Sputum

Sputum, a pathological secretion expelled from the respiratory tract via coughing movements. Analysis of sputum is a completely obligatory moment in clinical investigation. In a number of diseases of the respiratory apparatus it allows one to establish the diagnosis quickly and accurately. In particular, timely analysis of sputum is of serious significance in pulmonary tuberculosis. Detection of tubercle bacilli in the sputum sometimes makes it possible to establish the diagnosis in cases where clinical manifestations of the disease still do not allow it to be recognized. In view of the fact that tuberculous exacerbations often run under the picture of influenza, in all cases of acute diseases resembling influenza, examination of sputum is obligatory. Collection of sputum. For investigation it is best to take the first morning sputum obtained by coughing, not by expectoration, and to collect it without any admixture in a clean bottle with a stopper. If there is little sputum, it can be collected over the course of a whole day; the bottle must be kept in a cool place. In children who are unable to produce sputum, a cough reflex is induced by irritating the area of the root of the tongue and pharynx with a probe having a cotton tampon at the end; the sputum obtained in this way is collected immediately with a cotton tampon and preparations are made from it. General properties. The quantity of sputum secreted in a day may vary from insignificant amounts to 1–2 liters. In acute diseases the quantity of sputum is at first insignificant, but by the end of the resolution stage it becomes abundant. For certain diseases, especially with cavity formation, for example gangrene of the lung, bronchiectasis, ruptured empyema, and lung abscess, the characteristic feature is the discharge of a large amount of sputum. The quantity over a whole day is measured in a graduated cylinder. Odor. Usually fresh sputum has no odor.

572 In diseases accompanied by the decomposition of sputum within the body (for example, in lung gangrene, putrid bronchitis), sputum is already excreted with a fecal-putrid odor (putrid sputum); in bronchiectases—with a sweetish-putrid odor, etc. — Properties of sputum. In lung gangrene, bronchiectases, and putrid bronchitis, sputum soon after excretion divides into 3 layers: 1) the upper layer is foamy, greenish-yellow in color with floating purulent clots; 2) the middle layer—transparent, liquid, serous in character; 3) the lower layer—greenish in color, purulent in character. Sputum in a lung abscess divides into 2 layers: the upper layer is serous, the lower layer is greenish, thick, purulent. Foamy sputum is excreted in pulmonary edema. For further investigation, one can use either 1) freshly delivered sputum or 2) fresh, but previously sterilized sputum. This method is especially convenient in small laboratories without qualified assistants. For this, an equal or slightly greater amount of physiologic NaCl solution is added to the same jar in which the sputum was delivered; one or several jars are placed in a water bath at 80–100°, and left for 20–30 minutes; the sputum becomes uniformly liquid; then it is centrifuged or allowed to settle for several hours and the liquid layer is decanted. Preparations are made from the sediment. Almost all formed elements and bacteria are very well preserved in this process. When working with fresh sputum, it is poured out in a thin layer on a glass plate with a cover, and the plate is placed on a black background. The color of sputum can be different depending on its composition, namely: whitish or yellowish-gray, when mucus predominates in the sputum; greenish-yellow, if there is a lot of pus in the sputum; green, if the sputum is purely purulent without admixture of mucus, which is very rare; green sputum can also be due to the presence of blue-green pus bacilli in it or due to its decomposition (in the latter two cases, greening occurs upon standing of the already excreted sputum); brownish-green color—due to admixture of bile; blood-red—in foamy sputum during bleeding from an injured vessel (hysterical bleeding from mucous membranes, hemoptysis on the basis of capillary hemorrhages in the respiratory tract); rusty color is characteristic of lobar pneumonia, but is also encountered in other diseases, for example, in tuberculosis; is due to chemical changes in hemoglobin; dark, dark gray—in hemorrhagic infarction, in a terminating hemorrhage (most often of tuberculous origin); sputum the color of prune broth with simultaneous liquefaction in the presence of pneumonia indicates the beginning of gangrene or pulmonary edema; sputum the color of raspberry jelly occurs during hemorrhages on the basis of neoplasms; dirty brown or dirty green color in bloody decomposing sputum is obtained due to the conversion of hemoglobin into methemoglobin or due to the formation of heme (for example, in lung gangrene). — The color of sputum can also depend on the food taken and on accidentally inhaled particles, for example, paint, coal, etc. — Consistency can be (depending on the amount of mucus) liquid, semi-liquid, viscous, thick. Viscous sputum stretches when pulled out with a spatula, while thick sputum does not stick to the spatula. Sputum is especially viscous in lobar pneumonia, during an asthmatic attack, in all inflammatory processes of the respiratory tract; in these processes, sputum is initially scanty and viscous, but later becomes liquid. If sputum contains pure pus without admixture of mucus, it is slightly liquid (for example, in diseases of the lungs accompanied by purulent softening of the tissue). Character of sputum. 1. Mucous sputum consists mainly of mucus, may be of thick or liquid consistency; in the liquid form, there is usually a large amount of air bubbles forming foam; mucous sputum is transparent. 2. In mucopurulent sputum, mucus and pus are closely mixed, which indicates their origin from one area. 3. Purulent-mucous sputum contains more pus than mucus; purulent pieces are not mixed with mucus. Greenish-yellow, round, coin-shaped clots (sputum nummulare) merge upon standing, settling on the bottom, and form layered sputum. 4. Purulent sputum is rarely excreted, usually in an abscess; it is usually of semi-liquid consistency; upon standing it divides into 2 layers. 5. Bloody sputum can be 1) purely bloody; a large amount of pure blood of bright red color, liquid consistency, foamy, or in the form of thick clots is excreted; blood from the respiratory tract has poor coagulability; the place of hemorrhage cannot be determined by sputum; 2) sputum stained with blood; blood in the form of flakes and clods joins mucous, mucopurulent, or purulent-mucous sputum; sputum is grayish-pink in color, evenly stained, consists of mucus and a large amount of saliva with admixture of blood from the gums (often in hysterics); 3) sputum closely mixed with blood can have a different appearance: a) mucous-bloody sputum of yellow to rusty-brown color, viscous consistency is characteristic of inflammation of the alveoli and the smallest bronchi; b) serous-bloody sputum, liquid contains many air bubbles, dark brown color; c) purulent-bloody sputum indicates the presence of large cavities in which pus is mixed with blood. — Blood in sputum can occur in all diseases of the respiratory tract. Biermer points out the little significance of this sign for diagnostic purposes. Macroscopic examination. In macroscopic examination, one can discover in sputum: 1) fibrinous casts in the form of dense, tree-like branched formations of various sizes. The tree-like form of them depends on the shape of the bronchi, of which they are casts. Sometimes casts have the appearance of rings without branching (casts from the larynx and trachea). Branching is better revealed if the cast is shaken in a test tube with water. The color of the casts is whitish or reddish from blood. They are excreted in fibrinous bronchitis, pneumonia, tuberculosis, actinomycosis, lung abscess, and heart defects accompanied by pulmonary edema. 2) Koch's spirals—see Bronchial asthma. 3) Tissue fragments—pieces of dense consistency of various sizes, with dark stripes in the center, and diffusely stained yellowish around the periphery. In these pieces, connective tissue and lung vessels can be discovered. They are often found in the lower layer of gangrenous sputum, in lung abscess, rarely in tuberculosis. Pieces of bronchial glands (when a tuberculous gland breaks through into the lumen of the bronchi)—yellowish color, curdy consistency. Their exact character can be determined only under a microscope. 5) Bone fragments are excreted in bloody-purulent sputum, most often when they are swallowed, but they can also be bones of the body itself. 6) Cartilage fragments are excreted most often in laryngeal, tracheal, and large bronchial processes. 7) Pieces of neoplasms are rarely encountered in bloody sputum—during the breakdown of tumors. The character of the tissue can be determined only by patho-anatomical examination. 8) Concretions—stones of grayish-white color are usually encountered in tuberculous cavities. They are excreted independently rarely. Their shape, size, and consistency can be different depending on composition. The main component of the stone is calcium and magnesium phosphate, sometimes carbonates and silicic acid salts. To study the stone, it is decalcified with nitric acid, the remaining organic skeleton is embedded in paraffin. In sections, tubercle bacilli and elastic fibers are often found. 9) Plugs, a) Lentil-shaped plugs, see Koch's lens corpuscles (corpuscula oryzoidea)—grayish-white curdy formations up to the size of a lentil grain, excreted in mucopurulent sputum of tuberculous patients during strong tissue breakdown. Usually observed in the contents of cavities. Under a microscope, they consist of a network of elastic fibers, detritus breakdown, often crystals of fatty acids and a large amount of tubercle bacilli; the latter are very often observed in the lenses in the form of pure cultures. b) Dittrich's plugs and similar plugs from the tonsils (tonsillar plugs), grayish-white color, various sizes and soft consistency. When crushed, they sometimes emit a putrid odor. They are often found in the lower part of three-layered sputum. Dittrich's plugs are encountered in gangrene, bronchiectases, putrid bronchitis, lung abscess, and less often in the sputum of tuberculous patients. Under a microscope, Dittrich's plugs and plugs from the tonsils consist equally of cellular and bacterial breakdown, crystals of fatty acids, droplets of neutral fat, pieces of coal, various bacteria, Leptothrix fungi. Blood pigment and occasionally infusoria can be found in Dittrich's plugs. Plant parasites: 1) grains of actinomycosis—yellowish formations, the size of a grain of sand, of fairly dense consistency. Under a microscope, they consist of accumulations of characteristic mycelia (see Actinomycosis); 2) mold grains of grayish-black color; consist (under a microscope) of mold fungi. — Animal parasites. Simple organisms (Monas, Segmonas, Trichomonas), hooks or bladders of echinococcus (when localized in the lung and when it breaks through from the liver into the lung), larvae of nematodes for which the lung is a stage of migration (ascaris; ancylostoma, etc.) can be discovered in sputum.

Strongylata) and finally the eggs of the trematode Ragonimus Ringeri, which localizes in the lung. Food residues enter from interdental spaces, from the lacunae of the tonsils and folds of the oral cavity, as well as due to the fact that vomiting often joins the coughing movements. Physicochemical examination. In a test tube, sputum is warmed on a water bath at 60° until it turns into a uniform liquid; a funnel is inserted into the neck of the flask to prevent evaporation of the sputum; after this, the sputum is cooled to 15° and weighed in a pycnometer. The specific gravity of sputum depends on its composition, mainly on the amount of pus and blood serum; mucus has little influence. The specific gravity fluctuates approximately from 1,006.0 to 1,037.5. Reaction of sputum is determined by litmus paper. Usually the reaction is alkaline or neutral. An acidic reaction indicates a decomposition process. Protein is present in small quantities in any sputum, but in some diseases its amount sharply increases because the walls of the alveolar vessels become pinkish and begin to let blood serum through. Qualitative determination of protein. To 10 cm³ of sputum, 20 cm³ of 3% acetic acid are added, shaken well, filtered, and the mucus remains on the filter. The filtrate is subjected to a protein reaction. Quantitative determination of protein. The filtrate obtained by the method just mentioned is used for quantitative protein determination by the Esbach or Brandberg method (see Urine). The amount of protein in sputum is considered significant by some doctors: 1) in differential diagnosis between chronic bronchitis and tuberculosis: in bronchitis the protein reaction gives only a slight opalescence, in tuberculosis it gives flocculent precipitation; of course other diseases, such as gangrene, pneumonia, etc., must be excluded in this case. According to Levenboim and Bushman, a protein content of 1°/oo and higher speaks for tuberculosis, but a lower content does not exclude it. 2) For prognosis in tuberculosis, a large amount of protein is considered a bad sign. Reaction for the presence of bile. Bile can enter sputum from the blood and from the liver if there is a communication with the lung due to rupture. Bile pigments are determined by the Obermayer-Popper method. The sputum is shaken well with a double amount of alcohol, filtered, and 1-2 cm³ of the following reagent are layered on the filtrate: 625 cm³ of water, 125 cm³ of 95° alcohol, 75 g of NaCl, 12 g of potassium iodide, 3.5 cm³ of 10% iodine tincture; in the presence of bile, a green ring is obtained at the contact boundary between the sputum and the reagent, with a small amount of bile, a bluish ring. Reaction for blood - see Guaiac test. Microscopic examination of sputum. Preparation: several small, pus-like pieces of various consistencies of sputum, as well as suspicious flakes, are selected onto the center of a clean, flamed slide. All selected particles are covered with a cover glass without smearing them, and the center of the cover glass is pressed with a blunt instrument to distribute the sputum in a thin layer. The sputum should not protrude beyond the edges of the cover glass. Platinum or cobalt spatulas must be used for taking sputum; they are flamed to red heat before and after each use. In the absence of these instruments, two spatulas made of bast splints (a thin wooden plate about 1 cm wide, 12-15 cm long; the end used to take sputum is cut at an acute angle) can be used. After each use, these spatulas are destroyed. From the sediment of liquid or sterilized sputum (after centrifugation), a drop is taken with a sterile pipette and covered with a cover glass. First, the preparation is examined at low magnification (3 Leitz), and then at high magnification (6 or 7 Leitz); any suspicious place at low magnification is placed in the center of the field of view of the microscope and the magnification is increased. Cellular elements of sputum. Squamous epithelial cells of polygonal and rounded shape with one nucleus and granular protoplasm. They are found both singly and in whole layers. Old cells lose their nuclei, and their edges become much more noticeable (cornified cells); such edges in layers of cells are very easily mistaken for elastic fibers. Individual squamous epithelial cells are found in any sputum, but large quantities of them are found in mucous sputum, especially in the initial stages of catarrh of the upper respiratory tract. These cells originate from the oral cavity, pharynx, trachea and vocal cords. Ciliated columnar epithelial cells. Cells of pear-shaped form, the narrow end is equipped with a hair, and the wide end with a whole fringe of cilia, but these hairs can be seen only in very fresh sputum, as they break off very quickly. These cells have one nucleus and finely granular protoplasm. Ciliated columnar epithelial cells are found in the mucous sputum and are almost not encountered in purulent sputum. They are found singly and in groups. They are detected during catarrhal processes of the trachea and bronchi, during asthmatic attacks, during fibrinous bronchitis, less often during tuberculosis, lobar pneumonia, pulmonary infarction, simple bronchitis, as well as during diseases of the nose and larynx if secretions from the upper respiratory tract are admixed to the sputum. Alveolar epithelial cells enter sputum in a degenerated form, and it is not possible to recognize them with certainty. Alveolar cells are considered to be cells 5-6 times larger than a leukocyte, of various forms, with one or several nuclei. Their protoplasm is filled with matte-shiny myelin balls and much more rarely with fat droplets. These cells contain various inhaled pigments, for example soot, coal, paints, etc. Then they are called dust or pigment cells. It is possible that alveolar epithelial cells and dust cells do not originate from the alveoli, but from mesenchymal (reticulo-endothelial) elements; there are authors who generally consider that there are no epithelial cells in the alveoli. The described alveolar cells are also called Buhl cells, who considered them precursors of the tuberculous process and attached great importance to their presence in sputum. It is now proven that these cells occur in any irritation of the respiratory tract, as well as in the morning sputum of healthy people. Often myelin balls, freeing themselves from cells, merge, forming droplets of various shapes and sizes, and are found in a free state in sputum (so-called myelin formations). Heart valve defect cells represent large cells of the same type as dust cells; they are filled with brownish-yellow or ruby-red pigment - hemosiderin. The pigment either diffuses the cells or fills them in the form of granules. These cells are usually found in groups; free pigment often lies between the cells. Unlike dust cells, they contain iron and give a reaction for Prussian blue: to a piece of sputum in which the mentioned cells are present, 1-2 drops of 3% HCl are added, mixed well, and the same amount of 2% potassium ferrocyanide solution is added to it. A cover glass is placed. After a while, the pigment of the cells, if it is hemosiderin, will turn blue. The same reaction can, however, be given by dust cells if the inhaled dust contains iron. Individual cells of heart defects have no special significance. These cells occur in cases of stasis phenomena in the lungs on the basis of heart insufficiency (especially with mitral defects), during pulmonary infarction, acute and chronic bronchitis, asthma, during the chronic course of tuberculosis, in short, in all cases of the appearance of blood in the lung tissue and the lumen of the alveoli. (See also Brown induration of the lung.) Leukocytes are found in any sputum. In mucous sputum they are single, and if there are very many of them, they already constitute pus. In sputum - mainly in mucous sputum - leukocytes can be well preserved in their structure. Such leukocytes take the stain well. In purulent sputum, the contours of many cells are less clear, their structure is barely noticeable, they take the stain poorly; in this case the granularity of the protoplasm is so altered that it is difficult to decide from the stained preparation to which type of leukocyte a given cell can be referred. The further degree of decomposition of leukocytes is the transformation of the protoplasm into a detrital mass, among which intact nuclei are encountered, and then finally the nuclei also decompose. In such decomposition in pus from an old cavity, in lung gangrene, in bronchiectasis, abscess, etc., many small fat droplets can be detected (Sudan III stain). From leukocytes, neutrophils are contained in the predominant amount in any purulent sputum. Eosinophils can be in single specimens in any sputum. A particularly sharp increase in their number (up to 90% of all leukocytes) is observed in bronchial asthma. In addition, an increase in the amount of eosinophils in sputum can occur in acute fibrinous bronchitis and in tuberculosis. Some authors have tried to make prognostic conclusions upon the appearance of eosinophilic cells in tuberculous sputum; however, further observations did not confirm these conclusions. Lymphocytes: an increased amount of lymphocytes is contained in sputum at the beginning of the tuberculous process, while the sputum has not yet become purulent.

According to the studies of Wolff-Eisner, the number of lymphocytes may reach 80% in such cases. In later stages of tuberculosis, the number of lymphocytes falls, while the number of neutrophils increases. Large mononuclear cells in the sputum are often impossible to distinguish from alveolar epithelial cells. Until now, their presence in the sputum is not attributed any specific significance, and their number is also insignificant. Leukocytes can, like alveolar epithelium, engulf pigment. Erythrocytes. Single unchanged erythrocytes are present in every sputum and have no diagnostic value. In hemorrhages from the respiratory organs, they are present in large quantities. In sputum, erythrocytes can be unchanged, shrunken, and leached. Leached erythrocytes are most often of pulmonary origin. It happens that during macroscopic examination the presence of blood is clearly visible, while during microscopic examination erythrocytes are either not found at all or only single ones. This phenomenon occurs because the erythrocytes have already destroyed, while the blood pigment remains. Elastic fibers are found in sputum during the breakdown of lung tissue. They are found in lentils, as well as in purulent and caseous particles of sputum. Elastic fibers are found both in an unchanged form and in the stage of degeneration and even decomposition. 1) Unchanged fibers are shiny, double-contoured, smooth throughout their length, slightly pointed at the ends, and dichotomously dividing. They are isolated in bundles of different sizes. They can have a reticular or alveolar loop structure, or they can be arranged as smooth bundles without alveolar structure; in addition, individual fragments can be encountered, but then these fragments have such an uncharacteristic appearance that they cannot be considered elastic fibers. Elastic fibers are most often found in tuberculosis, but can also be found in a whole series of other diseases leading to the destruction of lung tissue: in lung gangrene and abscess, in neoplasms, less often in syphilis. Elastic fibers can come not only from the lungs, but also from the larynx and bronchial walls. In the latter two cases they are thinner and wavy, but in appearance it is still difficult to indicate the localization of the process. With very rare exceptions, the finding of tubercle bacilli always finds elastic fibers in the sputum. Along with typical elastic fibers, degenerated Coppen-Jones fibers can be found in the sputum [see separate table (st. 71--72), fig. 4], which usually occur in old cavities, in caseous particles of sputum and resemble the bark of old wood; in places they have bulbous endings; these fibers often fold in such a way that they resemble actinomycotic друзe. Due to their coarse appearance and location, they often have only a very distant resemblance to non-degenerated elastic fibers. These fibers are covered by a special dense substance, poorly soluble in fat. To dissolve these deposits, it is best to place such sputum in a thermostat for a long time. The deposits break down into small fat droplets, and the ordinary non-degenerated elastic fibers stand out sharply. Such fiber forms are usually found in pure chronic forms of the tuberculous process of the lungs. 2) Calcified fibers have the appearance of thick, coarse, not too shiny fibers; they are isolated in not very large bundles. Ehrlich attaches great importance to these fibers, especially if along with fibers remnants of blood vessel walls, cholesterol crystals, and tubercle bacilli are found. This quartet, in his opinion, is characteristic for a cicatricial process which, for some reason, has melted again. If, however, the elastic fibers cannot be found in a native preparation, and the case is suspicious, the sputum can be processed by the Bizzozero method: 1-2 tablespoons of sputum (can be less) are mixed with an equal amount of 10% caustic potassium; the mixture is poured into a test tube and heated in a water bath until the sputum turns into a uniform liquid; in a later modification, after heating, it is recommended to add a few drops of 1% (in 70% alcohol) eosin to the resulting liquid sputum; the liquid is distributed in centrifuge tubes, centrifuged, and preparations are made from the sediment. The alkali dissolves everything except elastic and some types of plant fibers. Elastic fibers are stained in a shiny pink color, all other fibers are either not stained at all or if stained, they do not have such a shine. The diagnostic value of elastic fibers is very great, their presence always indicates the destruction of lung tissue. Fibrin consists under the microscope of parallel bundles or a network of thin fibers. To distinguish fibrin fibers from similar mucus fibers, 30% acetic acid is added to the preparation, from which the fibrin fibers become transparent, while the mucus becomes more turbid and sharply striated. Fibrin dissolves easily in chloroform, sodium sulfate solution, and lime water. Amyloid bodies (corpora amylacea) are occasionally found in sputum in lung gangrene and abscess. Under the microscope they have the appearance of round or oval homogeneous, shiny, colorless or slightly yellowish formations. They have, but not always, concentric striation and a darker core in the middle, consisting of clods. From iodine they are sometimes stained yellow, sometimes blue and blue-violet. From sulfuric acid some bodies dissolve, others do not, from alkalis the same. Some authors explain this unequal attitude to chemical substances by the influence of the sputum itself, others by the age of the bodies. - Cells of neoplasms. Several cases of finding individual cells in carcinoma and sarcoma have been described. It is very difficult to make a diagnosis based on individual cells, since in sputum these cells already appear in a changed form. Pieces that have separated and are noticeable macroscopically must be examined histologically. - Giant cells in sputum are described as large cells with 5-8 nuclei and granular protoplasm. - Plant parasites. The thrush fungus, Monilia albicans, blastomycetes (in old terminology Oidium albicans) are most often found in the mucous sputum. 1) as accidental contamination, 2) in diseases of the oral cavity, 3) in weak patients, most often in tuberculous ones, in which the mucous membranes of the respiratory tract and esophagus are affected, 4) according to recent literature, in concomitant or independent diseases of the lungs. They are stained with all basic dyes. In preparations, dichotomously branching threads with a double contour are visible; among the threads one can see conidia of spherical and cylindrical form. - Yeast fungi are often found in large quantities in the oral cavity and from there mix with the sputum, but they can also be parasites, either having caused the disease independently, or as a secondary infection in bronchiectases, gangrene, lung abscess, and tuberculosis. The place of residence of yeasts is often cavities. They are characterized by their budding forms. Mold fungi can cause independent diseases of the respiratory tract, similar to tuberculosis, but are more often observed as a secondary infection in tuberculosis, actinomycosis, and other diseases. In sputum, molds of the Aspergillus and Mucor aspergillus types appear; under the microscope they are presented in the form of threads, hyphae, which at the ends sometimes carry thickenings (fruiting hyphae) in the form of bulbs, studded with twigs with chains of spores. The fruiting hyphae of Mucor end in bags filled with spores (sporangia). Free spores, having the appearance of shiny balls, are scattered in large quantities over the preparation. - Among animal parasites, helminth eggs were described in sputum: Schistosoma haematobium, Schistosoma japonicum (in Moscow recently 2 cases of Paragonimus Ringeri were described; both in Koreans). Sharkey-Leyden-Roben crystals (syn.: asthmatic, protein Pavec-Lanker, Bizzozero-Neumann) (see. Bronchial asthma, clinic). They are found in the sputum of asthmatics, usually during periods free from attacks; in acute, stagnant and putrid bronchitis, in pneumonia, in tuberculosis, especially with difficult breathing, and also in parasitic hemoptysis. Hematoidin crystals have the appearance of rhombuses and bundles of different sizes of red-yellow color. These crystals are found in sputum in bronchiectases, in pus from an empyema that has broken into the lung, after tuberculous hemorrhages, in gangrene and abscess. The appearance of these crystals is usually observed as a result of previous hemorrhages. - Tyrosine crystals - bundles of the finest needles of greenish color; leucine crystals - balls of different sizes; they have radial and concentric stripes. Leucine crystals are stained yellowish-brown. Both types of crystals are usually found in sputum together and very rarely in cases when sputum stagnates for a long time in the lungs (in gangrene, lung abscess, and bronchiectases). Tyrosine dissolves in weak solutions of mineral acids, in alkalis, ammonia; leucine is soluble in alkalis, insoluble in HCl, alcohol, and ether. - Cholesterol crystals - very thin colorless tablets, most often with one broken corner. They are found under the same conditions as tyrosine and leucine.

When concentrated sulfuric acid is added to them, the crystals dissolve at the periphery, while the center becomes carmine-red; if Lugol solution is added to this as well, a blue, red, green, and violet coloration is obtained. - Crystals of fatty acids; single ones may be present in any sputum, but a large number is found during putrid decomposition of sputum in the lungs, in Ditrich plugs, and in tonsillar plugs. They can easily be mistaken for elastic fibers, but fatty acid crystals melt into droplets of fat upon heating. - In addition to the crystals listed, phosphate of tripel, calcium phosphate and calcium carbonate, as well as oxalates, are found in sputum. Schurmann's spirals are most often found in greenish-yellow worm-like, dense pieces of sputum that are difficult to crush. Under the microscope they appear as spirally twisted, very thin fibers; these fibers resemble a twisted braid in their arrangement, and a more brilliant central thread is sometimes visible in the middle; the spiral is usually covered with pus and epithelial cells; often crystals of Charcot-Leyden are also visible on it. Sometimes the spiral is found only as a central thread. Large Schurmann spirals can be observed with low-power microscopes, while small ones and the central thread are better seen with high-power microscopes. Bacterioscopic and bacteriological studies of sputum; experiments on animals. To determine the bacterial flora of sputum, dry stained preparations and an oil immersion system of the microscope are used. For this, opaque purulent pieces are taken onto a slide on which sputum has been poured, a piece is torn apart using two spatulas, and a small piece is taken from the very middle and placed on the middle of the slide; then a second piece is taken and the same is done, and so on. 7-8 pieces are collected on a slide and rubbed between two slides, or the smear is spread in streaks on one slide with a platinum spatula (this method was proposed by Pfeiffer). Koch and Kitazato recommend washing a piece of sputum successively in several dishes with sterilized water, each time shaking it in the water with a platinum needle. With each washing, the piece decreases in volume and finally disintegrates into the finest particles. From these particles a preparation is made by the method indicated above. The prepared preparations are dried in air and fixed by passing three times slowly through fire. Microorganisms, in particular tubercle bacilli, do not die under such a method. Scraping tuberculous sputum from a fixed preparation, when injected into an animal, causes a tuberculous process in it. For bacterioscopic studies of sputum, it is necessary to prepare a minimum of 2 preparations: one for staining tubercle bacilli, and another for Gram staining to determine other microbes, which may be a secondary infection in tuberculosis, and which may also cause independent diseases. If microorganisms in sputum lie on large flat epithelial cells, they are not taken into account, since it is quite clear that these are saprophytic microorganisms of the oral cavity and nasopharynx; the exception is tubercle bacilli, which are unlikely to be saprophytic. If a large number of various microbes from the deep respiratory passages is present in sputum, it is necessary to find out whether we are dealing with fresh sputum and whether these microbes were present in it at the moment of its discharge (putrid sputum) or multiplied accidentally while standing. Koch's test for tubercle bacilli (1882). A carefully prepared preparation (preferably on a new slide) is fixed and stained. The most commonly used staining method to this day can be considered the Ziehl-Neelsen method (see Ziehl-Neelsen method). There is a whole series of modifications of this method; for example, Gabbutt proposed after Ziehl's fuchsin to use 1/25% methylene blue on 25% sulfuric acid for 1/4 of a minute, i.e., to combine the bleaching process with additional staining. Wexelsbaum used saturated alcoholic methylene blue for 1 1/2-2 minutes for the same purpose. Sinev recommends instead of Ziehl's fuchsin solution to impregnate filter paper with 2% alcoholic fuchsin, dry it, cut it into pieces the size of the preparation; during staining, lay such a paper on the preparation, moisten it with a 5% phenol solution, and then stain as usual. - At present, the Spengler method is quite widely used for staining tuberculous bacilli: 1) heat the preparation with carbolic fuchsin until vapor appears, pour off without washing; 2) for 2-3 seconds picric acid alcohol (50 cm³ saturated aqueous ac. picronitricum + + 50 cm³ absolute alcohol); 3) rinse with 60° alcohol; 4) decolorize to a pale yellow color in 15% HN0₃; 5) rinse with 60° alcohol; 6) re-stain with picric alcohol until the preparation has a clearly yellow coloration. With all the indicated methods, tubercle bacilli will be stained red [see separate table (appendix to article Urine), figure 1]. In methods where methylene blue is used as the second stain, the background and other microbes are stained blue. According to Spengler, the background is yellow, and other microbes are not stained. Tubercle bacilli in sputum can be of the most diverse forms: long, short, thick, thin, straight and curved, clearly segmented and solid, with inclusions of small and large grains, red and black; they are rarely found in the form of unevenly stained threads and branched bacilli. In addition to true bacilli, small formations ranging from blue to black-red color can sometimes be found in sputum, which Spengler considers to be fragments of bacilli (Splitter). Using a modified Gram stain, Muh discovered an unstainable granular form of tuberculous bacilli according to Ziehl. Muh's grains resemble the finest grains of various sizes, bluish-black in color, located singly, in clumps or rows, and retaining the structure of bacilli. The practical significance of finding Muh's grains in sputum is none, since it is almost impossible to distinguish them from the cocci usually present. The type of bacilli (human, bovine or avian) in sputum cannot be determined from the bacterioscopic picture alone. To diagnose tubercle bacilli in sputum, it is necessary to be sure that they are not only acid-fast but also alcohol-resistant, since in sputum, although relatively rarely, acid-fast bacilli are encountered that have nothing in common with tubercle bacilli except acid resistance, which are easily decolorized by alcohol. These bacilli are found in the crevices of tonsils, as well as in rhinoscleroma, pulmonary gangrene, bronchiectasis and putrid bronchitis, but sometimes these bacilli are also difficult to distinguish from true tubercle bacilli both by morphological picture and by their fairly strong alcohol resistance. It is necessary to resort to cultures and experiments on animals to clarify this. The bacilli in sputum are arranged singly and in groups. Quite often they can be found in the cytoplasm of leukocytes; attempts were even made to attach prognostic significance to this sign. The amount of tubercle bacilli in sputum can be extremely variable. Sometimes in a series of preparations it is possible to find 2-3 bacilli, sometimes they cover the preparation like a pure culture. In the same portion of sputum, there may be no bacilli in one part, and many in another. Excretors of bacilli may not excrete them in all portions. In weak 19 patients who are unable to cough sputum from a cavity, bacilli may not be found at all. With hemoptysis, when pure blood, not sputum, is discharged, and with tuberculosis diseases that have joined with abundant sputum discharge, sputum from the cavity does not have time to be discharged, and Koch's bacilli may not be detected at all. Finally, in the cavities themselves and in long-stagnant sputum, tubercle bacilli may undergo autolysis, etc., therefore one cannot trust a single examination of sputum. - In cases where bacilli are not found with ordinary methods, sputum can be subjected to special treatment. Many methods have been proposed for this, but they all come down to converting sputum into a liquid state, so that single bacilli, scattered in different parts of purulent sputum, can be collected by sedimentation into a small sediment, from which smears are made. Sputum liquefaction can be obtained 1) by applying high temperature, 2) by the method of digestion, 3) by combining high temperature treatment with chemical treatment, and 4) by treating sputum with chemical substances. Where smears are carefully prepared and examined, sedimentation methods give very little advantage; much better results are given by repeated examination of different portions of sputum. - Biedert's method: 1 tablespoon of sputum is diluted with twice the amount of water and 4-8 drops of caustic alkali solution, well mixed, after which it is left to stand for 5 minutes; then, with gradual stirring, another 4-6 tablespoons of water are added; the mixture is boiled until it turns into a uniform liquid; the resulting liquid is either poured into a conical glass for prolonged settling, or poured into centrifuge tubes, centrifuged, and from the sediment preparations are made in the usual way, previously smearing the slide with the same, but untreated sputum, or with a 1% aqueous solution of egg white, so that the sputum smear adheres to the glass.

Ulenhut's method: 1 part of sputum in a sterilized jar is mixed with 1-2 parts (depending on the viscosity of the sputum) of a 25% antiformin solution (a mixture of sodium hypochlorite and caustic alkali taken in a certain ratio); it is shaken vigorously. The mixture can be left at room temperature in an incubator or on a water bath at 56° until completely dissolved, after which destilled water is added in an amount of x/5 of the volume of the mixture and centrifuged for x/2 hours in a centrifuge giving 2,000 revolutions per minute. The liquid layer is poured off by quickly inverting the test tube, after which, without bringing the test tube back to a normal position (holding the conical end upward), it is left for 5-10 minutes on a pad of filter paper to extract as much water as possible from the sediment. A not too thin preparation is made from the resulting sediment and stained in the usual way. Antiformin (even a 2.5-5% solution of it) dissolves mucus, pus, and all bacteria very quickly, except for tubercle bacilli, which die only after 12-24 hours in a 15-20% antiformin solution. Kozlov's method -- see Antiformin. Bacteriological research. The technique for obtaining tubercle cultures from sputum is so simple that the application of this research method should find the widest distribution in everyday practice in all suspicious cases where tubercle bacilli have not been detected by bacterioscopy; the percentage of positive findings increases significantly in this case. For obtaining tubercle cultures from sputum, Gon's method (Nouhin) is best to use. This method is based on the ability of tubercle bacilli to withstand the action of acids of high concentration, in which other microorganisms die quickly. For sowing, Luebennau's egg medium is used, which is prepared as follows: very fresh eggs are wiped with alcohol and opened with a sterile forceps. The contents of the egg are poured into a flask with glass beads. It is shaken thoroughly and 30% by weight of 5% glycerine acid broth (pH 6.3) is added. The mixture is shaken thoroughly, poured into test tubes, which are placed in an incubator in an inclined position for 2-3 hours at 85° (to coagulate the mixture). Then 0.5 cm³ of artificial condensation water in the form of the same broth is added to each test tube that has dried during coagulation. The medium is placed in an incubator for two days to check sterility; 2-3 cm³ of fresh sputum (if only a small amount of sputum is delivered, less can also be used) is collected in sterile utensils, poured into a sterilized jar with glass beads or broken glass, and 5-6 cm³ of 8% sulfuric acid is added to it; if there are many foreign microbes in the sputum, the percentage of acid is increased (up to 12-15%), the mixture is shaken vigorously to break up individual lumps. When the sputum turns into a homogeneous mass, it is poured into a centrifuge test tube and centrifuged. From the moment the acid is added until the end of centrifugation, 20 minutes must pass, after which the liquid layer is poured off by quickly inverting the test tube, and the sediment is sown on 6-8 test tubes with Luebennau's medium using a platinum loop or a sterile Pasteur pipette; it is necessary to ensure that condensation water is present in the nutrient medium (condensation water can be renewed, each time checking the test tubes in the incubator). Since the sowing stands in the incubator for a long time, the test tubes with the sowing are closed with wax cakes so that the nutrient medium does not dry out. For this, a mixture of 2/3 wax and x/3 paraffin or pure wax is melted in a saucepan and poured little by little onto a clean glass plate. The liquid, spreading, hardens into round cakes. By heating the edge of the test tube with the sowing from above, the cake is glued on. Growth at 38° is usually obtained between the 7th day and the 3rd month. If no visible growth is obtained in this period, a scraping is still made from the nutrient medium and the preparation is stained according to Ziehl-Nielsen, because there are cases where even in the absence of visible growth, tubercle bacilli cultures were obtained in the preparations. Subcultures from such cultures give good growth. It is also possible to isolate tubercle bacilli cultures from sputum on glycerine potato according to the method of Levantein and Sumiyoshi, but this method is more complex. Experiments on animals. For experiments, it is most convenient to use half-grown guinea pigs; they become infected with tuberculosis faster than adults. Since there are usually foreign microorganisms in the sputum that can kill the animal before the tuberculous process begins, the sputum must be preheated for half an hour on a water bath at 53-55°; with the warmed sputum, a suspension is made in sterile physiological solution and injected under the skin of the groin. Bloch recommends crushing the regional lymph nodes (in this case the inguinal nodes) between the fingers before injecting the suspicious material under the skin; after 7-14 days, a lymphadenitis with tubercle bacilli is usually obtained (the glands can be punctured or removed and examined). After three months, the animal is killed and its organs are examined, in which usually both noticeable macroscopically tubercles and tubercle bacilli can be detected by microscopic examination. From the organs, smears and sowing can be made according to Gon's method. When animals are infected with acid-resistant, non-tuberculous bacilli, the animals survive. When the animals are killed, neither the histological nor the bacterioscopic picture characteristic of tuberculosis can be detected. To determine other microorganisms in the sputum, which may be either the causative agents of a mixed infection with tuberculosis or cause independent diseases, preparations stained according to Gram are used. It should be said that in sputum a single species is rarely encountered, but almost always, along with a large number of microbes of one species, others are also found, often as a mixture from the upper respiratory tract. In particular, in cavities in tuberculosis, as long as the cavity is very small and does not communicate with the external air, there are usually only tubercle bacilli; but as soon as the cavity becomes open, microbes inhaled with the air get into it, which find here a favorable soil for their development. In non-tuberculous cavities, the causative agent of the disease multiplies first, and later it may be displaced by putrefactive microorganisms. For diagnostic purposes, in most cases bacteriological research of sputum (isolation of microbes by sowing) is not required, but cultures of the causative agents of the disease are often needed for the preparation of autovaccines. For sowing, selected lumps of sputum, preferably purulent ones, are washed in several portions of sterilized 0.85% physiological NaCl solution to wash away mucus and saprophytic microorganisms of the oral cavity, and sowing is made from the washed sputum: 1) when a vaccine is not required from a single species, but from all existing species, sowing is made on several test tubes with blood agar and with glycerine agar (these two media are suitable for most microbes); 2) if a certain species is needed, then a special nutrient medium for this microbe is taken, poured into 2-3 Petri dishes and sowing is made; a piece of washed sputum is smeared over the surface of one dish; this same piece is transferred to the second dish, and so on. The required microbes are subcultured from the dishes onto test tubes to obtain the given microbe in pure form. Pneumococci [lanceolate, capsular diplococcus of Frankel-Wexelbaum (see standard table, attached to the article Urine, figure 3)] - see Diplococci, Microorganisms, Pneumococci. Streptococcus pyogenes (see Microorganisms, Streptococci) is contained in sputum in lobar pneumonia, bronchitis, lung abscess, lung gangrene, and in bronchiectasis, both as the causative agent of secondary infection in tuberculosis and as a saprophyte on the squamous epithelial cells of the oral mucosa; in sputum short and long chains are encountered. Hemolytic, pyogenic and erysipelas streptococci (Streptococcus erysipelatos, S. haemolyticus, S. pyogenes), mucous (Streptococcus mucosus) are encountered in the mouth and pharynx. Staphylococci (see) are detected in sputum in the same cases as streptococci. Hafk's tetracocci (see Microorganisms) are most often encountered as a secondary infection in tuberculosis, especially in the cavitary process, as well as in bronchiectasis, gangrene, abscess, and in simple and fibrinous bronchitis. In the mucous layer of sputum, as saprophytes, Micrococcus catarrhalis Pfeiffer (see Microorganisms) are encountered together with other pathogenic microbes (tuberculosis, influenza, whooping cough). Friedländer's diplobacilli (1882) are found in sputum in lobular and lobar pneumonias, bronchopneumonias, pleurisies, bronchiectases, and in tuberculosis as a secondary infection. As saprophytes they are permanent inhabitants of healthy mucous membranes, usually of the nose. Influenza bacilli (Bacteria influenzae, see Influenza) are usually present in large quantities in sputum; the impression is given that the preparation is strewn with them. They are located intra- and extracellularly. Gram-negative. They are stained in additional dyes paler than other microbes and cell nuclei, but more sharply than mucus. Very fresh sputum is required for sowing.

One may resort to the method of cough sowing (Hustenaus-saatmethode): a cup with prepared potato broth is held 10 cm from the patient's mouth and the patient is asked to cough. The diphtheria bacillus (see) is stained only at the poles with toluidine blue: 5.0 g of toluidine blue of Grüber, 100 cm³ of absolute alcohol, 500 cm³ of distilled water are diluted on cooling, 500 cm³ of 5% carbolic water are added; it is left to stand for 1-2 days and filtered. It is stained for 2-3 seconds. The Pseudomonas aeruginosa (Vas. pyocyaneus Jessar) (see Microorganisms) is encountered as the causative agent of secondary infection in tuberculosis. Sputum containing this bacillus is colored in an unusual green and emits a characteristic odor. The anthrax bacillus (see) is encountered in anthrax pneumonia. Plague bacilli (see) are usually detected in bloody or serous-bloody sputum in pulmonary plague. Sputum preparations obtained are recommended to be fixed by the Zobelheim method: absolute alcohol is poured dropwise onto the preparation for one minute, the alcohol is ignited and quickly extinguished. They are stained with the following composition: 2.0 g of methylene blue, 0.5 g of borax, 100 cm³ of distilled water. According to Gram, plague bacilli are decolorized. It is most convenient to isolate plague bacilli on neutral or weakly alkaline gelatin at 20-22°, since other microbes present in the sputum do not multiply at such a temperature. Clumps of sputum without washing are applied to the surface of the gelatin. After 2-3 days, fat-like colonies with a coarse-grained center, rising above the surface of the medium, appear on the surface of the gelatin. A rough, transparent, jagged border is present around it. The gelatin does not liquefy. - Animal experiments. Suspected material is rubbed into the shaved skin of the abdomen of a guinea pig. After four to five days the animal dies. Material for sowing can be obtained from the buboes after 24-48 hours. The obtained culture is recognized by agglutination. In addition to the listed microbes, intestinal bacilli may be detected in sputum during pneumonia (most often in infants), typhoid bacilli (typhoid pneumonia), diphtheria bacilli, leprosy bacilli, meningococci, and gonococci. In putrid sputum [see separate table (appendix to article Urine), Fig. 2], which is already excreted in a decomposed form in pulmonary gangrene, putrid bronchitis, and bronchiectasis, together with the true causative agents of the disease, numerous spindle-shaped bacilli (Vas. fusiformis), Proteus bacilli, pseudodiphtheria bacilli, diphtheroids, acid-fast bacilli, and spirochetes are encountered in large numbers, and usually several different species of microbes are detected together. With such decomposed sputum, a Gram-stained preparation is especially necessary, since with all other methods microbes from such sputum do not stain at all. The material may give the impression of being completely sterile, but upon Gram staining the abundance of microorganisms is striking. - Pulmonary gangrene can be caused by a homogeneous microbe: the bacillus of gas gangrene (Vas. perfringens) of Welch, morphologically similar to the anthrax bacillus. According to Gram, it stains positively only in tissues and young cultures: preparations from a three-day culture already give decolorized bacilli. Fungi. Actinomycetes are described in man by Israel (1878); they are encountered in mucopurulent and purulent-mucous sputum, often with admixture of blood. To study the fungus in its stained state, the conidia are rubbed between two glasses and stained according to Gram [see separate table (appendix to article Urine), Fig. 4]. In its stained state the fungus gives a network of Gram-positive filaments, sometimes clearly segmented, of varying thickness; some filaments give branching. The filaments end in bulbous thickenings. Upon additional staining with eosin the bulbs are colored violet in the center and pink at the periphery. Some filaments break down into bacilli. Liske also refers Streptothrix, which does not form bulbs at the ends and does not form conidia, to actinomycosis. Branched filaments without bulbs are frequently encountered in sputum from the upper respiratory tract as saprophytes, but they are also encountered as a pathogenic fungus. - Actinomycetes in sputum are encountered both as aerobes and as anaerobes. The isolation of actinomycetes in culture was very difficult until recently, but in 1930 Helzer proposed an extremely simple and good method for isolating culture: the examined sputum in a sterile vial with glass beads is covered with approximately twice the volume of glycerin, shaken well, and left for 24 hours in a thermostat at 37°, after which a sowing is made from this mixture onto 3-4 agar plates or into several test tubes. For sowing onto a nutrient medium, large quantities of glycerin with sputum must be poured, spread over the surface, after which the excess material is drained as much as possible. The plates are placed in a thermostat at 37° for 2 days as usual. Among the relatively small number of other colonies, small porcelain-like white colonies are noticeable, which are easily separated from the nutrient medium; they consist of clearly segmented individuals resembling streptococci, sometimes giving branching and ending in thickenings; they grow less frequently in the form of pseudodiphtheroids and even less frequently in the form of branched filaments; but in the latter case the colonies are leathery, firmly adherent to the nutrient medium and difficult to smear.