Inquiry 5 страница

Gram staining. Fire-fixed smear is covered with a strip of blotting paper, and carbolic solution of methyl violet is placed on top. The paper is removed in 1.5—2 minutes and the smear is covered with Lugol's solution for 2 minutes. The solution is then removed and the Preparation is placed in 96 per cent alcohol for 30-60 seconds (until the stain stops dissolving). The preparation is then rinsed in water and given another staining by carbon fuchsine solution (1: 10 dilution) for a minute.

Several microbes can be differentiated by Gram staining. These are Gram-positive capsulated pneumococcus, streptococcus, staphylococcus, Websiella (gram-negative capsulated Friedlaender's diplobacillus, Plate y)> Pfeiffer's bacillus, etc. All these microorganisms occur in small quan­tities in the sputum of the respiratory ducts of healthy persons and only become pathogenic under certain unfavourable conditions when they cause

\1

Special Part

Chapter 5. Respiratory System

pneumonia, lung abscess, bronchitis, etc. Their amount in the sputum then increases markedly. But the quantitative factor is however not decisive for their pathogenicity.

If bacterioscopy does not reveal the expected causative agent, the sputum is used to inoculate the nutrient medium. Bacteriological studies help identify the microbe and determine its virulence and drug-resistance. This is necessary to prescribe a correct medicamentous therapy. Finally, if the described simple procedures fail to identify the causative agent, the sputum is used to infect an experimental animal.

Study of the pleural fluid. The amount of fluid contained in the pleural cavity of a healthy person is insignificant. Its composition is close to that of lymph. The fluid serves as a lubricant to decrease friction between the pleural membranes during respiration. The volume of pleural fluid may in­crease in disordered circulation of the blood and lymph in the lungs. This can be either transudate (of non-inflammatory origin) or effusion (occurr­ing in inflammatory affections in the pleura). Effusion can also be due to clinical causes such as primary infection of the pleura or it can be a symp­tom attending some general infections and some diseases of the lungs or mediastinum (rheumatism, infarction, cancer and tuberculosis of the lungs, lymphogranulomatosis, etc.). The pleural fluid is studied in order (1) to determine its character (transudate, effusion, pus, blood, chylous fluid); (2) to study the cell composition of the fluid in order to obtain infor­mation concerning the character of the pathology and sometimes its diagnosis (when cancer cells are detected); (3) to reveal the causative agent of an infectious disease and to determine its sensitivity to antibiotics. Analysis of the pleural fluid includes macroscopic, physicochemical, microscopic and sometimes microbiological and biological analysis.

The appearance of the pleural fluid depends mostly on its cell composi­tion and partly on the chemical composition. Fluids of the following character are differentiated: serous, serofibrinous, fibrinous, seropurulent, purulent, putrefactive, haemorrhagic, chylous, and chylous-like.

Transudate and serous effusion are clear and slightly opalescent. Tur­bidity of the fluid may be due to abundance of leucocytes (seropurulent and purulent effusion), erythrocytes (haemorrhagic effusion), fat drops (chylous effusion) or cell detritus (chylous-like effusion). The character of the cells can be determined by microscopy. The chylous character of the ef­fusion is determined by an ether test (opacity disappears in the presence of ether). This fluid can be due to congestion of lymph or destruction of the thoracic duct by a tumour or an injury. The chylous character is given to the pleural fluid by fatty degeneration of cells contained in ample quantity. Fat is stained with Sudan III in both cases.

The colour of transudate may be pale yellow, serous effusion from

yellow to golden, and in jaundice it may be deep yellow. Purulent effusion is greyish or greenish-yellow; in the presence of blood it becomes reddish or, more frequently, greyish-brown. The putrefactive effusion is of the same colour. Depending on the amount of the haemorrhage and also on the time of blood retention in the pleura, the haemorrhagic fluid can be pink to dark red or even brown. In haemolysis it may have the appearance of lacquer. Chylous effusion looks like thin milk.

The consistency of pleural effusion is usually liquid. Purulent fluid can be thick, cream-like, and sometimes it enters the puncture needle with dif­ficulty. Pus of the old encapsulated empyema can be of puree consistency, with grains, and fibrin flakes.

Only putrefactive effusion has offensive smell (gangrene of the lung). The smell depends on protein which is decomposed by anaerobic enzymes.

Physicochemical studies of the pleural fluid include determination of relative density of the fluid and protein; these are the main criteria for dif­ferentiation between the effusion and transudate. Relative density of the pleural fluid is determined by a hydrometer; a urometer is normally used for the purpose (see " Analysis of Urine"). Relative density of the tran­sudate is about 1.015 g/cm³ (1.006-1.012), and of the effusion is slightly higher, i.e. 1.018-1.022.

Protein content is lower in transudate than in the pleural fluid, i.e. not higher than 3 per cent (usually 0.5-2.5 per cent). The pleural effusion con­tains from 3 to 8 per cent of protein. A refractometric method is more suitable for determining protein in the pleural fluid, but some other methods can also be used, such as biuretic, gravimetric, Roberts-Stolnikov method (see " Analysis of Urine") and others. The composition of protein fractions of the pleural fluid is about the same as of blood serum. Albumins prevail in transudate while fibrinogen is absent or almost absent for which reason transudate does not clot. The fibrinogen content of pleural effusion is lower than that of blood (0.05-0.1 per cent) but its quantity is sufficient to clot spontaneously most of them. The total protein content of transudate rarely reaches 4-5 per cent and additional tests are therefore used to differentiate it from the pleural effusion. Rivalta's reac­tion; a cylinder is filled with water acidified with a few drops of acetic acid; 1 or 2 drops of the punctate are added; as effusion sinks to the bottom it leaves a cloudy trace (like cigarette smoke), while in case of transudate the reaction is negative. Lucaerini test: 2 ml of hydrogen peroxide (3 per cent solution) are placed on a watch glass (against a black background) and a drop of the punctate is added: opalescence appears in case of the positive reaction. Both reactions are used to detect the presence of seromucin in ef­fusion. This is a mucopolysaccharide complex which is absent from tran-sudates.

Special Part

Chapter 5. Respiratory System

Microscopy is used to study the precipitate of the pleural fluid obtained by centrifuging. The fluid may clot before or during centrifuging, and the precipitate becomes unsuitable for examination because most of its cells will be captured in clots. To preclude clotting, sodium citrate or heparin is added to the test fluid. Precipitate cells are studied by several techniques Studied are native preparations, dry smears stained after Romanovsky-Giemsa or Papanicolaou. Fluorescence microscopy, histological studies of the precipitate in paraffin, or cell culture are used to detect tumour cells.

In order to prepare a native preparation, a drop of the precipitate is placed on an object glass and is covered by a glass. The preparation is ex­amined with a dry system in a simple or a phase-contrast microscope. The quantity of formed elements is then assessed (many, moderate number, few). An accurate calculation of leucocytes and erythrocytes is unimpor­tant because their quantity in the preparation depends largely on the dura­tion and speed of centrifugation. A small quantity of erythrocytes can be contained in any punctate because of puncturing of the tissues. Their number is high in haemorrhagic effusion in patients with tumours, injuries, and hemorrhagic diathesis. The leucocyte count is high in bacterial infec­tions of the pleura. Leucocytes are scarce in transudates, which contain many mesothelium cells. Exudates sometimes contain cells suspected for tumour, but it is difficult to determine their nature in native preparation. The precipitate containing minimum supernatant liquid is used to make a smear. The elements of the precipitate, i.e. neutrophils, lymphocytes, eosinophils, monocytes, macrophages, mesothelial cells and tumour cells, can be differentiated by colour.

Leucocytes of pleural fluids look like those of blood. Mesothelial cells are large, round, spheric or polygonal, rarely with 2 or 3 nuclei. The round nucleus, with a rather gentle chromatin network, sometimes contains a nucleolus. The cytoplasm is blue and often vacuolized. Macrophages differ from monocytes by the presence in the cytoplasm of products of phagocytosis. The properties of tumour cells are the same as described in the section " Study of Sputum". It is very difficult to determine tumour cells in the pleural fluid, because in long-standing and sometimes acute pleural affections (and also in transudates) mesothelial cells acquire many properties that are inherent in blastoma cells. Luminescent microscopy helps in this situation: when stained with rhodamine, acridine orange or some other fluorochromes, tumour cells luminesce differently than the normal cells.

Neutrophils are found during the first 5-7 days in effusions of any aetiology. In tuberculous and rheumatic pleurisy, neutrophils are replaced by lymphocytes. Effusions rich in neutrophils are characteristic of pleural affections with pyogenic flora. Effusions containing considerable (sometimes prevailing) quantities of eosinophils also occur.

Transudates used for microbiological studies are as a rule sterile but _tney can be infected during repeated paracenteses. Effusion may be sterile (e.g- iⁿ rheumatic pneumonia or lung cancer). Mycobacteria are usually not found bacterioscopically in serous effusion of tuberculous aetiology, but inoculation of the nutrient medium of guinea pigs with the effusion gives sometimes positive results. The bacteria can often be detected by bacterioscopy of Gram-stained smears in pleurisy caused by pyogenic flora. Otherwise inoculation of media is required. In addition to pneumococci, streptococci, staphylococci, and enterococci, effusions con­tain also Klebsiella organisms, Pfeiffer's bacilli, colibacilli, etc. In order to prescribe a correct therapy, the revealed microbes are tested for antibiotic sensitivity.

Study of pleural washings. This is necessary to reveal tuberculosis mycobacteria in them (e.g. in patients who do not expectorate sputum), or to detect malignant tumour cells. The patient should lie on the affected side. His pharynx and larynx should be anaesthetized with a dicaine solu­tion, and then 10-12 ml of warm isotonic sodium chloride solution are slowly injected into the larynx and the trachea using a laryngeal syringe. The solution irritates the bronchial mucosa to cause cough and expectora­tion of mucus. The expectorated washings are collected in a sterile vessel. Mycobacteria are detected in them by the flotation method or by inocula­tion of a nutrient medium. To prepare material for cytological studies, the washings are centrifuged and native preparations and smears are prepared from the precipitate. Native preparations are inspected in a common phase-contrast microscope, or in a fluorescence microscope (after fluorochrome treatment). Smears are stained after Romanovsky-Giemsa (or by fluorochromes) for fluorescence microscopy.