Vital staining ppt notes

vital stains

1. Vital stains - Dr. Anil Kr

2. I ntroduction Most oral cancers are preceded by precancerous lesions and early cancers that can be identified by visual inspection of the oral cavity. Conventional oral examination is useful in the discovery of some oral lesions but it does not identify all potentially premalignant lesions, as some are not readily apparent to visual inspection alone . -Adjunctive techniques have emerged that may facilitate early detection of oral premalignant and malignant lesions

3. S taining of tissue There are basically three ways in which we impart color to tissues . They are 1. Staining with dyes 2. Impregnation with metallic salts. 3. The formation of colored compounds in situ by means of chemical reactions.

4. Vital stains Vital staining is defined as staining of the cells or tissues in a living state C lassified as- Intra vital staining :staining done in the body (in vivo) by injecting colloidal solution of dyes. Supra vital staining : staining of living cells out side the body (in vitro) usually applied to slide preparation of detached cells.

5. Intravital and supra vital stains 1- Trypan Blue 2- Vital New Red 3- Indian Ink 4- Congo Red 5- Toluidine Blue 6- Lugol’s Iodine 7- Methylene Blue 8- Acetic Acid 9- Rose Bengal 1-Ehrlich’s Methylene Blue 2-Janus Green 3-Neutral Red 4-Tetrazolium Salt 5-Fluorochrome 6-Pinacynol 7-Acridine Orange 8-Thionin 9-Nile Blue, A zo Blue

6. Intra vital stains Intra vital staining is usually confined to the demonstration of pinocytosis and phagocytosis by the cells of Reticuloendothelial system. A colloidal solution of the dye is injected and after an interval the tissue is removed and then fixed, processed and sectioned. The type of fixation and processing depends on the solubility of the dye employed.

7. Intravital staining

8. 1- Toluidine Blue Toluidine blue may be used as an additional aid to assess at risk of cancer lesions. The mechanism of increase uptake in dysplastic and malignant lesions may be due to increase DNA synthesis in malignant cells by greater diffusion through increased intercellular channels in tumor cells. The assessment of dye uptake depends on clinical judgment and experience.

9. Composition : 100 ml of 1% TB contains- 1 gm of toluidine blue powder , 10 ml of 1% acetic acid, 4.19 ml of absolute alcohol and 86 ml of distilled water, pH maintained at 4.5.

10. TISSUE STAIN APPLICATION SEQUENCE- A pplication of 1% acetic with Q-tip (20 sec) (to remove soapy saliva) Rinse in water A pply toluidine blue 1% with Q-tip (10-20 sec) Decolorize with 2% acetic acid (to reduce mechanically stained stains)

11. Toluidine blue staining

12. ADVANTAGES Helps to determine the extend of biopsy site. Easy to perform. Non invasive. Inexpensive. Helps to monitor treated cancer patients for recurrence. Sensitivity of 72-100% Specificity of 45-93%

13. DISADVANTAGES Not recommended for patients with physical or mental disability. Acetic acid may irritate the mucosa. Equivocal dye retention. Variable mode of application. 30% false positive results noted.

14. 2-LUGOL’s IODINE Lugol’s iodine consists of iodine , potassium iodide and distilled water in contrast to TB. Lugol’s iodine is retained in normal squamous epithelial cells but not in dysplastic or malignant epithelial cells. As there is enhanced glycolysis in cancer cells, do not promote the iodine–starch reaction. Lugol’s iodine solution produces a brown blac k stain by reaction of the iodine with glycogen. Normal mucosa contains higher amount of glycogen than abnormal mucosa and produces brown black stain.

15. Lesions after being stained with Lugol's iodine solution

16. 3- Acetic acid staining In the past, 3-5% acetic acid was used as a vital staining for the detection of oral cancers. Composition : Of 1% acetic acid rinse – 1 ml of glacial acetic acid and 99 ml distilled water Application of acetic acid causes reversible coagulation of cellular proteins particularly in abnormal squamous epithelial areas. Aceto-whitening is seen distinctly as compared with the normal pinkish color of the surrounding normal squamous epithelium.

17. (a)Before application of the vital stain. (b) After application of vital stain, asterisk showing biopsy site .

18. Acetic acid staining in later al border of tongue formazan

19. 4 -Trypan blue 1% aqueous solution of trypan blue Procedure- inj. o f trypan blue sol. i s done and then after an interval of 10 min the biopsy is taken. Fixation in formalin and processing by paraffin wax embedding . This is a valuable stain is taken up by the RES . By injection in to circulatory system ,it is employed for staining the uriniferos tubules.

20. Trypan blue A) Trypan blue exclusion test shows unstained live cells (L) and dark-blue stained dead cells (D)

21. uses Trypan blue is commonly used in   microscopy  (for cell counting) and in  laboratory mice  for assessment of tissue viability .  The method cannot distinguish between   necrotic  and  apoptotic  cells. It may be used to observe fungal  hyphae . T rypan blue is also used in ophthalmic cataract surgery.  

22. 5 - C ongo red test for amyloidosis Bennhold in 1922 Highly selective affinity for amyloid and subsequent green birefringence when viewed with polarizing microscope. After intravenous injection of the dye all the amyloid deposition in the body are stained bright red. More than 80% retention aft 1-4 hrs.

23. Staining of amyloid by Congo red is through hydrogen bonding as apposed to the electrostatic bonds formed between the dye and most other tissue components . In aqueous solution Congo Red stains many tissue components.

24. Localized amyloidosis of the upper gingiva

25. Vital staining of macrophages and osteoclasts: The identification of macrophages under experimental conditions were greatly facilitated by the development of vital staining which as it names implies ,means a method in which living cells become stained because of some vital activity ( Phagocytosis) Dye trypan blue injected either directly in to CT or administered IV or into peritoneal cavity

26. Dye phagocytized by macrophages and be seen as blue material in their cytoplasm. Such vital stains are colloidal dyes and it is because they are in the form of macromolecular aggregates that they are phagocytized. Colloidal silver and diluted I ndian ink are also frequently employed for vital staining of macrophages.

27. Osteoclasts and vital staining When the method of vital staining was first developed ,many experiments were performed not to determine the origin of osteoclasts but to see if they were phagocytic. Whereas it was shown readily that the cells we term macrophages would take up vital stains such as trypan blue or particulate matter such as Indian ink , osteoclast examined in same animals showed no significant uptake.

28. Since at that time osteoclasts were generally believed to develop from osteogenic cells or osteoblasts and since neither of these cell types was shown to be phagocytic it caused little surprise that osteoclasts evidenced no phagocytic abilities when tested by this method.

29. In 1963 Jee and Neelson using bone charcoal as suitable particulate matter for vital staining showed that material could be osteoclasts but only after enough time had elapsed for macrophages that had taken up the markers by phagocytosis to have fused and formed osteoclasts This proven some proof of two concept – -the osteoclasts were derived from macrophages -The osteoclasts themselves were not phagocytic.

30. 6 -Vital new red 5% aqueous solution of the dye is used. Tissue may be fixed in saturated aqueous solution of mercuric chloride ,processed by paraffin wax tech. 7 -Indian ink- commercial Indian ink is diluted with distilled water and after filtration sterilized in autoclave it with stands routine fixation and processing in paraffin wax.

31. SUPRA VITAL STAINING Staining of living cells after removal from organism( in vitro) usually applied to slide preparation of detached cells. eg . Ehrlich’s methylene blue ,Janus green stain.

32. Mechanism –supravital staining involves the application of specific dyes that penetrate all the cells and color certain cellular tissue components,

33. A dye used for supra vital work should be able not only to enter the cell but also diffuse through the protoplasm without the killing the cell and to color pre-exiting cell inclusions, distinctively. or color the whole cytoplasm of the particular cell strongly enough that these cells stand out prominently from surrounding inter cellular material and other cells Vital dyes stains blood cells in vitro while they are still alive . A number of these stains are employed in hematology.

34. 1)Ehrlich’s methylene blue technique By this method nerve endings in dissociated tissue (e.g. muscles) can be demonstrated. Method- tissue is teased out in warm saline solution in a watch glass. Small pieces are transferred to dilute (0.025-0.25%)solution of methylene blue in normal saline solution either on a slide or in another watch glass for 10-15 min. Having the watch glass inside a dish containing a piece of wet cotton wool controls evaporation.

35. The preparation should be examined under the microscope from time to time . if staining is sufficient or absent after 45 min ,the strength of the stain should be increased and the technique repeated.. Note - the staining fades quite quickly if the preparation is covered (oxygen is excluded ) treatment with saturated ammonium molybdate for 2 hrs. before mounting in glycerin helps to preserve the color . RESULTS - Nerve fibers and nerve ending – blue other tissue constituents- colorless to pale blue

36. 2 - Supra vital staining of leucocytes or other living cells: A solution of J anus green and N eutral red are used as a vital stain in hematological lab for the stains of leukocytes to differentiate between acute leukemia of the blastic type. RESULT– mitochondria in myeloblasts are small and tend to aggregate whereas in lymphoblast they are large and diffuse.

37. The technique is based on the affinity of J anus green B for mitochondria and neutral red for the neutral red vacuoles . Both dyes are toxic and their strength must be carefully controlled. Slide preparations are used and must be maintained at a constant temperature of 37oC.

38. Whitby & Hynes method: for supra vital staining with Jasus green and neutral Red stain. Mechanism - the selective staining of the mitochondria and of intra cytoplasmic granules is probably the result of their enzymatic activity .

39. Stock solution – Neutral red - 0.2 gm. in 50 ml absolute alcohol Janus green -0.25 gm. in 50 ml absolute alcohol. working solution- Janus green solution- .25 ml (.025%) Neutral red solution -0.8 ml (0.4%) Absolute alcohol -5 ml

40. A clean microscopic slide is flooded with stain and dries in horizontal position one small drop of blood not anti coagulated or bone marrow placed on the slide and covered with a cover slip .this will allow specimen to form a thin film ,allow to stain for 20 min.

41. RESULT - Intra cytoplasmic granules and vacuoles stain deep purple to orange . Mitochondria-green Cytoplasm- pale yellow to pale green blue Nucleus- colorless Neutrophils- yellow , Eosinophils - orange Basophils- maroon

42. 3- Tetrazolium salt- For the demonstration of many oxidative enzymes, tetrazolium salts are used as a hydrogen acceptor. on reduction they will form colored deposit. Two tetrazolium salts are in common use at the present time - monotetrazolium and ditetrazolium.

43. Ditetrazolium salt ,ditetrazolium chloride –nitro known as NBT (nitro blue tetrazolium) produces highly colored formazan deposit that is insoluble in lipid . Formazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases . monotetrazolium produces a finely granular formazan which is soluble in lipids.

44. The soluble yellowish ,oxidized, Nitro Blue Tetrazolium (NBT) salt is reduced to dark blue insoluble formazan when normal phagocytic cells ( granulocytes and monocytes ) are incubated with NBT.

45. In healthy individuals less than 10 % of phagocytic cells are NBT positive . A much higher percentage of positive cells results if bacteria stimulate the cells in vitro or in vivo and then incubated with (NBT ). S ince granulocytes exposed to bacteria have a markedly stimulated metabolic activity.

46. formazan

47. 4- Fluorochrome : They are basic dyes ,that fluorescence when excited by UV light. The stained living cells are examined by a combination of fluorescence and phase microscopy. Fluorochromes can be used to identify cytoplasmic inclusions e.g. mitochondria, Golgi apparatus , DNA staining. Also used to demonstrate acid fast bacteria in smears and stains.

48. Fluorochromes for DNA Staining and Quantitation

49. 5-Pinacyanol and Neutral red stain Pinacynol stains the mitochondria deep blue , has the advantage over Janus green ,that it does not fade for many hours and can successfully be combined with neutral red. REAGENT : Stock solution- Pinacyanol 0.03 % in absolute alcohol. Neutral red 0.4% in absolute alcohol. Working sol : 1 ml of each solution added to 5 ml of absolute alcohol. RESULT- Mitochondria-deep blue

50. 6- Acridine Orange supravital stain : Tech - Living cells are stained with acridine orange and are examined under fluorescence microscope. Reagent- Acridine Orange 0.001 % in N saliva pH -6-8 Procedure - 1 drop of fresh blood or bone marrow in several drops of stain on a slide is added ,cover slip applied and slide allowed to incubate at room temp. for 3 min and examined under fluorescence microscope.

51. stained with acridine orange

52. RESULT- living nuclei (DNA) – fluorescence yellow green Nuclear +cytoplasmic RNA – fluorescence orange red PMNL granules- Bright red Eosinophilic granules- orange Mast cell - Red Dead Nuclei - dull red

53. 7- Supra Vital staining of reticulocytes Reticulocytes are large nuclear cells in bone marrow when stained with vital dye ( new methylene blue ) seen to contain a network of bluish granules( ppt ribosomes) responsible for the name.

54. Advantage of vital stain : 1)- Elimination of staining artifacts (since living) 2)- Undamaged cells are examined. Disadvantage of vital stains: 1)- the preparation is not permanent and must be examined fresh before they dry out. 2)- the living cells are extremely fragile and therefore any error in technique is detrimental. 3)- the stain fades after short time

55. Limitations of vital staining 1)- Only a few specific elements may be demonstrated . 2)- Nuclear material not stained. 3)- the strength of dye is very critical Too weak- there is little or no staining Too strong- the cells are killed. .

56. 4)- Intra vital staining is mostly confined to the demonstration of the Reticuloendothelial system. Dye particles from a colloidal solution are ingested by the phagocytic cells and are subsequently found loose in the cytoplasm ,no specific elements being colored .

57. 5)- some of the method (ex. Ehrlich’s )are not truly vital in that staining will take place after the death of tissue. 6)- vital staining dye like methylene blue will retain their color only if there is excess of oxygen. and too bright light fade the color.

58. 7)- temperature of the solution is also critical , inta vital and supra vital stain must be done under controlled temp conditions. 8)- vital staining has limited application in electron microscopy as it can lead to alterations in metabolism ,which will be manifest as change in fine structure .

59. THIONIN: 1)- it is useful vital stain added to culture media ,it serves to differentiate species of Brucella . 2)- it has greatest value at the present time in the staining of frozen sections of fresh animal or human tissue ,particularly in the study of tumors.

60. NILE BLUE : Used to stain hydrae ,protozoa ,yeasts and for staining bone sections . It stains lipid vesicles in the cell and gives red colour under flouro microscopy AZO BLUE : Vital staining of protozoa .it can replace Indian ink in demonstration of capsule of bacteria . ALIZARIN RED : 1)- Vital Stain for nervous tissue 2)- An important application is for the gross staining of skeletons especially in fetus.