eChapters

Object: To measure the rate of transpiration by using Ganong’s potometer

Material required: Ganong’s potometer, beaker, wax or vaseline, stop watch and twig or a branch of a plant.

Theory: The amount of water absorbed is almost equal to the amount of water transpired. The difference between the two amounts is negligible. 

Method: Potometer consists of wide mouthed tube fitted with a hard cork in which a twig or branch of a plant is inserted. Other end of tube is dipped in beaker. One half of the tube is graduated. The apparatus is filled with water using the reservoir. An air bubble is inserted in the tube as shown in figure.

Object: To find out the relation between transpiration and absorption. 

Material Required: Wide mouthed glass bottle, with a narrow graduated side tube, split rubber cork, water and a plant with intact root system.

Theory: The loss in weight of plant indicates the amount of water transpired. And fall in water level of the side tube indicates the amount of water absorbed. This shows that amount of water transpired is normally equal to the amount of water absorbed by the plant roots.

Object: To demonstrate unequal transpiration by the two leaf surfaces from four leaf method. 

Material required: four leaves of china rose or any dorsiventral leaves, two stands, thread and grease or vaseline. 

Theory: Transpiration is a process in which loss of water occurs in the form of water vapour from the aerial parts of the living plant. 

Method: Take four leaves of equal size from a plant (e.g. China rose). Now apply or smear the vaseline on the four leaves as follows:

Object: To demonstrate that light and carbon dioxide are essential for photosynthesis (Moll’s half leaf experiment). 

Material Required: A potted plant, a wide mouthed bottle, caustic potash, splits of rubber Cork and vaseline etc. 

Object: To demonstrate that O₂ is evolved during photosynthesis. 
 

Material required: Hydrilla plant, beaker, test tube, funnel, pond water and pyrogallol. 
 

Theory: Oxygen is a byproduct of photosynthesis, which is evolved in presence of light and chlorophyll during photosynthesis.

Object: To demonstrate anaerobic respiration. 

Material required: Soaked pea or gram seeds, mercury, test tube stand, forceps and KOH pellets. 

Theory: Generally, plants at certain stage have the capacity to respire in absence of oxygen i.e. anaerobic respiration. In this process, oxidation of carbohydrate takes places, ethyl alcohol and CO2 being end products and only a little amount of energy is liberated-

Object: To demonstrate that oxygen is used up during respiration. 

Material required: A conical flask, some germinating seeds, glass tube having two bends at right angles, mercury, beaker, small tube and caustic potash (KOH).

Theory: Plants at some stage have a capacity to respire more rapidly in presence of oxygen i.e. aerobic respiration. In this process, oxidation of carbohydrate occurs resulting in conversion of carbohydrate into carbon dioxide (CO₂) and water with release of large amount of energy.

Object: To determine respiratory quotient of different types of respiratory substrates.

Object: To demonstrate the optimum conditions for seed germination. 

Material required: Beaker, glass slide, bean seeds, thread and water.

Theory: Germination is a process by which the embryo in the seed becomes activated and begins to grow into a new seedling under favorable conditions. Food is stored in the seeds in dry conditions but the developing embryo cannot utilize this dry food. Food can be utilized in liquid form only and seeds can utilize only dissolved oxygen. Excess water stop germination because once all the dissolves oxygen is utilized by the seed, further germination is not possible as the life supporting oxygen is lacking. Suitable temperature is necessary because low temperature retards the embryo activity and high temperature destroys the delicate embryo tissue. Seed usually germinate between 0oC to 50oC temperature and the optimum temperature is lies between 25 to 30oC. During germination rapid cell division takes place. Energy is required for cell division. This energy is obtained from oxidation. The oxygen is required for oxidation is supplied by the air.

Object: Determination of the viability of seeds by tetrazolium test. 

Material required: Healthy seeds, tetrazolium solutions, beakers, water and petridishes. 

Theory: Viability of seeds shows their potential towards germination.

Object: To demonstrate the measurement of growth in plants with the help of Arc auxanometer. 

Material required: Potted plant, Arc auxanometer, rope and weight.

Theory: Growth is a permanent change in plants with respect to its size, form, weight and volume.

Object: Effect of ethylene on regulation of stomata.

Material required: Plant leaves, ethaphon, ACC, double distilled water, measuring cylinder, beaker and electric balance.

Theory: Ethylene is a plant hormone that regulates many aspects of growth and development. Despite the well-known association between ethylene and stress signalling, its effects on stomatal movements are largely unexplored. Here, genetic and physiological data are provided that position ethylene into the Arabidopsis guard cell signalling network, and demonstrate a functional link between ethylene and hydrogen peroxide (H₂O₂). In wild-type leaves, ethylene induces stomatal closure that is dependent on H₂O₂ production in guard cells, generated by the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase AtrbohF. 

Veterinarians will be often required to mention the specific animal husbandry terms for various purposes like writing certificates for various schemes, postmortem, attending court of law in veterolegal cases etc. Hence this chapter aims in refreshing the vets with the common terms associated with different domestic animals and animal husbandry activities. 

The basic knowledge of normal physiological values in different animal species is essential for selection of the animal, diagnosis of any abnormality if any and its correction. The chapter provides the normal physiological values viz temperature, pulse, respiration etc in different domestic animals.

The different biochemical values serve as an indicator of the correct health status, viz important organ functions,  the alteration in values if any, helps in identifying the specific organ damage and exact diagnosis of the  abnormal health condition or disease. Some of the important biochemical values of the animals, which may assist in diagnosing the specific disease  conditions are listed in this chapter.

The  information on normal reproductive traits in domestic animals like calving interval, age at first calving etc, is essential for getting the maximal reproductive efficiency of the animal by the farmer. It also helps in improving the reproductive efficiency of the animals by different required means viz nutrient/mineral supplementation, antibiotic, hormonal therapy etc.

The table gives the approximate date of parturition, based on the date of breeding/mating in the year as a easy ready reference table.

The spermatogenesis is a continuous process in male animals but affected by following factors. 
    
1.     Genetic makeup of male animal: Certain sperm defects like Diadem defect, knobbed sperm, decapitated sperm, sterilizing tail stump, corkscrew mid piece and narrow head are inherited defects and reported in certain males. Few inherited genetic factors are also associated with poor libido and poor fertility. Inherited sperm defects affect sperm motility, fertilization and embryo formation.

Immediately after use, all sorts of items are thoroughly washed with water and the rubber catheters are washed with the force of tap water to remove the residual diluted semen. The glass wares are kept in 1% chromic acid solution for overnight to remove cloudiness of glass surface. Then the articles are washed with lukewarm laboratory grade detergent (ex. Labolene®) with a brush to make the articles grease free. All the items are thoroughly washed several times under distilled water and kept inverted position in a clean trough then the openings of glassware wrapped with aluminum foil / paper.

1.    Wilt
        
2.    Stem rot
        
3.    Alternaria leaf spot

Metals are toxic and non biodegradable pollutants released into the environment through effluents from agriculture waste, mining, metal processing industries, pharmaceutical industries as well as domestic and natural activities. Metallic pollutants tend to persist indefinitely and eventually get accumulated in the ecosystem. Metals especially arsenic, copper, cadmium, chromium, lead, mercury, nickel, selenium and silver etc. possess a risk to living systems. Conventional processes applied to remediation of metallic pollutants are often restricted due to technical and economical constraints. Particularly when the metal contamination is at very low concentration the conventional methods become inefficient. In this situation application of bioremediation techniques is a technology of choice for environmental protection and clean up.

There are three types of systems for expressing the energy value of feeds and many examples have been depicted in each of these systems the world over but here, one example has been taken up in each of these categories.

Digestible Nutrient Type

For example, Total digestiblenutrients (TDN) system: Digestibility coefficients of various organic nutrients like carbohydrate, fat and protein are determined by digestibility trials, which can be utilized to be involved for total digestible nutrients as a measure of nutritive value of feeds. Here only faecal enegy loss has been considered barring losses from other channels. Hence roughages are overestimated bysuchcalculations.Otherwise, thismethodissimple, economical and has some basis for animals to be fed on such standard in India. The TDN value is expressed in percentage as following :

% TDN = % dig. C.P. + % dig. C.F. + % dig. N.F.E. + [% dig. E.E. X 2.25]

The animal bodycan be imagined as a machine, which is gettinginput of desired or available feed given by the farmer and this animal body after processing through various metabolic activities, output of product is given off. Feed is composed of organic nutrients-carbohydrate, fat and protein and after reaching in the animal body, used for constructive work, for example synthesis of body tissues and other associated work. Later, milk or egg is produced as an output. Now putting the same perspective in terms of energy, that is, these organic nutrients of the feed contain chemical energy and after reaching in the animal body, used as mechanical energyin muscles or heat energyin metabolism. The energy in the body would be utilized for the synthesis of body tissues or other productive output, for example, draft work by bullocks. Therefore, energy is primarily required for the maintenance of the body and then, for the productive purposes :

• Growth for all the farm animals

• Milk from cow, buffalo, sheep, goat • Eggs from poultry, duck, turkey

• Wool from sheep

• Meatfromfatteninganimals under the law, for example– mutton, chevon, pork, chicken.

Patients are unable to consume foods and fluids orally when they are suffering fromillness. Under surgical conditions, severe burns, severe infectious neonatal infants, congenital anomalies, anorexia, coma or dysfunction of elementary tract etc., special feeding methods should be used for feeding such patients.

Objectives

• To provide water and electrolytes to prevent dehydration and correct electrolyte imbalance.

• To make up the loss of tissue protein.

• To provide energy to meet the daily needs of the patients.

The nutrients used in intravenous feeding are :

a) Water and electrolytes : The daily minimum water requirement of an adult on anaverage is 1.5 liters.Additional waterwillbe requiredto correct dehydration that might have occurred before therapy. The water loss made up in about 24 hour and the urinary excretion restored to about 300-400 ml in every 8 hrs.

b) Carbohydrates and alcohol : Glucose, Fructose and Sorbitol are used in intravenous feeding. Three per cent alcohol @ 8 g/h can also be given; because this provides 7.1 kcal energy/ g. Glucose is usuallyadministered as 5 % solution. Fructoseis preferred to Glucose, as it can be administered at a concentration of 10-15 % without any adverse effect.

c) Amino acids : Solution form of Protein hydrolysates, Synthetic amino acid mixture and a mixture of L-amino acids are available for intravenous feeding. Protein hydrolysates prepared from casein, free from peptides and fortified withTryptophan are commonlyusedfor intravenous therapy.

d) Whole blood or plasma : One litre of whole blood provides about 180 gm proteins including haemoglobin. Blood is used mainly to combat circulatoryfailure andloss of bloodfrombody. One liter of plasma contain about 60-70 gm of protein. Plasma infusion is given as a measure for increasing plasma protein level when it is low.

e) Vitamins: All the vitamins (twice daily requirements) should be added to intravenous fluid to meet the daily needs of the patient.

Chemistry of Alkanes

In living plants, hydrocarbons are universally distributed in the waxy coatings on leaves and other plant organs. Alkane fraction is commonly a mixture of hydrocarbons of similar properties. The qualitative pattern is relatively similar from plant to plant, but there are considerable quantitative variation. Alkanes are saturated long chain hydrocarbons. They are usually present in the range of C to C carbon atoms. Examples are n-nonacosane, C H and n-hentriacontane, C C . In general, odd-numbered carbon chains occur predominantly in the cuticular wax of plants and are substantially indigestible.

How alkanes are “user friendly” !

Following are some points to ponder :

• Alkanes are long carbon chains found naturally in plants and each plant species has its unique compliment of these different alkanes which act like “fingerprints”. Forage intake can be estimated from ‘faecal grab samples’, if the ratio between these endogenous plant alkanes and exogenous alkanes delivered from “a rumen slow - release gelatin capsule” is calculated.

• Alkanes can also be used for determiningdigestibility and characterizing thespecies compositionof thediet, makingalkanes an extremelypowerful tool for dairy research.

• Anenexpensive, reliable and simpletechnique offers tremendous potential for research in the area of low cost dairy production from forages. Such data may be used by “slow–release marker” to estimate intake and digestibility.

• Alkanes offer a potentially simple technique for the animal scientist and it is feasible to estimate alkane concentrations using Near Infra-Red scanning [N.I.R.]. It is a rapid analytical method, thus making alkane technology an extremely ‘user friendly’ tool to be widely adopted on behalf of the dairy industry.

For economical ration -preparation, selection of cheap feed amongthe locally available sources, is an important point to ponder. For this purpose, a list of available feeds is prepared alongwith their latest costs on dry matter basis. The cost per kgof dry matter is divided by the amount of the nutrient (as a decimal), it will give us a good idea of the :

• Cost of Crude Protein in ??Protein supplements and,

• Cost of Energy in ??Energy feeds.

For example, if alpha alpha hay costs Rs 3.93 per kgof dry matter, the cost per kg of Crude Protein = 3.93/0.171 = Rs 22.98 per kg.

In this way, it is possible to prepare a list of protein sources in the order of “Ranking” from which feeds can be selected at lower costs.

Effect of Differences in Dry Matter

Feeds are purchased on the basis of Dry Matter due to variation of moisture content. For example :

A Corn of 89 % Dry Matter costs Rs 7 per kg,

Another type of corn of 75 % Dry Matter can’t be comparable on this cost This can be done as following 

Several substrates, like proteins (enzymes), are very sensitive to alteration in the concentration of the solution they are placed in. Hence is the importance of a buffer which controls the chemical properties of the medium. Literally a “buffer” has the ability to resist changes in the hydrogen ion concentration. Although, buffers do contain other molecules, that might, in addition to the hydrogen ion concentration, influence the ionic strength, the activity of enzymes, and other parameters of the experiment.

In any biological experiment, appropriate buffer must be selected based on their effect on the experiment. Ideal buffers control pH and ionic strength without interacting adversely with the experiment undertaken. For example, although phosphate is a common buffer, it may not be suited when phosphate is a substrate or product of the reaction being studied. Also, some proteins interact poorly with some buffers. For example, Tris is not ideal as it exhibit high pKa and the large change in pKa when temperature changes, although it is cheap and most proteins are stable in Tris buffers, moreover it seldom reacts with reaction substrates hence, Tris buffer is commonly used in biochemical experiments.

Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, and decant supernatant.

Resuspend cells in 400 µL TE by vortexing, add 50 µL 10% SDS, 50 µL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^oC.

Shear DNA by 3-5 passages through a 26 G needle.
Extract twice with 500 µL phenol: chloroform (1:1), and twice with 500 µL chloroform. 
Precipitate nucleic acids by adding 25 µL 5 M NaCl and 1 mL 95% EtOH, vortex, and centrifuge for 10 min, decant supernatant. Dry pellet.

Reagents

Nuclease-Free Water: 14µl

10X Restriction Buffer: 2µl

Acetylated BSA (1mg/ml): 2µl

DNA (~1µg): 1µl

Restriction Enzyme (10u): 1µl

Final Volume: 20µl

Reagents

Restriction enzymes of choice, such as BamH1 and EcoRI

Restriction enzyme reaction buffer,

70 % Ethanol

100 % Ethanol

3 M Sodium acetate (pH 5.2)

Distilled water

Plasmid DNA isolated from bacteria, such as pGFP(R) and pBC(R)

Start with a 1:2 molar ratio of vector: insert DNA when cloning a fragment into a plasmid vector. This ratio will vary with other types of vectors, for example, cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment.

Principle

Plasmids requires hosts to replicate, hence it has to be inserted into bacteria. But efficiency of uptake of plasmid DNA by bacteria is low and will not do so without getting degraded. Therefore, to enhance the probability of uptake of the plasmid DNA by bacteria it is necessary to make the bacterial cells “competent” to absorb the plasmid DNA. One of the methods to increase efficiency of cell competence is given below. It should be noted that competent cells are more fragile than normal bacteria. Vortexing the cells, heating the cells above 42ºC or to 42°C for prolonged periods, or exposure of the cells to abusive treatments may kill them

Transformation

The uptake of DNA by competent cells is called transformation. Transformation involves:

Mix a small volume of DNA (usually 1-3 µl) with 100 µl of competent cells.

The mixture is incubated on ice for 30 minutes to allow the cells to take up the DNA.

The cells are then heat-shocked at 37 to 42°C for 1 minute, and cold shocked on ice for 2 minutes.

The heat shock/cold shock reverses the effect of the competent cell process, by healing the damage inflicted by the competent cell solution.

Add 0.5 ml of LB culture mediumz

Incubate the cells for 10 - 30 minutes at 37°C to aid the healing process.

This cell/medium mixture is then spread onto bacterial culture plates containing agar, LB, and a selection mechanism.

Principle

The principle of Electrophoresis involves the motion of a particle in a fluid medium, where the gravitational attraction (on a flowing liquid) is the sedimenting force, and the frictional force is the opposing force and is proportional to the velocity of the particle. These forces balance each other acting in opposite directions and hence balance each other. This will lead to the uniform motion of the particle in the mobile liquid medium (i.e. the particle moves at constant velocity). In case the external field is an electric field instead of a gravitational field, a macromolecule will respond to the external field in two ways. In case the molecule is charged, it will migrate in an electric field to the electrode of opposite charge. Under this principle the technique of electrophoresis functions.

All electrophoresis operations are governed by the principle:

here are many methods for extracting DNA from gel apart from using commercial kits, some methods however yield cleaner DNA than others.

1. Freeze squeeze

Excise band and place pieces of it in a home-made spin-column (a piece of filter placed in a PCR 0.5ml eppendorf, pierced the bottom of the PCR tube with a needle). Place the PCR tube at -20 °C for 15-20 mins or freeze in liquid N2. Then place the PCR tube into the 1.5 ml eppendorf and spin at top speed in a microfuge for 15 mins, collect DNA solution which can be EtOH ppt or use directly.

2. Powdered silica

Melt excised bands at 70°C cool and add equal volume of TE-buffered phenol and mix. Spin for 5 min, remove aqueous phase, repeat and EtOh ppt. the DNA.

3. Electroelution

Denaturing polyacrylamide/urea gels in TBE buffer

Prepare 20ml of a 5% polyacrylamide gel containing 7M urea by adding:

47.5% acrylamide:

2.5% bis-acrylamide solution 2ml

10M urea 14ml

10X TBE buffer 2ml

10% freshly prepared ammonium persulfate 0.2ml

deionized water 1.8ml

The principle of silver nitrate staining of proteins is based on the reduction of silver ions to its metallic form. (Silver ions in alkaline conditions bind with proteins through å-amino group of lysine and sulphur groups of cysteine and methionine residues). When the complexed silver ions are reduced in the presence of formaldehyde, they become metallic silver, which is seen on the gel as bands. Staining of proteins with silver nitrate after SDS-PAGE is quicker and ~ 10 times more sensitive than Coomassie brilliant blue staining and that is the reason it is widely used in research laboratories these days.

The procedure is as follows:

Cell Lysis Procedure

Pellet the cells in a microfuge tube: add 1.5 ml culture, centrifuge for 15 seconds at maximum speed, discard the supernatant, and then repeat the procedure. This will result in the cells from 3 ml of culture in a single tube.

Resuspend the cells in 1 ml Lysis buffer.

Take 300 µl aliquot for Bradford protein assay.

Add 50 µl lysozyme, and mix gently.

After 10-20 minutes at room temperature, microfuge for 10 minutes to pellet cell debris.

Use supernatant for LDH assay.

Media Preparation

Before mixing medium, determine the quantity of each medium and the amount of plant growth regulator needed for each experiment. Make slightly more medium than is required. A media check off list is helpful in making medium to keep track of ingredients.

Remove media stock solutions and plant growth regulator stock solutions to be used from the refrigerator. Make up fresh stock solutions if adequate quantities are not available or if solutions have precipitated.

Plant growth regulators stock solutions are prepared biweekly and stored at 4º C in the dark. Cytokinins (thidiazuron and BA) are dissolved in approximately KOH prior the addition of deionized water. Gibberellins and auxins (2,4-D, IBA and NAA) are dissolved in 100% ethanol prior the addition of water.

Fill the volumetric flasks upto ½ to ¾ with deionized water. Add proper a of stock solution and plant growth regulators and mark each item on the check off list. Add 30 g/l sucrose, stir until dissolved. Make up the volume of the flask with deionized water and later por into a larger beaker.

After preparing the desired DNA molecule using E.coli, the plasmid can be transferred into Agrobacterium by this method. Efficiency is low (~10³ transformants per mg of DNA) but the method is reliable and very rapid. This method also eliminates much of the plasmid rearrangement that occurs during triparental mating.

Procedure

Grow an Agribacterium strain containing an appropriate helper Ti plasmid in 5 ml of LB medium 16 hours at 28ºC.

Add 2 ml of the culture to 50 ml LB medium in a 250 ml flask and shake (~250 RPM) at 28ºC until an OD₆₀₀ of 0.5 to 1.0 is obtained.

Chill the culture on ice. Centrifuge the cell suspension at 1000 g for 30 minutes at 4ºC.

Discard the supernatant. Resuspend the cells in 1 ml of 20 mM CaCl₂ (ice cold). Dispense 100 mL aliquots into pre-chilled 1.5 ml tubes.

Add about 1 µg of the desired plasmid DNA (pure or crude) to the cells in each tube.

Bacteria are morphologically different. This characteristic is helpful in the identification of bacteria. Routine study of bacterial morphology should include examination of stained preparations. Motility should be examined by hanging drop preparation. According to the requirement, certain cell structures may be stained. Stained preparations should be made preferably from fresh agar slant cultures. Smears of Streptococcus should be made from a liquid culture. Morphology of bacteria may be described under the following terms:

1.Shape: Spherical, coccobacillary, short or long rods, filamentous, comma- shaped, curved or spiral forms.

2.Size: Spherical are measured in diameter and rod-shaped in length and breadth. Measurement is expressed in microns (µ).

3.Sides: Straight, concave, bulging, parallel or irregular.

4.Ends: Round, pointed, truncate or concave.

5.Axis: Straight or curved.

6.Arrangement: Singly, in twos, in groups of four or eight, in grape-like bunches, in long chains, or scattered irregularly, may be in bundles, angular arrangements or in Chinese letters.

7.Pleomorphism: Bacteria may differ from their typical shape. Some of the older cultures may depart greatly from their typical forms, and may show spindles, clubs, and filaments, swollen and branching forms. Variation may be both in shape and size.

8.Capsule: May or may not be present. When present, size should be noted. This requires special staining.

Joint supply : Some products or production processes have more than one use. For instance, cows can both provide milk and be eaten.

Keynesian : A branch of economics, based, often loosely, on the ideas of keynes, characterized by a belief in active government and suspicion of market outcomes. 

Quality Adjusted Life Years (QALYs) : Calculated by adjusting the estimated number of life-years an individual is expected to gain from an intervention for the expected quality of life in those years. The quality of life score will range between 0 for death, to 1 for perfect health, with negative scores being allowed for states considered worse than death

X-efficiency : Producing output at the minimum possible cost. This is not enough to ensure the best sort of economic efficiency, which maximises society’s total consumer plus producer surplus, because the quantity of output produced may not be ideal. For instance, a monopoly can be an X-efficient producer, but in order to maximise its profit it may produce a different quantity of output than there would be in a surplus-maximising market with perfect competition.

Years of potential life lost : A measure of the impact of premature mortality on a population, calculated as the sum of the differences between some predetermined minimum or desired life span and the age of death for individuals who died earlier than that predetermined age.

Zero-sum game : When the gains made by winners in an economic transaction equal the losses suffered by the losers.

Proper records can be maintained and individual performance can be noted.

Disadvantages: A number of males should be maintained which is not economical.

Advantages of polygamous mating: Economical because 1 male can be used for 4-5 females. We can use postpartum estrus effectively.

Immediately after parturition after 7 days they come to heat. Breeding can be done 7 days after parturition after separating the young ones. [Weaning age 21 days]

But this is usually not followed because the number of young ones born reduces subsequently.

Disadvantages: Proper records cannot be maintained.

NATIONAL LEVEL FACILITIES OF LABORATORY ANIMALS

• National Laboratory Animal Centre, Central Drug Research Institute, Lucknow.
• National Centre for Laboratory Animal Sciences (NCLAS) National Institute of Nutrition, Hyderabad

INSTITUTIONAL LABORATORY ANIMAL FACILITIES AT

I. FILL UPS

1. Somatic embryo encased with artificial endosperm and protective cover is called _____.
2. The term artificial seed was coined by_____.
3. The artificial seed technology was patented by_____.
4. The most common gelling agent used for artificial seeds is_____.
5. In desiccated system, the artificial seeds are coated with _____.