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20.4: Materials and Procedures - Biology

20.4: Materials and Procedures - Biology


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Materials

  • Student Stock Cultures and saline spore solutions:
    • Bacillus cereus
    • Bacillus subtilis
    • Sporosarcina ureae
  • Glass Slides
  • Stain tray and slide holder
  • Stain kit:
    • Malachite Green
    • Safranin
    • Filter paper rounds
    • Hot plate set-up: small beakers of DI water with boiling stones
    • Glass bowls with Sanisol

Procedures

  1. Prepare heat-fixed slides of each culture.
  2. Place a filter paper round on the slide on top of the smear.
  3. Cover the filter paper and slide with Malachite Green and at the hot plate set-ups, place the slide across the top of a steaming beaker of DI water. Steam for 5-10 minutes. While steaming, add additional stain as needed to keep the filter paper wet. DO NOT let the slide dry out.
  4. Remove and discard the filter paper. DO NOT discard in the sink!
  5. Gently wash with DI water (this is essentially the decolorizer step; there is no separate decolorizer).
  6. Counterstain by covering the smear with Safranin for 2 minutes.
  7. Gently wash with DI water, blot, and observe under oil immersion.

Results

Draw and color (or indicate colors of) your Endospore stain. Label vegetative cells and endospores.


Biological Value

The BV of protein is determined by measuring the amount of N consumed and that excreted. First, the obligatory losses of urinary and fecal N must be determined, which requires the feeding of nitrogen-free diets. This is followed by a determination of the amounts of urinary and fecal N with consumption of the test protein. The differences in N excreted between the two dietary conditions is expressed as [Δ fecal N] and [Δ urinary N]. The Greek capital delta conventionally means “the change in.” The formula for the BV is

The BV represents the fraction of amino acids absorbed by the gut that is retained by the body. The BV ignores that part of the dietary protein that is not absorbed and appears in the feces. The change in fecal N occurring with the two diets is subtracted from the values of N intake, in both the numerator and denominator. The equation seems to state, “Let's ignore that part of the dietary protein that is poorly digested and appears in the feces.” The equation seems to ask, “As far as the absorbed amino acids are concerned, how suitable are they for the needs of the animal?” A high BV indicates that the amino acids occur in proportions that are highly compatible with the needs of the body. A protein that is completely lacking in one of the indispensable amino acids would be expected to have a BV close to or equal to zero. The formula for the BV yields a fraction. This fraction may be multiplied by 100 to express the BV as a percentage.


Virtual Rat Dissection

Step 1: In the biology lab, you will be working with specimens that have been preserved in chemicals and you will be working with sharp instruments.

Before you start, obtain safety goggles, and nitrile gloves. Nitrile gloves come in different sizes, most women will wear a medium and most men will wear a large. This can vary though. Try not to waste gloves by choosing the correct size.

Check to see if your station has the equipment you will need to dissect the rat. This includes a dissecting pan, scalpel, scissors, probes, and pins.

Rats are ordered from biological supply companies. They come stored in a preserving fluid, either in bags or in a bucket. Always use latex or nitrile gloves when handling your rat and safety goggles are required to protect your eye from chemical splattering or debris.

The rats do have fur, though they can vary in color. Many lab rats are white, but this does not mean they are albino. Some rats are white with a black stripe, sometimes called "hooded rats." Other rats are dark brown or gray.

These rats are stored in "carosafe" a chemical that keeps them preserved. Over time, rats stored in buckets become bloated with the chemical, use caution when opening the body cavity of these rats as the chemical is prone to spray and splatter. Once an incision is made, the rats can be drained of fluid.

Place your rat in a dissecting tray and examine the external features. The rat (and all vertebrates) has anatomical regions to help locate structures.

cranial region – head | cervical region – neck
pectoral region - area where front legs attach
thoracic region - chest area | abdomen - belly
pelvic region - area where the back legs attach


Safety of Equipment in Laboratory: Precautions and Procedures

Read this article to learn about some of the precautions and procedures to be observed with some commonly used laboratory equipment for its safety.

Equipment Safety:

Whenever lab equipment is purchased, preference should be given to equipment that:

i. Limits contact between the operator and hazardous material, and mechanical and electrical energy

ii. Is corrosion-resistant, easy to decontaminate and impermeable to liquids

iii. Has no sharp edges or burrs.

Every effort should be made to prevent equipment from becoming contaminated.

To reduce the likelihood of equipment malfunction that could result in leakage, spill or unnecessary generation of aerosolized pathogens:

i. Review the manufacturer’s documentation. Keep for future reference.

ii. Use and service equipment according to the manufacturer’s instructions.

iii. Ensure that anyone who uses a specific instrument or piece of equipment is properly trained in set-up, use and cleaning of the item.

iv. Decontaminate equipment before it is sent out for repairs or discarded.

The following sections outline some of the precautions and procedures to be observed with some commonly used laboratory equipment.

Centrifuges:

Improperly used or maintained centrifuges can present significant hazards to users. Failed mechani­cal parts can result in release of flying objects, hazardous chemicals and bio-hazardous aerosols. The high speed spins generated by centrifuges can create large amounts of aerosol if a spill, leak or tube breakage occurs.

To avoid contaminating your centrifuge:

i. Check glass and plastic centrifuge tubes for stress lines, hairline cracks and chipped rims before use. Use unbreakable tubes whenever possible.

ii. Avoid filling tubes to the rim.

iii. Use caps or stoppers on centrifuge tubes. Avoid using lightweight materials such as aluminum foil as caps.

iv. Use sealed centrifuge buckets (safety cups) or rotors that can be loaded and unloaded in a biological safety cabinet. Decontaminate the outside of the cups or buckets before and after centrifugation. Inspect O-rings regularly and replace if cracked or dry.

v. Ensure that the centrifuge is properly balanced.

vi. Do not open the lid during or immediately after operation, attempt to stop a spinning rotor by hand or with an object, or interfere with the interlock safety device.

vii. Decant supernatants carefully and avoid vigorous shaking when re-suspending.

When using high-speed or ultra-centrifuges, follow the additional practices:

i. Connect the vacuum pump exhaust to a trap.

ii. Record each run in a logbook, keep a record of speed and run time for each rotor.

iii. Install a HEPA filter between the centrifuge and the vacuum pump when working with bio-hazardous material.

iv. Never exceed the specified speed limitations of the rotor.

Electrophoresis Equipment:

i. Ensure that electrophoresis equipment is properly grounded and has electrical interlocks. Do not bypass safety interlocks.

ii. Inspect electrophoresis equipment regularly for damage and potential tank leaks.

iii. Locate equipment away from high traffic areas, and away from wet areas such as sinks or washing apparatus.

Heating Baths, Water Baths:

Heating baths keep immersed materials immersed at a constant temperature. They may be filled with a variety of materials, depending on the bath temperature required they may contain water, mineral oil, glycerin, paraffin or silicone oils, with bath temperatures ranging up to 300°C.

The following precautions are appropriate for heating baths:

i. Set up on a stable surface, away from flammable and combustible materials including wood and paper

ii. Relocate only after the liquid inside has cooled

iii. Ensure baths are equipped with redundant heat controls or automatic cut-offs that will turn off the power if the temperature exceeds a preset limit

iv. Use with the thermostat set well below the flash point of the heating liquid in use

v. Equip with a thermometer to allow a visual check of the bath temperature.

The most common heating bath used in laboratories is the water bath. When using a water bath:

i. Clean regularly a disinfectant, such as a phenolic detergent, can be added to the water

ii. Avoid using sodium azide to prevent growth of micro-organisms sodium azide forms explosive compounds with some metals

iii. Raise the temperature to 90°C or higher for 30 minutes once a week for decontamination purposes

iv. Unplug the unit before filling or emptying, and have the continuity-to-ground checked regularly.

Shakers, Blenders and Sonicators:

When used with infectious agents, mixing equipment such as shakers, blenders, sonicators, grinders and homogenizers can release significant amounts of hazardous aerosols, and should be operated inside a biological safety cabinet whenever possible. Equipment such as blenders and stirrers can also produce large amounts of flammable vapours.

The hazards associated with this type of equip­ment can be minimized by:

i. Selecting and purchasing equipment with safety features that minimize leaking

ii. Selecting and purchasing mixing apparatus with non-sparking motors.

iii. Checking integrity of gaskets, caps and bottles before using. Discard damaged items.

iv. Allowing aerosols to settle for at least one minute before opening containers

v. Covering tops of blenders with a disinfectant-soaked towel during operation, when us­ing bio-hazardous material

vi. When using a sonicator, immersing the tip deeply enough into the solution to avoid creation of aerosols

vii. Decontaminating exposed surfaces after use.

Ovens and Hot Plates:

Laboratory ovens are useful for baking or curing material, off-gassing, dehydrating samples and drying glassware.

i. Select and purchase an oven whose design prevents contact between flammable vapours and heating elements or spark-producing components.

ii. Discontinue use of any oven whose backup thermostat, pilot light or temperature controllers have failed.

iii. Avoid heating toxic materials in an oven unless it is vented outdoors (via a canopy hood, for example).

iv. Never use laboratory ovens for preparation of food for human consumption.

v. Glassware that has been rinsed with an organic solvent should be rinsed with distilled water before it is placed in a drying oven.

Analytical Equipment:

The following instructions for safe use of analytical equipment are general guidelines consult the user’s manual for more detailed information on the specific hazards:

i. Ensure that installation, modification and repairs of analytical equipment are carried out by authorized service personnel.

ii. Read and understand the manufacturer’s instructions before using this equipment.

iii. Make sure that preventive maintenance procedures are performed as required.

iv. Do not attempt to defeat safety interlocks.

v. Wear safety glasses and lab coats (and other appropriate personal protective equipment’s as specified) for all procedures.

Scintillation Counters:

i. Use sample vials that meet the manufacturer’s specifications.

ii. Keep counters clean and free of foreign materials.

iii. To avoid contaminating the counter and its accessories with radioactivity, change gloves before loading racks in the counter or using the computer keyboard. Verify on a regular basis (by wipe testing) that the equipment has not become contaminated.

Atomic absorption (AA) spectrometers:

Sample preparation for atomic absorption procedures often requires handling of flammable, toxic and corrosive products. Familiarize yourself with the physical, chemical and toxicological proper­ties of these materials and follow the recommended safety precautions. Atomic absorption equip­ment must be adequately vented, as toxic gases, fumes and vapours are emitted during operation.

Other recommendations to follow when carrying out atomic absorption analysis are:

i. Wear safety glasses for mechanical protection.

ii. Check the integrity of the burner, drain and gas systems before use.

iii. Inspect the drain system regularly empty the drain bottle frequently when running or­ganic solvents.

iv. Allow the burner head to cool to room temperature before handling.

v. Never leave the flame unattended. A fire extinguisher should be located nearby.

vi. Avoid viewing the flame or furnace during atomization unless wearing protective eyewear.

vii. Hollow cathode lamps are under negative pressure and should be handled with care and disposed of properly to minimize implosion risks.

Mass Spectrometers (MS):

Mass spectrometry requires the handling of compressed gases and flammable and toxic chemicals. Consult MSDSs for products before using them.

Specific precautions for working with the mass spectrometer include:

i. Avoid contact with heated parts while the mass spectrometer is in operation.

ii. Verify gas, pump, exhaust and drain system tubing and connections before each use.

iii. Ensure that pumps are vented outside the laboratory, as pump exhaust may contain traces of the samples being analyzed, solvents and reagent gas.

iv. Used pump oil may also contain traces of analytes and should be handled as hazardous waste.

Gas Chromatographs (GC):

Gas chromatography requires handling compressed gases (nitrogen, hydrogen, argon, helium), and flammable and toxic chemicals. Consult product MSDSs before using such hazardous products.

Specific precautions for working with gas chromatographs include:

i. Perform periodic visual inspections and pressure leak tests of the sampling system plumb­ing, fittings and valves.

ii. Follow the manufacturer’s instructions when installing columns. Glass or fused capillary columns are fragile handle them with care and wear safety glasses to protect eyes from flying particles while handling, cutting or installing capillary columns.

iii. Turn off and allow heated areas such as the oven, inlet and detector, as well as connected hardware, to cool down before touching them.

iv. To avoid electrical shock, turn off the instrument and disconnect the power cord at its receptacle whenever the access panel is removed.

v. Turn off the hydrogen gas supply at its source when changing columns or servicing the instrument.

vi. When using hydrogen as fuel (flame ionization FID and nitrogen-phosphorus detectors NPD), ensure that a column or cap is connected to the inlet fitting whenever hydrogen is supplied to the instrument to avoid build-up of explosive hydrogen gas in the oven.

vii. Measure hydrogen gas and air separately when determining gas flow rates.

viii. Perform a radioactive leak test (wipe test) on electron capture detectors (ECDs) at least every 6 months for sources of 50 MBq (1.35 mCi) or greater.

ix. Ensure that the exhaust from (ECDs) is vented to the outside.

x. When performing split sampling, connect the split vent to an exhaust ventilation system or appropriate chemical trap if toxic materials are analyzed or hydrogen is used as the carrier gas.

xi. Use only helium or nitrogen gas, never hydrogen, to condition a chemical trap.

Nuclear Magnetic Resonance (NMR) Equipment:

The superconducting magnet of NMR equipment produces strong magnetic and electromagnetic fields that can interfere with the function of cardiac pacemakers. Users of pacemakers and other implanted ferromagnetic medical devices are advised to consult with their physicians, the pacemaker’s manual and pacemaker manufacturer before entering facilities which house NMR equipment.

Pre­cautions for work with NMR include the following:

i. Post clearly visible warning signs in areas with strong magnetic fields.

ii. Measure stray fields with a gauss meter, and restrict public access to areas of 5 gausses or higher.

iii. The strong magnetic field can suddenly pull nearby unrestrained magnetic objects into the magnet with considerable force. Keep all tools, equipment and personal items con­taining ferromagnetic material (e.g., steel, iron) at least 2 metres away from the magnet.

iv. Though not a safety issue, advise users that the magnetic field can erase magnetic media such as tapes and floppy disks disable credit and automated teller machine (ATM) cards, and damage analog watches.

v. Avoid skin contact with cryogenic (liquid) helium and nitrogen wear a protective face mask and loose-fitting thermal gloves during Dewar servicing and when handling frozen samples.

vi. Ensure that ventilation is sufficient to remove the helium or nitrogen gas exhausted by the instrument.

vii. Avoid positioning your head over the helium and nitrogen exit tubes.

viii. NMR tubes are thin-walled handle them carefully and reserve them for NMR use only.

High-pressure Liquid Chromatography (HPLC) Equipment:

HPLC procedures may require handling of compressed gas (helium) and flammable and toxic chemicals. Familiarize yourself with the hazardous properties of these products, as well as recom­mended precautionary measures by referring to MSDSs.

i. Inspect the drain system regularly empty the waste container frequently when using or­ganic solvents.

ii. Ensure that waste collection vessels are vented.

iii. Never use solvents with auto ignition temperatures below 110°C.

iv. Be sure to use a heavy walled flask if you plan to use vacuum to degas the solvent.

v. Never clean a flow-cell by forcing solvents through a syringe: syringes under pressure can leak or rupture, resulting in sudden release of syringe contents.

vi. High voltage and internal moving parts are present in the pump. Switch off the electrical power and disconnect the line cord when performing routine maintenance of the pump.

vii. Shut down and allow the system to return to atmospheric pressure before carrying out maintenance procedures.

Liquid Chromatography (LC/MS) Equipment:

LC/MS requires the handling of compressed nitrogen and flammable and toxic chemicals. Consult product MSDSs before using them.

Take the following specific precautions for working with LC/ MS equipment:

i. Verify gas, pump exhaust and drain system tubing and connections before each use.


20.4: Materials and Procedures - Biology

When writing a lab report, it is often a good idea to begin by writing the Materials and Methods section. This section is usually very straightforward, and writing it first helps many people establish the proper thought process and understanding of the work that will allow the rest of the report to flow more smoothly. Following this section, it is generally recommended to write the Results section, followed by the Discussion, and finally the Introduction. Although this strategy is only a recommendation, and although it may seem illogical at first, many have found this approach very effective for writing scientific papers.

The Materials and Methods section is a vital component of any formal lab report. This section of the report gives a detailed account of the procedure that was followed in completing the experiment(s) discussed in the report. Such an account is very important, not only so that the reader has a clear understanding of the experiment, but a well written Materials and Methods section also serves as a set of instructions for anyone desiring to replicate the study in the future. Considering the importance of "reproducible results" in science, it is quite obvious why this second application is so vital.

There are several common mistakes that are often found in the Materials and Methods section of a lab report. One major concern is deciding upon the correct level of detail. (Pechenik, p. 55) It is often very easy for a writer to get carried away and include every bit of information about the procedure, including extraneous information like the number of times heshe washed their hands during the experiment. A good guideline is to include only what is necessary for one recreating the experiment to know. Keeping this in mind will lead to a Materials and Methods section that is thoroughly written, but without the kind of unnecessary detail that breaks the flow of the writing. Another common mistake is listing all of the materials needed for the experiment at the beginning of the section. Instead, the materials and equipment utilized during the experiment should be mentioned throughout the procedure as they are used. Enough detail should be included in the description of the materials so that the experiment can be reproduced. Finally, it is generally recommended that the Materials and Methods section be written in past tense, in either active or passive voice. Many are written in third-person perspective but check with the professor to be certain what verb tense and perspective the report should use. This is demonstrated throughout the example of a well written Materials and Methods section.

Materials and Methods examples

Sample 1 : In preparing the catecholase extract, a potato was skinned, washed, and diced. 30.0 g of the diced potato and 150 ml of distilled water were added to a kitchen blender and blended for approximately two minutes. The resulting solution was filtered through four layers of cheese cloth. The extract was stored in a clean, capped container.

Four individually labeled spectrophotometer tubes were prepared using different amounts (as represented in Table 1) of the following reagents: a buffer of pH 7, a 0.1% catechol substrate, and distilled water. The wavelength of the Spectronic 20 spectrophotometer was set at 540 nm. To calibrate the specrophotometer at zero absorbance, a blank control tube prepared with no catechol substrate and labeled "tube 1" was inverted and inserted into the spectrophotometer.

It is important to note that the extract to be tested was added to each tube immediately before placing the tube into the spectrophotometer. 1.0 ml of catecholase extract was pipetted into tube 2. Tube 2 was immediately inverted and placed in the spectrophotometer. The absorbance was read and recorded for time zero (t0), the ten minute mark (t10), and each minute in between. Tube 2 was removed from the spectrophotometer and the same measurements were taken for tube 3 and tube 4 using the same protocol.

Sample 2 : A potato and a knife were obtained for this experiment. Also, distilled water, a blender, cheese cloth, a clean container with a cover, and eight spectrophotometer tubes were used. A Spectronic 20 spectrophotometer was used for this experiment, as were buffers of pHs 4, 6, 7, and 8. Catechol substrate, Parafilm coverings, KimWipes, a black pen, and pipettes were also obtained for this experiment. Finally, a pencil and pad were obtained for recording results.

Sample 3 : In preparing the catecholase extract, a potato was skinned, washed, and diced. A balance was used to obtain 30.0 g of the diced potato. 150 ml of distilled water was poured into a beaker. The water was added to the diced potato. The cover of a kitchen blender was removed. The potato and water were added to the blender. The solution smelled like potato. The cover was placed on the blender and the power button was depressed. The clock was observed until the second hand circled twice. The power button was pushed again to stop the blender. The resulting solution was filtered through four layers of cheese cloth. The extract was stored in a clean, capped container.

Explanations of the Example Links

Diced potato : In sample one the writer gives enough detail about the procedure so that is can be understood, but not so much that there is an excess of unecessary detail. (return to sample 1)

Calibrate : Calibration is a small but important detail to include in this section so that the experiment would be able to be repeated by anyone reading the report. Keep this in mind while deciding what to include in this section. (return to sample 1)

Distilled water : This example has a list of materials at the beginning which are not necessary in the materials and methods section. The body of the section should mention the materials and equipment used during the experiment so that it is not necessary to list them in order to know what was used for the procedure. (return to sample 2)

Extraneous detail : This is extraneous detail that is not needed to explain the procedure. The reader would know how to turn the blender on and off without being told that a button was pushed, and knowing that the solution smelled like potato is completely unrelated to knowing how to perform the experiment. (return to sample 3)

All citations from Pechenik, Jan A. A short guide to writing about Biology. pp. 54-102, Tufts University: Harper Collins College Publishers . 1993.


4 Main Steps of Tissue Culture Techniques | Biotechnology

The following four main steps of tissue culture techniques. The steps are: 1. Inoculation of Explant 2. Incubation of Culture 3. Sub-Culturing 4. Transplantation of the Regenerated Plant.

Step # 1. Inoculation of Explant:

Successful control of contamination largely depends upon the precautions taken to prevent the entry of microorganisms at the time of transferring the sterilised explants on the nutrient medium. Dust, hair, hands and clothes are the potential sources of contami­nation. The inoculating chamber should be dust free the operator should wear sterile headgear and clothes (aprons) before entering the culture area.

The hands should be wiped with 95% alcohol and the transfer area also should be cleaned and wiped with 95% alcohol before starting the transfer process. Talking or sneezing should be avoided during transfer of explant into the media. The neck or mouth of culture container should be flamed, the transferring instruments also to be flamed and dipped in alcohol.

Care should be taken so that the explant should not touch the edge of culture vessel, and after trans­ferring the mouth should be closed by cap or by cotton plug and petridishes to be sealed by ‘Parafilm’. During transfer it is also to ensure that the plant tissue should be exposed to the media properly.

Step # 2. Incubation of Culture:

After inoculation, the cultures are incubated in culture room or in a BOD incubator at 25±2°C temp. For certain plant or for some particular culture type below or above 25°C is needed.

Some tissues grow well under low light condition (approx. 1000 lux), for regenera­tion light and dark periods are needed, and for regenerated plantlet well lighted (approx. 3000 lux) condition and 16h light with 8h dark period is needed. The illumination in the culture room is provided by cool white fluorescent light placed approx. 18″ above the cul­ture racks.

Low humidity causes the quick desiccation of culture medium and high humidity is favourable for contamination of culture medium. Specific relative humidity (20-98%) is to be maintained in the culture room and uniform air circulation is to be done properly.

For cell suspension culture agitation and aeration is secured by the use of shaker sys­tems open platform orbital shakers or the orbital incubators fitted with fluorescent lights to provide different day/night regimes.

Step # 3. Sub-Culturing:

The growth and development of tissues cultured in vitro are generally monitored by observing the cultures at regular intervals in the culture room or incubators.

Based on the observations either with hand-lens or with the aid of simple microscope under aseptic con­ditions, the explants may be required to transfer to new media (freshly prepared) or with new ingredients or hormone composition depending on the state of growth of cell or tissue.

The same precautions and full aseptic conditions are maintained during the transfer process also. The delaying of this process may lead to inhibition of proper development of tissues and also delaying the regeneration of plantlets.

In case of suspension culture the change of media or fresh inoculation at quick intervals is needed and also for callus culture the sub-culturing of the callus tissue is needed to get the callus tissue in dividing conditions.

Step # 4. Transplantation of the Regenerated Plant:

Plants regenerated from in vitro tissue culture are transplanted to soil in pots. Prior to transfer to pots the acclimatization of these regenerated plants are needed. The plants at this time develop adequate root systems and cuticular leaf surface structure so that it can withstand the field environmental condition.

The process of acclimatization needs the humid chamber and a slow process to make the plantlet habituated from high humid con­dition to normal atmospheric humidity. The greenhouse or the growth chamber should have artificial light system also which includes a mixture of fluorescent and incandescent lamps designed to provide balanced wavelengths of light for plant growth and photosyn­thesis.

The greenhouse facilities are needed for winter crops and summer crops different­ly for maintenance of proper temp, required, air circulation and the relative humidity. The potted plants are grown in field for further observation, flowering and normal seed setting to get the next progeny.


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Primary Cell Culture: 3 Techniques (With Diagram)

Primary culture broadly involves the culturing techniques carried following the isolation of the cells, but before the first subculture. Primary cultures are usually prepared from large tissue masses. Thus, these cultures may contain a variety of differentiated cells e.g. fibroblasts, lymphocytes, macrophages, epithelial cells.

With the experiences of the personnel working in tissue culture laboratories, the following criteria/ characteristics are considered for efficient development of primary cultures:

a. Embryonic tissues rather than adult tissues are preferred for primary cultures. This is due to the fact that the embryonic cells can be disaggregated easily and yield more viable cells, besides rapidly proliferating in vitro.

b. The quantity of cells used in the primary culture should be higher since their survival rate is substantially lower (when compared to subcultures).

c. The tissues should be processed with minimum damage to cells for use in primary culture. Further, the dead cells should be removed.

d. Selection of an appropriate medium (preferably a nutrient rich one) is advisable. For the addition of serum, fetal bovine source is preferred rather than calf or horse serum.

e. It is necessary to remove the enzymes used for disaggregation of cells by centrifugation.

Techniques for Primary Culture:

Among the various techniques devised for the primary culture of isolated tissues, three techniques are most commonly used:

1. Mechanical disaggregation.

2. Enzymatic disaggregation.

3. Primary explant technique.

An outline of these techniques is depicted in Fig. 36.1, and the procedures are briefly described:

Technique # 1. Mechanical Disaggregation:

For the disaggregation of soft tissues (e.g. spleen, brain, embryonic liver, soft tumors), mechanical technique is usually employed. This technique basically involves careful chopping or slicing of tissue into pieces and collection of spill out cells.

The cells can be collected by two ways:

i. Pressing the tissue pieces through a series of sieves with a gradual reduction in the mesh size.

ii. Forcing the tissue fragments through a syringe and needle.

Although mechanical disaggregation involves the risk of cell damage, the procedure is less expensive, quick and simple. This technique is particularly useful when the availability of the tissue is in plenty, and the efficiency of the yield is not very crucial. It must however, be noted that the viability of cells obtained from mechanical techniques is much lower than the enzymatic technique.

Technique # 2. Enzymatic Disaggregation:

Enzymatic disaggregation is mostly used when high recovery of cells is required from a tissue. Disaggregation of embryonic tissues is more efficient with higher yield of cells by use of enzymes. This is due to the presence of less fibrous connective tissue and extracellular matrix. Enzymatic disaggregation can be carried out by using trypsin, collagenase or some other enzymes.

Disaggregation by trypsin:

The term trypsinization is commonly used for disaggregation of tissues by the enzyme, trypsin.

Many workers prefer to use crude trypsin rather than pure trypsin for the following reasons:

i. The crude trypsin is more effective due to the presence of other proteases

ii. Cells can tolerate crude trypsin better.

iii. The residual activity of crude trypsin can be easily neutralized by the serum of the culture media (when serum-free media are used, a trypsin inhibitor can be used for neutralization).

Disaggregation of cells can also be carried out by using pure trypsin which is less toxic and more specific in its action. The desired tissue is chopped to 2-3 mm pieces and then subjected to disaggregation by trypsin. There are two techniques of trypsinization-warm trypsinization and cold trypsinization (Fig. 36.2).

Warm trypsinization (Fig. 36.2A):

This method is widely used for disaggregation of cells. The chopped tissue is washed with dissection basal salt solution (DBSS), and then transferred to a flask containing warm trypsin (37° C). The contents are stirred, and at an interval of every thirty minutes, the supernatant containing the dissociated cells can be collected. After removal of trypsin, the cells are dispersed in a suitable medium and preserved (by keeping the vial on ice).

The process of addition of fresh trypsin (to the tissue pieces), incubation and collection of dissociated cells (at 30 minutes intervals) is carried out for about 4 hours. The disaggregated cells are pooled, counted, appropriately diluted and then incubated.

Cold trypsinization (Fig. 36.2B):

This technique is more appropriately referred to as trypsinization with cold pre-exposure. The risk of damage to the cells by prolonged exposure to trypsin at 37°C (in warm trypsinization) can be minimized in this technique.

After chopping and washing, the tissue pieces are kept in a vial (on ice) and soaked with cold trypsin for about 6-24 hours. The trypsin is removed and discarded. However, the tissue pieces contain residual trypsin. These tissue pieces in a medium are incubated at 37°C for 20-30 minutes. The cells get dispersed by repeated pi-pettings. The dissociated cells can be counted, appropriately diluted and then used.

The cold trypsinization method usually results in a higher yield of viable cells with an improved survival of cells after 24 hours of incubation. This method does not involve stirring or centrifugation, and can be conveniently adopted in a laboratory. The major limitation of cold trypsinization is that it is not suitable for disaggregation of cells from large quantities of tissues.

Limitations of trypsin disaggregation:

Disaggregation by trypsin may damage some cells (e.g. epithelial cells) or it may be almost ineffective for certain tissues (e.g. fibrous connective tissue). Hence other enzymes are also in use for dissociation of cells.

Disaggregation by collagenase:

Collagen is the most abundant structural protein in higher animals. It is mainly present in the extra­cellular matrix of connective tissue and muscle. The enzyme collagenase (usually a crude one contaminated with non-specific proteases) can be effectively used for the disaggregation of several tissues (normal or malignant) that may be sensitive to trypsin.

Highly purified grades of collagenase have been tried, but they are less effective when compared to crude collagenase. The important stages in collagenase dis­aggregation, depicted in Fig. 36.3, are briefly described hereunder.

The desired tissue suspended in basal salt solution, containing antibiotics is chopped into pieces. These pieces are washed by settling, and then suspended in a complete medium containing collagenase. After incubating for 1-5 days, the tissue pieces are dispersed by pipetting. The clusters of cells are separated by settling. The epithelial cells and fibroblastic cells can be separated.

Collagenase disaggregation has been successfully used for human brain, lung and several other epithelial tissues, besides various human tumors, and other animal tissues. Addition of another enzyme hyaluronidase (acts on carbohydrate residues on cell surfaces) promotes disaggregation.

Collagenase in combination with hyaluronidase is found to be very effective for dissociating rat or rabbit liver. This can be done by per-fusing the whole organ in situ. Some workers use collagenase in conjunction with trypsin, a formulation developed in chick serum, for disaggregation of certain tissues.

Use of other enzymes in disaggregation:

Trypsin and collagenase are the most widely used enzymes for disaggregation. Certain bacterial proteases (e.g. pronase, dispase) have been used with limited success. Besides hyaluronidase, neuraminidase is also used in conjunction with collagenase for effective degradation of cell surface carbohydrates.

Technique # 3. Primary Explant Technique:

The primary explant technique was, in fact the original method, developed by Harrison in 1907. This technique has undergone several modifications, and is still in use. The simplified procedure adopted for primary explant culture is depicted in Fig. 36.4, and briefly described below.

The tissue in basal salt solution is finely chopped, and washed by settlings. The basal salt solution is then removed. The tissue pieces are spread evenly over the growth surface. After addition of appropriate medium, incubation is carried out for 3-5 days. Then the medium is changed at weekly intervals until a substantial out­growth of cells is observed. Now, the explants are removed and transferred to a fresh culture vessel.

The primary explant technique is particularly useful for disaggregation of small quantities of tissues (e.g. skin biopsies). The other two techniques mechanical or enzymatic disaggregation however, are not suitable for small amounts of tissues, as there is a risk of losing the cells.

The limitation of explant technique is the poor adhesiveness of certain tissues to the growth surface, and the selection of cells in the outgrowth. It is however, observed that the primary explant technique can be used for a majority of embryonic cells e.g. fibroblasts, myoblasts, epithelial cells, glial cells.

Separation of Viable and Non-Viable Cells:

It is a common practice to remove the non­viable cells while the primary culture is prepared from the disaggregated cells. This is usually done when the first change of the medium is carried out. The very few left over non-viable cells get diluted and gradually disappear as the proliferation of viable cells commences.

Sometimes, the non-viable cells from the primary cultures may be removed by centrifugation. The cells are mixed with ficoll and sodium metrizoate, and centrifuged. The dead cells form a pellet at the bottom of the tube.

Medical Ethics and Safety Measures in Culture Techniques:

Since the culture techniques involve the use of animal or human tissues, it is absolutely necessary to follow several safety measures and medical ethics. In fact, in some countries there are established legislation/norms for selection and use of tissues in cultures. For example, in United Kingdom, Animal Experiments (Scientific Procedures) Act of 1986 is followed.

The handling of human tissues poses several problems that are not usually encountered with animal tissues. While dealing with fetal materials and human biopsies, the consent of the patient and/his or her relatives, besides the consent of local ethical committee is required. Further, taking any tissue (even in minute quantities) from human donors requires the full consent of the donor in a prescribed format.

The following issues need to be fully considered while dealing with human tissues:

1. The consent of the patient and/or relatives for using tissues for research purposes.

2. Ownership of the cell lines developed and their derivatives.

3. Consent for genetic modification of the cell lines.

6. Patent rights for any commercial use of cell lines.

In the general practice of culture techniques using human tissues, the donor and/or relatives are asked to sign a disclaimer statement (in a prescribed pro-forma) before the tissue is taken. By this approach, the legal complications are minimized.

Handling of human tissues is associated with a heavy risk of exposure for various infections. Therefore, it is absolutely necessary that the human materials are handled in a biohazard cabinet. The tissues should be screened for various infections such as hepatitis, tuberculosis, HIV, before their use. Further, the media and apparatus, after their use must be autoclaved or disinfected, so that the spread of infections is drastically reduced.


20.4: Materials and Procedures - Biology

The scientific format may seem confusing for the beginning science writer due to its rigid structure which is so different from writing in the humanities. One reason for using this format is that it is a means of efficiently communicating scientific findings to the broad community of scientists in a uniform manner. Another reason, perhaps more important than the first, is that this format allows the paper to be read at several different levels. For example, many people skim Titles to find out what information is available on a subject. Others may read only titles and Abstracts . Those wanting to go deeper may look at the Tables and Figures in the Results , and so on. The take home point here is that the scientific format helps to insure that at whatever level a person reads your paper (beyond title skimming), they will likely get the key results and conclusions.

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The Sections of the Paper

Most journal-style scientific papers are subdivided into the following sections: Title, Authors and Affiliation, Abstract, Introduction, Methods, Results, Discussion, Acknowledgments, and Literature Cited, which parallel the experimental process. This is the system we will use. This website describes the style, content, and format associated with each section.

The sections appear in a journal style paper in the following prescribed order:

Experimental process

Section of Paper

What did I do in a nutshell?

Abstract

What is the problem?

Introduction

How did I solve the problem?

Materials and Methods

What did I find out?

Results

What does it mean?

Discussion

Who helped me out?

Acknowledgments (optional)

Whose work did I refer to?

Literature Cited

Extra Information

Appendices (optional)

Section Headings:

Main Section Headings: Each main section of the paper begins with a heading which should be capitalized , centered at the beginning of the section, and double spaced from the lines above and below. Do not underline the section heading OR put a colon at the end.

Example of a main section heading:

INTRODUCTION

Subheadings: When your paper reports on more than one experiment, use subheadings to help organize the presentation. Subheadings should be capitalized (first letter in each word), left justified, and either bold italics OR underlined .

Example of a subheading:

Effects of Light Intensity on the Rate of Electron Transport

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Title, Authors' Names, and Institutional Affiliations

  • The title should be centered at the top of page 1 (DO NOT use a title page - it is a waste of paper for our purposes) the title is NOT underlined or italicized .
  • the authors' names (PI or primary author first) and institutional affiliation are double-spaced from and centered below the title. When more then two authors, the names are separated by commas except for the last which is separated from the previous name by the word "and".

ABSTRACT

  • the question(s) you investigated (or purpose), ( from Introduction )
    • state the purpose very clearly in the first or second sentence.
    • clearly express the basic design of the study.
    • Name or briefly describe the basic methodology used without going into excessive detail-be sure to indicate the key techniques used.
    • report those results which answer the questions you were asking
    • identify trends, relative change or differences, etc.
    • clearly state the implications of the answers your results gave you.
    • lengthy background information,
    • references to other literature,
    • elliptical (i.e., ending with . ) or incomplete sentences,
    • abbreviations or terms that may be confusing to readers,
    • any sort of illustration, figure, or table, or references to them.

    INTRODUCTION

    • Establish the context of the work being reported. This is accomplished by discussing the relevant primary research literature (with citations) and summarizing our current understanding of the problem you are investigating
    • State the purpose of the work in the form of the hypothesis, question, or problem you investigated and,
    • Briefly explain your rationale and approach and, whenever possible, the possible outcomes your study can reveal.
    • Begin your Introduction by clearly identifying the subject area of interest. Do this by using key words from your Title in the first few sentences of the Introduction to get it focused directly on topic at the appropriate level. This insures that you get to the primary subject matter quickly without losing focus, or discussing information that is too general. For example, in the mouse behavior paper, the words hormones and behavior would likely appear within the first one or two sentences of the Introduction.
    • Establish the context by providing a brief and balanced review of the pertinent published literature that is available on the subject. The key is to summarize (for the reader) what we knew about the specific problem before you did your experiments or studies. This is accomplished with a general review of the primary research literature (with citations) but should not include very specific, lengthy explanations that you will probably discuss in greater detail later in the Discussion. The judgment of what is general or specific is difficult at first, but with practice and reading of the scientific literature you will develop e firmer sense of your audience. In the mouse behavior paper, for example, you would begin the Introduction at the level of mating behavior in general, then quickly focus to mouse mating behaviors and then hormonal regulation of behavior. Lead the reader to your statement of purpose/hypothesis by focusing your literature review from the more general context (the big picture e.g., hormonal modulation of behaviors) to the more specific topic of interest to you (e.g., role/effects of reproductive hormones, especially estrogen, in modulating specific sexual behaviors of mice.)
    • What literature should you look for in your review of what we know about the problem? Focus your efforts on the primary research journals - the journals that publish original research articles. Although you may read some general background references (encyclopedias, textbooks, lab manuals, style manuals, etc.) to get yourself acquainted with the subject area, do not cite these, becasue they contain information that is considered fundamental or "common" knowledge wqithin the discipline. Cite, instead, articles that reported specific results relevant to your study. Learn, as soon as possible, how to find the primary literature (research journals) and review articles rather than depending on reference books. The articles listed in the Literature Cited of relevant papers you find are a good starting point to move backwards in a line of inquiry. Most academic libraries support the Citation Index - an index which is useful for tracking a line of inquiry forward in time. Some of the newer search engines will actually send you alerts of new papers that cite particular articles of interest to you. Review articles are particularly useful because they summarize all the research done on a narrow subject area over a brief period of time (a year to a few years in most cases).
    • Be sure to clearly state the purpose and /or hypothesis that you investigated. When you are first learning to write in this format it is okay, and actually preferable, to use a pat statement like, "The purpose of this study was to. " or "We investigated three possible mechanisms to explain the . (1) blah, blah..(2) etc. It is most usual to place the statement of purpose near the end of the Introduction, often as the topic sentence of the final paragraph. It is not necessary (or even desirable) to use the words "hypothesis" or "null hypothesis", since these are usually implicit if you clearly state your purpose and expectations.
    • Provide a clear statement of the rationale for your approach to the problem studied. For example: State briefly how you approached the problem (e.g., you studied oxidative respiration pathways in isolated mitochondria of cauliflower). This will usually follow your statement of purpose in the last paragraph of the Introduction. Why did you choose this kind of experiment or experimental design? What are the scientific merits of this particular model system? What advantages does it confer in answering the particular question(s) you are posing? Do not discuss here the actual techniques or protocols used in your study (this will be done in the Materials and Methods) your readers will be quite familiar with the usual techniques and approaches used in your field. If you are using a novel (new, revolutionary, never used before) technique or methodology, the merits of the new technique/method versus the previously used methods should be presented in the Introduction.

    MATERIALS AND METHODS

    • the the organism(s) studied (plant, animal, human, etc.) and, when relevant, their pre-experiment handling and care, and when and where the study was carried out ( only if location and time are important factors) note that the term "subject" is used ONLY for human studies.
    • if you did a field study , provide a description of the study site , including the significant physical and biological features, and the precise location (latitude and longitude, map, etc)
    • the experimental OR sampling design (i.e., how the experiment or study was structured. For example, controls, treatments, what variable(s) were measured, how many samples were collected, replication, the final form of the data, etc.)
    • the protocol for collecting data , i.e., how the experimental procedures were carried out, and,
    • how the data were analyzed (qualitative analyses and/or statistical procedures used to determine significance, data transformations used, what probability was used to decide significance, etc).
    • NOTE: For laboratory studies you need not report the date and location of the study UNLESS it is necessary information for someone to have who might wish to repeat your work or use the same facility. Most often it is not . If you have performed experiments at a particular location or lab because it is the only place to do it, or one of a few, then you should note that in your methods and identify the lab or facility.
    • NOTE : Very frequently the experimental design and data collection procedures for an experiment cannot be separated and must be integrated together. If you find yourself repeating lots of information about the experimental design when describing the data collection procedure(s), likely you can combine them and be more concise.
    • NOTE : Although tempting, DO NOT say that you " recorded the data ," i.e., in your lab notebook, in the Methods description. Of course you did , because that is what all good scientists do, and it is a given that you recorded your measurements and observations.
    • Statistical software used : Sometimes it is necessary to report which statistical software you used this would be at the discretion of your instructor or the journal
    • how the data were summarized (Means, percent, etc) and how you are reporting measures of variability (SD,SEM, 95% CI, etc)
      • this lets you avoid having to repeatedly indicate you are using mean ± SD or SEM.
      • any other numerical (e.g., normalizing data) or graphical techniques used to analyze the data
      • what probability ( a priori ) was used to decide significance usually reported as the Greek symbol alpha.
      • NOTE: You DO NOT need to say that you made graphs and tables.

      Here is some additional advice on particular problems common to new scientific writers.

      Problem : The Methods section is prone to being wordy or overly detailed.

      • Avoid repeatedly using a single sentence to relate a single action this results in very lengthy, wordy passages. A related sequence of actions can be combined into one sentence to improve clarity and readability:

      Problematic Example : This is a very long and wordy description of a common, simple procedure. It is characterized by single actions per sentence and lots of unnecessary details.

      "The petri dish was placed on the turntable. The lid was then raised slightly. An inoculating loop was used to transfer culture to the agar surface. The turntable was rotated 90 degrees by hand. The loop was moved lightly back and forth over the agar to spread the culture. The bacteria were then incubated at 37 C for 24 hr."

      Improved Example : Same actions, but all the important information is given in a single, concise sentence. Note that superfluous detail and otherwise obvious information has been deleted while important missing information was added.

      "Each plate was placed on a turntable and streaked at opposing angles with fresh overnight E. coli culture using an inoculating loop. The bacteria were then incubated at 37 C for 24 hr."

      Best: Here the author assumes the reader has basic knowledge of microbiological techniques and has deleted other superfluous information. The two sentences have been combined because they are related actions.

      "Each plate was streaked with fresh overnight E. coli culture and incubated at 37 C for 24 hr."


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      • Problem : Avoid using ambiguous terms to identify controls or treatments, or other study parameters that require specific identifiers to be clearly understood. Designators such as Tube 1, Tube 2, or Site 1 and Site 2 are completely meaningless out of context and difficult to follow in context.

      Problematic example : In this example the reader will have no clue as to what the various tubes represent without having to constantly refer back to some previous point in the Methods.

      " A Spec 20 was used to measure A 600 of Tubes 1,2, and 3 immediately after chloroplasts were added (Time 0) and every 2 min. thereafter until the DCIP was completely reduced. Tube 4's A 600 was measured only at Time 0 and at the end of the experiment."

      Improved example: Notice how the substitution ( in red ) of treatment and control identifiers clarifies the passage both in the context of the paper, and if taken out of context.

      "A Spec 20 was used to measure A 600 of the reaction mixtures exposed to light intensities of 1500, 750, and 350 uE/m2/sec immediately after chloroplasts were added (Time 0) and every 2 min. thereafter until the DCIP was completely reduced. The A 600 of the no-light control was measured only at Time 0 and at the end of the experiment."

      1. Function : The function of the Results section is to objectively present your key results, without interpretation, in an orderly and logical sequence using both text and illustrative materials (Tables and Figures). The results section always begins with text, reporting the key results and referring to your figures and tables as you proceed. Summaries of the statistical analyses may appear either in the text (usually parenthetically) or in the relevant Tables or Figures (in the legend or as footnotes to the Table or Figure). The Results section should be organized around Tables and/or Figures that should be sequenced to present your key findings in a logical order. The text of the Results section should be crafted to follow this sequence and highlight the evidence needed to answer the questions/hypotheses you investigated. Important negative results should be reported, too. Authors usually write the text of the results section based upon the sequence of Tables and Figures.

      2. Style : Write the text of the Results section concisely and objectively. The passive voice will likely dominate here, but use the active voice as much as possible. Use the past tense . Avoid repetitive paragraph structures. Do not interpret the data here. The transition into interpretive language can be a slippery slope. Consider the following two examples:

      The duration of exposure to running water had a pronounced effect on cumulative seed germination percentages (Fig. 2). Seeds exposed to the 2-day treatment had the highest cumulative germination (84%), 1.25 times that of the 12-h or 5-day groups and 4 times that of controls.

      • In contrast, this example strays subtly into interpretation by referring to optimality (a conceptual model) and tieing the observed result to that idea:

      The results of the germination experiment (Fig. 2) suggest that the optimal time for running-water treatment is 2 days. This group showed the highest cumulative germination (84%), with longer (5 d) or shorter (12 h) exposures producing smaller gains in germination when compared to the control group.

      Things to consider as you write your Results section:

      What are the "results"? : When you pose a testable hypothesis that can be answered experimentally, or ask a question that can be answered by collecting samples, you accumulate observations about those organisms or phenomena. Those observations are then analyzed to yield an answer to the question. In general, the answer is the " key result".

      The above statements apply regardless of the complexity of the analysis you employ. So, in an introductory course your analysis may consist of visual inspection of figures and simple calculations of means and standard deviations in a later course you may be expected to apply and interpret a variety of statistical tests. You instructor will tell you the level of analysis that is expected.

      For example, suppose you asked the question, " Is the average height of male students the same as female students in a pool of randomly selected Biology majors ? " You would first collect height data from large random samples of male and female students. You would then calculate the descriptive statistics for those samples (mean, SD, n, range, etc) and plot these numbers. In a course where statistical tests are not employed, you would visually inspect these plots. Suppose you found that male Biology majors are, on average, 12.5 cm taller than female majors this is the answer to the question.

      • Notice that the outcome of a statistical analysis is not a key result, but rather an analytical tool that helps us understand what is our key result.

      Differences, directionality, and magnitude : Report your results so as to provide as much information as possible to the reader about the nature of differences or relationships. For eaxmple, if you testing for differences among groups, and you find a significant difference, it is not sufficient to simply report that "groups A and B were significantly different". How are they different? How much are they different? It is much more informative to say something like, "Group A individuals were 23% larger than those in Group B", or, "Group B pups gained weight at twice the rate of Group A pups." Report the direction of differences (greater, larger, smaller, etc) and the magnitude of differences (% difference, how many times, etc.) whenever possible. See also below about use of the word "significant."

      Organize the results section based on the sequence of Table and Figures you'll include. Prepare the Tables and Figures as soon as all the data are analyzed and arrange them in the sequence that best presents your findings in a logical way. A good strategy is to note, on a draft of each Table or Figure, the one or two key results you want to addess in the text portion of the Results. Simple rules to follow related to Tables and Figures:

      • Tables and Figures are assigned numbers separately and in the sequence that you will refer to them from the text.
        • The first Table you refer to is Table 1, the next Table 2 and so forth.
        • Similarly, the first Figure is Figure 1, the next Figure 2, etc.
        • Each Table or Figure must include a brief description of the results being presented and other necessary information in a legend.
          • Table legends go above the Table tables are read from top to bottom.
          • Figure legends go below the figure figures are usually viewed from bottom to top.
          • When referring to a Figure from the text , "Figure" is abbreviated as Fig.,e.g.,
            Fig. 1 . Table is never abbreviated, e.g., Table 1 .

          The body of the Results section is a text-based presentation of the key findings which includes references to each of the Tables and Figures. The text should guide the reader through your results stressing the key results which provide the answers to the question(s) investigated. A major function of the text is to provide clarifying information. You must refer to each Table and/or Figure individually and in sequence (see numbering sequence), and clearly indicate for the reader the key results that each conveys. Key results depend on your questions, they might include obvious trends, important differences, similarities, correlations, maximums, minimums, etc.

          • Do not reiterate each value from a Figure or Table - only the key result or trends that each conveys.
          • Do not present the same data in both a Table and Figure - this is considered redundant and a waste of space and energy. Decide which format best shows the result and go with it.
          • Do not report raw data values when they can be summarized as means, percents, etc.

          Statistical test summaries (test name, p- value) are usually reported parenthetically in conjunction with the biological results they support. Always report your results with parenthetical reference to the statistical conclusion that supports your finding (if statistical tests are being used in your course). This parenthetical reference should include the statistical test used and the level of significance (test statistic and DF are optional). For example, if you found that the mean height of male Biology majors was significantly larger than that of female Biology majors, you might report this result (in blue) and your statistical conclusion (shown in red) as follows:

          "Males (180.5 ± 5.1 cm n=34) averaged 12.5 cm taller than females (168 ± 7.6 cm n=34) in the AY 1995 pool of Biology majors (two-sample t-test, t = 5.78, 33 d.f., p < 0.001) ."

          If the summary statistics are shown in a figure, the sentence above need not report them specifically, but must include a reference to the figure where they may be seen:

          "Males averaged 12.5 cm taller than females in the AY 1995 pool of Biology majors (two-sample t-test, t = 5.78, 33 d.f., p < 0.001 Fig. 1) ."

          Note that the report of the key result (shown in blue) would be identical in a paper written for a course in which statistical testing is not employed - the section shown in red would simply not appear except reference to the figure.

          • Avoid devoting whole sentences to report a statistical outcome alone.
          • Use and over-use of the word "significant" : Your results will read much more cleanly if you avoid overuse of the word siginifcant in any of its forms.
            • In scientific studies, the use of this word implies that a statistical test was employed to make a decision about the data in this case the test indicated a larger difference in mean heights than you would expect to get by chance alone. Limit the use of the word "significant" to this purpose only.
            • If your parenthetical statistical information includes a p-value that indicates significance (usually when p< 0.05), it is unncecssary (and redundant ) to use the word "significant" in the body of the sentence (see example above) because we all interpret the p-value the same way.
            • Likewise, when you report that one group mean is somehow different from another (larger, smaller, increased, decreased, etc), it will be understood by your reader that you have tested this and found the difference to be statisticallysignificant, especially if you also report a p-value < 0.05.

            Present the results of your experiment(s) in a sequence that will logically support (or provide evidence against) the hypothesis, or answer the question, stated in the Introduction. For example, in reporting a study of the effect of an experimental diet on the skeletal mass of the rat, consider first giving the data on skeletal mass for the rats fed the control diet and then give the data for the rats fed the experimental diet.

            Report negative results - they are important! If you did not get the anticipated results, it may mean your hypothesis was incorrect and needs to be reformulated, or perhaps you have stumbled onto something unexpected that warrants further study. Moreover, the absence of an effect may be very telling in many situations. In any case, your results may be of importance to others even though they did not support your hypothesis. Do not fall into the trap of thinking that results contrary to what you expected are necessarily "bad data". If you carried out the work well, they are simply your results and need interpretation. Many important discoveries can be traced to "bad data".

            Always enter the appropriate units when reporting data or summary statistics.

            • for an individual value you would write, " the mean length was 10 m ", or, " the maximum time was 140 min. "
            • When including a measure of variability, place the unit after the error value, e.g., " . was 10 ± 2.3 m ".
            • Likewise place the unit after the last in a series of numbers all having the same unit. For example: " lengths of 5, 10, 15, and 20 m ", or " no differences were observed after 2, 4, 6, or 8 min. of incubation ".

            DISCUSSION

            1. Function : The function of the Discussion is to interpret your results in light of what was already known about the subject of the investigation, and to explain our new understanding of the problem after taking your results into consideration. The Discussion will always connect to the Introduction by way of the question(s) or hypotheses you posed and the literature you cited, but it does not simply repeat or rearrange the Introduction. Instead, it tells how your study has moved us forward from the place you left us at the end of the Introduction.

            Fundamental questions to answer here include:

            • Do your results provide answers to your testable hypotheses? If so, how do you interpret your findings?
            • Do your findings agree with what others have shown? If not, do they suggest an alternative explanation or perhaps a unforseen design flaw in your experiment (or theirs?)
            • Given your conclusions, what is our new understanding of the problem you investigated and outlined in the Introduction?
            • If warranted, what would be the next step in your study, e.g., what experiments would you do next?

            2. Style : Use the active voice whenever possible in this section. Watch out for wordy phrases be concise and make your points clearly. Use of the first person is okay, but too much use of the first person may actually distract the reader from the main points.

            3. Approach : Organize the Discussion to address each of the experiments or studies for which you presented results discuss each in the same sequence as presented in the Results, providing your interpretation of what they mean in the larger context of the problem. Do not waste entire sentences restating your results if you need to remind the reader of the result to be discussed, use "bridge sentences" that relate the result to the interpretation:

            "The slow response of the lead-exposed neurons relative to controls suggests that. [ interpretation ]".

            You will necessarily make reference to the findings of others in order to support your interpretations.Use subheadings, if need be, to help organize your presentation. Be wary of mistaking the reiteration of a result for an interpretation, and make sure that no new results are presented here that rightly belong in the results.

            You must relate your work to the findings of other studies - including previous studies you may have done and those of other investigators. As stated previously, you may find crucial information in someone else's study that helps you interpret your own data, or perhaps you will be able to reinterpret others' findings in light of yours. In either case you should discuss reasons for similarities and differences between yours and others' findings. Consider how the results of other studies may be combined with yours to derive a new or perhaps better substantiated understanding of the problem. Be sure to state the conclusions that can be drawn from your results in light of these considerations. You may also choose to briefly mention further studies you would do to clarify your working hypotheses. Make sure to reference any outside sources as shown in the Introduction section.

            Do not introduce new results in the Discussion. Although you might occasionally include in this section tables and figures which help explain something you are discussing, they must not contain new data (from your study) that should have been presented earlier. They might be flow diagrams, accumulation of data from the literature, or something that shows how one type of data leads to or correlates with another, etc. For example, if you were studying a membrane-bound transport channel and you discovered a new bit of information about its mechanism, you might present a diagram showing how your findings helps to explain the channel's mechanism.

            ACKNOWLEDGMENTS (include as needed) | FAQs |

            If, in your experiment, you received any significant help in thinking up, designing, or carrying out the work, or received materials from someone who did you a favor by supplying them, you must acknowledge their assistance and the service or material provided. Authors always acknowledge outside reviewers of their drafts (in PI courses, this would be done only if an instructor or other individual critiqued the draft prior to evaluation) and any sources of funding that supported the research. Although usual style requirements (e.g., 1st person, objectivity) are relaxed somewhat here, Acknowledgments are always brief and never flowery.

            LITERATURE CITED

            1. Function : The Literature Cited section gives an alphabetical listing (by first author's last name) of the references that you actually cited in the body of your paper. Instructions for writing full citations for various sources are given in on separate page. A complete format list for virtually all types of publication may be found in Huth and others(1994) .

            NOTE : Do not label this section "Bibliography" . A bibliography contains references that you may have read but have not specifically cited in the text. Bibliography sections are found in books and other literary writing, but not scientific journal-style papers.

            APPENDICES


            Function : An Appendix contains information that is non-essential to understanding of the paper, but may present information that further clarifies a point without burdening the body of the presentation. An appendix is an optional part of the paper, and is only rarely found in published papers.

            Headings : Each Appendix should be identified by a Roman numeral in sequence, e.g., Appendix I, Appendix II, etc. Each appendix should contain different material.

            Some examples of material that might be put in an appendix (not an exhaustive list) :

            • raw data
            • maps (foldout type especially)
            • extra photographs
            • explanation of formulas, either already known ones, or especially if you have "invented" some statistical or other mathematical procedures for data analysis.
            • specialized computer programs for a particular procedure
            • full generic names of chemicals or compounds that you have referred to in somewhat abbreviated fashion or by some common name in the text of your paper.
            • diagrams of specialized apparati.

            Figures and Tables in Appendices

            Figures and Tables are often found in an appendix. These should be formatted as discussed previously (see Tables and Figures), but are numbered in a separate sequence from those found in the body of the paper. So, the first Figure in the appendix would be Figure 1, the first Table would be Table 1, and so forth. In situations when multiple appendices are used, the Table and Figure numbering must indicate the appendix number as well (see Huth and others, 1994).

            Modified 3-7-11
            Department of Biology, Bates College, Lewiston, ME 04240


            Scaling up

            In theory—and in the lab—the possibilities seem endless. In practice, however, is another matter. The challenges of scaling up lab-based discovery into commercial manufacturing are daunting, but that’s what Gingko hopes to crack. “Aside from obvious targets of synthetic efficiency,” said Boyle, “a key aim is to bed down such manufacturing processes among the existing industrial ecology, for example, to make sure that the feedstock of a biological strategy is cheaply and abundantly available.” With canny planning, different processing tasks could support one another, the byproducts of one metabolic pathway being useful for another. After all, he said, the petrochemicals industry has evolved so that no fractionate of crude oil goes to waste. Creating such an economical, sustainable web of processes for mass production synthetic biology might, “cause a rethink of the entire supply chain.”

            Boyle draws an analogy with the founding of industrial synthetic chemistry in the 19th century, in which the byproduct of extracting gas from coal, called coal tar, which was thought to be a useless waste residue, became the crucial feedstock for the production of synthetic dyes. This led dye companies to diversify into pharmaceuticals and other fine chemicals and to establish R&D departments to develop basic science. Brian Yeh and Wendell Lim of the University of California, San Francisco, have pointed out how development in 19th-century organic chemistry made it turn toward synthesis, which then led to the fundamental understanding of structure and reactivity. Perhaps synthesis in biology will have the same effect. Reference Yeh and Lim 23

            This comparison teaches another important lesson. The discovery of coal-tar dyes was a serendipitous accident (arising from an attempt to make the antimalarial quinine), taking applied synthetic chemistry in a direction no one had anticipated. The same might be true of synthetic biology as a manufacturing technology: no one has yet found the “killer app.” “Where we’re going may be quite different from where it seems today,” said Boyle. Just like evolution, really.


            Watch the video: Δομή και βιολογική δράση των ιών - Βιολογία (July 2022).


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