Session 2 - Preparing Soil Samples

Question 1

If you have a test tube with markings beginning at 1 ml how much of your soil sample should be tapped down into the bottom of the test tube?

Answer 1

1ml

Question 2

2. How many times should you tap down the soil sample?

Answer 2

10 times

Question 3

If you are making a 1:5 dilution, with a test tube having a 1 ml mark, how much water will you add to the soil sample?

Question 4

What is the dilution factor? How is this different from the dilution ratio?

Answer 4

The dilution factor is the total amount of solution (denominator) in your test tube, so if the dilution ratio is 1:5, the dilution factor is 5. The ratio tells the amount of solid sample to the total amount of the solution.

Question 5

If the dilution ratio is 1:100, what is the dilution factor?

Answer 5

100

Question 6

Do you record the dilution factor or the dilution ratio into the sMApp?

Answer 6

Dilution factor

Question 7

What is an appropriate water source for adding to your sample? Spring water, bottled drinking water, rain water.

Answer 7

NOT distilled water or municipal water that has been treated with chlorine and chloramine.

Question 8

Where should the water align on the test tube relative to the meniscus?

Answer 8

The bottom of the meniscus should be at the top of the mark/line that you want. So for a 1:5 dilution, the bottom of the meniscus (think of this as the “smile”) should rest just above the 5 ml line.

Question 9

9. How long should you shake the test tube?

Answer 9

9. How long should you shake the test tube? 30 seconds

Question 10

What procedure should you use to shake the test tube?

Answer 10

Your arm should
move smoothly and slowly from 9:00 o’clock to 12:00 o’clock

Question 11

How long should you ‘rest’ the sample?

Answer 11

Approximately 10 seconds. It is important that you are consistent with how long you wait.

Question 12

If you are making a 1:5 dilution with a test tube having a 2 ml mark, how much water will you add to the soil sample?

Answer 12

Approximately 8 ml, you will add water up to the 10 ml mark.

Question 13

If you want to further dilute your 1:5 dilution to make a 1:25 dilution, what is the procedure?

Answer 13

Take 1 ml of your 1:5 dilution, put it in a clean test tube, then add water up to the 5 ml mark.

Question 14

If you want to make your 1:5 dilution into a 1:10 dilution, what is the procedure?

Answer 14

Add more water to the 1:5 dilution, up to the 10 ml mark in the same test tube.

Question 15

Can you put a liquid sample on a slide undiluted? If yes, what would the dilution factor be for that sample?

Answer 15

Yes. The dilution factor would be 1.

Question 16

When would you need to dilute the sample?

Answer 16

If you could not see the organisms clearly because of too much debris in your sample.

Question 17

How do you do a 1:2 dilution with a liquid sample? What about a 1:3 dilution?

Answer 17

For a 1:2 dilution, you would fill the test tube up to the 1ml mark with the sample, then add water up to the 2 ml mark. For 1:3, you would fill the test tube up to the 1ml mark with the sample, then add water up to the 3 ml mark.

Question 18

How would you convert the 1:10 dilution to a 1:50 dilution?

Answer 18

Take 1ml from the 1:10 dilution and put it in a clean, dry test tube and add water up to the 5ml mark.

Question 19

How would you convert the 1:50 dilution to a 1:500 dilution?

Answer 19

Add water up to the 10ml mark to make a 1:100 dilution. Then take 1ml from the 1:100 dilution and put it in a clean, dry test tube and add water up to the 5ml mark.

Question 21

f you started with a 1:5 dilution, and you wanted a 1:125 dilution, what would you do?

Answer 21

Take 1ml of the 1:5 and put in a clean, dry test tube and add water up to 5ml mark so you have a 1:25 dilution. Then take 1ml of the 1:25 and put in a clean, dry test tube and add water up to 5ml mark. Now you have a 1:125 dilution.

Question 22

If you started with a 1:125 dilution, what would your dilution be if you transferred 5 ml to a clean dry test tube and added 5 ml spring water?

Answer 22

1:250

Question 23

If you started with a 1:10 dilution, what would your dilution be if you transferred 2 ml to a clean dry test tube and added 8 ml spring water?

Answer 23

1:50

Question 24

Should you use the same dilution for all samples?

Answer 24

No, each sample will be different, so judge the dilution for each sample based on how well you can see the organisms.

Question 25

What factors should you consider in determining the appropriate dilution?

Answer 25

How well you can see the organisms.

Question 26

What organisms are you assessing?

Answer 26

Nematodes, actinobacteria, fungi, oomycetes, protozoa, and bacteria.

Question 27

Would you use the same dilution for a Main Assessment, the nematode scan, and a bacterial scan? Explain.

Answer 27

You may use the same dilution for the nematode scan and the main dilution. Since bacteria are so small and you need to count them, you will need to dilute more.

Question 28

What bacterial density should be visible in a FoV in an appropriate dilution? Why?

Answer 28

The bacteria should be around 30-50 in a 1⁄4, 1⁄2 or whole field of view in order to be able to count properly. Too many bacteria makes it difficult to count accurately.

Question 29

Is this for a whole FoV, a 1⁄2 FoV or a 1⁄4 FoV?

Answer 29

It can be for any of these. The microscopist chooses.

Question 31

Is it necessary to make a new sample for bacterial counts? Explain.

Answer 31

One needs to make a new dilution from the same sample unless the sample has been sitting around for a few hours, then it is best to prepare a new sample.

Question 32

When you draw a sample from the test tube, where in the test tube do you draw from?

Answer 32

Just below the organic matter and you should go to the same depth every time you sample.

Question 33

Explain how to apply the coverslip on the sample drop.

Answer 33

At a 45 degree angle, you should take the coverslip and spread the drop the same diameter as your coverslip before you actually drop the coverslip on the slide, to try to consistently achieve uniform distribution of the sample underneath the coverslip.

Question 34

What do you do if there is too much debris and organic material on the slide?

Answer 34

1) Take another drop, but wait for it to settle more, or
2) Dilute the sample more, or
3) If it’s a big chunk, then take a corner of your coverslip and move it out of the way.

Question 35

Describe the step by step procedures
for setting up your microscope using the 4x lens.

Answer 35

The stage should be at the lowest setting to start with the 4x
dialed in. After the slide is put on the stage and secured
with the stage clip, the stage should be brought up to the
highest setting. Looking through the eyepieces, make
sure that one sees just one circle of light and adjust accordingly. Make sure the light intensity is down so as not to irritate the eyes. Then using the coarse focus knob lower the stage until the sample is clear.

Question 36

What objective should be in the ‘clicked down’ position when beginning to get your slide in focus? How do you begin with focusing on your specimen?

Answer 36

4x, adjust the eyepiece to the width of your eyes, then use the coarse focus knob to lower the stage until the sample is in focus.

Question 37

What focus knob(s) should you use when using the 4x objective? Why?

Answer 37

Coarse focus as you are setting up the scope so that all lenses will be aligned. Otherwise it will take ages to get into focus. From then on, you only need to use the fine focus knob.

Question 38

How do you make sure that when you look through the eyepiece lenses that you are only seeing a single circle of light?

Answer 38

Adjust the width of the eyepieces to match the width of your own eyes.

Question 39

What is the diameter of the Field of View using the 4x objective?

Answer 39

That will depend on your field number. If the field number is 18, your diameter will be 4,500 μm.

Question 40

When using the 10x, which focus adjustment should you use? Which not to use?

Answer 40

Fine focus knob, NOT the coarse focus knob.

Question 41

How do you adjust the condenser?

Answer 41

One takes a straight edge paper and puts it over the illuminator. Then using the condenser knob, adjust the condenser so the edge of the paper is sharply in focus. This will be the place right in between the red and blue colors and you are adjusting the knob.

Question 42

How do you know when the condenser is not properly adjusted?

Answer 42

When you look at the background and it looks marbled or mottled, then you need to adjust your condenser. You can do that any time you notice the background is not smooth.

Question 43

Should your iris diaphragm be open or closed when adjusting?

Answer 43

The iris diaphragm should be open when you are doing your first adjustments with the condenser.

Question 44

What is the diameter of the Field of View using the 10x objective?

Answer 44

If your field of view is 18, then your FoV diameter will be 1800 μm.

Question 45

What is the size of the Field of View using the 40x objective?

Answer 45

If you have a field number of 18, it is 450 μm.

Question 46

Where is the diopter located?

Answer 46

The diopter is the dial located on one or both eyepieces.

Question 47

How do you adjust the diopter?

Answer 47

One rotates the dial until the object that you are observing looks as focused as when you are looking with the other eye.

Question 48

What is the function of the diopter?

Answer 48

It functions to make sure the you are looking at the same plane with both eyes.

Question 49

How do you shadow?

Answer 49

You slowly close the iris diaphragm until the organisms “pop out” or are clearly visible.

Question 50

How do you know when you have properly shadowed?

Answer 50

When you have not shadowed properly the organisms appear “ghosty”. Once you shadow, the images are sharp and clear.

Question 51

What do the numbers mean in the above diagram?

Answer 51

The numbers define the reading areas.

Question 52

What do the yellow circles represent?

Answer 52

The circles represent the Fields of View.

Question 53

The example in the green bubble indicates ___ readings and ___
Fields of View?

Answer 53

5 readings, 3 FoVs

Question 54

Define “reading”.

Answer 54

A reading is the larger area you are looking at. When using the sMApp, one needs to have 5 readings per sample. Within each reading area, you could look at and do counts in 3-10 fields of view.

Question 55

Define “Field of View”.

Answer 55

A field of view is the area that is seen when you look through your microscope. Each time you move your slide, you are looking at a different field of view.

Question 56

How many readings do you do during a Main Assessment?

Answer 56

5

Question 57

How many Fields of View do you look at per reading?

Answer 57

This is determined by how many organisms you see. You should look at no fewer than 3 FoV per reading to get adequate data. 5 FoV is usually what most people look at, but 10 FoV per reading is the maximum you will do.

Question 58

How to shake?

Answer 58

30 seconds along a 90 degree axis, one inversion per second, not too harsh to smash, but also not too weak so aggregates break up and organisms are actually extracted (water needs to smack into cap and bottom)

Question 59

How long to let the sample settle?

Answer 59

10-15 seconds, be consistent with all samples

Question 60

How to draw with the pipette?

Answer 60

first coating of pipette: insert just below organic matter layer, draw liquid into pipette and then release gently along the side of the tube without causing too many big particles to be resuspended

then draw up the dilution again and release on drop on your slide

Question 61

Maximum area of sample outside the edges of cover slip?

Answer 61

5%

Question 62

How to clean the pipette?

Answer 62

Rinse several times in the “rinse water” cup, then transfer water from the “clean” cup to the “rinse” cup without touching the “rinse” water again

store pipette in “clean” cup with water drawn up inside

after microscope session rinse thouroughly with clean water, expell all liquid and leave the pipette to dry on rack

Question 63

How to spread sample with coverslip?

When necesseary to redo it?

Answer 63

use edge to spread material back and forth across before dropping at a 45 degree angle

large/too many bubbles, large chunks of materials prevent slip from lying flat, drops on top of slip, more than 5% liquid outside of the sip

Question 66

Setting up microscope:

Answer 66

• stage all the way down, 4x objective dialed in, iris diaphragm open
• turn on light
• place slide on stage
• bring the stage all the way up using the coarse focus knob (to oil the gears)
• adjust interpupillary distance until you see one circle, dim light if too bright
• then bring stage down until sample is focused, parfocal lense makes sure other objectives can be dialed in without colliding
• dial in 10x, use fine focus knob to bring it into crisp focus
• focus condenser (diaphragm open), raise it to the highest point, hold a piece of paper against the ilumination glass, focus condenser down until edges are sharp or until the blue red switch occurs
• focus diaphragm, close diaphragm until you can see sharp contours
• dial in 40x, adjust with fine focus, dimmer and iris diaphragm
• adjust diopters to have both eyes on the same focal plane to avoid headache