Chapter four MICROSCOPY * UHUO, C. A. * ; 4 Introduction 3 5 . . @ The study of the features of organism and its actual size under the microscope 18 termed microscopy. So, this will offer you the opportunity to study and acquire the knowledge and insight on how to use and care for the microscope. : Objective } When working with a very small/minute biological specimen, they is need you enhance the efficiency of the microscope. The oil immersion technique is one of the ways we do this. enaailian aint «Pembina Diagram of a typical student microscope 25(1) Eyepiece lens/ocular lens: 7x. 40. 15x. (11) Accessory lens (2) Tube (12) Condenser (3) Prism (13) Concave mirror (4) Clamp screw (14) Base foot (5) Nose-piece (15) Fine focus control (6) Nose-piece/turret (16) Coarse focus control (7) Objective 3.7x, 8x, 20x, 40, (17) Stock (house focusing mechanism) (8) Condenser clamp screw (18) Stage (keep clean and dry!) (9) Condenser iris (19) Stage mount (10) Filter holder (20) Stage clip (21) Limp Eyepiece lens: The lens used by viewers to look through the specimens. The eyepiece usually contains a 10X or 15X power lens. Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to correct for any difference in vision between your two eyes. Body tube (Head): The body tube connects the eyepiece to the objective lenses. Arm: The arm connects the body tube to the base of the microscope and enables the use! to carry the microscope. Coarse adjustment: Brings the specimen into general focus. Fine adjustment: Fine tunes the focus and increases the clarity detail of the specimen. Nosepiece: A rotating turret that houses the objective lenses. The viewer nosepiece to select different objective lenses. Objective lenses: One of the most important Parts of closest to the specimen. Teas four, or five objective lenses that range in power . 1 the microscope, be careful jective lens does!" Pong , be careful that the objective lens does touch the slide, as it could break the slide and destroy the ebcucied haat spins the a compound microscope is the lense from 26Specimen or slide: The specimen 1s the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass. The specimen is placed on the glass and a cover slip is placed over the specimen. This Hows the allows the slide to be easily inserted or removed from the microscope. It als specimen to be labeled, transported, and stored without dam Stage: The flat platform where the slide containing specimen is placed Stage clips: Metal clips that hold the slide in place or firmly. Stage height adjustment (Stage Control): These knobs move the stage left and right or up and down, Aperturs reach the specimen, On/off switel Mlumination light from an exte + The hole in the middle of the stage that allows light from the illuminator to This switch on the base of the microscope tums the illuminator off and on, The light source for a microscope. Older microscopes used mirrors to reflect I source up through the bottom of the stage; however, most microscopes now use a low-voltage bulb Iris diaphragm: Adjusts the amount of light that reaches the specimen. Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed, Base: The ba € supports the microscope and it’s where illuminator is located. Tr Oil Immersion Technique pes of Microscopy/Microscope When we are to examine small objects like red blood ll (7,um) and bacteria (1- um), an objective lens of 90x or 100x is most suitably required. This is because these lenses have a very short working distance and the diameter of the lower objective lens is very small to be compared with other lenses(x10, x40). Materials Microscope fitted with Abbe condenser and usual objectives Oil immersion objective lens Light source Immersion oil (eg. Glycerine) Lens tissue/ cleaner Xylene or dichloromethane Stained permanent slide with a thin cover glass, Blood specimen Method (1) Check that the oil immersion lens is clean. (ji) Polish the slide with lint-free cloth 27i denser | (ii) Set up the microscope. Espei he a _ ject of i ch i s ae fs Se ne nest fees then raise the nose piece and swing in the 90x or (vi) Use the coarse 100x oil immersion lens. (vii) Put a drop of the im 0 i (viii) Raise down until the oil immersion cover slip. | . - (ix) Now Jook down the microscope. Slowly raise uP using the fine dius knob. (x) The slide can be carefully moved to examine adjacent objects - for example to compare the structure of red blood cells and white blood cells. | ; , (xi) When you have finished, raising up and remove the slide and oil immersion objective. (xii) The oil is removed from the objective lens with dry lens-tissue. Ordinary tissue is suitable for the slide. : Avoid excessive use of the solvent as it is poisonous by skin contact and the slide. mmersion oil onto Iens enters the oil ai nd is very nearly touching the Precautio! inhalation. ‘Also it dissolves the content used to fix optical components in place. Since you are now familiar with the oil immersion technique, practice it and get the feedback from your counselor. (xiii) Return the clean lens to its storage container and the slide to its tray. Optical or light Microscopy/Microscopes: These microscopes use visible light (or UV light in the case of fluorescence microscopy) to make an image. The light is refracted with optical lenses. The first microscopes that were i ere invented belong to this cate ica microscopes can be further subdivided into several categories: ° Seon, Ones ‘Compound Mi : ° : icroscope: These microscopes are composed of two lens systems, an objective and an ocular (eye pi ye piece). The ee compound microscope is about 1000, usefull magnification of 2 Stereo Microscope (dissecti ecting microscope); ; about maximum 100 pe): These microscopes magnify up t0 Soot ae 10K and supply a 3-dimensional vie Eases daa ul for observing opaque objects, ww of the specimen. They are hame sugges 7 Bests, these micros pt all wavelength ones Bietoseopes use a bean of higher than is : ; therefore also higher and is 28F ge of x-Fa between the optical microscopes and electron microscopes. One advantage y microscopes over electron microscopes is, that it is possible to observe living cells. cound Scanning acoustic Microscopy/Microscope (SAM): These devices use focused soun waves to generate an image. They are used in materials science to detect small cracks or tensions in materials. SAMs can also be used in biology where they help to uncover tensions, stress and elasticity inside biological structure. Scanning Helium Ion Microscopy/Microscope (SHIM or HeIM): As the name suggests, these devices use a beam of Helium ions to generate an image. There are several advantages to electron microscopes, one being that the sample is left mostly intact (due to the low energy requirements) and that it provides a high resolution. | Electron Microscopy/Microscopes: Modem electron microscopes can magnify up to 2 million times. This is possible, because the wavelength of high energy electrons is very small. At the same time, the high energy electrons are pretty tough on the sample being observed. It may take a long time to completely dehydrate and prepare the specimen. Some biological specimens also need to be coated with a very thin layer of a metal before they can be observed. + Transmission electron microscopy (TEM): In this case, the electron beam is passed through the sample. The result is a two dimensional image. + Scanning electron microscopy (SEM): Here the electron beam is projected on the sample. The electrons do not go through the sample but bounce off. This way it is possible to visualize the surface structure of the specimen. The image appears 3 dimensional. Scanning Probe Microscopy/Microscopes: It is possible to visualize individual atoms with these microscopes. The image of the atom is computer-generated, however. A small tip measures the surface structure of the sample by rastering over the surface. If an atom projects out of the surface, then a higher electrical current will flow through the tip. The amount of current is proportional to the height of the structure. A computer will then assemble the position data of the tip and the current to generate an image. Conelusion: Microscopes can be classified based on the physical principle that is used to generate image. Different microscopes visualize different physical characteristics of the sample (eg. elasticity can be visualized with acoustic microscopes). Image contrast, resolution (which determines magnification) and destructiveness of the sample are other relevant parameters. Exercise State the function of each of the following parts of a microscope 1, Ocular: 2. Objective: 293. Rotating nosepiece: 4, Diaphragm: What is the primary contribution by each of the following men? 5. Zacharias Janssen: 6. Robert Hooke: 7. Leeuwenhoek: Explain the differences in each of the following pairs of terms: 7. light microscope and electron microscope 8. coarse adjustment knob and fine adjustment knob 9. simple microscope and compound microscope Fill in the blanks of the following questions: We the ability of a microscope to show details clearly. 12. If microscope had a 20x ocular and a 40x objective, the total magnification would be equal to. 13. The diameter of the 10x field of view is 14. The diameter of the 40x field of view Label the parts of the microscope in the dra 2. 3. 1 2 4. 3 5. : 6. x , ‘ 7. » 1 Be 8. | 9. 8, 10. ” i ML. : © 30Chapter five METHODS OF PREPARATIONS OF REAGENTS AND STAINS USED COMMONLY IN BIOLOGY ABORATORY ANI CHIJIOKE Many experiments involving chemicals are used in solution form. That is, two or more substances are mixed together in known quantities. This may involve weighing a precise amount of dry material or measuring a precise amount of liquid. Preparing solutions accurately will improve an experiments safety and chances for success. Using percentage by weight (w/v) The percentage concentration refers to the mass or volume of solute in a final volume of solution. That is the formula for Weight percent (w/v) is: Mass of solute (g) / Volume of solution (cm*) x 100. Example 1: To prepare 1 % glucose solution. | g of glucose will be dissolved in 95 mL of solvent (water in this case) and the resulting solution will be made up to 100 ml with water. Example 2: A 10 % NaCl solution has 10 g of sodium chloride dissolved in 80 cm? of water and make up to 100 cm? once the NaCl dissolved. Stains Stains are coloured dyes usually organic salts with positive and negative ions used in making thin sections of biological tissues more clearly visible through the microscope. If the colour comes from the negative ion (organic anion), the stain is described as acidic eg eosin but if the colour comes from the positive ions (organic cation), the stain is described as basic eg haematoxylin. Common Stains in Biology Laboratory (for Microscope Slide Stains) . Singesmost biological structures are transparent, we need a technique to distinguish cleaily, the parts as we observe under the microscope. This is possible by employing some means by which contrast between different structures can be obtained. Sometimes adjusting light helps but the most common method is staining, Stains such as methylene blue in low concentrations does not harm the tissues and so can be safely used on living materials, Such stains are called vital stains. For making temporary slides stains such as methylene blue, iodine, aniline hydrochloride, safranin etc are used. 31si d the colour are as follow ni i a ag jn plant and animal specimens Some common stains, their uses sponydrates in P ; Stains Lugol's iodine solution (0.1M Ih): brown or blue- black and stains glycogen red. Preparation Ingredients, Potassium iodide 108 Distilled water 100 mL odine crystals 5g Procedure istilled water. Slowly add 5 g iodin Dissolve 10 g potassium iodide in 100 mL et distilled new aii iy Ie i ing. Fil ina tightly stopper - crystals, while shaking. Filter and store in a tig! F reas maine blue (pH indicator and stain): Stains acidic cell parts (ike nucleus) blue. Use on animal, bacteria and blood specimens and can be used as a substitute for Janis B green, Preparation Dissolve 1 g of methylene blue in 74 mL of distilled water. Then dilute to 100 mL Phenolphthalein (pH indicator): Is use as an indicator in acid-base titrations. It tums colourless in acidic solutions and magenta in basic solutions. Preparation Dissolve 1 g of phenolphthalein in 50 mL of 95 % ethyl alcohol. Dilute to 100 mL with 95 % ethyl alcohol. Eosin Y stains: Stains alkaline cell parts (like cytoplasm) pink. Use on plants, animals and blood. Can be used as a substitute for Congo Red and Carmine Preparation Eosin Y stock Solutions. Ingredient: Water soluble eosin Y Double-distilled H:0 95 % ethanol Procedure 208 40 mL 160 mL. Add 2.0 g of water-soluble eosin . Y to 40 . . sissolved. Then add 160 mL of 95% ethanal ge wot Aisilled HO and mix utili’ 7 thar i Tate Y ota eo nol and mix. Store at room temperature. Ingredients: Eoxin ¥ stock solution 9 00 80 % ethanol ea a Glacial acetic acid 4mL 32Procedure ‘Add 200 mL of eosin Y stock solution to 600 mL of 80 % ethanol and mix well. While working in a fume hood, add 4 mL of glacial acetic acid and mix well. Store covered at room temperature. Safranin O staining solution: Mainly used for sections of plant tissues, stains red. Preparation Ingredient: Safranin O Stain 20 mg Distilled water 20 mL Procedure Transfer 20 mg of Safranin O Stain in one vial into a 100 mL beaker and add 20 mL of distilled HzO into the beaker and dissolve the stain by stirring to make 0.1 % Safranin O staining solution. Leishman’s stain: Stains nucleus of WBC blue and blood cells pink. Preparation Ingredients: Leishman stain powder 0.15 g Acetone-free methanol 20. mL Procedure Weigh 0.15 g of Leishman stain powder into a Glass Mortar and grind it then put it into a bottle through a funnel. Gently pour in about 20 mL of acetone-free methanol through the same funnel, ensuring that all the dry stain is washed into the bottle. Re-cap the bottle and shake it in a circular motion for 2-3 minutes to start dissolving the stain crystals in Methanol solution, Warm the content at 50 °C for 15 minutes with occasional shaking. Add the remaining amount of methanol to the mixture through the funnel and make the volume 100 ml, ensuring that even the last drop of the methanol washes in the funnel into the stain mixture. Tighten the screw-cap on the bottle and continue shaking for few minutes, Do not open the bottle and do not filter the content for about a week. After a week it is ready to use. Filter the content before use. Label the bottle clearly as Leishman Stock Solution with date of preparation and date of expiry. Tighten the screw-cap to prevent absorption of water vapors from the air, Do not store the solution near bottles containing acid. If stored correctly Leishman Stain solution is stable approximately for 3 months.Dilute the stock solution of Leishman stain with buffer solution according to the specific requirement for your work (ideally in 1:2 ie. 1 part stain and 2 parts buffer solution). Crystal Violet: Stains bacteria purple 33 at.sce CIenoN © SPITE Labeled organism B from 1-21. ore tae oF essay 9, Sate the functions of the parts labelled 4, 6, 9, 10 and 12 Send fi celwods * State the type of metamoi a in organism D and the stages wan oo) on. V7 bebe Mise! fl lite ‘pet, (mel ol a li at hriee too ell Lefeak mek, and baat bi Bur ne Oat tg ie Kk ortxbes Arce heres a Ca & Keg ee) |aoa a3 en anh Gran di on n oy fab Cicer Qipes Mt fhe ods > Haw would you control oud 's between them and mention @)w- Femur: (EW used) For bet “cavcacle: (et ged $e APAME ours (cased BAG ony! x th fol speTO. (aM disease does organism C transmit wo organisms 1 G. Give two difference: 11. Name the ¢ F from 1-10 using the key in the box. S. abel the organism int = bebe mo(qu Me en dees FE 2S ion» de @ Cont BH Manse an OMonil\ae 2 Taxus: (0) Gh. Foe, 4) To tc @ora ( Adee Oe us B ap Gras topfer 76Cong C Wdabek Lifcages Such as Ooywirre Menvy ri. wid goon Label the Mosquito nodes etrepalpes Const neers gy C be wl A ? ot b tyres, ior ALC eee ALVA, wg 3 Oo bus eee NEL ia Fe rabus aphzde_! morquafe «é o Chem a) ee AU lawn and ae ae Udy) mel ale 3 abeiae java ay bre tid song ids Myo (w iminale a eee ratio YY wy fe teste be weak he: Q)Ue ack ee [bel Dellaneiainy te } ow Es —— mosqui Othe type 7 peat & b & b ir coaploR nfomePhoss Stog so A. Rege fee Lara _—» Puja —PaduleRRA RAE OR Or rap spider Scorpion External AnatomyG>Oonkpede REFERENCES Taylor, D.J., Green, N.P.O and Stout, G.W (1998). Biological,Science. 3" -edition. Cambridge University Press, UK pp. 65-70 Mitchell, L.G., Mutchmor, J.A. and Dolphin, W.D. (1987). Zoology. The Benjamin/Cummings Publishing Company, Inc. pp. 609-647. Carol, V. (2012). Help Your Kids with Science. Penguin Random Housé, Darling Kindersley Limited, London. Jordan, EL. (2016). Invertebrate Zoology. Revised edition S. Chad And Company Limited, New Delhi. Pp.847-1022. 79Chapter Ten DISSECTION c C.N.MGBABU , THE Phylum: Mollusea (Snails) Dee hel of the giant up to 7.8 inches in length and 2.7-3§ P inches in height. An adult weighs about 32 grams. The body has two short tentacles ae two long ones that bear the eyes. | o | The shell has an appearance conical and narrow, with 7 to 9 spiral visible on its surface. The colour is not always the same; it depends on the site where the snail dwells. It is usually dark brown or reddish stripes. The opening is relatively small. An important part of the anatomy 0! gue, which is called the radula. It has small teeth that allow African snail reaches the environmental conditions of F with yellowish vertical O S f this snail is a structure in the mouth similar tO snails to scrap the food a ton s before eating it. They have a “muscular foot” that helps them move releasing a mucus while they’ move to reduce friction and avoid damage to their tissues. po" The shell is the location where the giant African land snail takes refuge from’ predators. They will also spend time inside their shells when the temperature begins to dip” too low at night for their comfort. AN Habitat: D In Aftica, it lives along the edges of forests but can live on the banks of rivers and™ streams, shrublands, agricultural area: i 2 s s, plantat : pi sites, ; plantations, gardens, wetlands and in various urban wi Feeding: eeding: OF This herbi — eee ime dos not discriminate between living or dead plant matters. The ats leaves, flowers, fruits, stems, barks, wood, seeds, grains “ttt seaweeds, and even lichens, fungi , fungi and other snail: senveats and ven ich, ails. They also need calcium to keep and maaan hey will consume more, some types of pl. eon : ey aren’t able to tat howe es, ey nh feed on bor get enough calcium in their di . eee carcasses, sand or small stones to gi 7 their diet from plants, they ™yy fe giant African land sni 10 get it. | when they are going to mate, moving, eating and resting ails don’ i i eat font seem to interact with each other except of t produce any sounds, and they spend their tim? 80Sometime these snails aestivate in the summer month e) cil . jonths i . conditions re = eep their body moist by creating a = Na avoid the hot ; 7 i nucus that their body creates. In the case of severe drought, thi ith a thin layer of , this process can take as Jong as tree Years. geproduction? trae giant African land snails are hermaphrodite, which means that they have the ative ora fo both male and female They have the capacity to self-enilize, but 12 do not usually do that, There is copulation which leads to exchange of male nes tiassifiation of the Giant land snail - ‘ant land snail, Archachatina marginata, is classified in the following way: The gi Phylum: Mollusca Class: Gastropoda Subclass: Pulmonata Order: Stybommatophora Family: Achatinidae Genus: Archachatina Species: marginata Orientation of snails The two types of orientation found in snails ai said to exhibit dextral orientation if the openin; with the apex pointing upwards and the holder faces 1 xeuts ifthe opening is to the left when placed in the same position, Orientation of snail is mportant when dissecting a snail. This is because the left and right sides of a snail body ue found if one places the snail in its normal crawling position with the head pointing ay from the holder. dissection of giant land snail Method of killing Asphyxiation of the land snai vith Water, The animal is immersed in wa f the animals, The snail becomes insen lissolved oxygen in the stoppered jar. Great he Visceral mass remains enclosed in the inner whorls. : , The following organs to be identified before the shell is removed include the foot, the v0 pai : F : ening of the Wo paits of eyes, the common genital openinss the mouth and the ventral opening of the an mucous gland duct. ‘od of removing the snail bo * Pick up the snail between two facing the right and the apex towards Yous re dextral and sinistral orientation. A snail 1g of the shell is to the right when it is held he opening. Sinistral orientation 1 jg achieved by drowning them in stoppered jars filled ‘er for about 24-36 hours depending on the size sitive and fully extend when it used up the ter part of the snail lies outside the shell while e shell. dy from th f your left fingers o| hand with the shell’s aperture 81a strong forceps: (dextral) oF left (sinistral) out Using your right hand, grip the body firmly with er not to destroy some of the «Loosen the body carefully by screwing it to the right of the shell. Be careful not 10 pull too much in ord organs. ; Potassium aluminium sul J the snail already freed from shell before the examination 0) ‘The body of snail is di The mantle covers the visceral mass, ¢! when the animal is retracted. phate (Alum) is used 10 was f the external features. vided into two regions. incloses U ‘They are the food and the visceral mass, the head and the tentacular appendages shell gE cerebral gangie salivary duct Diagram of external anatomy of the soft body of snail Method of opening the mantle cavity ; To open the mantle cavit - head points towards a ti hee snail, orientate the snail on its foot so that the ig * le pneum« following the white columella —- }ostom and cut backwards with scissors Cut across the bod; ; y followi i undemeath the mantle, the conection of the mantle border and the body Cut continuous! ly and pull th wteflexed the ie mantle backw: F + Then fix tn body vine roof. This exposes an coe The roof of the ith pins on the dissectit ial organs. eof o mantle cavit . issecting board wi . fa is pinned to the ate kidney and the heart are ene ‘idney, heart, hermaphrodite i le. This exposes the ee the left side while arized mantle (lung), rect lucts, ¢« i ephalic aorta and kidney duets. 82Genital pore + mantle cr . Pa fi Ta Body wall Kidney duet E Cephalic aorta Position of ‘common hermaphrodite duct Lotte hermaphrodite duet Foot Digestive gland or ‘hepatopancreas is, Snail with the mantle cavity pened to display it’s content The alimentary canal The alimentary canal of land snails consists of three sections namely: |. The fore-gut which include the bucal mass, oesophagus (anterior and posterior and the crop). 2. The mid-gut consisting of the stomach and the intestine. 3. The hind-gut or the rectum, Alimentary canal of land snail is exposed by making mid-dorsal longitudinal ittsion through the floor of the mantle cavity starting from behind the point where the ‘kr had previously been cut. The incision should go forward along the mid-dorsal Pigmented line to the tip of the snout and backwards along the dotted line as shown in the “’eram above, . : | Remove the skin to expose the buccal mass, nerve ring, oesophagus, paired salivary Sands, ang duets, crop, penis retractor muscle, spermathecal ducts, etc. 83puceat mass sativary and vagine tractor ™. Penis re! Stheeal duct orm cre a Male au a we ative ‘ian Diagram of the alimentary canal of snail ‘ONOCERUS VARIEGATUS ‘All invertebrates are, dissected from the dorsal or lateral side. side is preferred in disserting Z. variegates. "The following are steps to take: 1. Anaesthesize the insect in a closed chamber containing few drops of chloroform for 3-5 Dissection from the lateral minutes. 2. Remove the specimen; limbs. 3. Remove the wings and legs ofthe insect and make a longitudinal cut on the lateral side that your scissor does not of the body wall from the anal opening 10 the head. Ensure damage any ofthe internal organs Iying freely in the haemocoel. 4. Pin the insect down on waxed ilist/soft dissecting board on the edge of one side of the cut. Grasp the other edge of the body wall, extend it and pin it out, thus exposing the ‘observe for anaesthetic effect by non-movement of wings and perivisceral cavity and its contents. 5. Examine the transparent heart on the mid-dorsal line of each segment. 6. Display the alimentary canal and it associated structure and pin out. Identify oesophagus, Crop, gizzard, gastric caeca,mid gut, colon, rectum, as well as the excretory organ- malpighian tubules. 7. Clear the fatty tissue and identify the nerve cord as well as the thoracic segment. 8 Study the reproductive system and identify the sex of the specimen. 84 ‘ernate commen. camila BaeBBroaie ittie hermapnrodits SsLa pet maglion Gastric cox; agli Third tharcie ganglion sto ‘Ast audminal gamelion Hay tuaulee Rectua, Fig. 16 General dissection of female Zonocerus variegates Features of Biological Interest |. The alimentary canal, made up of foregut (stomodaeum), midgut (mesenteron) and hindgut (proctodaeum), serves nutritional purpose. 2. The malpighian tubules are used for excretion. The intestine acts as site for absorption while the rectum isused for reabsorption of Water, 4. The nervous system, which consists of the cerebral ganglia, the suboesophageal Bnglia from which the double longitudinal connective tissue to the abdominal “ements, are important in coordination. The ovaries contain eggs for reproduction. / : The spiracles and silvery branching tracheae are important in gaseous exchange. 85Chapter Six TEMPORARY SLIDE PREPARATION TECHNIQUES Nwonumara, G. N., Ambrose, F.C and Ukwueze, F.C Temporary preparations of materials for light microscopy are made for quick preliminary investigation. The nature of the specimen determines the stages that are involved. The stages include sectioning, staining and mounting but sectioning is excluded if the specimen is liquid. The specimen is mounted in water or dilute 1,2,3-propanetriol (glycerol) or other fluid of low volatility and is discarded after examination. To prepare a temporal slide, the materials required include: slides, cover slips, droppers or pipets, razor or microtome (if the specimen is solid and large), stain or any other chemicals such as glycerine, filter or blotting paper, and the specimen. Slide or Microscopic slide: This is a flat rectangular piece of soda lime glass, borosilicate cover glass or plastic, with ground edges. Slide is an instrument used in holding a microscopic specimen for viewing in a microscope. It could be flat glass slide, and depression or well glass slide. Both measure approximately 1 x 3 inches (25 x 75 mm) and between | mm-1.2 mm thick. Depression slides have an indentation in the center to hold a drop of liquid and are usually used without a cover slip. A cover slip or cover glass is a very thin square piece of glass (or plastic) that is placed over the water drop. It measures 18 or 20 mm square with variety of thickness (Number 1 - 0.13 - 0.17 mm thick recommended for oil immersion or high resolution work and Number 2 slips with 0.17 - 0.25 mm thickness and are used for general applications). Because of surface tension, the water drop alone tends to sit in a thick dome. With a cover slip in place, the drop is flattened out allowing the investigator to focus with high power very close to the specimen. The cover glass also confines the specimen to a single plane and thereby reduces the amount of focusing necessary. The cover glass protects the objective lens from immersion into the water drop. , Glass cover slips can be rinsed and reused many times if carefully handled. General method of making a temporary slide A temporary slide is usually made and observed as a wet mount, The slide is prepared by placing a cell suspension in liquid on the slide or by placing the specimen directly on the slide after which water, glycerin or stain is added to it. Thereafter, the 39sir is not trapped, The g that ensuring er slip. fully covered with @ oo Je are as follows specimen is careful smporal slide are king a te th tee a rocedures in mal or of thes general p' f liquid at the center oe - Place a drop 0 tid and large) 2 tweezers (a tool for picking - Section the specimen (if sol liquid using ecimen on the = Position the sample or spect small objects; : - Atan angle, place one side of the co’ the outer edge of the liquid drops slip slowly avoii - Lower the cover slip slow ¢ ‘ Remove excess liquid with the blotting pape! F Je making contact with sainst the slid wer slip aga! ding air bubbles; 3 Lower Coverslip Stowly Some of the techniques used in some tem| i. Smear technique for cheek scraping, Materials: porary preparation are; Slides, cover slip, cover slip labels Disposable spatula ot tooth Pick * NaCl (9 per cent) * Methylene blue stain + Filter paper Procedure * Rinse your mouth well with water, 40Gently scrape the inside of your cheek with the broad end of a clean tooth pick or a sterilized | disposable spatula. Spread the cells on a clean slide, Add a drop of 9 % and a drop of methylene blue. Cover with a cover slip and gently press it to flatten the cells Add stain by irrigation method (see below). Put the slide under the microscope for viewing. 11 or physiological saline Staining by irrigation technique Squash Technique for Onion root tip Squash technique is a simple method widely used for the study of chromosomes. This technique consists of applying gentle pressure on a small piece of stained tissue to spread the chromosomes in the cells. This technique is used to study dividing cells showing either mitosis or meiosis and suitable tissues for this are onion root tip, grasshopper testis or anther buds. The onion root for the preparation is put in acetic alcohol solutions for 12 - 24 hours (fixation). Thereafter, the tissue can be transferred to 70% alcohol. Material © Onion root tips ‘* Forceps and dissecting needles 41© Slides and cover slips © Acetic acid © Filter paper © Nail polish * 70% alcohol ch glass beaker © Pipette, glass droppe © 2N Hydrochloric acid © Acetocarmine/aceto-oreeit Procedure © Transfer the roots from the fixative or storige solution (70% alcohol) in a watehgl = Wash in water until the roots sink. = Drain off the water using a pipette and add a few drops of 2N HCI and leave for 10 minutes at room temperature or for a minute over a spirit lamp. While hydrolyzing by moving the watchglass. On the other over the flame be careful not to overl over a beaker of boiling water till the liquid steams. hand, warm the watehgla = After hydrolysis drain off the HCLand wash root tips in water. * Remove w: and add 1% acetocarmine or aceto - orcein to the root tips for ining. The staining requires 10 - 1 5 minutes. - 7 the res fer 2 -3 root tips on a slide, cut above 2 mm from the pointed end and discard Place a drop of 45% acetic acid on the root tip and carefully place a coverslip. Remove excess acetic acid by the edge of a filter paper. Place the slide between 2 layers of filter paper and gently tap the cov back of a pencil (o get an even spread of chromosomes, + Ifair bubbles get trapped in the cove slip by the lip add a few drops of glycerol or acetic 3 to the edge of the coverslip, = Seal the edges of the Pee coverslip by applying nail polish so that the fluid evaporation is minimum and obs rve the slide under a mic iti, Whole mount technique for Unicellular or Chlamydomonas, Paramecium, Protoy a organism or structure ‘a microscope slide, Materials + Petridishes, Beakers, § : * Paramecium culture, Volvos re ies Slide labels ms (eg Amoeba, small an). In whole-mounts, the entire Snough oF thin enough to be placed ditectly ont?= Methyl cellulose = Methyl green in ethanoic acidlacetocarmine = Acetic acid = Albumin glycerin * Alcohol series for dehydration « Xylene = DPX mountant * Noland's solution = Todine solution Procedure (for temporary slide) on Wet mount technique Put a drop of Paramecium culture on a clean slide and cover with a coverslip. ‘Add equal amount of methyl cellulose to a drop of the culture to slow the movement of paramecia. Irigate the slide with either 1% methyl green in ethanoic acid or acetocarmine. Both fix the organism and stain the nuclei green and red, respectively. Then observe under the microscope. Procedure (for permanent slide) Take a clean dry slide, put a small drop of albumin glycerin in the centre of the slide and make a thin film of it by spreading with the tip of the finger. ‘Add 1-2 drops of Paramecium culture on the slide using a glass dropper and observe under low power of the microscope. Dry the slide under an electric lamp to slow the movement of paramecia. Open a bottle of acetic acid and quickly pass the slide right side down over the mouth of the bottle. This will fix the protozoan. Keep the slide in a petridish of 6" diameter with the right side up. Add a few drops of acetocarmine stain so as to fully cover the culture. Stain the slide for about 5 - 7 minutes. Drain off the excess stain by a blotting paper. Wash the slide with 30%, 50%, 70%, 90% alcohol in ascending order. Always keep the petridish covered by another petridish. This will gradually remove the water from the culture. Wash the slide twice with absolute alcohol to complete the dehydration process. Ensure that the petridish is covered tightly so that no atmospheric moisture gets in. Remove the alcohol and add a few drops of xylene on the slide. This will clear the protozoan so that it is more visible under the microscope. If the slide is turbid (not clear) under the microscope after the addition of xylene, it show that the 43dehydration process is not complete. ‘And should be repeated with absolute aleop, and the xylene. = Puta drop of DPX mountant over so as to trap no air bubbles. ; Label the slide and keep the slide overnight in an incubator. With this, the ready for use. the culture and lower a coverslip carefully oyg Y Over Dry Mount:in a dry mount, the specimen is placed directly on the slide. A cover slip may be used to keep the specimen in place and to help protect the objective lens. Dry mounts are suitable for specimens such as samples of pollen, hair, feathers or plant materials. Basic materials for dry mount © Slides © Coverslips * Toothpicks «Forceps (optional) e Clear Nail Polish (optional) ¢ Scissors e Razor Blade * Microscope Materials for dry mount viewing e Feather « Hair © Small Insect or Insect Body Part © Newspaper or Magazine (cut to size) © Cloth (cut to size) Plant Matter (such as leaf, seed, bark) Procedure i. Use a toothpick or forceps to gather the dry subject material. |. Place the material on the slide. iii, Carefully lower a coverslip onto the slide, NOTE:If you wish, make the permanent by adding a drop of clear fingernail polish to the dry mate placing the coverslip. iv. Examine the slide under low and high power of the microscope. slide se rial belo 44v. Record your observations: write a journal entry, or draw or photograph them. vi. For a temporary slide, separate, then wash and dry the slide and coverslip. Exercise i What is the technique used in gram staining? .... ii The materials and reagents required in gram staining includ [SIN_] Materials Vii [i Ik ii Reagents. iit I iv i \v li vi W vi | ili Outline the basic steps in gram staining iv. Outline the general procedures in temporal slide preparation SMEAR TECHNIQUES | In the area of zoology and botany, especially in hematology and microbiology, examination of specimen are mostly done under microscopes. Specimens could be of 45ygment to whole organ different sources and also of different sizes, rai oF even organism, Due to these variations in sourve and Variation in slide preparation before specimen is viewed \ he specimen onto the slid, specimens are smeared before viewed. Smear i prepared to fx the specimen o is smear, and also prevent the sample from being lost while staining. eae ee evenly across the slide, it becomes easier to distinguish individ hich mig io be applied, it assists in making available details of the specimen which mi invisible, © of specimen, there ig ¢ the microscope. Fluid ht otherwise be Materials - Slide, label, cover slips, disposable spatula, methylene blue stain, iodine stain, pipette and paper towel. Procedure 1) Gently collect a ‘specimen (blood or saliva) with spatula or pipette. 2) Slowly spread or smear the specimen on a clean slide. 464) Remove excess liquid with paper towel and allow the prepared slide to dry in an environment of steady and moderate temperature. The prepared slide can now be viewed under a microscope. Clearer observation of specimen will be made if too much light is not shone on the slide. Stain application Staining is a method used in microscopy to increase contrast in a microscopic image. The basic reasons for staining are to enhance visualization of the cell or certain cellular components under a microscope, to differentiate between dead and living cells in a sample, to determine biomass in an environment of interest, to define and examine bulk tissues, to mark cells in flow cytometry, and in gel electrophoresis. Some common biological stains include carmine, coomassie brilliant blue, crystal violet, haematoxylin, cosin and iodine among many others. Therefore, to the prepared wet or dry slide, add small drop of stain at one edge of the cover slips. Position a paper towel to soak the liquid after capillary action has pulled the stain across the slant slide, staining the specimen. Caution Ensure there is no contact between the fluid and the experimenter since this can introduce living cells into the cell. Also, some diseases are transmitted through human fluid, hence, Contact with such specimen could be detrimental. Exercise 1) The importance of staining a prepared slide include; 472) State one reason for ensuring air is slip? Ans:., PREPARATION OF TEMPORARY OR WET MOUNT SLIDES FOR FUNGAL SAMPLE Instrument and materials ~ Compound microscope, microscope slides, cover slip, fungal sample with cleistothecium, dissecting probe, 5 % KOH solution, scalpel, hand lens, dissection microscope and dropping pipette with bulb. [ | Place 1 drop of 5 % KOH to the centre of the slide Procedures: 482. Transfer the fungal sample using the scalpel and drop ~~ onto 5 % KOH drop on the centre of slide Use a hand lens used to identify the location of "~~ cleistothecia on the sample by gently scraping it over the 5% KOH drop to allow cleistothecia to fall from the =] sample to the slide. | Gently place the cover slip over the 5 % KOH drop and = the fungal sample. Apply the cover slip by placing one edge down first and then lowering the rest of the cover slip down slowly, to reduce the number of air bubbles that will be trapped between the microscope slide and the cover slip 3. If air bubbles do occur, the prepared microscope Rita slide can be gently waved over an alcohol bumer, proximately six inches from the flame for only a few second. Do not allow the sample to boil 49HOW TO PREPARE A TEMP* ORARY WET MOUNT OF SPIROGYRA (ALGagy INSTRUMENT AND MATERIALS: Specimen to be mounted (Spirogyra), Microscope, Microscope glass slide, Wate) pecimen to glass, Cover slips, Petri dish, Filter paper, Brush/Forceps PROCEDURES: of Spirogyra on the microscope 1. With the use of forceps, place several filaments slide. 5 Add few drops of water on the slide. "At an angle, place one side of the cover slip against the slide making contact with outer edge of the liquid drop Lower the cover slowly, avoiding air bubbles. ; Remove excess water if present on the slide with the help of filter paper. This is to obtain a clearer view on the microscope. Observe your mount under a microscope. PRECAUTIONS: 1. Don't use excess amount of water. 2. Hold cover slip gently. Cover slips break easily so handle them with care. 3. Use proper staining technique, 4. Don't crush the mount too much. 5. Use brush/forceps to transfer mount to slide from watch glass, Things to consider when making mounts of fungus material 1. Selection of material to be mounted 4. Be sure you know what you are going to mount, if you do not know, ask for help. 50h Use the dissecting mic >pe or hand lens first, especially if you are working with a sultare plate or small sample. Ask for help if'you are unsure of what to select for your slide preparation. 2. Preparation of material for mount a, Much of the material you will examine with the compound microscope will come from cultures. Sometimes you will be making a s livi tion of fresh or dried tissue. Whatever mounting medium and place the material n it carefully. Otherwise you run the risk of not being able to see anything, especially if ou have lots of air bubbles, ». Selecting a mounting me« Once you have obtained a clean slide, the next step is + determine what you are going to use as a mounting medium. You should select the ‘oun'ing media before you select the material for mounting. Using water as a mounting ted: on is easy and is often sufficient for satisfactory preparations. Water mounts dry up tuic!y, however, and do not allow any particular structures to be viewed better than oth. To overcome these problems, many different mounting media have been used. Although the preparation and use of mounting media is a specialized and rather personal matter, there are a few that are in routine use in most laboratories because they offer distinct and well-known advantages. Some of these mounting media include: Congo Red Solution, KOH + phloxine, Lacto-fuchsin, Melzer's Solution, Shear’s mounting medium, Water + wetting agent, Lacto phenol e.t.c. c.Be sure the material is spread out very well: It is easier to observe this with the dissecting scope. If the preparation is too thick it will be difficult to see, especially with an oil immersion lens. Use fine transfer needles to tease the hyphae apart and spread them out for viewing. ic preparation, it is important to use the cor Exercises |. With the use of a fungal culture, prepare a temporary mount and view on a microscope. 2. Draw the diagram of what you observed in the microscope on a plane sheet and attach to your practical manual 3. Using Spirogyra scooped from any available pond around your locality, prepare an algal wet mount. REFERENCES Bennett, C. (1995), Laboratory Aspects of Medical Mycology, Laboratory diagnosis: 45-78, Conn H. J (2002). Biological stains; A Handbook on the Nature and Uses of the Employed in the Biological Laboratory. BIOS Scientific Publishers Ltd. Oxford, UK. 51