Displacement Pipetters: Their Care and Use

Displacement Pipetter, labeled

Undepressed thumb plunger

These instruments have been developed in recent years and have been valuable in manipulating tiny volumes, particularly of solutions of DNA and enzymes used in genetic engineering. They function by drawing fluid into the pipet tip using a thumb-driven piston whose movement is calibrated to draw up a specified volume of liquid. The units of volume are in microliters (µL), also termed lambdas ( l). Since there are a million µLs in a liter, 0.1 mL is 100 µL. The plastic disposable tip is discarded after a single use. The tips are color coded according to their capacity: white for 10 µL, yellow for up to 200 µL, and blue for up to 1000 µL.

The advantage of single-use disposable tips in genetic engineering is that there is no possibility of contamination with endonucleases which would destroy DNA samples. Also, the tips are much less expensive than single-use pipettes. However, the instruments are very expensive (about $200 each), and must be handled and cared for properly. NEVER allow fluid up into the body of the pipetter.

Illustrate a displacement pipette and include all of the following features:
body
thumb plunger
calibrated stop
blow out stop
lock ring
volume of aliquot (in window) capacity range indication
thumb knob (to adjust volume)
shaft
tip
tip ejector button
ejection ring

1. Select the pipetter whose range is appropriate for the volume you wish to measure out.
   2. Set the volume as follows:
   a. Loosen the lock ring at the base of the thumb plunger.
   b Rotate the thumb knob until the desired number of µL appears in the window . [Do not go above the specified range of
   the pipetter or you may damage the instrument.]
   c. Snug the lock ring. (Do not over-tighten.)
   3. Attach a fresh tip to the pipetter:
   a: Open the box of sterile tips.
   b: Press the pipetter firmly down onto one of the tips, tap once or twice to firm the fitting.
   c: Pull out the attached tip, immediately close the box to maintain sterility.
   4. Draw up the desired volume of fluid:
   a: Before you insert the tip into the sample solution, gently depress the plunger to the first (calibrated) stop and hold in
   place. DO NOT GO PAST THE CALIBRATED STOP.
   b: Insert the pipetter into the sample solution so that the tip is about 3 mm below the surface.
   c: Allow the plunger to slowly and smoothly return to its original position. Note that fluid is drawn up into the tip (no
               bubbles). DO NOT LET FLUID ENTER PIPETTER BODY.
   d: Withdraw the pipetter from the sample vessel, flame and re-cap vessel to maintain sterility.

Undepressed thumb plunger

Pressed to calibrated stop

Pressed to blowout position

5. Deliver the fluid into the receiving vessel:
   a: Insert the pipet into the receiving vessel so that the tip a: is 3 mm below the surface of the receiving fluid, or b: touches
   the bottom of the empty vessel.
   b: Depress the plunger slowly and smoothly until the first stop is felt.
   c: Press more firmly on the thumb plunger to blow out the remainder of fluid. You should see a bubble expelled.
   KEEP THE PLUNGER HELD DOWN.
   d: Withdraw the pipetter from the vessel, dragging it along the side to leave any adhered fluid.
   e: Allow the plunger to return to its normal position.
   6. Discard the used tip (unless you are to deliver additional aliquots of the same fluid):
   a: Hold the pipetter over a discard vessel.
   b: Depress the tip eject button with the thumb until the tip is ejected

PRACTICE USING THE PIPETTER’S FEATURES

Per desk for practice by two students:

2 20-200 uL pipetters
2+ non-sterile tips in a tray
test tube rack with:
2 13 × 100 mm test tubes, half full of water
4 13 × 100 mm test tubes, empty

1. Unlock the lock ring, select the volume by rotating the thumb knob. Do NOT exceed the range of the pipet as indicated on the handle top. Relock (gently).
2. Gently press the plunger to the first calibrated stop, press more firmly to feel the blow out stop.
3. Pick up a tip from a non-sterile practice box by tapping the pipetter firmly into the tip, then return tip to a different hole in the box, ejecting the tip by pressing the tip ejector button.
4. Set the pipetter to 100 µL, and draw up this volume of dH₂O, deliver into a clean test tube. Change setting to 10 µL, deliver to a second clean test tube. Note how small a volume this is.

Buccal Smear

Buccal Smear Lab

The cells which line the inside of your cheeks are classified as a stratified squamous epitheliumtissue and are the surface of a mucous membrane. These flat, scale-like buccal cells (pronounced “buckle”) are shed constantly as the tissue is renewed. By gently scraping the inside of your cheek, these cells can be harvested, and when smeared and stained, may be used to illustrate a number of important biological phenomena including cell and tissue structure, oral bacterial floraand morphology, etc. This tissue is non-keratinized and therefore the surfacecells are living and still possess their nuclei, in contrast with shed epidermalcells. See DiFiore’s Atlas of Histology, 9th Ed, pp 326&327 for similar tissue found lining the vagina. (Note that the keratinized surfacecells of the epidermis have no nucleus.) Here is a labeled image of a buccal smear stained with methylene blue.

Compare the following steps with those in the Bact. Smear and Staining Protocol.

Equipment and Supplies

Soap and Water
Microscope Slides
Dropper Bottle of dH₂O
Dropper Bottle of 0.3% Methylene Blue
Toothpicks (optional)
Bunsen Burner or Alcohol Lamp, Striker
Bibulous Paper or Paper Towel

Preparation of Slide and Fingernail:

Wash Slide

 

Clean a microscope slide well with soap & water, dry with a non-linty paper towel.

 

 

Clean Finger Nail

 

Cleanse very thoroughly under the nail of your index finger.

 

 

Add Small Drop of Water

 

Place a small drop of dH ₂0 in the center of the very clean slide.

 

 

Harvest the Cells, Prepare the Smear:

Collecting Cheek Cells

GENTLY scrap the inside ofyour cheek to pick up some of the shed stratifiedsquamous cells. Do NOT scrape chunks. A toothpick may be used if you have no fingernails. Gentle scraping is the watchword, there should be no discomfort.

 

Expressing the cells

 

 

 

 

Press Cells onto Slide

 

Express the material from under your nail by pressing with your thumb, and press the material into the drop of water on the slide, mix and spread the material around to the size of a dime.

 

Spread Material on Slide

 

 

 

 

Fix the Smear

Fix Specimen on Slide

 

Pass the slide through the flame several times to fix the smear. Do NOT heat the slide above a temp which is comfortable. You are merely “gluing” the smear to the slide.

Stain the Smear

Add Methylene Blue

Place a drop of 0.3% methylene blue on the specimen. Let sit for 1 minute.

 

 

Rinse off Slide

Rinse off the excess stain with tap water. (Do not splash on your white shirt!)

 

 

Blot Dry Slide

Blot dry with an non-linty paper towel or bibulous paper. Do not rub.

 

 

Blot Dry Slide

 

 

 

Dry Slide

Flame again briefly to dry slide.

 

 

Examine Under Microscope

Buccal Smear

Examine first with the 4x objective, scanning the entire field to find a well-distributed region. Avoidregions where cells may be piled up to thickly . Then view with the 10x and 40x objectives, illustrating the view at both powers. Note 1) the nucleus, 2) nucleolus, 3) cell boundary and 4) the variety of bacteria colonizing the surface of the cells.
For Microbiology only: you will be instructed on oil immersion use , then illustrate bacterial morphologies with the 100x oil immersion objective.

Clean Up

Washed Slides

When finished, scrub the slide well in hot soapy water, rinse well and drain dry in a plastic test tube holder.

Use of Contact Paper for Mounting Handouts and Specimens

Cut contact paper larger than the trimmed handout.

Position the handout with contact paper over page

Handout sheets should be permanently mounted in your notebook using contact paper. This forms a protective, transparent cover, and ensures that you will not lose them. You may also permanently mount flat specimens in the same way.

Materials:

Use clear, transparent contact paper which has a smooth surface (no embossed pattern). Otherwise, the specimen will be obscured. The most suitable brand was “Adhere”, but appears to be no longer available. “Con-Tact” Clear and “Kwik Kover II” are readily available and are suitable. You may wish to try a different brand. Use these criteria for evaluation: clarity of detail of mounted specimen, ability to reposition an incorrectly placed specimen, ability of the contact paper to be written upon with ink (especially your TOMBO Pen, or india ink), cost, tendency of adhesive to creep out beyond edge of the contact paper, resistance to yellowing.

Protocol:

Date and title the page in your notebook (date = day protocol is mounted). Add any written notes relating to the handout first since writing on contact paper is difficult. Cut away all excess paper on printed handout.

You may trim the excess paper as close as possible without removing any of the printing.

Placed trimmed handout on top of unrolled contact paper , cut contact paper so that at least 1/2 inch margin projects beyond the specimen. The final dimensions should be smaller than the size of the notebook page. (Some prefer to cut rolls of contact paper in half which then approximates the desired page height.)

Strip off the backing from the contact paper without creasing the contact paper . (Start it by slightly tearing backing paper.) Lie it on the table sticky side up.

CRITICAL STEP: Hold trimmed specimen above contact paper so that the printed side is down, and margins are even. Bow the sheet and lower center down onto adhesive. Roll down specimen evenly onto contact paper, avoiding bubbles, creases, wrinkles, etc. Press out from the middle out to adhere to adhesive . Do not attempt to pull printed material off contact paper, the ink will stick to the adhesive (what a mess).

Trim any excess margin from around the specimen, but try to preserve the 1/2 inch margin.

Position contact-papered specimen into place in notebook , as close as possible to the bottom of the page, leaving space at the top for title, date & cross references. Make sure the sticky edges do not project beyond the edge of the page. Press from the middle out to adhere edges.

For handouts which have lines for cross referencing to the page in your notebook where the items is to be found, leave the lines uncovered with contact paper so that you can enter the page numbers as the items are entered into your notebook.

Here is the inside front cover with a Lab Schedule and the facing page with a Handout Table of Contents.

Here is a view of the guidelines drawn on the edges of the closed book. The TITLE goes between lines 1 and 3, the cross reference goes on line 6, and the body of the entry goes below line 9.

 

 

 

 

 

 

 

Index to Micro Slides

Clostridium botulinum, etiological agent of botulism, has terminal “slipper” shaped spores.

Trichinella spiralis, encysted in skeletal muscle (pork?) is a eukaryotic nematode

Please keep these slides in the correct order in their case. Carefully wipe all immersion oil off of them before you return them to the case. Sign your name (very small) and the year on the label on the front of the slide box to show that you received them in good order.

Slide No)    Speciman                    Catalog Number¹
1) Letter “e”                                                    Z 1
2) Bacteria, three types                               90 W 0151
3) Bacillus anthracis                                     90 W 2021
4) Mycobacterium tuberculosis                90 W 2054
5) Proteus vulgaris-flagellar stain           90 W 0542
6) Bacterial capsules (negative stain)     Ba 016A
7) Neisseria gonorrhoeae                            Ba 133
8) Pseudomonas flourescens                     Ba 165
9) Azotobacter aerogenes                           90 W 0529
10) Rhizobium leguminosarum                 Ba 169a
11) Brucella abortus                                       Ba 37E
12) Enterobacter aerogenes                        90 W 0521
13) Streptococcus lactis                                90 W 0559
14) Staphylococcus aureus                          90 W 2079
15 ) Bacillus subtilis                                       90 W 0533
16) Clostridium botulinum                          Ba 45
17) Clostridium tetani                                   Ba 60
18) Rhodospirillum rubrum                        90 W 0551
19) Giardia lamblia                                         PS 210
20) Trichomonas vaginalis group              PS 250
(with flagellum clearer)
21) Trypanosoma lewisi                                PS 300
22) Trypanosoma gambiense                      PS 310
23) Plasmodium vivax                                   PS 620
23) Trichinella spiralis                                  PS 2430
24) Mixed Protozoans                                    Z 50

¹ All slides beginning with a “90 W” are from Wards. The rest are from Carolina Biological: Z = zoology, Ba = bacteria. PS = parasites.

Immersion Oil Microscopy

 

 

Applying oil to the slide

100x objective has been rotated into the oil.

The most powerful lens of the light microscope is the 100x oil immersion objective. Because light is refracted every time it passes through a medium with a different refractive index, (air to glass or vice versa) the quality of the image is reduced with each passage. Thus, by reducing the number of such passages to a minimum, the clarity, brilliance and resolving power is preserved. You can see the difference between 400x and 1000x in the image to the left.

Immersion oil has been formulated so that it has a refractive index identical to that of glass. (It is written on the label of the immersion oil container as n _D ²⁵ : record it in your notebook.) Thus there is no refraction of light when it passes from glass to oil and vice versa. You can see the effect of this by removing the glass dropper rod from the oil, and re-immersing it. What happens to the image of the glass rod? How do you explain this observation?

Thus, two changes in refractive index can be eliminated by placing a drop of immersion oil on the specimen, and immersing the 100x oil immersion objective directly into the drop. You should be struck by the clarity that results.

Illustrate the four stage process of using the oil immersion lens:

Focus very carefully with the 40x objective over the stained specimen on the slide. Once focused, do not alter focus for the next three steps!

Rotate turret half way so that the 40x and 100x objectives straddle specimen.

Apply a small drop of oil directly on the slide over the specimen.

Rotate 100x objective into the immersion oil.

Three important rules attend the use of this lens:

1. Never use an oil immersion lens without the oil.
2. Never get oil on any other lens.
3. Clean up all oil when finished.

Protocol:
1. Focus at low power on a region of a smeared and stained specimen which is well-spread and stained (not too thin, nor too thick).

2. Rotate turret to 40x objective, locate desired portion of specimen in the center of the field. Refocus very carefully so that the specimen is focused as sharply as possible. (Do not alter focus for the following steps )

3. CRITICAL STEP: Partially rotate turret so that 40x and 100x objectives straddle the specimen.

Place a small drop of oil on the slide in the center of the lighted area. (Take care not to dribble on the stage.)

Note the small drop of oil directly over the area of the specimen to be examined.

4. Rotate turret so that the 100x oil immersion objective touches the oil and clicks into place.

Focus only with fine focus. Hopefully, the specimen will come into focus easily. Do not change focus dramatically. If you still have trouble, move the slide slightly left and right, looking for movement in the visual field, and focus on the object which moved.
5. With more than one specimen on a slide, do not alter focusing, rather, place a drop of oil on the second specimen, and slide the slide laterally until it is in place.
6. Never go back to the 10x or 40x objectives after you have applied oil to the specimen since oil can ruin the lower power objectives. [The 4x objective can be used because it is high enough to be above the oil.]
7. Clean up!: When you have finished for the day, wipe the 100x oil immersion objective carefully with lens paper to remove all oil. Wipe oil from the slide thoroughly with a Kimwipe. Cleanse stage should any oil have spilled on it. Recap the immersion oil container securely, replace in drawer.

Microscope Storage and Grade Sheet

Proper preparation of the microscope for storage reduces chances of damage to the instrument, and ensures that it will be ready for the next use without unwanted surprises. Students should know and follow these steps to proper storage. There will be unannounced occasional graded inspections of microscopes following their use.

Note the grade sheet, which will be used for that purpose, at the bottom of the page.

Microscope CORRECTLY vs. INCORRECTLY stored.

CORRECTLY PREPARED: Correctly stored with respect to correct objective, retracted mechanical stage and power cord securely wrapped.

INCORRECTLY PREPARED: 4x objective is not returned to lowest setting.

Microscope objective not on lowest setting.

INCORRECTLY PREPARED: 4x objective is not returned to lowest setting.

Microscope cord wrapping, Correct vs. Incorrect

INCORRECTLY PREPARED: 4x objective is not returned to lowest setting.

INCORRECTLY PREPARED: sharp bend in cord.

CORRECTLY PREPARED: Cord is neatly wrapped around the base.

Problems in the Storage Cabinet:

Dust cover not replaced and arm is positioned away from you in the storage cabinet.

 

 

 

 

 

 

 

Microscope Storage Grade Sheet:

 

Microscope Storage Grade Sheet-DOCX

OR

Microscope Storage Grade Sheet-PDF