Special stains useful in Microbiology laboratory

Special stains useful in Microbiology laboratory

1. Special stains useful in diagnosis in Microbiology laboratory DR.YOGITA Mistry GMC,Surat

2. Name of special stains Acridine orange Calcofluor white Aura mine phenol Toluidine blue Wright’s – Giemsa stain Albert’s stain LPC

3. Stains for flagella Leif son stain Gray method Silver technique Stains for metachromatic granules Albert stain  Neisser stain  Leoffler methylene blue Ponder’s stain Stains for spirochetes Fontana  Levaditi  Silver Impregnation  India ink negative stain 

4. For Chlamydia Gimenez  Machiavello Giemsa For rickettsia Castaneda Machiavello Giemsa

5. WRIGHT’S GIEMSA STAIN Mainly stains the cellular eliments of the peripheral blood , has little use for staining bacteria. Used primarily to detect the intracellular yeast form of Histoplasma capsulatum or intracellular amastigotes of Leishmania species or Trypanosoma cruzi, malarial parasites. Also helpful for demonstrating certain intracellular viral inclusions. There are two ways of staining-rapid and slow method.

6. RAPID METHOD: Useful for staining the malarial parasite, schuffener’s dots and also for trypanosoma demonstration. Method: Fix films in methyl alcohol for 3 min. Stain in a mixture of 1 part stain and 10 parts buffer solution for 1 hr. Wash with buffer solution(buffered water ,ph 7-7.2), allowing preparation to differentiate for 30 seconds. Blot and allow to dry in air.

7. Rapid method with heat application is useful for demonstrating spirochetes. Method: Fix the smear with absolute alcohol foe 15 min. or by drawing three times through flame. Cover a fixed film with the diluted stain. warm till steam rises , allow to cool for 15 seconds, then pour off and replace with fresh stain and heat again. Repeat the procedure 4-5 times , wash in distilled water , dry and mount.

8. Histoplasma cysts

9. Ring form of P . falciparum

10. SLOW METHOD: Useful for demonstrating objects difficult to stain in the ordinary way such as spirochetes. Principle is to allow the diluted stain to act for a considerable period. But care has to be taken as fine precipitate can de deposited. Procedure: Fix the film in methyl alcohol for 3 min. Mix 1 ml stain with 20 ml buffer solution. Place a piece of thin glass rod in the stain in the dish. Leave to stain for 16-24 hr. Wash the slide for in a steam of buffer solution. Allow to dry in air , mount and examine.

11. spirochetes

12. STAINS FOR METACHROMATIC GRANULES Albert stain  Neisser stain  Ponder’s stain Loeffler’s methyline blue ALBERT’S STAIN Albert’s staining is specially demonstrates the presence or absence of the metachromatic granules, a characteristic feature of Corny bacterium diphtheria. During gram staining if a smear appears as purple rods with straight or slightly curved with clubs at the end, with a characteristic V shape then it is suspected as Corny bacterium diphtheria. The further confirmation can be done by Albert’s staining technique

13. ALBERT’S STAIN . This techniques employ two stains: Albert’s solution Toluidine blue 1.5 gm Malachite green 2gm Glacial acetic acid 10ml Alcohol 20ml Distilled water 1 liter

14. Albert’s iodine Iodine 6gm Potassium iodide 9gm Distilled water 900ml Procedure Cover a heat fixed slide with Albert's stain and allow to act for 3-5 min. Wash in water and blot dry. Cover with Albert's iodine for 1 min. Wash and dry. Granules stain bluish black , protoplasm green and other organisms light green.

15. Neisser's Stain Method Stain with Neisser's methylene blue for 3 min. Rinse rapidly with water.  Stain with dilute iodine for 1 min. Wash rapidly in water. Counter stain with neutral red for 1 min. Results: Cytoplasm appears pink and granules deep blue

16. Reagents Neisser’s stain solution For use, mix 20 ml of solution A and 10 ml of solution B Neisser’s Solution A:  Methylene blue 0.1 gm;  95% ethanol 5 ml; glacial acetic acid 5 ml  distilled water 100 ml. Dissolve the dye in the water and add the acid and ethanol Neisser’s Solution B :  Crystal violet 0.33 gm; 95% ethanol 3.3 ml  distilled water 100 ml. Dissolve the dye in the ethanol- water mixture

17. LPC STAIN FOR FUNGUS Fungi can be stained with lacto phenol cotton blue. Composition: The composition of lacto phenol cotton blue is as follows: Phenol crystals 20g Lactic acid 20 ml Glycerol 40 ml Distilled water 20 ml Cotton blue (or methyl blue) 0.075 g A drop of 95% alcohol is applied on the "slide. The fragments of the culture of the fungus is placed on the slide. It is teased out gently with needles. When it has spread well on the slide, allow the alcohol to evaporate. Then add a drop of the stain ( lacto phenol cotton blue). A cover slip is applied carefully without allowing air bubbles to form. A gentle pressure is applied and the excess stain around the cover slip is removed with the edge of a blotting paper. Now the slide can be viewed under the microscope to see the details of the fungus.

18. TOLUIDINE BLUE Mainly use for identification of cysts of Pneumocystis jiroveci ( carinii). Reagents: Sulfation reagent; 45 ml of glacial acetic acid is poured into a Coplin jar which had been placed into a plastic tub filled with cool tap water (not below 10°C). A 15-ml portion of concentrated sulfuric acid is slowly added with a glass pipette, being careful not to produce splashing. The solution is gently mixed with a glass rod. The sulfation reagent can be kept at room temperature and could be used for 1 week. .

19. Toluidine blue 0 solution: 0.3 g of Toluidine o 60 ml of distilled water 2.0 ml of concentrated hydrochloric acid 140 ml of absolute ethyl alcohol. The solution is stored at room temperature and can be used for 1 year. Staining was performed with all solutions in Coplin jars. For other reagents: two Coplin jars each is needed for 95% ethyl alcohol, absolute ethyl alcohol, xylene.

20. ( i ) With forceps, slides are placed in the sulfation reagent for 10min. The reagent is mixed with a glass stirring rod immediately after insertion of the slides and again after 5 min. (ii)The slides removed from the sulfation reagent with forceps, placed in a glass slide holder, and washed gently under cold running tap water for 5 min. The excess water is then drained. (iii) The slides are placed in toluidine blue 0 for 3 min. (iv) The slides are dipped in and out of 95% ethyl alcohol (in two Coplin jars) for approximately 10 mins until clean, with most of the blue dye being removed in the first jar. (v) The slides were dipped in and out of absolute ethyl alcohol (in two Coplin jars) for approximately 10 mins for further decolorizing. (vi) The slides are dipped in and out of xylene (in two Coplin jars) for approximately 10mins until clean. (vii) The back and frosted areas of the slides were wiped dry with a paper towel. (viii)The slides are examined with 20x and 40x objectives; examination under oil was is not necessary but could be done once the cover-slipped slides had dried. The staining procedure itself took approximately 20 min. Up to 4 slides could be stained readily simultaneously.

21. Calcofluor white stain Colorless dye Valuable flurochrome stain for rapid detection of fungi in wet mounts, smears , and tissue sections. it fluorescence when exposed to long wave length UV and short wave length visible light. Most use full in detecting yeast cell , hyphae , pseudohyphae in skin and mucous membrane scraping Fungal structure will display a brilliant apple green or a blue white ,depending on the wave length of the exciter light and filter combination

22. Preparation of stain: Stain is prepared by dissolving 1 gm of flurochome in 100 distilled water. From this a working solution is made by diluting 10 ml in 90 ml of 0.05% Evan's blue stain. For use, 1 drop is mixed with 1 drop of 20% KOH.

23. ACRIDINE ORANGE STAIN This stain is now use with increase frequency to detect bacteria in smears prepare from fluids and exudates in which bacteria are expected to be in low concentration. Use: It has been recommended for rapid identification of trichomonas vaganalis ,yeast cells , clue cells in vaginal smears. Also helpful to detect intra cellular gonococci , meningococci , and bacteria perticularly in blood cultures. Use of AO for staining acid fast bacilli and examined under UV light can be more rapid and efficient screening method. With UV light, RNA component orange red and DNA component yellow-green seen.

24. To make 500 ml of acridine orange acid stain: Acridine orange 0.13gm Acetic acid , glacial acid 10 gm Distilled water 490 ml Alcohol saline solution: Ethanol/methanol 5ml Physiological saline 245ml Method: Cover the unfixed dried smear with acridine orange acid stain for 5-10 seconds. This stain also fix the slide. Wash off the stain and decolorize the smear with alcohol saline solution for 5-10 seconds. Rinse the smear with physiological saline and place the slide in draining rack. Add a drop of saline or distilled water to the smear and cover with a cover glass. Examine by using florescence microscope with BG 12 exciter filter and No.44 and No.53 barrier filters. Alternatively ,use LED fluorescence.

25. T. vaganalis orange red with yellow green nucleus Yeast cell orange Bacteria orange Leucocytes yellow green Epithelial cells yellow green

26. AURAMINE PHENOLE STAIN Mainly useful for detection of M.TB in sputum. As the smear can be examine at lower magnification , shorter time is required for a slide than Z N technique. Also few AFB are more likely to get detected by this stain. With recent development of LED florescence system , I is now possible to examine smear inexpensively and easily in district labs. When required, aura mine stained smears can be restained by Z N stain by first treating the smear with 5% oxalic acid for 2 mins followed by washing in water.

27. Aura mine phenol stain : Phenol crystals 15gm Aura mine o 1.5gm Distilled water 500 ml 1%acid alcohol Ethanol/methanol 693ml Distilled water 297ml HCL 10ml Potassium permanganate 0.5 gm in 500 ml distilled water t0 make 0.1% of it.

28. Cover heat fixed dried smear with the filtered aura mine phenol stain for 15 min.(this stain binds to mycolic acid of TB bacilli) Wash with clean water. Decolorize with acid alcohol for 3-5 minutes. Wash off with clean water. Cover the smear with potassium permanganate solution for 15 seconds and clean with water.(provides the dark back ground) Tubercle bacilli fluoresce bright-yellow against a dark back ground.

29. Reporting of aura mine stained sputum smear examined using 10 and 40 * objectives based on WHO system: 1-19 bacilli in 40 fields scanty 20-199 bacilli in 40 fields + 5-50 bacilli per field ++ More than 50 bacilli per field +++

30. LOEFFLER METHYLENE BLUE STAIN Use full stain for identification of gram negative organism such as H. influenza and N.meningitidis which often do not stand out against red staining background of gram stain. It stains the polymorph nuclear cells blue and bacterial cells deep blue , back ground light gray in color. This stain should be consider as an adjunct to gram’s stain in laboratories where inaccessibility to a fluroscence microscope present. It is also helpful in respiratory secretion to detect Pneumocystis jiroveci( carinii ) rapidly. Helpful to see PMN cells in inflammatory diarrheal disease and mononuclear cells in stool in pt of typhoid.

31. Methylene blue 0.5 gm Ehtanol 30ml Potassium hydroxide 0.1ml Distilled water 100ml Procedure: Place a drop of methylene blue stain on a slide. Mix a small amount of specimen with the stain , cover with a cover slip. Examine under 40* objective for leucocytes , pus cell, RBCS.

32. NEW METHYLENE BLUE New methylene blue 1gm Sodium citrate 0.6gm Sodium chloride 0.7gm Distilled water 100ml It will detect the presence or absence of bacteria , their number and shape.

33. Iodine preparation for stool sample The value of wet preparations lies in the fact that certain protozoa trophozoites retain their motility which may aid in their identification. Definitive identification however may not be possible, especially for amoeba, since the nuclei of trophozoites and cysts are often not clearly visible. Wet preparations on fresh unpreserved liquid stool should be performed and examined as soon as possible (within 30 minutes of passage) Reagents Normal saline (0.85%) Lugols iodine potassium iodide 10gm powdered iodine crystals 5gm distilled water 100ml

34. Eosin stain: Eosin powder 0.5 gm Distilled water 100ml Procedure: Place a drop of fresh physiological saline on one end of a slide and a drop of eosin stain on other side. Using a piece of stick or wire loop , mix a small amount of fresh specimen with each drop. Cover each preparation with cover slip. Smear should not be much thick otherwise amoebae or cysts will not be seen. Examine under 10 and 40* objectives. Useful to see motile E.histolytica trophozoites containg red cells , motile G. lamblia trophozoites , stongoloides larvae, eggs and cysts of parasite.

35. FLAGELLAR STAIN Because of their extreme thinness, flagella are best demonstrated with the electron microscpoe,metal shadowed films or film made with phosphotungstic acid for negative staining. But when electron microscope are not available , it is possible to demonstrate the presence and arrangement of flagella by special staining methods for the light microscope. To be resolvable, flagella must be thickened at least 10 fold by a superficial deposition of stain procured by the action of mordant, usually tannic acid. An easier , cleaner and more reliable method is wet mount flagellar stain.

36. WET MOUNT FLAGELLAR STAIN : Mordant solution: 5% phenol 10 ml Tannic acid 2gm Aluminum potassium sulphate, 10 ml Saturated aqueous solution Ryu’s stain Crystal violet , saturated ethanoic solution 10ml Mordant solution 100ml

37. Procedure: After growing bacteria on medium, touch a loopful of water onto the edge of a colony and let motile bacteria swim into it. Then transfer loopful of water on a slide to get a faintly turbid suspension and cover with a cover slip. The bacterial suspension is thus prepare with a minimum of agitation , which would detach the flagella. After 5-10 min. when many bacteria have attach to the surface of the slide and cover-slip , apply two drops of Ryu’s stain to the edge of cover slip and leave the stain to diffuse in to film. Examine with microscope after 5-15 min.

38. Leifson flagella stain              Solution A:             Sodium chloride                       1.5 g             Distilled water                        100 ml                        Solution B:             Tannic acid                              3.0 g             Distilled water                           100 ml                Solution C:             Pararosaniline acetate               0.9 g             Paraosaniline hydrochloride     0.3 g             Ethanol, 95%              100 ml Mix equal volumes of solutions A and B; then add 2 volumes of the mixture to 1 volume of solution C. The resulting solution may be kept refrigerated for 1 to 2 months.  

39. For staining from liquid cultures, Leifson recommends two rounds of centrifugation and final suspension in distilled water to remove any medium components. Place 100  ml of the liquid culture in a micro centrifuge tube, centrifuge, and remove spent medium. Resuspend in 100 ml of distilled water by gently vortexing, again centrifuge, and remove supernatant. Form a slightly cloudy emulsion by resuspending in ~200 ml of distilled water. Gently vortex.  Again, emulsions should be only slightly cloudy prior to proceeding to staining. Optimization of the washing procedure will most likely be necessary to maximize quality of flagella stain.  

40. Leifson flagella stain 1.      Take a prepared slide and using a wax pencil draw a rectangle around the dried sample. Place slide on staining rack. 2.       Flood Leifson dye solution on the slide within the confines of the wax lines. Incubate at room temperature for 7 to 15 minutes . The best time for a particular preparation will require trial and error.  3.      As soon as a golden film develops on the dye surface and a precipitate appears throughout the sample, as determined by illumination under the slide, remove the stain by floating off the film with gently flowing tap water. Air dry. 4.      View using oil immersion, at 1,000x magnification, by bright-field microscopy. Bacterial bodies and flagella will stain red.

41. When a bacterial culture is stained with Liefson stain, the tannic acid component of the stain produce a colloidal precipitate which can be taken up by the bacterial flagella will become colorized which can be easily visualized using microscopy. The concentration of the tannic acid and dye is important in staining the bacterial flagella while the alcohol concentration in the Liefsons stains helps in maintaining the solubility of the components. On microscopic observation, the bacterial cells and flagella will stain red and the flagellar arrangement can be visualized easily. The age of the bacterial culture, condition of staining solutions, concentrations of the staining solution etc can also affect the staining reaction.

42. STAINS FOR SPIROCHAETES Fontana’s stain for films and levaditi stain for sections. Large spirochaetes such as borrelia stain by ordinary method ,including gram’s stain(giving a negative reaction) But smaller ones such as treponemes and leptosira are too thin to be seen , can be best observe by dark ground microscope, where their bright appearance and motility draw attention to them.

43. Fontana’s method for films : Solutions Fixatives: Acetic acid 1ml Formalin(40%) 2ml Distilled water 100ml Mordant: Phenol 1gm Tannic acid 5gm Distilled water 100ml Ammoniated silver nitrate Add 10% ammonia to 0.5% solution of silver nitrate in distilled water.

44. Procedure: Treat the film three times,30 seconds each times with fixatives. Wash off the fixatives with absolute alcohol and allow the alcohol to act for 3 min. Pour on the mordant ,heating till steam rises, and allow it to act for 30 seconds. Wash well with distilled water and dry again. Treat with ammoniated silver nitrate , heating till steam rises, for 30 seconds , when the film becomes brown in color. Wash well in distilled water , dry and mount in Canada balsam as some immersion oils causes the films to fade at once. Spirochetes are stained brownish black on brownish yellow back ground.

45. Levaditi’s method for tissue sections: Fix the tissue , which must be in small pieces 1 mm thick ,in 10%formaline for 24 hr. Wash the tissue for 1 hr in water and there after in 96-98% alcohol for 24 hr . Place the tissue in 1% solution of silver nitrate for 2 hr at room temperature, and there after at 50 degree C for 4-6 hr. Then rapidly wash in 10% pyridine solution. Transfer to the reducing fluid which contain 4% formalin 100 part Acetone 10 part Pyridine 15 part ,which are added immediately before use. Keep in this solution for 2 days at room temperature in dark. After washing well , dehydrate the tissue with alcohol embed in paraffin. Mount it in Canada balsam.

46. Silver impregnation Use to see the spirochetes in tissue section where dark field microscopy not possible , and when they are not in sufficient concentration. Warthin-Starry, Dieterle, Steiner silver impregnation method are used. They all perform equivalently. It is also useful for flagellar demonstration. Reagent A 100 ml distilled water 5 g tannic acid, 1.5 g ferric chloride, 2.0 ml formalin 1.0 ml 1 % sodium hydroxide. Reagent B, ammoniated silver nitrate solution: 100 ml of 2% silver nitrate . About 10 ml of this volume is removed and saved; to the remaining 90 ml, ammonium hydroxide is added drop wise until the heavy precipitate that is formed is dissolved. From the 10ml previously removed, 2% silver nitrate is added drop wise until a slight clouding appears and persists. At this point, the pH is adjusted to 10.0 with the ammonium hydroxide . Reagent B is relatively unstable, and must be used within 4 hr of preparation

47. The smears are covered by reagent A for 2 to 4 min; they are then rinsed in distilled water. . After the water rinse, reagent B (pH 10.0) is added for about 30 sec.. The smears are immediately washed with distilled water, air-dried, and examined under oil immersion. Leptospires are stained dark-brown to black .

48. CAPSULAR STAINS  Positive Capsule Staining   Since capsule is water soluble in nature, it is too difficult to stain the capsule with normal staining methods. The positive capsule staining method ( Anthony Method ) uses two reagents to stain the capsular material. The primary stain Crystal violet is applied over a non heat fixed bacterial smear so that both the bacterial cells and capsular material take up the color of the primary stain. The ionic nature of the bacterial cell binds the crystal violet stain more strongly while the non-ionic nature of the capsule get adhere with the crystal violet stain. When the decolorizing agent copper sulfate is added over the bacterial smear, the loosely adhered crystal violet stain is washed off from the capsular material without removing the tightly bound crystal violet from the cell wall. The capsular material absorbs the light blue color of the copper sulfate in contrast to the purple bacterial cell.   Negative Capsule Staining   Another simple method to visualize the bacterial capsules is by using negative staining Technique. During staining the non heat fixed bacterial smear with the acidic stains such as Nigrosin will not penetrate the bacterial cells (since both acidic stain and bacterial surface has negative charge). Instead the acidic stain deposits around the bacterial cells and create a dark back ground and the bacteria appear as unstained with a clear area around them, capsule. Note : If you heat fix the bacterial smear for capsule staining, the cells will shrink creating a hallow zone around the bacterial cell and will be mistaken for the capsule.

49. India ink preparation: Mainly use for Cryptococcus neoformans, because of its large polysaccharide capsule, can be visualized by the India stain. Organisms that possess a polysaccharide capsule exhibit a halo around the cell against the black background created by the India. Method of Use:   Mix the specimen with a small drop of India on a clean glass slide . Place a cover slip over the smear and press gently. The preparation should be brownish, not black. Using reduced examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells.

50. LIMITATIONS It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification. The diagnosis of  C. neoformans  by negative staining should be considered a presumptive result. Leukocytes, fat droplets, and tissue cells are sometimes confused with  C. neoformans  cells. Leukocytes and tissue cells may be dissolved by adding a drop of 10% KOH. Some strains of  C. neoformans , as well as other cryptococci may not produce discernible capsules  in vitro .

51. The modified technique employs 2% chromium mercury . A small drop of CSF is placed on a clean glass slide. Then, a small drop of 2% chromium mercury is mixed with the CSF on the slide. Immediately, a small amount of India ink is added. Finally, the cover slip is mounted and the preparation is observed with a bright-field microscope . Thus, three layers from the outer capsule that have previously been discerned only by electron microscopy can be distinguished, namely, the lucent stratum of the capsule, the fibrillar material of the capsule, and the light zone .

52. Congo red stain Materials Congo Red stain Acid fuchsin stain Acid alcohol Congo Red Capsule Stain Procedure 1. Place a loop-full of Congo Red on a slide 2. Mix a small amount of your organism into the drop of Congo Red . Spread the organism/dye suspension well on the slide 3 . Let the slide thoroughly air dry . 4. Fix the dried slide with acid alcohol for 15 seconds. 5. Rinse with distilled water and cover the slide with acid fuchsin for 1-5 minutes. 6. Rinse with water and allow to air dry. 7. Examine the slide under oil immersion. Cells stain red/pink, and the capsules appear as colorless halos against a dark red background

53. Congo red

54. STAINS FOR CHAMYDIA C. trachomatis  is an obligate intracellular pathogen ,that causes   urethritis ,  proctitis , trachoma , Infertility   prostatitis  and  epididymitis  in men   cervicitis ,  pelvic inflammatory disease  (PID),  ectopic pregnancy . The characteristic inclusions are H P bodies( Halberstaedter prowazek bodies) . Staining method to these bodies are: Giemsa Castaneda machhiavello

55. Giemsa stain for Chlamydia

56. Machiavello stain: Reagents: 1% Methylene Blue 1.0 gm Distilled water 100ML shelf life: 3 months storage: room temperature 0.25% Basic Fuchsin 0.25 gm Basic Fuchsin 100 ml Distilled water (H2O) shelf life: 1 month storage: room temperature 5% Citric Acid Solution 0.5 gm Citric Acid (H3C6H5O7) 95 ml Distilled water (H2O) shelf life: 2 months storage: room temperature

57. Procedure: 1. Deparaffinize and hydrate to distilled water. 2. Stain in 1% methylene blue solution overnight. 3. Decolorize in 95% alcohol. 4. Wash quickly in distilled water. 5. Counter stain in 0.25% basic fuchsin solution for 30 minutes. 6. Decolorize rapidly in 0.5% citric acid solution for 1 or 2 seconds , never more than 3 seconds. 7. Differentiate rapidly in absolute alcohol. 8. Dehydrate in 95% alcohol and absolute alcohol 3 changes each. Clear in xylene, 3 changes. 9. Mount with Permount. Results: Chlamydia bright red Nuclei blue

58. Castaneda stain  Castaneda's staining solution     Solution A        Potassium dihydrogen phosphate,   1 g        Disodium hydrogen phosphate                        25 g        Distilled water                                           1000 ml        Formalin                                                       1 ml        Dissolve the potassium dihydrogen phosphate in 100 ml distilled water and the disodium hydrogen phosphate in 900 ml distilled water. Mix the two solutions to give a buffer pH 7.5, and add formaldehyde as a preservative.

59.     Solution B        Methylene blue    1 g        Methanol        100 ml     Staining solution        Solution A      20 ml        Solution B    0.15 ml        Formalin         1 ml     Safranine-acetic acid        Safranine (0.2% aqueous solution)     1 part        Acetic acid (0.1% aqueous solution)   3 parts

60.  Procedure.    * Prepare films from infected tissue and dry in air    * Apply the stain for 3 min .    * Drain, do not wash    * Counter stain for a 1-2 seconds in safranin -acetic acid    * Wash in running water, blotdry . Elementary bodies : blue. Cell nuclei and cytoplasm: red.

61. SUMMARY: Special stain are used to see the structures of cell which are not able to stain by routine methods. Also helpful for provisional diagnosis of disease. Fluoresce method will increase the sensitivity and time saving method. Also useful to proceed further before any expensive diagnosis method is used .

62. REFERENCES Monica Cheesbrough Koneman Ananthanarayan Mackie & McCartney

63. THANK YOU