THE RETARDATION OF LYTIC PROCESSES BY COLLOIDAL .sx SILICA SOLUTION .sx S. L. CUMMINS AND C. WEATHERALL .sx From the Department of Tuberculosis , Welsh National School of Medicine , Cardiff .sx Received for publication June 13th , 1931 .sx SILICA , in colloidal solution , is known to possess curious and , at first sight , anomalous properties in relation to the lysis of red blood-corpuscles .sx On the one hand , Landsteiner and Jagic ( 1904 ) have shown that silica sol may act like a haemolytic " amboceptor , " so altering erythrocytes that they dissolve on the subsequent addition of fresh active rabbit serum ; and , on the other , Landsteiner and Stankovic ( 1906 ) have proved that a mixture of colloidal silica and ox serum inhibits the complementary action of a haemolytic system .sx One of us ( S. L. C. ) showed ( 1922 ) that colloidal silica completely protects typhoid bacilli from the bactericidal action of fresh human serum , and , in attempting to explain the mechanism of this protection , carried out experiments which suggested that colloidal silica solution prevented specific haemolysis by interfering with the action of complement , and drew the conclusion that its anti-bactericidal action is due to the same property .sx It seems probable that the factor at work in all these experiments is the fine precipitate produced by the addition of silica sol to serum the " kieselsaureeiweissfallung " of Landsteiner and Stankovic .sx As a preliminary to further investigations , it seemed desirable to study more closely the anti-bactericidal properties of colloidal silica solution in regard to relatively serum-soluble organisms on the one hand , and relatively serum-fast organisms on the other .sx The technique adopted was that used in the earlier experiments with typhoid bacilli in 1922 , and need not be described again here .sx It suffices to say that our more recent experiments , on precisely similar lines to those of 1922 , have proved conclusively that the addition of colloidal silica solution , under the condition of our experiments , completely prevents the lysis of B. typhosus ( Rawlings ) , B. coli communis and B. dysenteriae ( Flexner ) by normal human blood or fresh human serum .sx On the other hand , we have found that relatively serum-resistant organisms , including staphylococci , streptococci and tubercle bacilli , are just as numerous after exposure to the action of blood without admixture of silica , as after exposure to a mixture of blood and silica sol .sx These germs , being naturally resistant against the solvent action of blood and of blood-serum , derive no advantage from the presence of a substance capable of inhibiting " complement .sx " Another series of experiments with B. typhosus , in which the blood was left out and a similar volume of nutrient broth substituted , served to show that colloidal silica solution possesses no growth-stimulating properties in vitro .sx These preliminary tests confirmed the conclusion already formed that the action of colloidal silica solution , in preventing the destruction of serum-soluble bacteria , depends upon an interference with the lytic action of " complement .sx " Earlier unpublished observations on tryptic digestion had , however , suggested that silica sol might possess anti-lytic powers apart from this interference with complement , and so we decided to examine its effects upon the lysis of pneumococcus in ox bile a reaction in which complement is not known to play a part .sx Experiment I. .sx A fairly strong suspension of Type I pneumococcus culture in sterile normal saline emulsion was prepared and a supply of fresh ox bile obtained .sx The solution of colloidal silica employed was prepared by Dr. Tytler from water-glass ( Boots ) , according to the method described by Gye and Purdy ( 1922) .sx This solution , used throughout all the following experiments , was found to give a residue of 0.878 per cent .sx when dried , and its activity was proved by its power to agglutinate erythrocytes in salt solution , and its antibactericidal action in relation to typhoid bacilli .sx The following mixtures of the above reagents were set up in small test-tubes :sx All three mixtures were placed in the incubator at 37 C. for one hour , after which they were removed and examined .sx Naked-eye appearances :sx From this experiment it was clear that the presence of silica sol interfered to a marked extent with the lysis of pneumococci by ox bile .sx This finding , which suggested that colloidal silica solution was capable of interfering with a lytic process regarded as being independent of the presence of complement , led us back to the earlier unpublished experiments on the interference of silica sol with tryptic digestion .sx An attempt was now made to repeat these as follows :sx Experiment II .sx Allen & Hanbury's trypsin solution ( " Bacteriological in Ampoules " ) was used in a 1.8 per cent .sx solution of sodium phosphate as the digestive fluid .sx To this , in final concentrations of 1/20 and 1/40 , slices of coagulated egg-white 3 mm .sx square , and cut so thin as to be almost transparent , were added , with and without 0.5 c.c. silica sol .sx It was found that , especially with the weaker solutions of trypsin , the digestion of egg-white was markedly retarded .sx Experiment III .sx This experiment was carried out with freshly prepared fibrin .sx About 1 c.c. of freshly drawn human blood was stirred with a thin glass rod during clotting .sx The fibrin , collected on the end of the glass rod , was washed in running water until nearly free from colour .sx Two masses of fibrin , so prepared , and of approximately equal size , were added to trypsin diluted 1/25 with phosphate , with and without an equal volume of silica sol .sx The result , after two hours at 37 C. , showed that whereas the control without silica was digested , with silica the fibrin was merely broken up .sx These experiments indicated that tryptic digestion was interfered with to a considerable extent by silica sol , but suggested that the action was a retardation rather than an actual inhibition of lysis , the time factor being of importance .sx Similar conclusions were drawn from experiments in which the degree of tryptic digestion was estimated with Congo-red fibrin and also in the case of peptic digestion .sx In view of the known effects of inhaled silica dust in leading to lung fibrosis and lymphatic obstruction in those exposed to it for sufficient periods of time , and in view of the theory enunciated by Gye and Kettle ( 1922 ) , and now generally accepted , that " the fibrosis brought about by finely divided silica " is " due to the slow formation in the tissues of silica sol , " these anti-lytic properties appertaining to or associated with colloidal silica solution appear worthy of consideration in connection with the pathology of silicosis .sx All those who have examined sections from silicotic lungs are familiar with the collections of dust-cells to be seen packed tightly into , and apparently blocking up , the perivascular and peribronchial lymphatics .sx These cell collections may be seen months or years after the miner has ceased to be exposed to dust , and the question arises as to how the cells come to persist .sx Mavrogordato ( 1922 ) suggests that " there are two reactions characteristic of free silica :sx That it exerts a preservative action on the cells that take it up .sx That cells laden with free silica tend to flock together and are .sx preserved en masse , i. e. tend to agglutinate and form pseudo- .sx tubercles .sx " To explain this preservation he suggests the following hypothesis :sx " That the fine particles of free silica are slightly soluble in the alkaline tissue juices and that there is a drift of alkaline silicate through the invaded cells , the principle being similar to that of the preservation of eggs by water-glass .sx Tissue death is associated with acid autolysis and the alkaline silicate will be precipitated as silica acid , thus , perhaps , preserving the cells as ` mummies ' by impregnation .sx " The experiments above described appear to add significance to the suggestion of Mavrogordato as to the possibility of a prevention of acid autolysis of dust-cells by silica in solution .sx As to the agglutination of these cells into masses , another curious property of colloidal silica comes to mind .sx Landsteiner and Jagic ( 1904 ) called attention to the agglutination of red blood-corpuscles by silica sol in the presence of salt in appropriate amount .sx One of us ( S. L. C. ) ( 1922 , b ) independently observed and reported the same phenomenon , and he takes this opportunity of expressing his regret that , being at that time unaware of the earlier work of Landsteiner , he failed to refer to it in his paper .sx In view of these anti-lytic and cell-agglutination properties of colloidal silica , the following experiment , designed to throw further light on its effects upon a suspension of leucocytes and dust-cells , was carried out :sx Experiment IV .sx A heavy suspension of silica dust was made in sterile saline solution .sx The dust used was part of a sample given us by Dr. Mavrogordato , and represented the insoluble residue recovered from the lungs of a gold miner dying from silicosis on the Rand .sx Two c.c. of this suspension were inoculated intraperitoneally into guinea-pig No .sx 1936 on January 6th , 1931 .sx This animal was killed next day , and a sample of peritoneal fluid removed with sterile precautions , and mixed with an equal volume of sterile 3.8 per cent .sx solution of sodium citrate to prevent clotting .sx Mononuclear cells larger than the usual blood monocytes were a conspicuous feature in " wet " preparations .sx Examined under " crossed Nichols , " many of these large cells were seen to contain doubly refracting particles , presumably phagocyted silica dust .sx This suspension of leucocytes and dust-cells was divided into two portions , one , labelled A , being mixed with an equal volume of sterile normal saline solution , and the other , labelled B , with an equal volume of colloidal silica solution .sx These mixtures were incubated in sealed capillaries , and pairs of these were examined from time to time .sx The results were as follows :sx Examination of Fixed Films .sx Cell enumeration in Fuchs and Rosenthal Cytometer .sx The " counts " in Series B were always difficult owing to the large number of cells present and their agglutination in clumps .sx This agglutination seemed to become rather less marked from January 29th , 1931 , onwards , but the counts were never satisfactory .sx This experiment , by indicating that the cells were better preserved in Series B than in Series A , suggested that it was the added colloidal silica solution rather than the phagocyted silica particles which had led to the interference with the normal cytolysis observed .sx It seemed probable that the cells lost their vitality shortly after removal from the body , and that the tendency to slow solution of silica particles in phagocytes , postulated by various workers to occur in vivo , was not operative under the conditions of our experiment .sx To obtain further information on this point , the following experiment was now carried out :sx Experiment V. .sx Two guinea-pigs were inoculated intraperitoneally , one with anthracite dust in saline and the other with silica dust .sx Both these animals were killed in 24 hours , and their peritoneal fluid pipetted off with sterile precautions , citrated , and the fluid from each guinea-pig put up in two series , Series A being without and Series B with added colloidal silica solution .sx The results in both animals were similar to those obtained in Experiment IV , showing that it was the added colloidal silica solution which was operative in agglutinating and preserving the cells , rather than any silica which might have been dissolved from the quartz dust used to produce the cellular exudate .sx It was , of course , improbable that the time allowed in these experiments would suffice to allow of the slow solution of silica particles which appears to take place in the silicotic lung .sx It was for this reason that we depended on the added silica sol to obtain the effects noted .sx CONCLUSIONS .sx Following upon an earlier observation that the addition of colloidal silica solution preserves typhoid bacilli from lysis in human blood or serum , we have shown that this property extends to other serum-soluble bacteria .sx We have obtained evidence that colloidal silica solution is also able to preserve Type 1 pneumococci from lysis in ox bile .sx Our experiments show that both tryptic and peptic digestion of aIbuminous substances is markedly retarded by the presence of colloidal silica solution .sx We find that the addition of colloidal silica solution to a cellular exudate produced by the intraperitoneal injection of dust particles leads to the agglutination of the leucocytes and dust-cells and to the preservation of a large proportion of these cells , intact though probably dead , for a period of two months and upwards , beyond which time our observations have not been extended .sx