Qualitative Soil Assessment Step by Step 2016 Microscope Requirements • Bright Field Microscope

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Qualitative Soil Assessment Step by Step 2016 ! Microscope Requirements • Bright Field Microscope - binocular • 4X, 10X, 40X objective lenses • ABBE Condenser • Numerical Aperture 1.25 • Iris diaphragm • LED light source recommended • Digital Camera • Microscope cover • ***Resolution is the determinant expense for a microscope. To test the microscope, count ! bacteria, if there is a challenge then seek better resolution. Additional Laboratory Materials • Microscope slides: 25 X 75 mm, 1 mm - 1.2 mm thick • Cover Slips - 0.13-0.17 mm thickness, 22x22 mm is preferred but no smaller than 18x18 mm • Pipette or eye dropper with vial to hold it — the eye dropper should not hit the bottom of the vile because it can break, resulting in inconsistent drop size • 3+ Test tubes or medicine vials with mL increments starting at 1 mL • 1/4 teaspoon • Lens cleaning paper and a dust free cloth • Towel or paper towel • Spring water • Lab Squeeze Bottle: 500 mL ﬁlled with spring water • Test tube rack • 8 ounce vessel for chlorinated water — to rinse eye dropper before placing it back in the eye dropper vial • Empty vessel — to squirt out excess liquid from rinsing eye dropper • Access to hand wash station with soap !• Computer and spread sheet ! Determining Sample Locations • What do you want to know and how will you treat according to your sample data? • Micro-environments - Where are the most productive and least productive zones? Where are the dry zones? Weed zones? Soggy zones? Compaction zones? !***Sample numbers will depend on your resources of time and ﬁnance as well as land size. ! ! ! ! ! ! ! !

Sampling Single Plants of Concern It matters if samples are from a specific plant of concern such as a potted plant or a tree. It also !matters if samples are taken from orchard and vineyard systems, pastures and annual fields. In the case of specific plants, samples should be taken from between the drip line and stem or !trunk. Te larger the plant, the more samples are needed. Draw what this could look like. ! ! ! ! ! ! ! ! Draw an illustration of what sampling from a field or pasture would look like. ***Hint: Most pro- !ductive and least productive zone? Weed patch? Compaction? ! ! ! ! ! ! ! ! ! ! ! ! ! ! !Draw an illustration of sampling from an orchard, vineyard or perennial system. ! ! ! ! ! ! ! ! ! ! ! !

Collecting Samples • Materials: • Buckets - numbering in zones or bring a cloth to wipe the buckets clean during the sampling process • Soil core or shovel • Ziplock bags • Permanent marker ***Te more samples you take from the zone, the more accurate the average will be. Take at least 3 samples from a zone, 5 is better, 20 is best. • Remove signiﬁcant plant debris from soil surface • Use soil core (1/2" to 2" diameter wide) or shovel to gather soil from surface to 3 inches down • Place same zone samples into labeled bucket or • If samples are to be sent away for assessment • Mix samples together, crumbling open aggregates • Place 2 cups or about 1 lb. of this mixture into a 1 gallon ziplock bag • Leave as much air in the bag as possible • Each bag must be labeled with clients name, date collected and zone name ***It is ideal to send the samples on the same day you gathered them. If you won't be sending the soil the same day you gathered it, leave the unmixed soil in an uncovered bucket, store in a cool dark place until your ready to ship. On the day your ready to ship, mix the soil contents of each bucket thoroughly. Each zone will go into it's !own labeled bag. Random sampling Example 1: Consider land as a whole grid. Tis works well for land that has even plant growth, soil types and elevations. • Print a map of the growing site • Pick 20 random sites throughout the designated area • Number the site points • Randomly draw 5-15 numbers out of a hat • Sample from those points !• Combine all samples, label, package !Or Example 2: Consider the topography of the land — this is the preferred method Sample for topography: • Split ﬁeld into habitat areas • Productive areas • Non productive areas • Wet areas • Dry areas • Weed patch • Ridge line • Grid each area • Number each grid section

• Pick 3-15 numbers for each grid section • Sample from each point, mix, package, label ! ! Simple Dilution • 1:5 • 1 unit volume solute + 4 unit volumes solvent =! dilution factor of 5 Serial Dilution • A series of simple dilutions which ampliﬁes the dilution factor • Te source of the dilution material of the previous step • Total dilution factor: Te product of the individual dilution factors in each step up to it.

Assessing Solid Material (Compost or Soil) • A 1:5 dilution is ofen appropriate for assessing compost but not soil. Tis is due to the fact that there is a signiﬁcant mineral component in soil that is not as prominent in composts. • Soil will likely require making serial dilutions, consider beginning at a 1:10 dilution (see Serial Dilutions below). ! Assessing Liquid Material (Extract or Compost Tea, Probiotics…) • Material does not need to be diluted, apply appropriate amount of drops to the slide !• For qualitative analysis, dilute this material to do bacterial counts. See Serial Dilutions below. Simple Dilution, 1:5 • Measure 4 mL of solvent (material that is diluting — spring water) into medicine spoon

• Measure 1/4 tsp or 1 gm solute (material to be diluted — soil or compost) place into vial or medicine spoon. • Break open the aggregates in the 1:5 dilution by shaking the vial for 30 seconds (count one one ! thousand, two one thousand…). Keep elbow at 90º to your torso. Do not shake vigorously. Serial Dilutions • A “must” for counting bacteria • Beginning with a 1:5 dilution, add 5 mL spring water to the vial. Mix for 30 seconds as with the 1:5, the result is a 1:10 dilution • Fill a new medicine spoon or vial with 1 mL from the 1:10 dilution • Te addition of 1 mL water will result in a 1:20, adding 2 mL = 1:30, 3mL = 1:40, 4 mL = 1:50, add 9 mL it will be a 1:100 dilution. • Take 1 mL from the 1:100 dilution and place it into a new medicine spoon. Adding 4 mL will result in a 1:500 dilution, adding 9 mL will = 1:1000 dilution (preferred dilution for counting bacteria). ***Note: Everything increases by a factor of 10 ! Preparing a Slide • Clean slide and cover slip with lens cleaning paper so that both are perfectly clean • Shake the vial, count to 10, place eye dropper or pipette 1 mL into vial and draw the sample from the vial. • Place appropriate amount of drops on your slide — do not touch the pipette to the glass.… In general: 1 drop for 20 X 18, 20 X 20 and 18 X 18, 2 drops for 22 X 22. • Before placing pipette back into the cup, rinse it in the cup with chlorinated water by sucking water into pipette and emptying it into the empty cup. • Place cleaned pipette into the holder and suck up water into the eye dropper. Do this every time pipette is used to keep both the pipette and holder clean, they are hard to clean AND this will keep from contamination will alter the results of the assay. • Hold cover slip by the edges to keep it clean, place the edge of the coverslip on the slide and drag it gingerly across the drop to spread the liquid over the slide • Drop cover slip on slide — there should be zero air bubbles and zero liquid oozing out on the slide • If you cannot see through the material on the slide, it’s too dense, now it’s time to make a serial dilution !***Note: You can use the same slide at least two times without have to clean it Focusing the Microscope • Wipe down scope — stage, eye pieces, condenser, light source with lens cleaning paper or a microﬁber cloth • Stage is all the way down • Begin at the 4X objective lens (40X TM) • Use the course focus knob to bring stage up and bring sample into focus • "Bull's Eye" eye pieces • Focus condenser

• Setting the mood — address the lighting through light control dial and iris diaphragm • Beyond the 4X lens use the fine focus only!!! • Go through 10X objective lens (100X TM) to fine focus the sample and then to 40X objective lens (400X TM). !• Custom focus binoculars Steps for a Qualitative Assessment 1. Scan For Nematodes 10X lens (100X TM) • Identify morphology — zoom in to 400X TM (using the 40X lens) record findings appropriate- ly • Make note of: • Organic Matter (OM) present — roots, strands, materials • Acids (fulvic/humic) present or not • Aggregation - simple or complex • Microarthropods • Larvae • Bacterial diversity ***Note: Afer becoming more confident with identifying nematodes, scan at the 4X lens one is used to using the microscope and identifying organisms, scanning the slide can be done with ! greater speed using the 4X lens. 2. Accounting Te Soil Food Web 40X lens (400X TM) • Make note of diversity in bacteria • 20 random FOV (Fields Of View) • Account for the presence or absence of: • Actinobacteria (% length of F.O.V.) • Fungi (% length of F.O.V., color, diameter) • Oomycetes (% length of F.O.V., color, diameter) • Protozoa • Other indicators of + or — such as anaerobic indicators, oomycetes, lack of diversity of fungi, ! bacterial clumping…. 3. Bacterial Counting • Make a 1:100 dilution (typically) • Count 5 FOV • If standard deviation (SD) is at/below 20% of the mean count no more!!! !• If SD is above 20% of the mean, count more FOV or recount the outlying number. ! ! ! !

© 2016 Haviland Earth Regeneration 6 Qualitative Soil Assessment Step by Step 2016 ! ! ! ! Adjust the Spreadsheet Dilution Factor !• Change under column X for all organisms Testing for your dropper !• Count the number of drops it takes from the specific dropper to fill vial with 1 mL of water. Change Field of View and Number of Drops • Change the field of view factor for specific cover slip size and number of drops it takes for ! specific pipette to fill 1 mL with water • Enter FOV and # of drop values into Y13, Y18, Y21, Y28, Y35, Y36, Y37, Y41 ! ***Note: You will not enter the number of FOV for nematodes ! ! Resources • Compost Tea Quality: Light Microscope Methods by Elaine Ingham, Ph.D • Te Microbe Manual: Qualitative Assessment of Microorganisms by Elaine Ingham, Ph.D. and Carole Ann Rollins, Ph.D • Te Microbe Poster: Microorganisms Found in Soil, Compost, Castings, and Compost Tea by ! Elaine Ingham, Ph.D. and Carole Ann Rollins, Ph.D ! ! !