MACERATION AND SMEARS

MACERATION AND SMEARS

1. MACERATION & SMEARS

2. MACERATION A methods by which tissues are separated into individual celled or their group by dissolving the middle lamella with which the cells are held. The separation of fixed plant or animals material through the hydrolysis of their interstitial tissue or cement is known as maceration (Peter Gray)

3. Maceration is resorted to when attempting to visualise a three dimensional nature of structural elements, to make a study of individual cell and group of cells. ADVANTAGES OF MACERATION Helps to view cells in their entirely, which is not possible while sections are used Helps to study the nature of cell wall thickening including pores

4. The reagent and technique used in the process depends upon the middle lamella. The middle lamella in herbaceous plants consists of mainly pectic substances, which can be hydrolysed by mere boiling in water. In wood tissue the middle lamella is more ligneous and can be hydrolyzed by treatment with alkali, acid or enzymes THREE COMMON METHODS ARE GIVEN BELOW : SCHULTZE’S METHOD : The material is treated with concentrated nitric acid and few crystals of potassium chlorate(kclO3) and warmed (avoid fumes). When the materials turn completely bleached was thoroughly water

5. HNO3+ KClO3  KClO4 potassium per chlorate-> highly reactive & toxic ……. So, aviod fumes This method is used for very hard , woody materials. JEFFREY’S METHOD Cut fry/fresh material into small slices (.5mm thick) Boil & cool repeatedly Treat with mixture of equal volume of 10% chromic acid , 1-2 day at 30-40°C Tap material gently using a glass rot with rounded end to loose the cells

6. Wash throughly in water to remove the acids(usually the macerated tissue along with the fluid mixture is poured into a large volume of H2o & the macerated bits are picked up with the brush) Stain with suitable stain Mount in 50% glycerine Temporary /semi-permanent preparations Staining with 1% safranin for 6 hours Rinse thoroughly with water Dehydrate by rapid addition of hygrobutol Give 2 more changes of hygrobutol

7. Add a little balsam highly diluted with hygrobutol Evaporate it down to a mounting consistency Mount on a neat glass slide Used for woody & herbaceous materials Aviod inhaling the, vapour since HNO3 vapour since HNO3 vapour is highly toxic HARLOW’S METHOD : The material is treated with chlorine water for 15-30 min. wash again and is macerated to obtain cells. These cells are dehytrated by using hygrobutol & is later infit treated by canada balsam & then mount in the same balsam

8. SMEARS: Cytological method used to study the internal details of individual cells especially nuclear structure. Smears are prepared from cells which are not firmly attached by middle lamellae Frequent materials for this study are sporocytes of plants which give pollen grains after meiosis & cells of testis in animals This can be easily prepared because it doesnot need sectioning In plants smears are usually prepared from the microsporocytes of the anther of rheo discolor or chlorophylum Smears are prepared by smearing a material under study on a clean glass slide to which is fixed and mounted

9. 2 common used staining methods are the acetocarmine method and Feulgen method Acetocarmine method This method, originally developed by BELLING involves killing, fixing and staining all in one process and easy also The preparations reveals details regarding structure and association of chromosome to a great extent Carmine dye is obtained by adding alum to cochineal(a yellowish red powder obtained by grinding the fried bodies of female Dactyl opius cacti insect)

10. PREPARATION OF ACETOCARMINE Take 200cc, dil.acetic acid(90cc acetic in 110cc dis.H2o) Add 1g carmine dye to it Cool &decant Add a few drops of aqu.ferric acetate, till the colour becomes dark wine red Keep it in well- stoppered dark bottle

11. 2 . FEULGEN METHOD This method was originally developed in 1924 by feulgen and Rossenbeck . Later Lille adopted a modified error proof technique using Schiff’s reagent for feulgen stain PREPARATION OF BASIC FUCHSIN/FEULGEN STAIN Dissolve 1g basic fushsin & 1.9g sodium metasulphite in 100ml 0.15N HCl in conical flask & shake well for 2hrs Add 500mg activate charcoal & shake Filter & store in dark bottles

12. MAKING SMEARS PERMANENT Invert the smear slide in a petridish containing a mixture of 10 parts acetic acid & 90 parts alcohol to separate cover glass from the slide(1:9-acetic acid:alcohol ) Take coverslip using a pointed forceps Flood the coverslip with absolute alcohol & drain off the alcohol. Repeat until the coverglass is clear Flood with methyl benzoate & drain off this fluid & invert the cover glass over a drop of DPX mountant or Euparol on a clean warm slide Follow the same procedure with the slide & finally mount with a fresh clean & warm cover glass.