Mikroteknik

Table of ContentsPreface ..................................................................................... i Preface Expert ........................................................................ ii Table of Contents .................................................................. iii Chapter 1 Material Safety Data Sheet ................................... 1 1.1 Reading chemical label on MSDS and Pictogram ............2 1.2 International norm of MSDS ............................................8 Chapter 2 Clearing and Whole Mount in Plant ................... 13 2.1 Clearing definition ......................................................... 13 2.2 Clearing by dissolution of protoplasm ............................ 14 2.3 Clearing tissue without removing protoplasm ................ 19 Chapter 3 Smears and Squash.............................................. 22 3.1 General procedure of smear or squash ............................ 23 3.2 Smear ............................................................................ 23 3.3 Squash .......................................................................... 30 Chapter 4 Paraffin Embedding Method in Plant ................ 33 4.1 Collecting ...................................................................... 33 4.2 Fixing ............................................................................ 34 4.3 Dehydrating ................................................................... 35 4.4 Infiltration and Embedding ............................................ 36 Chapter 5 Paraffin Embedding Method in Animal ............. 42 5.1 Fixation ......................................................................... 42 5.1.1 Fixatives may be classified: ..................................... 43 5.1.2 Fixatives Components: ............................................ 43 5.2 Dehydration ................................................................... 44 5.3 Clearing ......................................................................... 44 5.4 Infiltration ..................................................................... 44 5.5 Embedding/Blocking ..................................................... 45 5.6 Cutting/Sectioning ......................................................... 45 5.7 Mounting the trimmed block on a paraffin table ............. 46 5.8 Floating out sections, Affixing and Deparaffinizing ....... 48 5.9 Staining (Hematoxylin and Eosin) ................................. 49 5.9.1 Two staining procedure that common use in animal technique ......................................................................... 50 5.9.2 Varies Stain Compositions ...................................... 52 5.10 Mounting .................................................................... 53 Chapter 6 Animal Whole Mount ......................................... 57 6.1 What is whole mount? ................................................... 57 6.2 An important note on fixation ........................................ 58 6.3 Obtaining images .......................................................... 58 6.4 Choosing the age of the embryo .................................... 58 6.5 Whole mount fluorescent immunohistochemistry .......... 59 6.6 Antibody staining of whole mount Drosophila embryos 61 6.7 Zebrafish whole mount immunohistochemistry ............. 63 6.8 Troubleshooting tips – whole mount ............................. 64 6.8.1 High background .................................................... 64 6.8.2 No signal ................................................................ 65 6.8.3 Patchy staining........................................................ 66 6.8.4 Morphology of the tissue is not good. ..................... 67 Chapter 7 Blood Smear ........................................................ 69 7.1 Making a smear ............................................................. 69 7.2 Preparing staining buffer ............................................... 71 7.3 Blood Smear Preparation with Romanowsky Staining ... 72 7.3.1 Slide Preparation Procedural notes .......................... 74 7.3.2 Biologic causes of a poor smear .............................. 75 7.3.3 Staining Procedure .................................................. 75 7.3.4 Low power (10x) scan ............................................ 76 7.3.5 High power (40x) scan ............................................ 76 References ............................................................................. 79 Appendix: Material Safety Data Sheet ................................. A CHAPTER 1 MATERIAL SAFETY DATA SHEET Objective After read this chapter we expect you will be able to: 1. Explain why people have to read MSDS before work with hazardous substance 2. Recognize and understand symbol and code number of hazardous substance according NFPA or HIMIS 3. Understand pictogram of hazardous substance 4. Summarize some important information regarding with personal and environment safety from MSDS Person or people that working using hazardous chemical or dangerous goods have to know the risk substance in order to organize safety for her/his self and environment. To obtain safety information, people have to read material safety data sheet (MSDS) first. The warning of safety level can read from container of substance. Material safety data sheet (MSDS) which is also called safety data sheet (SDS) is document that contain information about the potential hazards (health, fire, reactivity and environmental) of the chemical and how they affect health and safety in the workplace. MSDS can be obtained from companies that produce the substance or internet by typing name substance as key word. For example: HCl + MSDS. The aims providing MSDS are to give information around proper storage of a substance, first aid, spill response, safe disposal, toxicity, flammability, and additional useful information. By reading and understanding some information that stated in MSDS, at least three points can be deduced: a. How deep hazardous associated with the substance? b. What should I do to protect myself, area around me, and environment? c. What should I do if an accident occurs? Previously, MSDS was common consumed by hygienist and safety professional, but now the user of MSDS is also researcher, employers, supervisors, nurses, doctors, and emergency personnel. MSDS give those peoples procedures for handling or working with that substance in a safe manner. 1 1 Reading chemical label on MSDS and Pictogram In the scope of hazardous substance.1. MSDS also contain symbol that represent type and level of particular hazard. National Fire Protection Association (NFPA) and Hazard Materials Information System (HMIS) respectively. Blue = Health hazard Red = Fire hazard Yellow = Reactivity hazard White = Special hazard or personal protective equipment (PPE) Figure 1. The symbol is a shortcut to raise awareness about hazardous a substance (Figure 1). NFPA has diamond format (Figure 2A) and HMIS has rectangle format (Figure 2B). Label notifications give warning regarding hazardous degree of a substance. Each color in both format represent a different type of hazard. Red. Blue. each level has meaning as below: 0 = minimal hazard 1 = slight hazard 2 = moderate hazard 3 = serious hazard 4 = severe hazard 2 . The number move from 0-4. and Yellow color also contain number that denote degree of hazard. Generally two style-symbols are applied in MSDS. and Hazard Materials Information System (HMIS) (B) on 2-Mercaptoethanol according ScienceLab. Reactivity of figure 2A at yellow place has number 1. 3 The substance could cause serious temporary or irreversible injury. In above figure. major or permanent damage may result from single or repeated over exposures Major injury likely unless prompt action is taken and medical treatment is given Temporary or minor injury may occur . The substance could cause death or irreversible injury. Explanation of hazardous degree according National Fire Protection Association (NFPA) and Hazard Materials Information System (HMIS).Health Risks 4 The substance is a severe health risk if the substance is not handled safely. in the other hand figure 2 B has number 0. Table 1. we can see slight different between figure 2 A and B.A B Figure 2. Style hazard symbol based on National Fire Protection Association (NFPA) (A). 2 The substance could cause temporary incapacitation. NFPA The Blue Section . Apparently NFPA and HMIS have a little bit different consideration of hazardous degree related with number denotation on the same substance (Table 1). 3 HMIS Life-threatening.com MSDS. temperature conditions. or selfreaction at normal temperature and pressure 3 The substance may Materials that may form 4 . 0 There is no health hazard. Materials may ignite spontaneously with air 3 A flammable liquid or Materials capable of ignition solid which can be readily under almost all normal ignited. moderately heated or exposed . 2 The substance must be Materials which must be heated for ignition.1 The substance could cause irritation. Includes flammable liquids with flash points below 73 °F (23 °C) and boiling points above 100 °F (38 °C). and boiling points below 100 °F (38 °C). The Red Section .Fire 4 A flammable vapor or gas which burns readily. or very volatile flammable liquids with flash points below 73 °F (23 °C). reaction. to high ambient temperatures before ignition will occur. as well as liquids with flash points between 73 °F and 100 °F. Includes liquids. solids and semi solids having a flash point above 200 °F (93 °C) 0 There is no fire hazard Materials that will not burn The Yellow Section . detonation or .Reactivity Hazards 4 The substance is readily Materials that are readily capable of detonation or capable of explosive water explosive reaction. polymerization. Includes liquids having a flash point at or above 100 °F (38 °C) but below 200 °F (93 °C) 1 The substance must be Materials that must be preheated before ignition preheated before ignition will can occur occur. Irritation or minor reversible injury possible No significant risk to health Flammable gases. explosive decomposition. decompose. protective gloves.The White Section . Materials may react nonviolently with water or undergo hazardous polymerization in the absence of inhibitors The substance is stable. Materials may polymerize. selfreact. a laboratory 5 . Materials may react violently with water or form peroxides upon exposure to air. decompose. changes at normal temperature and pressure with low risk for explosion. condense. The substance is readily Materials that are unstable and capable of non-explosive may undergo violent chemical reaction. and will not react with water. or selfreact. polymerize. and a laboratory apron should use COR = Corrosive D: A face shield.personal Special Hazards protection*) OX = Oxidizer A : safety glasses ACID = Acid B: Safety glasses and protective gloves ALK = Alkali C: Goggles. protective gloves. even under fire conditions. unstable (self-react) at high temperatures and pressures. Non-explosives The White Section .2 1 0 detonate when exposed to explosive mixtures with water heat or an ignition source and are capable of detonation or explosive reaction in the presence of a strong initiating source. Materials that are normally stable. or undergo other chemical change at normal temperature and pressure with moderate risk of explosion. goggles. The substance may Materials that are normally become unstable at high stable but can become temperatures. apron. Pictogram is hazardous label that convey specific information about hazard of chemical by using a symbol plus other graphic elements. and an exhaust fume hood (for reactivity hazard no. First. it label during delivery or transportation the dangerous goods. a laboratory apron. protective gloves. Pictogram has three destination labeling. The pictogram according to Euro style include as below : 6 . or color . = Use no water = Radioactive NOTE *) . The second. such as a border. And the third. and an exhaust fume hood H: Splash goggles. it label on container or work place. and an exhaust fume hood should be used when handling this material. background pattern. it appear on MSDS sheet. protective gloves.3 or 4 E: Safety glasses. The similar pictogram is developed by Work Health and Safety (WHS) Australia at 2012.C E F Corrosive Explosive Highly Flammable F+ N O Extremely flammable Dangerous for the environment Oxidizing T T+ Xi Toxic Very toxic Irritant Xn Harmful Figure 3. The new labeling system derived from 7 . Pictogram according to European style. manufacturer or distributor who provides the chemical. Occupational Safety and Health Administration (OSHA) and American National Standards Institute (ANSI) formats. The style as following table: Section Item Section 1: Identification Section 2 : Hazard(s) identification Section 3 : Composition and information on ingredients Explanation This section links the chemical name on the label to the MSDS.2 International norm of MSDS Writing format for MSDS has two style formats. Pictogram according to Work Health and Safety (WHS) Australia. The following table shows these new pictograms and the types of hazards that they represent : Severe health hazards Health hazards Acute toxicity Explosi ve Flamma ble Oxidising Corrosi ve Gases under pressure Environmen tal hazard Figure 4. Mostly MSDS format comply ANZI style. This section discusses the health effects one may encounter when exposed to the material. address and the phone number of the company. 1. This section must identify all the hazardous ingredients of the material. The 8 . The MSDS also lists the name.the United Nations’ Globally Harmonised System (GHS) of Classification and Labelling of Chemicals. The procedures will be written so that untrained individuals can understand the information.section will describe the appearance of the material. target organs that could be affected. and so on. and general fire-fighting instructions. the potential health effects and symptoms associated with exposure. This information may include how to contain a spill or the types of equipment that may be needed for protection. handling procedures. Section 6: Accidental release This section gives information measures on how to respond when a material spills. Topics that could be described are: general warnings to prevent overexposure. routes of entry. extinguishing items. The necessary personal protective equipment should be considered for 9 . Section 5: Fire-fighting This section will describe measures information on the fire and explosive properties of the material. leaks or is released into the air. and hygiene instructions to prevent continued exposure Section 8 : Exposure This section discusses controls/ personal protection engineering controls and personal protective equipment that would help reduce exposure to the material. Section 4: First-aid measures This section will describe possible first aid procedures for each route of entry. Section 7 : Handling and This section discusses storage information on handling and storage of the material. vapor density. reproductive effects. freezing/melting point. solubility in water and specific gravity or density. boiling point. vapor pressure. The following characteristics should be detailed: appearance. odor. skin protection and respiratory protection. It addresses chemical stability. target organ effects. This section will include information about the physical and chemical properties of the material. etc This section will help determine the environmental impact should the material ever be released into the environment.” The following information can be addressed: acute data. physical state. This section requires that potentially hazardous chemical reactions be identified.Section 9 : Physical and chemical properties Section 10 : Stability and reactivity Section 11 : Toxicological information Section 12 : Ecological information Section 13 : Disposal considerations eye/face protection. conditions to avoid. pH. hazardous decomposition and hazardous polymerization This section discusses data used to determine the hazards that are given in Section 3. This section gives important information that may be helpful in the proper disposal of the material. Indicate if these characteristics do not apply to your material. “Hazard Identification. incompatibility with other materials. recycling and reclamation 10 . carcinogenicity. The information can cover disposal. SELF-QUIZ 1. hazard class and the identification number (UN or NA numbers). yellow c. This section should include any other important information concerning the material. blue d. The basic shipping information could include: the hazardous materials description. To get a better understanding on the format of MSDS. refer to the appendix at the end of this book. Red b.  emergency procedures.Section 14 : Transport information Section 15 : Regulatory information Section 16 : Other information This section is designed to give basic shipping information. we hope they will more understand about MSDS and can learn to withdraw five major information from MSDS. After the students read the real example of MSDS.  safe handling and storage procedures. What alert hazard of following color of chemical hazard label? a. five main information should be underlined in MSDS. What is MSDS? 2. This section discusses information on the regulations under which the material falls. This information can include: hazard ratings. The information include:  the identity of the chemical. white 11 . preparation and revisions of the MSDS. By consideration that safety is a major concern.  health and physicochemical hazards.  disposal considerations. and label information. what you should put major attention when you read MSDS? 12 . Why should an individual working with chemicals understand the hazard coding system on a chemical label? 5. Regarding with safety for your self. What interpretation highest number or lowest number of chemical hazard label? 4.3. area around your working place. and environment. while mount has meaning attached to material surface. elucidating three-dimensional organization plant organ. All we know that nature of plant cell and 13 . 2. Whole has intact connotation . however. whole and mount. small bud. Describe role substance with high index refractive in clearing process 4. haustorium or hyphe of fungi in infected tissue. Distinguish clearing process category one and two 3. calcium oxalate type. principally material or specimen can be observed under microscope. Terminology whole not absolutely describe intact body or morphology such as individual bryophyte. and other organ also are considered as whole. The clear tissue is beneficial for studying of vascular anatomy. Define meaning terminology of whole mount and clearing in plant tissue 2.1 Clearing definition Literally. whole mount is composed two word. Understand clearing procedure using principle of removal cytoplasmic component 5. In nature plant tissue doesn’t transparent while some microscopy work require transparent condition. Understand clearing procedure using principle of without removal cytoplasmic component 6. and screening embryo or ovule mutant .CHAPTER 2 CLEARING AND WHOLE MOUNT IN PLANT Objective After read this chapter we expect you will be able to: 1. part of intact body for example small flower. In practice whole mount has a meaning placing a whole organism or specimen on slide for microscopic observation or examination. clearing has meaning easily to see because transparency of medium. How is large leaf? In whole mount. And it is compulsory that the material should be transparent in order allow light can pass to optic system of light microscope. Prepare whole mount specimen Based on the word root. Therefore some clearing method were introduced to clear plant tissue. silica cells. ergastic substance. c. laticifers. dark pigment formation due to three mechanisms: a. application 5% NaOH or KOH for 3 days or more produce cleared tissue. In the category two by contrast. furfural from carbohydrates. trichomes. In the category one. phenolic oxidation almost occurred with dark pigment result. peptides. In a cleared cell or tissue can stand out some character because of their special chemical or physical properties. Phenol-aldehyde condensation to give very stable polymers whose constitution depends on reaction conditions. There are many clearing method that already develop to make tick tissue or small organ visible or transparent.tissue contain varies pigment. stomata. a clearing is performed by remove cytoplasmic content using harsh chemical (e. involving quinoid phenol derivatives and amine-containing substances. Phenol-amine coupling. Affinities phenol polymeric to protein seem to protect tissue by making slow down NaOH attack. Making cytoplasmic component transparency degree is almost equal. crystals. b. 14 . According to Singleton (1972). a weak solution NaOH or KOH (2 %) is used for soft tissue to avoid cellulose degradation. Those substances and organelles have different refractive index and light scattering therefore the cell and tissue look opaque or semiopaque. organelles.g NaOH).g. tanniferous idioblasts. e. cytoplasmics component are not removed also tissue structure is unaltered by treatment process. e.2 Clearing by dissolution of protoplasm In normal standard.g. and even nuclei (in embryos). The characters include such feature vascular anatomy. During hydrolysis and oxidation by NaOH. However. schlereid distribution. Naturally-occurring aldehydes or those produced in protoplasm breakdown. These methods are classified into two categories. Finally the clearing agent work to make cell or tissue translucent both through their refractive index and though their abiliy dissolved cytoplasmic component. 2. may be involved. They need a clearing procedure to improve visualization. However refractive index of the components are changed to more uniform their refractive by chemical agent. Oxidative phenol-phenol coupling to give quinones and related compounds. acidified sodium chlorite. chlorine . drying tissue usually produce dark pigment resulting from air and enzymic oxidation of phenolic compounds.Dry . warm 95% ethanol. Even drying leaf has weak point. The phenolic pigment can be removed by repeated washing in succession with warm water.The phenolic pigment that produce during alkali treatment ban specimen transparency even though protoplasm already gone.Fixed Fixed tissue have varied responses to alkali treatment depend on kind of fixative. As consequence. hypochlorite .Formaldehyde reacts with many plant phenols. 15 . Therefore acid environment also both concentration and length incubation of bleaching agent are important to put in attention. However. However. application bleaching agent should be put in consideration for fragile tissue because they can dissolve cellulose and lignin especially in acid condition. commercial household bleach. chromium trioxide as in Stockwell's solution .Fresh . fixative containing formaldehyde produce contra clear effect because such follow: 1. warm 70% ethanol. Dark pigment that may be formed in earlier clearing stage is recommended to bleached using bleaching agent. And some bleaching agent that were known to work well are : hydrogen peroxide . Ruzin (1999) strong suggested to dry first before treating with NaOH.Formaldehyde make cross-linking protein in tissue which enable tissue resistant to alkali (NaOH or KOH) attack and the whole specimen may disintegrate because degrade the cellulose before it clears. 2. because they slightly accelerate clearing by extracting various constituent of protoplasm. to form dark polymers . especially the condensed tannins (leucoanthocyanins and catechins). Dry tissue is quicker to clear compare fresh tissue because of disruption protoplasm during drying. Carnoy or 70% alcohol can function as pretreatment event. Alkali treatment can be applied to three type tissue : . Continue dehydrate in 70%-95% and absolute of ethanol briefly to avoid de-staining. Clearing leaf tissue to observe Calcium oxalate crystal Clearing tissue of whole mount specimens has different method depend on the aim of research target.Example clearing component tissue by removing cytoplasmic A. 16 . 2. 4. The dry leaf. 2002): 1. 6. Put back in ethanol absolute to solve milky problem. Clearing leaf tissue in general A leaf as object of research may be in dry or fresh condition. dipping in chloral hydrate solution (2. Give additional clearing in xylene. If the leaf was not bleah/clear. then it dip in Stockwell solution for one to several hour. 7. The clearing method for observation crystal Ca-Ox mostly is conducted by dissolving protoplasm as basic principle. Mount leaf with solution that soluble in xylene such as Canada balsam or Enthellan B. Stockwell composition Water 90 ml Potassium bichromate 1g Glacial acetic acid 10 ml Chromic acid 1g The above solution has function to bleach tissue. if still contain water the specimen look milky white. Wash briefly to omit any left of pigment with water. The following section describe some example good clearing method for preparation calcium oxalate slide. however. Make sure the specimen dehydrated. Dehydrate tissue in 30% and 50% ethanol for 5 minute each. The fresh leaf may treat first in boil alcohol or Carnoy’s solution (Table 2) to remove chlorophyll (Khasim. The order steps of clearing and staining leaf tissue is described below (Khasim. 5.5 g/ml) is additional step to clear specimen. Submerge the specimen (treated fresh leaf or dried leaf) in 5% NaOH or KOH at 37oC for 24 h or more until transparent. Stain tissue in 1% safranin (in 50% ethanol) for few minutes. submerge in NaOH or KOH directly. 2002). 3. Mounting using Hoyer solution. usually take 24 h.1. 3. and 5 minute in 96% ethanol. Slice corm clearing Submerged specimen in 5% NaOH at 37oC for 24 hour. Fatmawati (2012) using A. petiole and corm) fix in farmer solution ( 70% ethanol : glacial acetic acid = 3:1) for while. 2. dehydrate in alcohol series 3050-70-80-100% for 10 minute each. After bleach. transferred to 30% alcohol for 1 h. move to 50% house hold bleaching for 1 h. move to 50% house hold bleaching for 1 h.muelleri The specimens (leaf and petiole) were submerged in in 5% NaOH at 37oC for 24 hour . Muelleri The specimen (leaf. wash three times with aquades.Campanulatus (domesticated) Leaf clearing Submerged specimen (leaf. 17 . Replace NaOH with 50% house hold bleaching . leave it there for 1 h. move to 80% house hold bleaching for 30 minute and wash in tap running water. wash in alcohol abs. Rohmiati (2012) using A. Then. Endriyani (2009) using A. remove them to 95% ethanol for 24 h. Repeating clearing using mixture of ethanol : xylene in varies proportion . wash using aquades for 5 minute. petiole: 1x1 cm2) in 5% NaOH at 37oC for 24 hour. 1x1 cm2) in 5% NaOH at 37oC for 24 hour. The process clearing is finish. The specimen ready to observe under microscope. wash once more before dehydrate in ethanol series 30-50-8095-100% for 10-15 minute each. wash three times with aquades. wash briefly before transfer to 10% NaOH until the specimen transparent.Campanulatus (wild) Leaf and petiole clearing Submerged specimen (leaf. then directly mounting in glycerin 4. Finally mounting the specimen in glycerin. transfer the leaf to 30% and 50% ethanol for 15 minute each. move to 50% house hold bleaching for 1 h. 3:1 – 1:1 – 1:3. then mounting using glycerine. Handayani (2009) using A. Petiole clearing Submerged specimen (petiole 1x1 cm2) in 5% NaOH at 37oC for 24 hour. dehydrate in alcohol series 25-50-70-80% for 10 minute each. Mounting the leaf using glycerin. Then mounting using Hoyer solution. dehydrate in alcohol series 25-50-7095% for 10 minute each. Slice corm clearing Submerged specimen in 5% NaOH at 37oC for 24 hour. 2011) C. move to 50% house hold bleaching for 1 h. The specimen ready to observe under microscope. Finally mounting the specimen using glycerin solution.6 Druse crystal (A) and Raphide crystal (B) in corm of A. Treat soy bean leaf or other dicotyledenous species with more concentrated NaOH (10% in water) for 2-4 weeks. move to 50% house hold bleaching for 20 minute. transfer to 30 and 50% alcohol for 10 minute each. Submerged specimen in 5% NaOH at RT for 20 minute. 1986) 1. Stain cleared tissue with chlorazol E black (1% in ethanol absolute) for 3-6 minutes 18 . Mounting the leaf using Hoyer solution. wash using aquades for 5 minute. 5.variabilis Leaf clearing Submerged specimen (leaf. move to 50% house hold bleaching for 1 h and wash in running tap water. A 10 μm B 30 μm Figure. then directly transfer to slide glass and mounting using glycerin. Sliced-corm clearing Fix the sliced corm in 70% ethanol for 30 minute. Aulia (2012) using A. Then. Clearing tissue for sieve tube (Lersten. Petiole clearing Submerged specimen (petiole 1x1 cm2) in 5% NaOH at 37oC for 24 hour. move to 50% house hold bleaching for 20 minute (shaking some times to omit bubble).5 g/ml) 2. then directly transfer to 30% ethanol for 15 minute and finally mounting using glycerin. transfer the leaf to 30% and for 10 minute. Continuing clearing in chloral hydrate (2.variabilis (Aulia. 1x1 cm2) in 5% NaOH at 3739oC for 43 hour. 2007). Infected chickpea hair by Ascochyta rabiei (Harijati. 2007) 19 .41/2 is 9 part of 41/2 solution and one part of benzyl benzoate (Ruzin. The Composition BB. BB41/2 is the famous one of clearing solution . The aim position is phloem more close with objective lens so that it will see first. Detail of BB-41/2 clearing solution is as below : 41/2 solution by weigh 85% Lactic acid 2 Chloral hydrate 2 Phenol 2 Clove oil 2 Xylene 1 BB-41/2 solution 41/2 solution Benzyl benzoate by weigh 9 1 Chloral hydrate itself is potential to clear tissue (Harijati. 2. Figure 5.3. Mount sample with lower epidermis upward. The Chloral hydrate solution was applied after clearing – staining infected leaf produced very nice whole mount leaf-hair slide ( Figure 5). There are many clearing solution formula already developed with chloral hydrate as a important component.3 Clearing tissue without removing protoplasm Acidified of chloral hydrate has been used successfully to clear tissue without remove cytoplasmic component in many experiment. 1999). 47 Xylene 1. strong solution of phenol and chloral hydrate or mixture of these substance. Even usually initial step for microtechnique is fixation. just leave the specimens in one of those 20 . It means that more vertical planes can observed in the microscope in a particular focal plane.43 Visikol 1.60 Glycerol 1.50 Methyl salicylate 1.33 Ethanol 1.54 Acidifi ed chloral hydrate in 1. introduce one stop clearing which involves infiltration of material with high refractive index. By watching staining. Garner (1975). The depth of field is proportionate to the refractive index.45 The high refractive index of medium can allow the light the medium without refraction between the boundary of the glass and objective lens of microscope. however.36 Chloral hydrate 1. Clearing agents with high refraction index allow more light more light to continue through the microscope to the observer. Those material include a lactic acid. Composition clearing-staining solution for infected tissue (Harijati .clearing solution we realize why the tissue gave clear appearance. Refractive index of some common microscope media Media Refractive index ( n D20 ) Water 1.43 glycerol Lactic acid 1. 2007) Component Volume or weight 300 ml 99% ethanol 300 ml 50 ml chloroform 50 ml 125 ml lactic acid 125 ml 450 g chloral hydrate 450 g 2 g trypan blue or chlorazol Black E 2g Table 3. Some component of solution has high refractive index (Table 3).Clearing-staining tissue also contain more chloral hydrate (Table 2). Table 2. A high refractive index also create improvement dept of field. In this procedure quite simple. or CRAF 2. diluted to 100 ml in deionized water) at 60oC for 12-14 hour. Please explain about whole mount in microscopy discipline! 2. What should do if you want omit protoplasmic component or not remove them? 3. change many times 5. Fix tissue in either FAA. There are many procedure available. In general Ruzin (1999) describe as below : 1.solution or mixture of substance until they are transparent then mounting them in medium which contain high refractive index as well such Hoyer medium.02 g in 2 ml 95% ethanol. Transfer to mixture ethanol acid solution ( EtOH : HCl = 3:1) for 1-5 minute 7. Example clearing tissue without removing cytoplasmic componen Combination of lactic acid and chloral hydrate is favorite solution without removal cytoplasmic component. Stain in basic fuchsin (0. Could you describe procedure to clear leaf tissue by aim to see Ca-Ox crystal? 21 . What do you think about substance with high refractive index? 4. 4. FPA. Wash 12 h in deionized water. Wash in 100% ethanol for 24 h 8. Clear in xylene and mount using Canada balsam SELF-QUIZ 1. Transfer the tissue to BB-41/2 clearing solution then continued transfer to 95% ethanol 3. Dehydrate to 95% ethanol for 12 hour 6. the following solution formula can apply to clean slide : Potassium dichromate Conc. Now the solution is ready to use. Prepare some solution for fixation and staining In general. Table 4. Cleaning slide is performed by immerse the slide for while (depend on dirty level of slid) and wash thoroughly under running tap water. add 100 ml H2SO4 slowly then stir low beat. Distinguish between smear and squash 2. Prepare clean slide glass when reuse slide glass 3. After potassium dichromate dissolve well. chromosome slide is prepared using smear or squash technique. Sulphuric acid Distillated water 20 g 100 ml 100 ml Dissolved 20 g potassium dichromate in 100 ml dH2O.CHAPTER 3 SMEARS AND SQUASH Objective After read this chapter we expect you will be able to: 1. And actually both smear and squash technique share in same process but there is a little deviation (Table 4). stop stirring and let the solution cool. The equation and difference between smear and squash smear Pre-treatment no Dissolving middle lamella yes The compactness of origin less tissue squash yes yes more Smear and squash definitely require clean microscopy slide because during preparation process of those technique not need adhesive. It recommended some time immerse in strong 22 . During that process will produce a heat. Conduct preparation cytology specimen using smear or squash technique 4. Therefore if someone feel worried about cleanness of slide. Also important note. The most popular example smear in plant is anther’s microsporocyte. 3. Harvest and fix anther in FAA solution (Table 5). For example anther of Tradescatia sp suppose harvest 15 days before pollen grain shedding. 3. root tip. remove anther wall and others debris 23 . squeeze out microsporocyte.ethanol containing few drops ammonia solution. give few drops acetocarmine staining (Table 6) using Pasteur pipette over the anther. young leaf and others meristematic tissue.2 Smear Such in blood smear. By press gently. Take anthers some days before pollen grain appear or disperse. Example of Iron acetocarmine method as below: Method I 1. The famous one is iron-acetocarmine. then wash in running tap water and drying in free dust area. harvest material is conducted in mid day in order to obtain good meiosis. To conduct smear or squash action.1 General procedure of smear or squash The source material that suitable for smear or squash is microsporocytes. plant smear has principal spreading evenly of material as thin as possible. Place fixed anther on a clean slide. 2. There is some method that applied for smear. flow chart below is general pattern to obtain specimen Killed and fixed tissue Hydrolysis Maceration Stain Cover 3. Put cover slip and remove excess stain using tissue paper 5. Pour Carnoy’s solution (Table 5) in petri dish that previously equipped with two or three glasses (Figure 1). Put few drop acetocarmine staining (Table 6) on smear and cover with cover slide 5. Place anther in center of clean slide 2. Method II 1. Keep it at least 10 minute.4. Rectangle petri dish equipped with rod glasses 4. The fixatives that common use in cytology studies Fixative name Carnoy (type I) or Farmer Carnoy (type II) CRAFT (chromic composition volume Absolute ethyl alcohol 15 ml Glacial acetic acid 5 ml Absolute ethyl alcohol 15 ml Glacial acetic acid 5 ml Chloroform 15 ml Chromic acid (1% chromium 20 ml 24 . Figure 7. Put another slide over anther. Remove excess solution with tissue paper and seal cover slip with transparent nail polish Table 5. press gently and spread evenly using that slide 3. Leave preparation for few days to facilitate the stain into microsporocyte. Put slide with invert face touch fixative solution. Seal cover slip with transparent nail polish 6. 5 ml 10 ml 41.5 ml 90 ml 5 ml 5 ml Table 6. Wok well during experiment 75ml 5ml 20 ml 10 ml 5 ml 65 ml 30 ml 20 ml 10 ml 40 ml 40 ml 30 ml 10 ml 20 ml 50 ml 35 ml 15 ml 1 ml 7. 70% ethyl alcohol acetic acid. add 85% EtOH. HCl to dH2O. HCl Distilled water 85% ethanol 4g 1 ml 15 ml 95 ml Preparation Add Carmine and conc. Mix well using magnetic bar gently while boil ( 10 minute).acid acetic acid formalin) (type I) trioxide) 1 % Acetic acid 37-40% formalin CRAFT (type II) Chromic acid (1% chromium trioxide) 10 % Acetic acid 37-40% formalin Water CRAFT (type III) Chromic acid (1% chromium trioxide) 10 % Acetic acid 37-40% formalin Water CRAFT (type IV) Chromic acid (1% chromium trioxide) 10 % Acetic acid 37-40% formalin Water CRAFT (type V) Chromic acid (1% chromium trioxide) 10 % Acetic acid 37-40% formalin Navashin 10% chromic acid 2 % osmic acid in 2 % chromic acid 10 % acetic acid Distillated Note : add 1% saponin to mixture FAA (Formalin. The staining that common use in cytology studies Snow’s solution Composition Carmine Conc. alcohol) * Glacial acetic acid Formalin *). Mix 25 . After cooled. Store in brown bottle Feulgen (30 mL) Composition Basic Fuchsin 150 mg 1 N HCl 4. Cool and add few drop ferric acetate until appear wine-red color.well then filtered. Fix anther in Famer’s solution for 12 hours or some week to enhance the staining ability of chromosome 2.metabisulfit) 0.45 g Aceto-orcein Composition Orcein Glacial acetic acid Preparation Dissolved 150 mg basic fuchsin using 30 ml boiling dH2O.g. 1 paper. dilute up to 45 % using dH2O Aceto-Carmine Composition Carmine 1g Glacial acetic acid 90 ml Conc Ferric acetate few drops dH2O 110 ml preparation Add 110 m dH2O to 90 ml glacial acetic acid.5 mL 1N HCl to filtered substance. Add and dissolve 0.45 g K2S2O5. add 1 g carmine. Norit). Let it for 24 h in dark to bleach. Place the anther in center of clean slide and put a drop of acetocarmine (Table 5) over it.5 ml dH2O 30 mL K2S2O5 (Pot. Add 0. Boiling the mixture up to boiling point in fume hood.2 g Orcein dissolve in 100 ml 100 ml glacial acetic acid. Storage in brown bottle preparation 2. Let it stand for 12 hour Filter and store as main stock Method III 1.2 g Dissolve 2.5 g activated charcoal (e. Gently boil the mixture while stirring Cool and filter Storage in brown bottle Note : when use . Cool the mixture to 50oC then filter through Whatman No. Stop boiling. 26 . shake 2 minute. Add 4. and filter. mix well. 3. Moving the slide in following jar for 1 minute each a. Jar 3 containing 1:1 alcohol absolute and xylene 7. Squeeze out microsporocyte by pushing gently . Jar 1 containing 1:3 acetic acid and alcohol absolute b. the root keep clean and easily to harvest. Jar 2 containing 1:9 acetic acid : alcohol absolute c. The root is prepared by germinate onion on water (Figure 9). Passing the slide over fire 5. By this technique . And in the morning (around 7-9 am) is good time to harvest the root because the root cells are active dividing mitoticly. Instead of anther. 27 . Mount with Canada Balsam or Enthelan (dilute with xylene). Avoid boil the cell Figure 8. Transfer slide to jar that containing acetic acid : alcohol absolute = 1:1 for 5 minute 6. The slide over fire gently for a second to spread cell flatten (Figure 8). The popular root tip experiment is onion’s root. root tip also is suitable as smear material. remove cell wall and debris 4. 3. Passed the slide over fire 4-6 times with 1 second each to allow cells to spread and flatten (Figure 8). Place the root in the center of clean slide glass and give 2 drops of acetocarmine staining (Table 5). fix using CRAF fixative (Table 4) 2. Put cover slip.method I 1. Fix roots in Famer’s solution for 12 hours or some week to enhance the staining ability of chromosome 28 . 4. Figure 10. wrap around the slide with tissue/towel paper (Figure 10) and press gently with finger or pencil bottom to spread cell. Remove the paper and seal cover slip using transparent nail polish Root. Cut root tip  2 mm length .method II 1. Sample spreading 5. Onion germination method Root.Figure 9. Please take a note : do not to allow the cell boiled. Place the root on the center clean slide glass. Remove the root tip using Pasteur pipette or other tool to test tube/watch glass containing 1 N HCl then digest at 60oC for 12 minute at constant temperature 3. 8.HCl = 1 : 1 for 5-30 minute. Jar 1 containing 1:3 acetic acid and alcohol absolute b. 5. This solution has function to dissolve middle lamella. add 1 ml Feulgen staining (Table 6) 29 . Seal cover slip using transparent nail polish Root. 7. Root. Mount with Canada Balsam or Enthelan (dilute with xylene). Cut root tip  3 mm length and fix in Farmer solution for at least 24 hour 2. 3. Put cover slip and passed the slide over fire (Figure 8) shortly (1 second) for 3-4 times. Place the root on the center clean slide glass. chopped with razor blade or scalpel or cutter.2. Take cover slip using forceps and put the slide in jar containing mix solution acetic acid: ethanol abs. Remove the root to container that already filled with mixture 95 % ethanol : conc. and pressed gently with the blade. = 1 : 1 for 5 minute. chopped with razor blade or scalpel or cutter. drops acetocarmine. Remove the root to container that already filled with mixture 95 % ethanol : conc. 6. 4. Remove HCl. Jar 2 containing 1:9 acetic acid : alcohol absolute c. Transfer the root to Carnoy solution type II (Table 5) for hardening tissue. 6. This solution has function to dissolve middle lamella.HCl = 1 : 1 for 5-30 minute. 4. and pressed gently with the blade. Avoid boil the material. Avoid boil the material. drops acetocarmine. Transfer the root to Carnoy solution type II (Table 4) for hardening tissue. 5. Moving the slide in following jar for 1 minute each a.method III 1. 3. Put the slide in a dish that contains 10% acetic acid aq until cover slip loose. Fix roots in Famer’s solution for 12 hours or some week to enhance the staining ability of chromosome 2. Jar 3 containing 1:1 alcohol absolute and xylene 9.method IV 1. Put cover slip and passed the slide over fire (Figure 8) shortly (1 second) for 3-4 times. 5 g 8-hydroxyquinoline in one liter of ddH2O .5 h produce the best result. remove un-stain root. Ice-cold water Pretreatment Ice-cold water is applied to plant that has a lot chromosome. 8-hydroxyquinoline is prepared by dissolved 0. 3. Colchicine Low concentration of colchicine (0. 3.5 %) is recommended for pretreatment (1-2 h.4.1-0. Pretreatment for barley’s root or wheat’s root are recommended 16 to 18 h and 24 h respectively. Keep in refrigerator for 12 to 24 hour. Place the soft root to 45% acetic acid on slide.  Contracts the chromosome length with distinct constriction  Increase the viscosity of the cytoplasm Numerous pretreatment has been applied and developed described below : 1.002 M 8hydroxquinoline solution for 3-5 h at 16-18oC. Soybean’s root that incubated for 1. Application high concentration of colchicines cause polyploidy. Put 1 drop 45% acetic acid on the center of clean slide glass 6. 7. Longer pretreatment will shorten chromosome. Cover the vial with a thick layer of ice. The material (for example root) put in vial that already filled cold water.3 Squash Pretreatment is beneficial in squash method. Add cover slip and press gently on cover slip using rubber part of pencil or other friendly tool to spread out the cells. 30 . 8-Hydroxyquinoline Pretreatment with 8-hydroxyquinoline is effective for plant with small. The pretreatment is suitable for cereal chromosome. 2. Warmer temperature cause sticky of chromosome. The root keep in 0. The pretreatment has some purpose :  Stops formation spindle  Increase number of metaphase stage by arresting the chromosome at metaphase plate.3-0. Incubate for 10 minute until the tip produce distinct dark coloring 5.size chromosome. at RT). Fix root tip or shoot tip or other material in Farmer’s solution (Table 5) or mixture of ethanol : glacial acetic acid: chloroform = 3:1:1 2. 5. because a heat accelerate maceration effect of HCl. Pretreatment the root tip in 0. Leave the specimen in this solution for 2030 minutes. let it un-cover in refrigerator overnight.5% is conducted at 60oC for overnight. Some example squash proceed is described below : Oxyquinoline –aceto-orcein squash method 1. 3. Chopped the dark part and cover with cover slip immediately and press gently to spread cell.03 % 8-hydroxyquinoline aqueous in cool room for 4 h. one hour for each wash. Paradichlorobenzene (PDB) Similar with 8-hydroxyquinoline. Prepared HCl-Orcein mixture: Mix aceto-orcein stain (Table 6) : HCl = 9:1 2. Wash in 70% ethanol three times . Do not heated. Remove the root to watch glass and give a few drops of HCl-Orcein solution. The pretreatment is suitable for wheat and barley chromosome.5 h at 15-20 oC or RT. The treatment require 1. 6.5 % PDB (in dH2O). Rinse material with water or 70% ethanol. Note : keep care heating. Passed gently on fire 3-4 times with interval 1 minute. move material in 45% acetic acid before squash. cut and omit undark part of root. 4.4. Pretreatment root is performed when the solution cool with length treatment 2-2. PDB is the best when applied for plant with small-size of chromosome. 31 . 5. Incubate in capped vial containing Snow’s solution (Table 5) at 50-60 oC for 24-48 h. Incubate root tip in aceto-orcein solution for 20-39 minute or longer for small chromosome (12-16 h). Dissolving of 1. α-Bromonaphthalene The effect of α-bromonaphthalene is similar with colchicines. Before cover slip sealed . Transferred the root to clean slide glass. 4. Alcoholic Hydrochloric Acid-Carmine squash method 1. 3. Pretreatment using α-bromonaphthalene is in saturated for 2h at room temperature. put a few drop mixture of Hoyer’s mounting medium : 40 % acetic acid = 1: 1. Put cover slip and squash the material. 6. What is function of Carnoy solution? 5. Why squash and smear technique are suitable for cytological studies? 2. placed the material in the center of clean slide glass. What condition /status plant tissue is suitable for cytological studies? 4. What is the advantage pretreatment? 3. SELF-QUIZ 1. the material have to dip in solution that contain mixture of concentrated HCl and ethanol? 32 . Why some time after fix in fixative solution.5. cutting/sectioning. 4. Conduct trouble shooting when facing with unexpected result Permanent slide is the top slide in plant microtechnique. collecting material is critical work that should put in attention. Because if people careless or do it the wrong way. Understand steps in preparation paraffin. dehydration.CHAPTER 4 PARAFFIN EMBEDDING METHOD IN PLANT Objective After read this chapter we expect you will be able to: 1. There are some material used to support tissue and the famous one is paraffin embedding. affixing. In fresh sectioning. Create a permanently microscopic slide of plant´s part by paraffin embedding method. embedding/ blocking. infiltration /impregnation. When we collect plant materials from field. deparaffinizing and staining. result will deviate from expected result. The subdividing of soft fresh material is 33 . healthy and representative plants.1 Collecting Plant materials which will make a permanent microscopic slide should be choose in “good” sense such as no wound. The paraffin embedding has several steps which include fixation. The soft or fragile tissue needs supporting material to restrain microtome’s knife compression for obtaining thin sectioning (5-15 m). Also it need tissue which is rigid physically in nature to allow razor blade cut the tissue as thin as possible. the slide only produce satisfy slide in short time (hours until some days).embedding method 3. The permanent slide can be kept longer for years and offered good quality slide specimen. clearing. Before run the first step. and mounting. It is important to recognize the size of plant material that will be cut for sample. place it between sheets of wet toweling paper and keep in closed container such as a tin can or a zip plastic. If the tissue soft and wet such Aloe vera. fresh sectioning in thin size is hard and often failure to obtain good anatomy observation even already cut in more 100 m thick. 2. Whole young plant. Table 7. Several kind of fixative solution are simple fixative solution such as ethanol 70 % and compound fixative solution such as FAA (Table 7). B. and other more or less cylindrical organs are usually cut into short sections or disks (Figure 11). Nawaschin or Craf (Table 8). A B Figure 11. Reagent of FAA No Stock solution 1 Ethyl alcohol (50 or 70 %) 2 Glacial acetic acid 3 Formaldehyde (37-40 %) Quantity (mL) 90 5 5 Table 8. etc. Broad leaves should be cut into small pieces. with the material placed on a sheet of wet blotting paper or held carefully against finger. Section of plant 4. Part of plant that will be used as sample. fern sori. When we fix plant materials we also evacuate the air of plant tissues with desiccator or aspirator that connected to vacuum pump (Figure 12). Herbaceous stems. may be cut into complete transverse pieces measuring 2 to 4 mm along the rib. The aim of fixing is to preserve the plant tissues as well as plant tissues alive. selected to include midrib. Narrow leaves that are not much over 5 mm wide. The aim of aspiration is to remove the air from air cavity of plant tissues. lateral veins.best done with a razor blade.2 Fixing Fixing is a process that fix the plant tissues in a fixative or killing solution. A. fungus pustules. Reagents of Nawaschin/Craf No Stock solution Nawaschin or Craf type (mL) Nawacschin I II III IV V (soft) (hard) 34 . roots. or other desired structures. petioles. Leaves are almost invariably cut into small pieces for processing. Reagents of ethanol series No Stock solution 1 50 % ethanol (if use 50 % ethanol in FAA) 2 60 % ethanol 3 70 % ethanol (if use 70 % 35 Volume (mL) 50 50 50 . Fixing and evacuating the air from air cavity of plant tissues. A. The aim of dehydrating is to extract the water from plant tissues. Vacuum pump that connected to aspirator jar. Section of plant that float to above fixative solution 4.1 2 3 4 5 6 1 % chromic acid 1 % acetic acid 10 % acetic acid Glacial acetic acid Formaldehyde (37 – 40 %) aqueous Water 75 20 - 75 - - 5 - - 20 5 5 - - 65 40 20 A 20 30 40 - - - 10 20 30 - - 10 10 50 35 15 - B Figure 12. Table 9.3 Dehydrating Dehydrating is a process to extract the water from plant tissues and to facilitate the fixative solution fills the plant tissues (Figure 13). B. Johansen solution (Table 10) and combination solution ethanol and TBA (Tertiary Butyl Alcohol) (Table 11). Several kind of dehydrating solution include ethanol series (Table 19). Reagents of combination ethanol and TBA No Stock solution Ethanol TBA (mL) Distilled equal with (mL) water ethanol (mL) 1 50 % 40 10 50 2 70 % 50 20 30 3 90 % 50 40 10 4 100 % 50 50 5 100 % 100 (three times replacement) Figure 13.4 Infiltration and Embedding Infiltration is a process of filling plant tissues with paraffin or other embedding agents gradually. the infiltration begin with TBA-paraffin (Table 15).ethanol in FAA) 80 % ethanol 90 % ethanol 100 % ethanol 100 % ethanol 4 5 6 7 50 50 50 50 Table 10. For example if we use ethanol series for dehydrating. The early solution of infiltration depend on the kind of dehydrating solution. Series of dehydration solution 4. the infiltration begin with combination of ethanol-xylene and then xyleneparaffin (Table 14). but if we use Johansen solution and combination between ethanol and TBA for dehydration. Paraffin that 36 . Reagents of Johansen No Stock solution 1 2 3 4 Water 95 % ethanol TBA 100 % ethanol Johansen type (mL) I II III IV V 50 30 15 40 50 50 45 10 20 35 55 75 25 Table 11. Pouring liquid paraffin in plastic box. or we can use plastic box (Figure 14) Table 14. Sample is embedding in paraffin block. All of infiltration and embedding process conducted in incubator. The container for sample embedding or sample block is called paraffin boat. In one block may contain one sample or more than one sample (Figure 15). B. D. Various type of plastic box . Embedding sample in plastic box. A. it is formed from slightly thick paper like a cube. Reagents of infiltration type 1 and embedding No Stock solution Ratio 1 100 % ethanol : xylene 3:1 2 100 % ethanol : xylene 1:1 3 100 % ethanol : xylene 1:3 4 Xylene : paraffin 3:1 5 Xylene : paraffin 1:1 6 Xylem : paraffin 1:3 7 Paraffin Two times replacement Table 15. C.use for embedding has melting point 58 ºC. Embedding sample in stainless steel box 37 . Reagents of infiltration type 2 and embedding No Stock solution Ratio 1 TBA : paraffin 3:1 2 TBA : paraffin 1:1 3 TBA : paraffin 1:3 4 Paraffin Two times replacement A B C D Figure 14. The most useful sizes range from 1 x 1 x 2 cm to 2 x 2 x 3 cm. A. Drop one drop of egg albumen on slide glass and place the paraffin ribbon on it. After that the sample block is mounted on wood block as a holder (Figure 16 and 17). Big paraffin block consist of more than one sample 4. The next step is sectioning the sample block. Adhesive for mounting usually egg albumen. Next stage is affixing. The piece of material to be sectioned is fastened to a mounting block. The thickness of sample section approximately 5 µm (Figure 18).5 Trimming. A 38 B . Inexpensive mounting blocks can be made of hard and porous wood. The angle of the razor-blade holder and the trimmed sample block holder in microtome is 45 º. Sectioning. Sectioning is cutting the sample block by rotary microtome. Then the slide glass is placed on hot-plate (40 ºC) until dry (Figure 19). it is mounting the paraffin ribbon that formed from sectioning on slide glass with an adhesive prior to staining. New slides should be cleaned. Sample of part of plant that embedded in paraffin block .A B Figure 15. and Affixing Trimming is a process to cut the part of sample block that no filled plant sample. Single paraffin block consist of one sample. The knife should be sharp and clean. which is clamped into the rotary microtome. although they may seem to be clean. Soak the wood blocks in hot canning wax. B. B. The result of paraffin block that has been trimmed. Paraffin block slices layer by layer. B. Mounting of paraffin block. The result of paraffin block that has been mounted on wood holder 39 . Paraffin block that is facilitated by hot cutter razor mounted on wood holder. A. Wood holder A B C Figure 17. Single paraffin block consist of one sample that begin to trimming although still in the plastic box. C. C.C D Figure 16. D. Cutter razor is heated above Bunsen. Trimming of paraffin block . A. B. Sectioning of paraffin block. A. Stain consist of basic base and acid base. left hand hold a rod to facilitate the paraffin ribbon. B.A B C D Figure 18. Affixing of paraffin ribbon on slide glass. Series of slide glass that contain sample above the hot plate . The paraffin ribbon that generated from sectioning process. Paraffin ribbon that contain sample on slide glass begin melting 4.6 Staining Staining is to stain the slide glass that contain sample of plant tissue. A. right hand to rotating the wheel of microtome . Good paraffin ribbon approximately 5 µm thick and no rupture. D. natural 40 . The aim of staining is to make a contrast among plant tissues. The angle of razor blade and the knife holder of microtome about 45 º. Sectioning process. A B Figure 19. C. the kind of stain. etc. What is fixation? 3.7 Mounting and Labeling Mounting is mount a slide glass with an adhesive and cover by cover slip. Labeling is give an identity to the slide glass. and the next dehydration to stain that dissolve in ethanol. 4. Staining step begin with xylene for deparaffin paraffin ribbon and then rehydration to stain that dissolve in water. What is dehydration? 4. involve name of plant specimen. Explain the steps of paraffin embedding method in plant! 2. In plant tissue usually use safranin-fast green stain. What is infiltration? 5. Safranin will stain unlignified cell wall. SELF-QUIZ 1. and in contrast fast green will stain lignified cell wall. the color is red.base and synthetic base. the kind of section. Explain the steps of staining! 41 . and finish in xylene again to clearing the slide glass. Examples of mounting agent are Canada balsam and entellan. the color is green. Embedding / blocking .protect tissues against shrinkage and distortion during dehydration.Infiltration / impregnation . infiltration and embedding 42 .poagulate cell contents into insoluble substances . Fixation also necessary to separate the solid phase of protoplasm from aqueous phase. to convert the cell parts into materials that will remain insoluble during susequent treatment and protect the cells from distortion and shrinkage when they are subjected to fluids. process animal tissue with paraffin method based on available protocols. Paraffin plays an crucial role.Clearing . it needs a series processing. tissues will be pressed or cracked and can not be examined properly. understand the steps of paraffin method in animal tissue processing 2.Affixing .1 Fixation Fixation is a process to prevent post-mortem changes of the tissues. Unfixed tissue elements have limited binding sites for dyes.Dehydration .penetrate the tissue rapidly . Without paraffin. fill intercellular spaces and embed the tissue.Cutting / sectioning . Fixation increase permeability and are more receptive to staining.Deparaffinizing and staining .CHAPTER 5 PARAFFIN EMBEDDING METHOD IN ANIMAL Objective After read this chapter we expect you will be able to: 1.Mounting 5. clearing.Fixation . One of methods for tissue processing is paraffin method. Paraffin method include some processses: . Fluids used for fixation are called fixing solutions or fixatives. Fixatives should: . Before animal tissues can be visualized using light microscope. aldehydes: formaldehyde. potassium permanganat.oxidizing agents: osmium tetroxide. picric acid 5.2 Fixatives Components: Aldehyde 10 % formalin ( 4 % formaldehyde) 40 % formaldehyde distilled/tap water Paraformaldehyde 2.protein-denaturing agents or coagulant: methyl alcohol.26 % NaH PO 2 100 ml 900 ml 41. potassium dichromat . 5.1 Fixatives may be classified: . ethyl alcohol.52 % sodium hydroxide 8.4 ml paraformaldehyde 2g Alcoholic fixative Carnoy’s fixatives absolute ethanol 60 ml glacial acetic acid 10 ml chloroform 30 ml Fixation : 1 – 5 hours Picric acid fixative Gendre’s fluid 100 % ethanol saturated with picric acid 80 ml 40 % formaldehyde 15 ml glacial acetic acid 5 ml Fixation: 4 hours Bouin’s fluid saturated aqueous picric acid solution 40 % formaldehyde glacial acetic acid Fixation: 24 hours Mercuric chloride-containing fixative Zenker’s fluid 43 75 ml 20 ml 5 ml .- allow cell parts to become selectively and clearly visible by means of dyes. glutaraldehyde .1. acetic acid miscellaneous:mercuric chloride.5 ml 4 2.1. distilled water potassium dichromate mercuric chloride glacial acetic acid Fixation : 4 – 24 hours 950 ml 25 g 50 g 50 g 5.2 Dehydration Dehydration is a process to remove water and fixatives from tissue and replace it with dehydrating fluid. Dehydration achieved in a series of gradually increasing percentage of alcohol in water. Gradual changing through 30%, 50%, 70%,80%, 90%, 95% and absolute alcohol reduce some tissue shrinkage. Dehydration can be done by following procedure: ................. 30 minutes - ethanol 30 % ................. 30 minutes - ethanol 50 % 30 minutes - ethanol 70 % …………… 30 minutes - ethanol 80 % …………... 30 minutes - ethanol 90 % …………… ethanol 95 % …………… 30 minutes 1 hour - ethanol(absolute) 1 ………….. ethanol(absolute) 2………….. 1 hour 5.3 Clearing Alcohol used for dehydration will not mix with paraffin, some fluid that miscible with both alcohol and paraffin must be used to remove alcohol. Paraffin can infiltrate the tissue if alcohol completely removed. Xylene and creosote usually used for clearing. One example of clearing procedure: - xyleneand absolute ethanol(1: 1) …… 30 minutes xylene 1 ………………………………….. 1 hour xylene 2 ………………………………….. 1 hour 5.4 Infiltration Infiltration is a process by which the tissue is filled with paraffin. Tissue directly tranferred from clearing agent to melted paraffin. If thin sections (5-7 microns) are desired, infiltration used paraffin with melting point 56-58 oC. For extremely thin sections ( less than 5 microns), hard paraffin with melting point 60-68 oC can be used to obtained the good result.Tissues are kept in melted paraffin 0.5- 1 hour in the oven. Two changes of paraffin are sufficient. Horny skin, bone and brain need third 44 changes and infiltration time may be extended to six hours or even overnight. 5.5 Embedding/Blocking Infiltrated tissues are immediately transferred to container or block and fill with melted paraffin to block the tissues. The tissues are oriented based on the tissue section desired. Placing and orienting the tissue is conducted in warm temperature to prevent paraffin harden rapidly. Hard paraffin block can be cooled by placing it in water. Cooled paraffin block is ready for trimming and cutting by using rotary microtome. Figure 20. Cooling paraffin block 5.6 Cutting/Sectioning After fixing, dehydrating, infiltrating the specimen and embedding it, a paraffin block containing the specimen surrounded with paraffin wax should be obtained. The surplus paraffin needs to be removed and the paraffin block trimmed in its proper form prior to sectioning.The paraffin layer surrounding the specimen is not too thick. The larger the surface of the paraffin block to be sectioned, the higher the risk of problems in section cutting and it serves no purpose to cut blank paraffin. Cutting cause microtome knives/blades dull at the end.Knife /blades is usedonly for sectioning the embedded specimen so that there should be minimize paraffin surrounding the specimen. If mounting several serial sections under a common cover slip, the surrounding paraffin should be kept to 1 or 2 mm. The thinner the surrounding paraffin coat, the more sections can be mounted under a single cover slip. For sections to be 45 mounted individually, specimen aresurrounded with a layer of 35mm paraffin. If only a single specimen is embedded in a single block, trimming amounts of paraffin wax off the block every slide with a scalpel or a razor blade gentlyuntil the specimen is surrounded with a thin layer of wax. Trying to cut too thick a layer of paraffin can cause the block and the specimen to crack. The specimen should be in the exact middle of the paraffin block.The upper and lower sides of the trimmed block should be parallel. Improper trimming of upper and lower block sides will result in curved ribbons of sections. Figure 21. Trimming the paraffin block using a scalpel 5.7 Mounting the trimmed block on a paraffin table Paraffin block has to be attached to paraffin table made of pieces of hardwood, aluminum or hard plastic that fit with specimen clamp (block holder). Figure 22. Paraffin tables made of hardwood about 3cm x 2,5 cm x1 cm. 46 and place it to paraffin table. The paraffin block should cool down completely. The trimmed blocks is put on warm knife. Paraffin block can be stabilized by melting some small paraffin shavings around the block.Final trim can be done after which the paraffin block is ready to be mounted on the microtome. Figure 23. Warming the knife in the flame of a Bunsen burner Figure 24.Mounting the trimmed paraffin block on the table can be done by warming the knife in the flame of a Bunsen burner. The trimmed block is put on warm knife 47 . Deparaffinizing can be done be by place section in xylene . Sectioning: creating a “ribbon” of sections 5. Slides kept in warm condition (40-45 oC)until water completely removed. Other folds and air bubbles can be removed when section attached to slide glass. 48 . 10 minutes each.8 Floating out sections. Dried slides are ready for further processing (deparaffinizing). it lay on the water surface to remove folds. The paraffin block is ready to be mounted on the microtome Figure 26. Affixing and Deparaffinizing Before specimen ribbon is mounted to slide glass. two times changes. Single or multiple sections can be separated from the ribbon drawn up onto the albuminized (with Mayer’s albumen) slides (affixing process).Figure 25. Eosin is formed by a reaction between bromine and fluorescein.9 Staining (Hematoxylin and Eosin) Hematoxylin is a basic dye. cell walls. . It is oxidized and combined with a mordant to allow it to bind to the cell structures. ribosome and endoplasmic reticulum). Picking up the ribbon onto the slide glass 5. Eosin is an acidic dye . stains basic structures (cytoplasm. There are two eosin: eosin Y which is slightly yellowish and eosin B which is slightly bluish. and extracellular fibres). Harris's hematoxylin and Mayer's hematoxylin are often used in histology staining. Hematoxylin is extracted from the logwood tree (Haematoxylincampechianum). stains acidic structure of the cell (nucleus. Floating out sections Figure 28. 49 .Figure 27. Stain in Mayer’shematoxylin solution for 8 minutes. Rinse in 95% alcohol.1 Two staining procedure that common use in animal technique StainingProcedure 1 (Mayer’shematoxylin): 1.Figure 29. 8. 2. 10 dips. Re-hydrate in 2 changes of absolute alcohol.9. Wash in warm running tap water for 10 minutes. 9. 5. 4. Hematoxylin chemical structure Figure 30. 3. 2 changes of xylene. 95% alcohol for 2 minutes and 70% alcohol for 2 minutes. 6. 10 minutes each. 50 . Rinse in distilled water. 5 minutes each. Counterstain in eosin-phloxine B solution (or eosin Y solution) for 30 seconds to 1 minute. Deparaffinize sections. Wash briefly in distilled water. Eosin Y chemical structure 5. 7. Mount with xylene based mounting medium. 5 minutes each. Bluing in 0. Rinse in 95% alcohol. 12. 10. 2 changes of absolute alcohol. 13. 10 minutes each. 11. 11. Results: Nuclei ---------------------------------------. 5 minutes each.2% ammonia water or saturated lithium carbonate solution for 30 seconds to 1 minute. Wash briefly in distilled water. 4. 5 minutes each. 2. Clear in 2 changes of xylene. 5 minutes each. StainingProcedure 2 (Harris’hematoxylin): 1. Dehydrate through 95% alcohol. 5 minutes each. Wash in running tap water for 5 minutes. 10 dips. 9.blue Cytoplasm ---------------------------------. 12.10. 2 changes of absolute alcohol. 5. Wash in running tap water for 5 minutes. Deparaffinize sections. Mount with xylene based mounting medium. Dehydrate through 95% alcohol. Clear in 2 changes of xylene. 14. 2 changes of xylene. 3. Differentiate in 1% acid alcohol for 30 seconds. Stain in Harris’hematoxylin solution for 8 minutes. 8. 95% alcohol for 2 minutes and 70% alcohol for 2 minutes. 15. Histology of kidney (H & E Staining) 51 .pink to red Figure 31. 6. Re-hydrate in 2 changes of absolute alcohol. Wash running tap water for 1 minute. 7. Counterstain in eosin-phloxine B solution (or eosin Y solution) for 30 seconds to 1 minute. Add the chloral hydrate and citric acid and boil the mixture for 5 minutes.9.800 ml Mix to dissolve and store at room temperature.1 litre • Mercuric oxide .2 Varies Stain Compositions Mayer's haematoxylin: • Haematoxylin . Coolandfilter.1g • Chloral hydrate .1 litre • Potassium or ammonium alum .1 g Distilled water --------------------------. • Glacial acetic acid .750 ml Glacial acetic acid (concentrated) ----. • Distilled water . Dissolve the haematoxylin in the alcohol and add.25%): Eosin Y stock solution -----------------.1g • Distilled water .50g • Citric acid .200 ml 95% Ethanol ---------------------------------. Eosin Y Solution: Eosin Y Stock Solution (1%): Eosin Y --------------------------------------.2. • 100% alcohol – 50 mls • Potassium alum . or by standing overnight at room temperature.40mls Dissolve the potassium alum in the water by warming and stirring.5 ml Mix well and store at room temperature. Cool. Eosin Stock Solution: Eosin Y -----------------------------------.100g. The Staining ready for use immediately.2g Add the haematoxylin. Eosin Y Working Solution (0.50g • Sodium iodate . Harris' haematoxylin: • Haematoxylin .100 ml 52 .5g. add the acetic acid and filter. Bring rapidly to the boil remove from the heat and add the mercuric oxide. potassium alum and sodium iodate to the distilled water and dissolve by warming and stirring.0.250 ml 80% Ethanol -----------------------------.5g. The solution is then ready for use.10 g Distilled water ------------------------------.5. The pH will be around 10.100 ml Phloxine stock solution -------------. Eosin-Phloxine B Working Solution: Eosin stock solution -----------------. a mountant should possess certain characteristics: 53 .--1000 ml Mix well.1000 ml Mix well and store at room temperature Differentiate 30 second to 2 minutes after hematoxylin staining Bluing Reagent Lithium Carbonate Solution (Saturated): Lithium carbonate ---------------------------.2% Ammonia Water Solution (Bluing): Ammonium hydroxide (concentrated) -----. Bluing for 30 seconds to 1 minute after hematoxylin staining and clearing/differentiation. Bluing for 30 seconds to 1 minute after hematoxylins taining and clearing/differentiation 0.2 ml Distilled water -------------------------------.54 g Distilled water -------------------------------. Mounting media is called mountant.4 ml Mix well.10 ml 70% ethanol ---------------------.100 ml Mix to dissolve.10 Mounting Mounting is the final stage in the preparation of tissues for microscopy.Mix to dissolve.100 ml Mix to dissolve and store at room temperature. 5.10 ml Ethanol (95%) ------------------------. Phloxine Stock Solution: Phloxine B ------------------------------1g Distilled water -------------------------.1. 5.9.3 Clearing (Differentiation) Reagent for H&E Staining 1% Acid Alcohol Solution: Hydrochloric acid ---------------.0 Store this solution at room temperature.780 ml Glacial acetic acid -------------------. Water. Canada balsam.miscible with dehydrant or clearing agent Mounting media are divided into categories: hydrophobic and hydrophilic. Which one of the following subtances is not a fixative? a. plasticiser. Too thick mountant can cause the image of tissue is not focusly examined.formaldehyde e.potassium permanganat c. xylene).able to completely permeate tissue .set without crystallising. hidrochloric acid 54 .. Coverslipping SELF-QUIZ 1. section is covered with mountants coverslip. Sections are mounted in hydrophilic media directly from water. and permount are hydropobic mountants.not react with or induce fading in stains and reaction products .resistant to contamination . glycerol and phosphate buffer glycerol are hydrophilic mountants. osmium tetroxide b. cracking or shrinking . Coverslip was pressed gently to ensure that mountants are not too much and all tissue parts are covered with mountant. DPX (distrene. Figure 32. After staining process. Hydrophobic mountants are used for sections that need to be dehydrated and cleared before they are applied. methanol d.colourless and transparent . miscible to water and ethanol e. Cutting too thick layer of paraffin block can cause: a. miscible with clearing agent e.stained tissue in tap water is to: a. except: a. miscible to water and paraffin 7. coloring tissue b. miscible to water b. canada balsam d. clearing tissue e. tissue can not be stained b. tranparent c. ethanol b. allowing cell parts to shrink 3. tissue poorly absorb stain 55 . water and ethanol e. blue the tissue b. xylene c. decolorize the tissue 8. fix the tissue d. tissue cracked c. induce stain fading d. Which one of the following substances is a clearing agent? a. ethanol and paraffin 6. Fixatives functions in animal tissue processing is : a. lithium carbonate 4. water b.2. Imersing haematoxylin. dehydrating tisssue c. protecting tissue against distortion d. paraffin d. aldehyde e. miscible to ethanol c. dehydrate the tissue e. permeable to tissue b. Clearing agent used in clearing process must: a. miscible to ethanol and paraffin d.Mounting media should posses the following characteristics. ethanol c. Which one of the following fluid should be absent in the end of infiltration process? a. set without shrinking 5. redden the tissue c. Which one of the following tissues needs a longest time during infiltration? a. can bind directly to the tissue b. tissue easily be deparafinized e. needs oxidation to bind the tissue d. bone e. stain acidic stucture of the cell 10. Haematoxylin: a.d. stain cytoplasm e. tissue hardly be examined 9. is acidic dye c. spleen 56 . kidney c. gaster d. liver b. 1 What is whole mount? Whole mount is placing a whole organism or specimen on a slide for microscopic examination. we will elaborate in brief about fixation. 57 . If an antibody has been used successfully on cryosections (IHC-Fr . than a normal section on a slide. image obtaining. without sectioning onto slides first. Understand general whole mount preparation 2. Whole mount staining is very similar to staining of cryosections or immunocytochemistry (ICC). usually embryos. and choosing embryo age. antibody. As introduction before describe in details some specific methods and example preparation whole mount. Researchers use different times. but the details in these procedures provide a guideline for optimizing the experiment at these stages if necessary. incubations for fixative. Therefore.this does not including paraffin embedded sections). Understand antibody staining of whole mount Drosophila embryos 4.CHAPTER 6 ANIMAL WHOLE MOUNT Objective After read this chapter we expect you will be able to: 1. Conduct trouble shooting when facing with un-expected result 6. then the antibody should work for a whole mount embryo. This is often used on embryos by stem cell and embryonic development researchers and also neuroscientists who are able to stain the whole embryos at various stages to follow the expression of target proteins through the development of the animal. The difference being that the sample being stained is much larger. permeabilization and substrate color development will need to be much longer to allow for permeabilization right into the centre of the sample. Understand whole mount immunohistochemistry of zebrafish 5. blocking buffer. Whole mount staining is the staining of small pieces of tissue. and thicker. Understand whole mount fluorescent immunohistochemistry 3. Know the real internal structure of embryo and to look for abnormalities of embryo without sectioning first 6. wash buffer. zebrafish embryo fixation and preparation requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilized.6. and the number of stained cells will make obtaining a clear image very difficult. we could perform antigen retrieval. In case zebrafish. If small enough. then there is a possibility the antibody is sensitive to the protein crosslinking. However. they can also be set in gelatin and sectioned if it is difficult to obtain a clear view of the staining through the whole embryo (particularly at larger late embryo stages or larger tissue samples). including fixative. 6. as the protein cross linking formed by the fixative may block access of the antibody to the epitope. In this case. antibody and developing solution will not be able to permeate to the centre of the sample. 6. most researchers use 4% paraformaldehyde (PFA). If PFA fixation does not work for the whole mount tissue. This is not possible on embryo samples as the heating procedure would destroy the sample.4 Choosing the age of the embryo This is important as the embryo grows. this will not be suitable for all antibodies. it will become too large to stain. rather than sectioning the whole embryo onto separate slides after staining.2 An important note on fixation Whichever fixative has been successfully used in IHC-Fr with the antibody you have chosen should be suitable for whole mount. 58 . The whole embryo can be imaged while floating in glycerol buffer in a petridish. Normally.3 Obtaining images Some researchers view and obtain images of embryos as they are. However. The various reagents. If immunofluoresent labeling is used. then confocal microscopy can be a useful tool to scan through the embryo. Although this concentration of PFA is very low. this has to be left on for a long period of time on whole mount samples to allow for permeabilization to the centre of the sample. the whole embryo can be mounted in glycerol before setting in a coverslip. before mounting. grease should be used around the corner of the coverslip to help keep it in place and prevent damage to the coverslip when using the microscope. in IHC-P. Therefore. However. and you will require another fixative. larger and older embryos can be dissected into segments before staining if necessary. Methanol is a popular second choice of fixative when optimizing whole mount procedures. Place embryo in a 5 ml bijous in 4% paraformaldehyde. Incubate the embryos twice for 1 hr in block (PBS 1% Triton + 10% FCS + 0. Procedure: 1. room temperature.Recommended ages:  Chicken embryos: up to 6 days  Mouse embryos: up to 12 days 6. Generally whichever fixative has been used successfully with the antibody when used in cryosections. 59 . We suggest trying between 2 hours and overnight. 20% DMSO) or other fixative of choice. this may require some optimization.2% Sodium Azide). Wash 3X in PBS 0. Obtaining the embryo: Chicken: Gently break the egg into a medium sized clean glass petridish. OR fix in m-DMSO (80% methanol. this fixative should be suitable for whole mount. 3.1% Triton thirty minutes each time. Leave to fix 4oC.e the fixative has permeabilized the whole sample) then it should sink to the bottom of the solution. This gives a clearer idea of where the target protein of interest is expressed within the tissues. Mouse: Operate on adult female to remove embryos. \ We recommend to remove as much embryonic membrane and excess tissue as possible as this can prevent the antibody perfusing into the embryo. Ensure the sample has sunk to the bottom of the fixative before proceeding.5 Whole mount fluorescent immunohistochemistry The advantage of using fluorescence to stain whole mount sections is that confocal microscopy can be used to section through the larger embryo or tissue sample without having to manually section onto slides. However. The embryo will naturally float to the top of the yolk.5 . Dissect the embryo in ice cold PBS removing as much unwanted tissue as possible. It will then be visible for careful removal using clean scissors and a Pasteur pipette with the tip removed (this prevents any damage to the embryo from the narrow end of the pipette). 2. 4. The time required will need optimization. When the sample is fully equilibrated with the fixative (i. 5. 75% glycerol has approximately the same density as gelatin which is used to mount and set the samples on a slide. If the sample is to be embedded 60 . Rinse three times in PBS once reaction and staining have reached desired intensity Mount and view embryo’s. 3. 17. 10. 9. Store at 40C until analysis.02% sodium azide to prevent microbial growth. 6. Place sample in 100% glycerol for 48 hours. 13. the antibody should be diluted in blocking buffer containing 0. 15. 8. Place in 50% glycerol until the sample sinks. the sample should sink).1% H2O2 diluted in blocking buffer) overnight 4oC. Mounting: 1. Wash embryo’s 3X 1 hr in PBS 1% Triton + 10% FCS0.2% sodium azide Wash 3X 10 minutes in PBS 1% Triton Wash well to remove traces of sodium azide as this will inhibit peroxidase activity when developing Add secondary antibody in blocking buffer (no sodium azide) Incubate for 2 to 4 days with gentle rotation 4oC Wash 3X 10 minutes in PBS 1% triton Incubate embryos in DAB substrate for 2 to 3 hrs RT. 12. Transfer embryos to a dish and add fresh DAB plus 5 ul H2O2 per 1 ml of DAB. Incubate embryos in peroxidase block (0. When sample is fully equilibrated with the glycerol (i. Wash embryos 2X in blocking buffer. It is recommended that as incubations can be very long in whole mount staining. 2. This incubation time will require some optimization depending on the antibody and also the size of the embryo. 7. Therefore. Transfer embryos using Pasteur pipette with the end cut off to a 2 ml tube. 11. Use grease around the edges of the cover slip for protection. Ensure the sample is at this stage before proceeding. when equilibrated. samples should be equilibrated in 75% glycerol after staining for approximately 1(5 minutes) (again.e it is fully perfused with the glycerol) it will sink to the bottom of the vial. 16. The embryo can be imaged at this stage. Add primary antibody at the required dilution /concentration. or mounted whole in the glycerol on a slide. 14. Incubate for 1 to 4 days on a gentle rotation devise at 4 oC. light rotations of the apple-juice plate will bring the dechorionated embryos to the surface.02% SDS or to fix the embryos in 500 μl picric acid / 500 μl nheptane. 61 . 2.6 Antibody staining of whole mount Drosophila embryos Procedure : 1. the sample should sink to the bottom. aim the flame at the middle of the tip and hold the end of the tip with two fingers (glass is a bad heat conductor therefore your fingers are safe). especially along the edges of the plate. Vortex the fixation mix at highest speed for 1 min. wash embryos in funnel by squirting water along the funnel three times. fill up the cap with 1 ml of methanol and vortex on highest speed for about 1min. Remove yeast and dechorionate embryos by covering the apple-juice plate with 100% bleach for 2min. place 20% gelatin pre-warmed to 65oC. When equilibrated in the gelatin. 4. dip brush with embryos into the fixation mix and whisk the brush with the cap lying on its side fix embryos for 15 min on a shaker with gentle rotation. This procedure will remove the extra embryonic membrane.5 ml cap: 400 μl PBS. To stop fixation remove about 80% of the lower phase of the fixation mix with the pipette. dechorionated embryos will adhere to the brush. Wash embryos into the funnel by squirting deionized water over the plate. 5. 3. 7.in gelatin and sectioned on a vibratome. During bleaching: close the narrow opening of a funnel with a nylon-mesh (diameter of holes: 40 μm). 100 μl 40% formaldehyde and 500 μl n-heptane. break away the long drawn out and very thin part from the tip. when the middle glows red remove the tip from the flame and immediately pull. 6. pick up the embryos with a very fine paint brush. As an alternative to the glass pipette it is possible to use a 1 ml Gilson pipette. For the detection of some extracellular antigens it helps to add 0. attach mesh by wrapping a rubber band around the funnel. Remove rubber band and mesh. 6. During fixation: heat the tip of a glass Pasteur pipette over a Bunsen burner. Leave for approximately 30 minutes to equilibrate before taking out the sample to mount. Prepare fixation mix in 1. 3% Triton added). If not all embryos sink to the bottom of the cap. 16. Add secondary antibody diluted in 10% NCS / PBT for 2 hr at room temperature. Do not vortex again. 11. 10min incubation with 30% NCS / PBT. 14. 2 min after vortexing the majority of the embryos will be at the bottom of the cap. Wash procedure: three rinses with PBT. Enhancement of very weak signals: use Vectastain ABC Elite Kit. three rinses with PBT. or TSA Kit Fluorescent detection: use secondary antibodies coupled to Alexa488. incubations can last from 2 hr at room temperature (high affinity antibodies) to overnight at 4oC (low affinity antibodies). 10. develop Alkaline phosphatase signal in the dark and take an aliquot of embryos out of the cap with a 1ml Gilson pipette to follow the staining. remove as much of the liquid as possible and refill the cap with methanol. Note that Triton only slowly dissolves in PBS i. lay cap on its side on a gently moving shaker. Non-fluorescent detection: use Diaminobenzidine assay (normal signal) or Alkaline Phosphatase assay (weak signals). Overnight incubation at 4oC aides the perfusion of the antibody. postfix embryos with 4% formaldehyde / PBT for 10 min to stabilize alkaline phosphatase signal. PBT can be kept at room temperature indefinitely. After most of the embryos are at the bottom of the cap. 9. Alexa568 or Cy5 62 . 15.02% sodium azide should be added.02% sodium azide. For (Abcam) ab290 2h at room temperature are sufficient. Repeat step 13. Rehydrate the embryos with three washes in PBT (PBS with 0. store the primary after the first incubation at 4oC for further stainings.8.e. Add primary antibody diluted in 10% NCS / PBT and 0. set up the solution about 20 min in advance. three rinses with PBT. remove all liquid and wash embryos three times with methanol. Calf serum should be stored in the fridge and 0. 13. incubate in 20% newborn calf serum / PBT on shaker for at least 10 min.e. most primary antibodies can be used at least three times i. Depending on the quality of the primary antibody.10 min incubation with 30% NCS / PBT. 12. diluted 1:1000 (Abcam) ab290 can be re-used five times. Permeabilize the embryos in ice cold acetone / PBS for 8 minutes only. Wash at least 4X for 5 min in PBS 1% triton. Incubations for fixative. wash buffer. replace 70% glycerol/ PBS with 90% glycerol / PBS and allow embryos to sink to the bottom of the cap. The acetone will help to permeabilize the tougher egg membrane.17. (this prevents any damage to the embryo from the narrow end of the pipette). blocking buffer. Procedure: 1. which is not necessary for other species such as chick or mouse 4. Incubate the embryos twice for 1 hr in block (PBS 1% Triton + 10% FCS). Use a Pasteur pipette with the tip removed to move the embryo from one vial to another or to add or remove reagents. Use 4% paraformaldehyde (PFA). requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilized. Incubate embryos in 50% glycerol / PBS until they sink to the bottom of the cap (about 10 min). Wash embryos 2X in blocking buffer 8. Transfer embryos using Pasteur pipette with the end cut off to a 2 ml tube. replace the 50% glycerol l /PBS with 70% glycerol / PBS wait again until embryos are at the bottom of the cap (about 1hr). 3. Wash at least 4X for 5 min in PBS 1% triton.7 Zebrafish whole mount immunohistochemistry Whole mount staining of Zebrafish embryos. 6. or perfix (this is very suitable for neuro markers). 5.1% H2O2 diluted in blocking buffer) overnight 4oC. 6. Place embryo in a 5 ml bijous in fixative for 1 hour. depending on what has been previously used successfully with the antibody in cryosections. The fixative should be chosen carefully. Add primary antibody atthe required 63 . room temperature. 2. Incubate embryos in peroxidase block (0. and also on the target protein. permeabilization and substrate color development will need to be much longer than normal immunocytochemistry / immunohistochemistry to allow for permeabilization right into the centre of the sample. 7. There is no need for any light protection for fluorescent staining. Embryos can be stored in 90% glycerol / PBS at 4oC for at least three years. now commonly used. antibody. As antigen retrieval methods are not recommended for whole mount (it can destroy the tissue). need to be much longer that in ICC or IHC to allow penetration through the sample.1 High background Fixative used is not suitable for the antibody. 6. This incubation time will require some optimization depending on the antibody and also the size of the embryo. Incubate for 1 to 4 days on a gentle rotation devise at 4oC.Transfer embryos to a dish and add fresh DAB plus 5 μl H2O2 per 1 ml of DAB. add 70% glycerol / PBS to cover the agarose and place a coverslip on the sample. 6. we would recommend checking the antibody datasheet to obtain information on fixation agents used successfully in whole mount sections with the antibody you are using. Wash embryo’s 3X 1 hr in PBS 1% Triton + 10% FCS Wash 3X 10 minutes in PBS 1% Triton Incubate embryos in DAB substrate for 2 to 3 hrs RT. ensure the concentration of this is no more than 4%. The usual alternative to PFA is methanol fixation. and PFA fixation will not be suitable for some antibodies. 10. This should cause fewer difficulties with protein cross linking. The following tips are more specific to whole mount staining. Once the agarose is set. the antibody should be diluted in blocking buffer containing 0. Some antibodies will still be sensitive to the small amount of protein cross linking at this lower percentage PFA. 13.02% sodium azide to prevent microbial growth. Most researchers use PFA for fixation. 15. . which will be much larger than a tissue section.8 Troubleshooting tips – whole mount Very similar difficulties to immunocytochemistry (ICC) and immunhistochemistry (IHC) can occur when staining whole mount tissue. However. and wash steps. 14. Store at 40C until analysis. 12. Most of these relate to the fact that incubation times for all reagents. fixatives used successfully in cryosections are 64 . Rinse three times in PBS once reaction and staining have reached desired intensity Mount and view embryo’s.8. 11.9. dilution /concentration. Mount in melted 1% agarose in PBS. It is recommended that as incubations can be very long in whole mount staining. If this information is not available. abcam. As antigen retrieval methods are not recommended for whole mount (it can 65 . This can lead to non specific background staining.usually successful in whole mount. antibody. wash buffer. Antibody left on for too long. 0. If this is not successful. Follow the wash step guidelines provided in the protocols.8. As the incubations in whole mount staining are very long.2% sodium azide can be added to antibody buffers and blocking buffers wherever possible. Most researchers use PFA for fixation. Triton rather than Tween is used as this is a stronger detergent which will permeate more easily. The recommendation when optimizing antibody concentration in whole mount is to start with 3 or 4 day incubation. Use clean glassware (preferably sterile) and fresh reagents. If this is not successful. Fixative used is not suitable for the antibody. Wash steps not sufficient. Incorrect incubation times. Please note this should not be added to peroxidase conjugated secondary antibodies as it can inhibit the enzyme activity.2 No signal Antibody not left on for long enough www. work backwards to 1 day (antibody will need to be on the sample for at least 24 hours to ensure full penetration through the sample). Wash steps will need to be long enough to permeate and wash through the whole sample. work backwards to 1 day (antibody will need to be on the sample for at least 24 hours to ensure full penetration through the sample).com/technical Antibody incubations should be much longer than when staining sections on slides to ensure adequate permeation to the centre of the sample. blocking buffer. Fixation time may also require optimization. 6. permeabilization and substrate color development will need to be much longer to allow for permeabilization right into the centre of the sample. The recommendation when optimizing antibody concentration in whole mount is to start with 3 or 4 day incubation. microbial contamination can become a problem. Incubations for fixative. Follow washing guidelines carefully before adding peroxidase conjugated secondary antibody to ensure any sodium azide is washed away. Microbial contamination. It is also used at a relatively high concentration of 0. However. Incubations for fixative. This will lead to patchy areas where staining is not sufficient. Fixation time may also require optimization. Whole mount staining of Zebrafish embryos requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilised. Air bubbles in the tissue / Inadequate mixing of reagent and sample.5 to 1%. or with full access to the antibody. In order to allow full permeabilization of reagents and antibody through the whole sample. antibody. we would recommend checking the antibody datasheet to obtain information on fixation agents used successfully in whole mount sections with the antibody you are using. there may be areas of the tissue not fully washed. and PFA fixation will not be suitable for some antibodies. The usual alternative to PFA is methanol fixation. This should cause fewer difficulties with protein cross linking. fully fixed. Mouse and chick – extracellular membrane not removed.3 Patchy staining Incorrect incubation times. wash buffer. 6. Some antibodies will still be sensitive to the small amount of protein cross linking at this lower percentage PFA.destroy the tissue). If any of these reagents have not penetrated the whole sample. blocking buffer. If this information is not available. Triton rather than Tween is normally used. Remove as much embryonic membrane and excess tissue as possible as this can prevent the antibody perfusing into the embryo. fixatives used successfully in cryosections are usually successful in whole mount. Zebrafish – egg membrane not permeabilized. Fix for 1 hour. Incorrect detergent used in buffers. We recommend following the zebra fish whole mount staining procedure provided.8. This is a stronger detergent which will permeate more easily. permeabilization and substrate color development will need to be much longer to allow for permeabilization right into the centre of the sample. ensure the concentration of this is no more than 4%. wash in PBS 1% triton then permeabilize the egg membrane in in ice cold acetone / PBS for 8 minutes only. Ensure the sample is placed on a gentle rotating or rocking devise whilst incubating to prevent formation of air 66 . Avoid use of forceps where possible. or to add or remove reagents. Sample is too large. 67 . This is a stronger detergent which will permeate more easily. It is also used at a relatively high concentration of 0. To move the tissue from one vial to another. The usual alternative to PFA is methanol fixation. rather than PFA. If any of the reagents have not penetrated the whole. However. It is sometimes possible to dissect the sample into sections which are more manageable. An embryo will grow to a stage where they are too large for whole mount staining with good results. Ensure the sample has been fixed for long enough to allow penetration of fixative through the sample. Sample has been heated or treated for antigen retrieval. 6.8. We suggest optimizing between 2 hours and overnight. The timing of fixation may require some optimization. or trapped air bubbles in which tissue will not be stained. Try using another fixative. there may be areas of the tissue not fully washed. we would recommend checking the antibody datasheet to obtain information on fixation agents used successfully in whole mount sections with the antibody you are using. as it destroys the structure of the tissue. use a plastic Pasteur pipette with the tip removed (this prevents any damage to the embryo from the narrow end of the pipette). Reagents will not be able to fully penetrate the tissue if it is too large. Triton rather than Tween is normally used. Heat treating the whole mount samples for antigen retrieval is not possible. and other types of sample will need to be kept within an optimized size range. fully fixed. or with full access to the antibody. This will lead to patchy areas where staining is not sufficient. Incorrect detergent used in buffers. fixatives used successfully in cryosections are usually successful in whole mount.bubbles and to ensure access of reagent to all the tissue. Fix at 4oC. Inadequate fixation or over fixation.4 Morphology of the tissue is not good. If this information is not available.5 to 1%. and the staining through the sample will be patchy. In order to allow full permeabilization of reagents and antibody through the whole sample. Sample has been crushed whilst handling. SELF-QUIZ Why whole mount staining is very similar immunocytoshemistry? Please explain your answer ! 68 to . CHAPTER 7 BLOOD SMEAR Objective After read this chapter we expect you will be able to: 1. Make and stain a blood smear with Giemsa staining 2. Make and stain a blood smear with Romanowsky staining . A well-made blood smear is a beauty to be hold, and likely to yield interesting and significant information for a research project. A poor slide is a torment. The extra time and care taken during the field season will be rewarded later when the smears must be scanned, and parasites identified and counted. Here, the methods for making and staining smears are given, as well as a list of sources for high quality slides, stain, and chemicals. Dried blood samples for genetic studies should always be made at the same time as the smears. The method is very easy and modern research must combine studies of morphology under the microscope with molecular methods. The technique for making and storing dried blood samples is given in the section “Dried Blood Samples”. 7.1 Making a smear 1. A single smear can be made per slide (smear running the length of the slide) or two (or even three) smears can share a slide, with the smears running the width of the slide. Putting two smears per slide saves on weight (glass is heavy) for field trips, and storage space. 2. It is easiest to use microscope slides with a frosted end, so that identifying information can be written there with pencil. Warning: Compare different pencils to find one that does not yield labels that rub off or wash off in the methanol dip. 3. Place a drop of blood approximately 4 mm in diameter on the slide (near the end if one smear is to be made, or at the proper location if two smears are to share a slide). See the drawing below. 4. Spread the drop by using another slide (called here the “spreader”), placing the spreader at a 45° angle and BACKING into the drop of blood. The spreader catches 69 the drop and it spreads by capillary action along its edge. To make a short smear, hold the spreader at a steeper angle, and to make a longer smear, hold it closer to the drop. Now, push the spreader across the slide; this PULLS the blood across to make the smear. Do not push the blood by having it ahead of the smearing slide! It should take about one second to smear the drop. A smooth action is required, with the edge of the spreader held against the slide. This will yield a nice, even smear. 5. If doing one smear per slide, the spreader then becomes the next slide to receive a smear. Thus, each slide serves two duties, as a spreader, then as a slide to receive a smear. If two smears are made per slide, be sure to flip over the spreader to use the other edge for the second smear produced. The spreader then is used to receive the next two smears. Warning: If there is surplus blood on the spreader, wipe it off carefully before flipping it over to make the second smear on the slide 6. For blood taken from mammals, a THICK blood film can also be made, but this is not possible with blood from birds or reptiles. Only mammals have erythrocytes that lack a nucleus. Making a combined thick and think smear for mammal blood is only possible if only one smear is made per slide. Make the thin smear starting about 1/3 from the non frosted end of the slide. Then, place another drop of blood at the clear end and use the edge of the smearing slide to spread the drop out to about a 1 cm circle. The thick smear will take longer to dry. Because the erythrocytes of mammals lack a nucleus, thousands of cells can be stacked, and parasites still seen (not for identification, but simply to detect an infected animal). 7. Smears should be air-dried, and then dipped into 100% methanol. A coplin jar with a screw top is best for this. We use a plastic version, which won’t break in the field, but has a poorly sealing top. Slides can be stored while drying in a small plastic slide box (holds 25 slides). Then, they are placed, two at a time, back-to-back, into the slots in the coplin jar. Thus, ten slides can be dipped at once. Be sure the alcohol does not reach the frosted end of the slide. After one minute, the slides are removed and placed on end to drain the alcohol. They can then be placed into a plastic slide box for complete drying. 70 8. In the field, we place the plastic slide box or boxes into a zip-lock bag with silica gel, and they are allowed to dry overnight. 9. To store slides during long field trips, and where many slides are to be made, they can be placed back into their original cardboard boxes, with a piece of index card or other clean paper between each slide. Figure 33. Making the blood smear 7.2 Preparing staining buffer Stock buffers (two) The alkaline stock is Sodium phosphate, dibasic anhydrous, H2HPO4. Mix 9.5 gm with distilled water to make 1000 mL. The acid stock is Potassium phosphate monobasic 71 To prepare at least five slide smears which are even. The plastic jar used in the field for dipping into methanol.3 Blood Smear Preparation with Romanowsky Staining Objectives: 1. The slide is allowed to air-dry and is then stained. Use blue plastic slide boxes that hold 25 slides. 7. so cleaning should not be required. A Romanowsky stain is any stain combination consisting of eosin Y or eosin B with methylene blue and/or any of its oxidations products. Coplin jars.anhydrous. The blood is streaked in a thin film over the slide. Giemsa stain will color skin for several days! Slide boxes. This plastic bottle has a pour spout that ALWAYS leaks. Not all Giemsa stains are equal in quality. 72 . Such stains produce the typical purple coloration of leukocyte nuclei and neutrophilic granules as well as the numerous blues and pinks found in other cell types. A second slide is used as a spreader. To stain and determine the acceptability of the Wright stained blood smear by evaluating that all formed elements are readily identifiable according to criteria outlined in the textbook. smooth and have an acceptable feathered edge. and adjust to ph 7 or 7. and 900 mL of distilled water. Other supplies Microscope slides.2 by adding the acid buffer stock to lower pH or alkaline to raise pH. KH2PO4.07 gm with distilled water to make 1000 mL Working buffer: Mix 39 mL of acid stock with 61 mL of the alkaline stock. Principles: Smear Preparation A small drop of blood is placed near the frosted end of a clean glass slide. Giemsa. Methyl alcohol is used as both a solvent and fixative in this procedure. So. mix 9. Good-quality slides seldom will retain any oil from machines used in their manufacture. store the bottle in a plastic bag and always handle the bottle through the bag. Just a very few mL should be necessary to reach the required pH. Wright’s Stain The Wright’s stain is a Romanowsky stain. Check pH. 2. 2. Allow the blood to spread almost to the edges of the slide. supplies.e. the sample accession number should also be on the slide. The wedge smear 1. Capillary tubes. smooth. 6. Blood smears can also be made from finger stick blood directly onto a slide. or date of birth).: patient name. For the purpose of this lab exercise. The spun smear requires an automatic slide spinner. Note: DIFF-SAFE® dispensers or wood applicator sticks can also be used to place the droplet of blood on the slide.Specimen: EDTA anti coagulated blood is preferred. approximately ½ inch from the frosted area of the slide. Fill a capillary tube ¾ full with the anti coagulated specimen. Place a drop of blood. A thin film of blood with a feathered edge will remain on the slide. 3 x 1 inch with frosted edge 2. and draw it back against the drop of blood. and hold the narrow side of the non frosted edge between your left thumb and forefinger. 3. 5. If given. 73 . Hold the spreader slide at a 30° angle. plain or DIFF-Safe® dispensers or wood applicator sticks Slide Preparation Procedure: Three methods may be used to make blood smears: 1) the cover glass smear 2) the wedge smear 3) the spun smear. and fluid motion. Glass slides. Label the frosted edge with the date and two patient identifiers (i. With your right hand. Reagents. we will use the wedge smear. 8. Place the slide on a flat surface. 4. Push the spread forward with one light. just in front of the blood drop. place the smooth clean edge of a second (spreader) slide on the specimen slide. 7. and equipment for Slide Preparation 1. about 2 mm in diameter. patient ID. ) 10. immature cells and abnormal cells 74 . b. e. 2. The WBCs are unevenly distributed and RBC distortion is seen at the edges. d. the thicker and shorter the smear). 7. If the hematocrit is decreased.Notify instructor once five acceptable slides are made. Large cells such as monocytes. Any delay results in an abnormal distribution of the white blood cells. the wedge method does not always produce a quality smear. c. the smear should be made without delay. Failure to keep the spreader slide at a 30° angle with the slide. Once the instructor has checked you off. The thickness of the spread when pulling the smear is determined by the 1) angle of the spreader slide (the greater the angle. Failure to keep the entire edge of the spreader slide against the slide while making the smear. Common causes of a poor blood smear : a. The moisture from your breath will cause RBC artifacts.3. Smaller WBCs such as lymphocytes tend to reside in the middle of the feathered edge. As soon as the drop of blood is placed on the glass slide. 3. If the hematocrit is increased. 2) size of the blood drop and 3) speed of spreading. (Do not blow to dry. the angle of the spreader slide should be decreased. 4.1 Slide Preparation Procedural notes 1.9. Although this is the easiest and most popular methods for producing a blood smear. Failure to push the spreader slide completely across the slide. 8. Allow the blood film to air-dry completely before staining. A good blood film preparation will be thick at the drop end and thin at the opposite end. The blood smear should occupy the central portion of the slide and should not touch the edges. 5. the student can proceed with staining two of the five acceptable slides. Drop of blood too large or too small. 6. the angle of the spreader slide should be increased. 7. Spreader slide pushed across the slide in a jerky manner. with many of the large white cells accumulating at the thin feathered edge of the smear. then remake the smear. There is nothing you can do to correct this. Staining Reagents and Supplies Slides can be stained manually or on automatic slide stainers. 2.3. For this lab. Raise the slide out of the stain and allow the majority of the stain to run off the slide. b.3 Staining Procedure 1. Clothes pin or forceps for holding the slide f. 7. we will use the manual method. 5. Commercial Wright’s stain b. Commercial buffer c. Attach a clothes pin (or use forceps) to the thick edge of the blood smear. Spun smears produce the most uniform distribution of blood cells. Place the slide upright on a slide drying rack with the feathered edge up and allow to air dry. Coplin jars e. 6. Cold agglutinin . Wipe off excess fluid from the back of the slide. Document the abnormality.3.2 Biologic causes of a poor smear a. When completely dry.RBC’s will form into stacks resembling coins. examine the smear with the microscope as follows: 75 . Place the slide in the first jar containing deionized water. Remove the slide carefully and dip several times in the second jar containing deionized water to rinse off the excess stain. Allow to stand 5-10 seconds. Drying racks for slides 7. Lipemia . Rouleaux . c.RBCs will clump together. Place the slide in the Coplin jar with Wright’s stain. There is nothing that can be done to correct this on whole blood. 7. a. 4. Allow to stand 10-20 seconds. Warm the blood at 37°C for 5 minutes. Deionized water d.holes will appear in the smear. NOTE: It may be necessary to change the DI water frequently if many slides are being stained. 3.can be found in the outer limits of this area. a. Determine the overall staining quality of the blood smear by evaluating cells. c. granular cytoplasm. 3. 7. Note clumps of similar cells in the feather because they may be representative of an abnormal population. a. There should be no stain precipitate present on smear. b. WBCs or platelets.7. The RBCs should have central pallor. Stain should not be too dark or too pale. Determine if there is proper distribution of the cells on the smear. bright red/orange granules dark purple nuclei and granules. b. There should be no area containing large amounts of broken cells or precipitated stain. WBCs pulled to the feather end of the blood slide should NOT exceed 2-3X the number of WBCs present in the examination.3. Scan the edges and center of the slide to be sure there are no clumps of RBCs. The area where approximately 50% of the RBCs show minimal overlapping and 50% are individually spaced. 2. Find an optimal area for the detailed examination and enumerations of cells.5 High power (40x) scan 1. dark purple nuclei with varying shades of blue cytoplasm dark purple nuclei with reddish. 4.3.4 Low power (10x) scan 1. 76 . This is sometimes seen in lymphoproliferative and myeloproliferative disorders. Cell Type RBCs Lymphocytes Neutrophils Monocyte Eosinophils Basophils Appropriate Appearance on Well-Stained Slide reddish pink. lighter purple nucleus with a grayblue cytoplasm. Reject slides that do not fit this requirement because large cells are disproportionately dragged to the feather end which may affect differential accuracy. Once the student has evaluated the quality of their smear. Nuclei and cytoplasm of WBCs should be the proper color.d. the student should ask the instructor to examine the smear based on stated criteria. e. If blood smear is used to look for abnormalities within the blood. What blood type should be focuses under microscope observation ? 77 . SELF-QUIZ 1. Platelets should be clearly visible. Why in each process on the blood smear preparation we should keep the slide always in wet condition ? Please explain your reason ! 2. 2. NOTES 78 . Dubai. London. Journal of Nematology 15(1):142143 Cui D.Lippincott Williams & Wilkins. USA. The Iowa State University Press.REFERENCES Aulia G. Malang. Skripsi. Ex Decne var. skripsi. sylvestris). Theory and Practice of Histological Techniques. Baltimore. Universitas Brawijaya. Jurusan Biologi. Jawa Timur. Melbourne. Malang. Karakterisasi Bentuk Dan Kerapatan Kristal Kalsium Oksalat Pada Iles-iles (Amorphophallus Variabilis Bl. Bancroft JD and Gamble M. Brown B. Edinburgh. Malang. Madrid. 1 st. 3 th ed. 1975. pages 96-97. Toronto. Ames. Fakultas Matematika dan Ilmu Pengetahuan Alam. Karakterisasi Bentuk dan Kerapatan Kristal Kalsium Oksalat pada Walur (Amorphophallus campanulatus (Roxb) Bl. Churchill Livingstone.Sydney. Stain Technology 50(2): 99-105 Gross K and Steinman H K.2011.f.adelphia. An Improved Technique for Clearing and Staining Plant Tissues for Detection of Nematodes. Lea & Febiger Publisher. St Louis. kirkpatrick T. Fakultas Matematika dan Ilmu Pengetahuan Alam.. Pengaruh pemberian dolomite terhadap ukuran dan kerapatan kristal kalsium oksalat pada helaian dan tangkai daun tanaman porang (Amorphophallus muelleri Blume). Gardner RO. Philadelpia Endriyeni E. Bybd DWJ. 79 . New York. Jurusan Biologi. 2012. 2009. Kecamatan Saradan. Cambridge. Universitas Brawijaya. Botanical microtechnique and cytochemistry. Fakultas Matematika dan Ilmu Pengetahuan Alam. Fakultas Matematika dan Ilmu Pengetahuan Alam.). 1983. 1976. 2009. Phi. Hematology: Principles and Procedures. Universitas Brawijaya.1993. Berlyn GP and Miksche JP. Tokyo Handayani . Cape Town.) di Klangon. Atlas ofHistologywith Functionaland Clinical Correlations. Singapore.São Paulo. Mohs Surgery and Histopathology: Beyond The Fundamentals. Skripsi. Skripsi. Jurusan Biologi. Analisis Anatomi Kristal Kalsium Oksalat pada Beberapa Varian Porang (Amorphophallus oncophyllus Prain ex Hook.Cambridge University Press. Ed. New York. Delhi. Ed. Fatmawati. Malang. Iowa. 2002. 5th edition. Jurusan Biologi. 1 st. 2012.2009. Madiun. An overview of botanical clearing technique. and Barker KR. Universitas Brawijaya. New York. Ruzin SE. School of Life Sciences Faculty of Science.Harijati N. Freeman & Company. and Simon JE. Koroch AR. 2002. 1999. Cells.gov. Clinical Hematology and Fundamentals of Hemostasis. Karakteristik Bentuk dan Kerapatan Kristal Kalsium Oksalat pada Suweg (Amorphophallus campanulatus var hortensis).H.. Sass JE. Common plant phenols other than anthocyanins. Lersten. 1940. Plant microtechnique. 1986. Department of Botany. Johansen DA. by Andras Nagy. in Manipulating the Mouse Embryo. La Trobe University. Ames. pages 606608. Fakultas Matematika dan Ilmu Pengetahuan Alam. Malang. In ‘The Chemistry of PlantnPigments’ CO Chichester (ed). Applications in Plant Sciences 1 ( 5 ): 1300016 Special reference source of MSDS http://www. adapted from “Techniques for Visualizing Gene Products. New York Singleton VL. Melbourne. Animal Tissue Techniques. Kristina Vintersten. NY. Joyner A and Nancy W. Khasim SM. Cold Spring Harbor. An improved clearing and mounting solution to replace chloral hydrate in microscopic applications . W. contributions to coloration and discoloration. Ed. Botanical microtechnique. McGraw-Hill Book Co. The Iowa State College Press. 2003. 2013. New York. 2 nd. Oxford University Press. Immunohistochemistry of Whole-Mount Mouse Embryos. Iowa. Humason GL. Re-investigation of secretory cavity development in Lysimachia (primulaceae. Cold Spring Harbor Laboratory Press. Marina Gertsenstein. Technology and Engineering. 102: 193-197 Rohmiati L. New Phytol. 2007. Tissues. A study of the resistance of chickpea (Cicer arietinum) to Ascochyta rabiei and the effect of age of plant tissue on disease development.). Third Edition. 2008. PhD Dissertation .au/sites/swa/whsinformation/hazardous-chemicals/sds/pages/sds 80 . F A Davis Co. Botanical microtechnique: principles and practice.co.1972. 2012. Australia Harmening D. Villani TS. and Organ Systems. Plant microtechnique and microscopy. 5th edition. 1958.” Chapter 16. Skrispi. 3rd edition. New Delhi. and Richard Behringer. Publisher. Capital Publ. Academic Press. 1967. Universitas Brawijaya. Jurusan Biologi.safeworkaustralia. Inc. San Fransisco. http://www.worksafe.gov.au/sites/swa/whsinformation/hazardous-chemicals/sds/pages/sds 81 .drs.gov/dsg/hazcom/msdsformat.edu/css/factsheets/msdss.au/safety-and-prevention/healthand-safety-topics/material-safety-data-sheets http://www.html http://www.au/workplace/hazards/hazchem/managi ng-risks/labelling-and-safety-datasheets/index.osha.ccohs.illinois.vic.html http://www.qld.safeworkaustralia.deir.gov.gov.htm#.ca/oshanswers/legisl/msdss.Uh7bU3_5mX8 http://www.aspx https://www. 82 . APPENDIX: MATERIAL SAFETY DATA SHEET A . B .                                                                                      .                                            .                                         .                                        .                                        .                 .