Mpa4

Question 1

Microscope care

Answer 1

Cover scope.
Don’t drag…lift by base.
Dial objectives with nosepiece, not obj.
Keep assembled and covered.
Use only optical tissue or soft cloths.
Clean once a yr or as necessary.

Question 2

Slide care

Answer 2

With breath, optical cleaner or soap and water.
Use soft cloth.
Don’t let wet glass touch each other. Keep in covered containers to protect from dirt etc.

Question 3

Pipettes and test tubes

Answer 3

2 containers marked clean and dirty.
Place drop on slide, then return excess to tube. Then draw up dirty water at least 3x. Then draw up clean and expel into dirty. Store then in clean.
Monitor insides. Discard if can’t be cleaned. Clean with soap and water if needed.
Empty and clean test tubes asap and rinsed. Store upside down to dry.

Question 4

Cross contamination.

Answer 4

Don’t touch. Use lab scoop.
Don’t mix liquids.
Wash and dry slides coverslips, pipettes and test tubes between uses.
Group bags from same location together but don’t store with other places.
Prepare from a single bag at a time. Keep others closed.
Keep a clean water jar for making dilutions. Store pipettes in separate container.
Change water in dirty glass when becomes cloudy.

Question 5

Calibrate pipettes

Answer 5

Use a test tube clearly marked at 1ml or 2 ml.
Use very little force for each drop. Allow gravity to pull it off.
Plastic pipettes may change with use.
Mark bulb with drops per ml
Calibrate each pipette 5x and take the average. sMapp accepts .5 numbers.
Watch for cracks in pipette seams. Also discard if water drops without any pressure.

Question 6

Sample handling.

Answer 6

Analyze asap.
Mail either same day or next day service
Finished compost or bare soil 5 days max. It isn’t active.
Anything active ie liquid samples, productive soils and active compost, in less than 24 hrs if possible
Store in similar conditions as the sample was taken from.

Question 7

Water.

Answer 7

Clean water uncontaminated by microbes or chemicals.
High salts will also destroy microbes. Lowsalts, ie distilled and reverse osmosis will destroy microbes.
Store in stainless steel or inert plastic containers.
Treat tap water with 1 drop humic acid per gallon to neutralize chlorine and chloramines. Or run through compost. Change compost when through flow loses tan color.
Rainwater is good but store carefully.
Bottled water check ingredients.

Question 8

Where to take soil samples

Answer 8

Define zone of interest. Record. May include climate or season, plant community composition, soil type, organic matter, food web biology, water dynamics as in wetland, riparian arid or semiarid.
Draw map. Identify areas of plant species, weeds, bare land, healthy plants. Mark management practices.
What questions do you want to answer?
Mark each area with numbered grid.
Select 3 to 5 grid squares from total number. Take 3 cores from around plant in center of each grid. Place all 3 in single bag. Label sample number and position on grid. Second sampling take from different plant from each grid.
Don’t take samples from what is not representative of that zone. Ie a road or fence line or pond.
First rule: All samples must come from root system of plants that are representative of sample area.
Second rule: be consistent with place of sample…halfway between trunk and dripline.
May divide a property if there are obvious differences in soil characteristics or plant health. Do separate tests for vegetables, row crops, gardens, fruit trees, lawns, etc.
If soil is uniform, a single set of 3 samples my adequately represent a field, or 3 fields. Soil conditions may be similar in spite of differences in texture etc.

Question 9

How to take liquid samples

Answer 9

Do not immerse hand or arm in liquid.
Take from top 1 to 2 in while being aerated. Take 10ml each time. Wait 10 to 15 sec and take more. Repeat 3 times.
Don’t fill container more than 1/3 full to allow oxygen exchange.
Assess immediately. If there must be delay, cover to avoid contamination. Sample may go anaerobic in 4 to 6 hrs.
In mailed samples the movement will help keep aeration, but assess same day as recieved.
No need to dilute unless very crowded.

Question 10

Compost samples

Answer 10

Teaspoon, Ziploc, marker.
Pile: collect a tsp from min 5 different areas and depths.
Windrow or lg pile: 20 tsp from different areas/depths
Put all in same bag.
Label outside date, location, type sample, person who sampled, etc
Fill max 1/3 full. Seal with air.
Labels never go inside bag.
Store in same temp and climate conditions and maintain humidity same.

Question 11

Microscope setup

Answer 11

Rest: stage down, 4x in, iris diaphragm open, covered.
1. Turn on.
2. Place slide on stage. Clamp.
3. Elevate stage all the way with coarse knob. 4x still in place.
4. Adjust interpupillary distance. Adjust light.
5. Focus with 4x.
6. Dial in 10x. Focus with fine knob.
7. Focus condenser with iris diaphragm open.
8. Shadow with iris diaphragm.
9. Dial in 40x. Focus and adjust Adjust light and diaphragm.
10. Adjust diopter

Question 12

Measure camera FoV

Answer 12

Properly size camera view in software. Fit to height usually gives max. Edges should be visible, barely.
Place micrometer on stage.
Set up microscope.
Line up outer edge of a mark on left side of camera FoV.
Looking at computer screen, count marks visible in horizontal view.
Multiply number of marks by units of measurement indicated on micrometer to get horizontal FoV.
Rotate camera if needed, to get vertical FoV.

Question 13

Sample and slide prep

Answer 13

Mix material in bag to make homogeneous.
Fill test tube to 3ml water. Don’t wet sides of tube.
Add 1ml of sample to 4ml line.
Add 1ml water to 5ml line.
Cap and shake 30x in 30seconds, from 9 to 12 o’clock.
If can’t see light through, may need to dilute further.
Let rest 10 seconds.
Pipette: draw sample into pipette couple of times to coat sides with critters.
If sample is very disturbed by this process, allow to settle 10 sec more.
Insert just below floating om.
Hold pipette vertically above slide and gently release 1 drop of liquid.
Be sure liquid covers below coverslip and no air bubbles.
Be sure not more than 5% is outside coverslip.
Use edge of coverslip to spread, then drop gently onto slide
Return unused sample to test tube.
Rinse pipette in dirty water, then draw up clean water and expel into dirty. Store in clean water with water drawn up.
And end of day rinse in clean water and store in rack to dry
Look at slide and determine if further dilution is needed.
If organisms get too random in sample you will need lower dilution.
Dilutions of 1.50 or even 1.500 may be necessary for dense clay.

Question 14

Dilutions

Answer 14

Dilution factor: 1:5=1 part sample, 4 parts water. Total 5.
Dilution ratio: 1:5=1part sample, 5 parts water. Total 6 ml.

1/5 x 1/5 =1/25. One ml of 1:5 in 4 more MLS of water makes 1:25 dilution.
1:25 x1/5=1:125.
1:125x1/2=1:250
1:10x1:10=1:100
1:100x1:5=1:500

Question 15

Reading areas, swimming lanes, face of a die.

Answer 15

Reading areas are the coverslip divided into 5.
Swimming lanes are used for main assessment.
5 faces of a die are for bacterial counts.
Each swimming lane has FoVs, determined by richness of sample.
Faces of die…1 FoV in each for total of 5 for bacterial count.

Question 16

Standard deviation

Answer 16

Average is sum of all samples divided by total number of samples measured
We use average to estimate number of organisms in a gram of soil.
When calculating average, the distribution of organisms should be close to uniform.
A weft of fungi would not be uniform.
Most soil or compost measurements are not expected to be uniform. So, we assess more then 1 FoV or 5 or 10, and use the average to get a meaningful estimate.
Bacterial counts are reasonably uniform after shaking, if sample is spread on slide correctly.
May need to remove FoV if obvious outlier…a weft of fungi etc.
If remove largest outlier, should also remove smallest.
Calculate standard deviation to express the average distribution of organisms. If SD of questionable FoV is more than the SD we know we can remove it and measure organisms in next FoV to replace data point that was obviously an outlier.
A narrow range indicates reasonably accurate data points.
Trim the mean:. If o e or more organism groups is outside 1 SD, examine highest and lowest values in the 5 FoVs. Removal of an outliers will reduce the SD in a group of samples, making the entire assessment truer to reality.
Percentage of the mean SD are:
20% of the associated population estimate is optimal.
50% is ok.
70% indicative of scarce or clumped populations.

Question 17

Trim the mean

Answer 17

If one or more organism groups is outside one SD, you may need to remove the outliers. Should remove the highest and lowest FoVs. We want low variability to be sure it’s as accurate as possible.

Question 18

Interpreting data variability

Answer 18

An SD value greater than 70% means those organisms are practically non existent. When there is a greater population they will appear with greater frequency, and the SD will be lower.

Question 19

Soil assessment results.

Answer 19

Look at the mean and SD for each group. If the SD is less than 50% for most of the organisms it means data can be considered reliable.
If any single group is higher than 70% statistically it’s not different than 0.
If SD for fungi oomycetes or actinobacteria greater than 70%, prepare a new slide and maybe even a new sample. If SD is still grater than 70% you can conclude there is not enough organisms to produce reliable data.

This does not apply to bacteria and protozoa!
Bacteria sd tends to be low because they readily disperse well through the sample.
Protozoa SD tends to be very large because they tend to stay associated with specific surfaces. You will find several clumped around organic matter, and then none for several FoVs.
We don’t want SD for ciliates, oomycetes, or actinobacteria to be very low. It indicates low oxygen levels.

Question 20

When to sample?

Answer 20

Before planting.
10-14 days after first application of organisms.
Every 6-8 weeks during spring and early summer while lots of activity.
Back off to every 2 months unless diseases or insects become a problem.

Question 21

How to take samples

Answer 21

Brush aside organic material. Take all samples at same depth…mostly 3”.
Take samples from 3 to 5 plants in each area. If economy needed combine cores from the 3 to 5 samples in 1 bag.
Use ziploc bags.
Label on outside of bag.
Take at least 100 MLS or a 1/4 cup to maintain conditions in bag.
Don’t mix cores in bag until ready to assess.
Leave air in bag.

Question 22

Guide your clients sample design

Answer 22

You can quantify the differences between treatments using measurements such as SD, if you have a good design.
Make sure map is marked well, including any areas that may have had different treatment, or no treatment.
Make sure client has a clear question they want answered, such as “Can I grow vegetables here?” Google maps can be used to create map.
Help them draw a grid on each area of interest and design a random sampling plan for each area. 3 to 5 samples from each treatment area. The more variable an area the more samples should be collected. Discuss pricing according to the sample plan.

Question 23

Assessing multiple samples

Answer 23

When multiple samples from the same general area each sample should be assessed separately. Then look at SD and mean between the samples. Then you’ll know if the 3 or 5 samples are representative of the area they are from.
See if SD overlaps between each sample. If one samples SD is very different, repeat assessment on that sample to see if there’s an error. If it remains an outlier, ask client if there’s a reason. If all other samples are similar then maybe an animal peed there etc.
Also possible that each sample seems very different from each other. Client should go to another, randomly chosen grid .
If you have at least 5 samples you may be able to take away the highest and lowest values, so client doesn’t have to get a new sample.

Question 24

Medium and large scale sampling

Answer 24

You can’t mix different kinds of plants during sampling. Each successional stage species must be considered separately. If all orchard trees are same species each tree will be considered a location.
If trees are all the same but some healthy, others not, may be able to just divide in 2 grids

Question 25

Sampling and shipping

Answer 25

Use detailed instructions .
Finished compost and out of vigorous growth season soil samples may be shipped 2-3 day mail.
Spring samples and liquid samples must be shipped to arrive same day or overnight.
Be sure you can do assessments the day samples arrive or next day at the latest. Prioritize overnight or same day shipping. Try to store samples at ambient temp of origin.

Question 26

Contaminated samples or foreign samples

Answer 26

Foreign samples may contain non native organisms. Autoclave or return sample to client after testing. To receive foreign samples you may need govt certification and an autoclave, laminar flow hood etc to deal with left over samples.
Samples with anaerobic conditions present may be oxygenated by mixing or drying when you dispose of it. If law permits you may send it to landfill or through sanitary system. Or autoclave. Inform client of anaerobic conditions.
When soil biology doesn’t produce adequate results soil may be contaminated with toxins heavy metals or radioactive materials. You should return samples if contamination is suspected.

Question 27

Microscope parts and uses

Answer 27

Eyepiece…allow us to see organisms via objectives.
Diopter…change focus on 1 eye to compensate for differences between eyes.
Headpiece connects eyepiece to objective lenses.
Body goes from head to base or bottom of microscope.
Nosepiece holds the objective lenses.
Objective lenses magnify objects.
Coarse focus is used with 4x to bring sample into focus.
Fine focus is used at 10x and 40x to bring organisms 8nto focus.
Stage holds the slide.
Clips secure slide so it doesn’t move about on the stage.
Stage control moves stage on x and y axes.
Iris diaphragm increases contrast by developing shadows.
Condenser focuses light on the sample.
H

Question 28

Microscope setup from scratch.

Answer 28

4x dialed in and stage at lowest level.
Place slide on stage and secure.
Raise to highest level with coarse focus.
Use coarse focus to move down till slide is in clear focus.
Adjust light.
Dial in 10x and focus using fine knob.
Focus condenser.
Close iris diaphragm until organisms pop.
Dial in 40x and focus. Adjust light and iris diaphragm if needed.
Focus eyepieces using diopter.