Staining Prepration - simple positive and simple negative
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Staining techniques in microbiology involve using dyes to color biological specimens, enhancing their visibility and contrast under a microscope. These techniques are used to identify and differentiate cells, tissues, and microorganisms. Common types include simple staining, differential staining, and special staining for specific structures like capsules and endospores.
Staining Prepration - simple positive and simple negative
- 1. Topic : Double staining method
- 2. Stains and staining The selected sections need to be stained. The stains help to distinguish different tissues, cells or inclusions from one another by developing specific colours. Acetocarmine, Aniline blue, Crystal violet, Erythrosir.c:, Hematoxylins, Fast green, Light green and Safranin are some of the commonly used stains. (See appendix for preparation)
- 3. Most of the stains are specific in reaction and are purposely used so that definite structures or subst<mces are stained. The following are some of th.e stains used for staining different structures. (A) Specificity :
- 4. Safranin or fast green is used alone to stain filaments of algae, fungi, sections of bryophytes, spores of pteridophytes, pollen grains of gymnosperms etc. Aniline blue or safranin is uitable for algae. (B) Single stains.
- 5. 1. The material is kept in a watch glass. A few drops of stain are added so that the material is immersed in the stain. 2. The material is allowed to remain so for a few minutes and allowed to take stain. The time required varies with materials. 3. After the stain is taken up, the excess of stain is washed off in water. The washing is repeated till stain stops coming out. Following is the common method of staining.
- 6. 4. In some cases, excess stain is removed by acid water or acid alcohol if water alone fails to do so. 5. The stained material is ready for mounting. Fungi are stained in cotton blue as given below. (i) A drop of cotton blue (prepared in lactophenol) is placed on a slide. (ii) Fungal hyphae is now placed in this drop. (iii) The slide is run over the flame of the spirit lamp so that the stain is warmed up. (iv) The preparation is now ready for
- 7. Commonly two or more stains are employed wherever tissue differentiation is found. Combination of acidic and basic dyes of contrasting colours is of general use. This permits the distinction of woody tissue from non- woody tissue. The following few combinations are commonly recommended- 1. hematoxylin and safranin, 2. safranin and fast green, 3. safranin and aniline blue, 4. safranin and crystal violet and 5. crystal violet and erythrosine (C.) Combinations.
- 8. There are two types of preparations - semi- permanent and permanent. The procedures differ in both the cases. These are given below. (D) Staining procedures.
- 9. (a) For semi-permanent & temporary preparations. Certain preparations are made for temporary use. The material is studied and the slide is then discarded. The method for staining them is given below. 1.The selected sections are transferred from watch glass containing water to another watch glass containing principal stain (e.g. hematoxylin, safranin or crystal violet). 2. The sections are allowed to remain in the stain for sometime (for about 4-5 minutes).
- 10. 3.Excess amount of stain is removed by washing the sections repeatedly with water. (This can he seen under the microscope. The stain should be taken either by lignified or nonlignified tissues. Otherwise the section should be washed till the stain disappears from one type of tissue).. 4.If destaining is not achieved, sections are washed with acid alcohol. In this case, further washing with water is necessary till traces of acid are removed.
- 11. 5.This is followed hy transfer of sections to a watch glass containing counter-stain (e.g. safranin, fast green, erythrosine). This stain acts on the tissue more rapidly than the principal stain. Therefore, section is kept in this stain for shorter duration (about a minute or two). 6.Excess of stain is removed hy washing stained sections with glycerine (15-20%). The seetion should distinctly hring out demarcation het ween tissue system while preserving the colour of the stain. 7. The section is now ready for mounting.
- 12. (b) For permanent preparations. In certain cases preparations need to he stored permanently as a future record. The method of preparation followed is descrihed below. 1.The section is first stained with principal stain (aqueous hematoxylin, safranin or crystal violet). 2.The section is then washed with water till no more stain dissolves and water remains colourless. 3.Section is passed through a graded series of alcohol for dehydration. A watch glass is filled with requisite amount of alcohol, (beginning with 30% alcohol) and the section is transferred to it.
- 13. This watch glass should always be covered with another larger one. In order not to disturb the section, used alcohol is removed by glass dropper. All the 30% alcohol is replaced with 50% alcohol. This procedure is repeated till 70% of alcohol grade is reached. 4.At this stage, counterstain is employed (e.g. safranin, fast green or erythrosine prepared in 80'Yr) or 90% alochol). 5.This stain acts quickly and as such section is washed immediately after the requisite time is over.
- 14. 6. Destaining is done by washing sections with 90% or 100% alcohol. 7. The section is now transferred to absolute alcohol to complete the dehydration.
- 15. 8.Clearing now begins with 25% of xylol (25 cc of xYlol and 75 cc of absolute alcohol). The sections are gradually passed through xylol series of 25%, 50%, 70%, 90% and finally transferred to pure xylol. If dehydration is not complete, pure xylol turns white or turbid. At ths stage section should be passed through reverse series. 9.Pure xylol is the last stage of clearing. Section is now ready for mounting. 10. Mounting is done in Canada balsam.
- 16. Specific schemes for staining combinations (for temporary and semi- permanent preparations.)
- 17. Specific schemes for staining combinations (for permanent preparations)
- 18. DOUBLE STAINING Prepare section Safrenin 10%Alcohol 30%Alcohol 50%Alcohol 70%alcohol 90%Alohol Fast green 100%alcohol xy:al(1:3) xy:al(1:1) xy:al(3:1) pure xylene DPX mounting
- 19. Reference : A TEXT BOOK OF PRACTICAL BOTANY VOL. II Bendre Kumar https://youtu.be/VfuOr7oT3YU?si=bP5QTCso 3C3TgZZo