk the rows of the common garden in the perscribed order with one person on each side of the row, scanning the row for Agapostemon virescens
2. When a bee is sighted, paint a circle circle on its thorax. After painting radio/yell to the other group and relay the color used, so as to avoid doubling up on colors.
3. Record data for each painted bee into the pendragon form on the visor

Depending on the success of our painting efforts in the morning, we will maybe begin to collect data for home range and population size estimates.

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Here is a draft for the video protocol. I'd love any useful comments you may have; it is definitely a work in progress, so if you read something and it isn't clear, please let me know and I will change it. Thanks, A.M.

Protocol for recording pollinators:
v.1.0 (Jun 27 2007)


List of heads to video
A few (~5) pin flags
Five 3 x 5 in. cards and a sharpie
Set of camcorders and battery packs
A radio

1. Get a list of heads that need to recorded from Andy the evening prior to the actual recording date. Each person will be responsible for 3-4 heads for that recording day.

2. Get to the farmhouse at 8am sharp so that you can start recording for sure by 9am.

3. Make a list of 'cue cards' for each head that you are to record. This involves writing:
plant location (row and position), color of twist tie and date on a 3 x 5 in. card. This is the first thing you will 'film' when going out to the CG, so that we can match up videos to the correct file.

4. Go to each head on the list and make sure that it is still flowering. If not, then add another plant (we'll supply > 5 heads per list), and make a note that the originally selected head is not flowering. If the plant IS flowering, then place a flag next to it so that you can find it easily. Go to the next head on the list.

5. Next, get the correct camcorder and battery (labeled A-J), put it on a tripod and put it in position over the inflorescence (head). The ideal distance is about 1 ft. away from the head with the camera zoom at about ½ max. zoom. You should be able to see the entire head; try to imagine identifying bees using your recorded image and adjust accordingly. Take a quick video of your 'cue card' for each head and then turn off the camera.

Set up all of your cameras first, before starting to record for pollinators. We want to start them all at the same time, so you will need to coordinate with other members of TEAM VIDEO to start synchronously (using your radios).

Make sure that there are no big branches, stems, twigs, etc. that could possibly wave in front of the camera, thus obscuring the inflorescence.

6. At more or less the same time, go to each camera and press the red 'record' button. Then, skedaddle away so that you don't influence the pollinators!

7. At 4pm, go and stop each camera. Disconnect the batteries and return the camcorders and batteries to Andy. He'll upload the video to a PC and re-charge the batteries for the next round of filming!


Mowing went well today. The crew did a good job. I have some notes:

Rows 10 - 33 got blasted from the east by the grass clipping etc from the mower.
Rows 35 - 56 got blasted from the west.
Row 34 didn't get blasted. Row 34 was chosen at random, row 38 was chosen last year. Each year before 2006 I blasted the whole garden from either the east or the west. That was too inefficient.

Here's the schedule of not-to-be-blasted rows for the next few years:

year row
2008 31
2009 39
2010 36
2011 28
2012 34
2013 27
2014 32
2015 29
2016 35
2017 37
2018 30

I ran over 4 flags (loose or bent) and didn't hit any rocks. There aren't very many new gopher mounds. Look for new mounds far N, Row ~ 35, Pos ~925, and Row 56. I don't think I ran over any Echinacea plants. I was running blind in R~40-42, up to P935, and P865 in R10-12. Also, I had to add flags in R 10 far N. The brome is flowering super thick this year. The CG looks so different from last year because of the brome. Some brome infl are eye-level W of the garden in pos <910! Those fence posts in R 13.5 and ~38.5 are annoying and must go. The cottonwoods need to go to too--too much shade. Deal with trefoil & phalaris.

I can think of some things I will do differently next year. I'll only do them next year if I remember. Next year I'll have to look at this flog to find the unblasted row. Here's the plan.

in fall, leave flags in the 98 garden or put in staples
get flags delivered in plenty of time (consider color coordination)
30" are much better in non-burn years
sharpen blade, buy gas
mow entry paths & set up stairs
flag perimeter & unblasted row
mow perimeter
mow aisle on both sides of unblasted row

Orientation (print maps beforehand):
wear safety glasses, ear protection optional
place flags 10cm N of each plant
search for plants or staples
emphasize that plants can be difficult to find, but the goal isn't to find every one (measure if necessary to get good coverage in thick areas)
walk E & W in unmowed areas & anywhere on mowed areas
lift legs over rows
pull pins & collect plastic
start flagging in positions 860, 935, 960, 983, then flag on either side of unblasted row
coordinate so rows are flagged before mowing
after a few rows, get folks working rows 50 - 56 & 10 - 16. Don't bother flagging cg96.

Remove duff from all plants in an organized fashion.

flag bags
meter sticks (we need more, we only have six)
safety glasses
ear protection
mower sharp blade
gloves for all & gloves for SW. Get the XL; L is too small.

Plan to spread mowing over two days to avoid exhaustion. Sharpen blade in between.
After duff is removed weed thistles, sweet clover, trim shrubs & trap gophers.

To do--cut cottonwoods, ashes in ditch, trees E of CG.