Frederick L. Gates

A Method of Proteolytic Enzyme Titration.

In 1906 Müller and Jochmann 1 proposed a simple method of determining the presence of proteolytic enzymes in biological fluids by erosion of the surface of a Loeffler serum plate under a drop of the fluid. Later this technique was modified by the use of a gelatin plate dyed with carmine, but at best it was a + or − method, probably because the substrate was not sufficiently uniform to permit quantitative estimates of enzyme action. If a photographic plate, or film, is fogged on both sides by equal exposures, is developed, fixed, washed, and dried, it presents a gelatin surface and texture of sufficient uniformity for quantitative tests. Proteolytic enzymes erode the surface and free the included silver, so that the light transmission of the exposed area increases as digestion proceeds, and can be estimated relatively by colorimetry, or directly by a calibrated thermopile and galvanometer. The method is as follows: Small paraffined chambers are filled with trypsin or pepsin solution so that the meniscus projects above the rim. Face down over each chamber is placed a small square of the photographic plate, which has been previously brought to the proper pH concentration in phosphate buffer solution and allowed to dry without rinsing. Due to the inverted position of the gelatin layer the products of proteolysis and the released silver fall away from the surface, and interfere less with the progress of the reaction than as if they accumulated there. Temperature is carefully controlled, and plates are removed at timed intervals, rinsed quickly in cold water, immersed in a bath of the opposite reaction to arrest enzyme action, and dried rapidly before a fan.

Some factors affecting the levels of the serum calcium and phosphorus of normal rabbits

At intervals throughout the past year, with the exception of the summer months, we had occasion to make a considerable number of determinations of the inorganic calcium and phosphorus content of normal rabbitssera. The results are of interest in that they reveal certain progressive variations due apparently to seasonal changes in the environment, and they also show an effect of caging that should be taken into account. As received from various dealers, 144 male rabbits showed a blood calcium level of 12.31 mg. per 100 cc. of serum. 1 This general average was obtained, however, from figures that varied considerably for different months of the year, (Table I.) From a low level of 11.74 mg. in January, high levels were reached in May and November of 12.92 and 12.88 mg. Similar variations were found in the blood calcium level of 82 normal rabbits (110 determinations) which had been caged, individually, indoors in a well lighted and ventilated room, and fed hay, oats and cabbage for periods of 1 to 7 weeks. The figures for these rabbits parallel those obtained on admission, but show the blood calcium to be at a uniformly higher level—13.12 mg. per 100 cc. serum—with the January low level at 12.40 mg., and the May and November peaks at 13.62 and 13.47 mg. respectively. We have already noted 2 that the caging of normal rabbits, under conditions that obtain with us, results in a definite rise in the blood calcium. It remains to be seen how the apparent seasonal variations in the blood calcium can be correlated with the changes in the size and functional activity of certain elements in the endocrine system which Drs. Brown, Pearce and Van Allen have observed at the same seasons of the year. 3

A preliminary survey of the effects of ultra violet light on normal rabbits.

With no prejudice regarding the results to be obtained we have exposed five series of normal rabbits to the quartz mercury arc lamp daily for periods of three to seven weeks and observed at autopsy the weights of certain endocrine glands in relation to the body weights of the animals. It was expected that this survey might orient us for further studies in the biophysics of ultra violet light. The survey was made possible by the cordial cooperation of Doctors Brown, Pearce, and Van Allen, who placed at our disposal unpublished figures from similar examinations on a large number of normal rabbits covering a period of two years. Many controls are required because of individual and especially seasonal variations in the gland/body weight ratio. Light colored or albino male rabbits, with ears shaved and backs clipped, in groups of eight (two controls), were exposed to the unfiltered radiations of a quartz mercury arc lamp for 30 minutes daily at a distance of one meter. This arbitrary dosage was probably too large at first and relatively ineffective during the final periods, when pigmentation had occurred. It caused a marked erythema with some desquamation, followed by moderate pigmentation. At the expiration of the chosen periods the rabbits were etherized and bled from the aorta. The following glands were weighed and examined in gross and microscopically: pineal, hypophysis, external parathyroids, thyroid, thymus, adrenals, and testicles. The weights of these glands were recorded in milligrams per kilogram net body weight after removel of the gastro-intestinal tract and its contained food and feces, and the expression of bladder urine. The changes in weight, per cent, compared with the figures for 15 to 36 normal control rabbits examined at the same season of the year are shown in the accompanying table.

The quantitative action of ultraviolet light on Staphylococcus aureus.

By means of a compensated thermocouple of extreme sensitivity kindly made for us by Professor Pfund of Johns Hopkins University, direct measurements have been made of the ultraviolet light energy, at various wave lengths, required to kill Staphylococcus aureus. The organisms were exposed on agar plates to the lines of the quartz mercury arc spectrum, isolated by a monochromatic illuminator with quartz lenses and prism. After incubation, counts were made of the colonies produced by surviving organisms in the exposed areas. Comparison with control counts from adjacent, unexposed, areas shows that the bacteria vary widely in individual susceptibility so that the most resistant may survive an exposure 12 to 20 times that to which the least resistant succumb. The average values of a number of observations at each wave length produce a smooth curve which shows the ratio between the incident energy in ergs per square millimeter and the percentage of organisms killed. The light energy required to kill 50 per cent of the exposed bacteria, at each wave length, has been taken as the mean value of the bactericidal effect of incident ultraviolet light. Measured thus, as incident energy, wide differences were found in bactericidal power at different wave lengths of the mercury arc spectrum. The lethal energy for 50 per cent of the exposed organisms ranged from 154 ergs per square millimeter at 2378 A.U., through 88 ergs at 2675 A.U, to 3,150 ergs at 3022 A.U. In a single determination the corresponding light energy at 3126 A.U. was 25,000 ergs per square millimeter. Intermediate wave lengths gave intermediate values in an orderly sequence and a line through the observed points produced a curve similar to, but not identical with, that for the absorption of ultraviolet light by S. aureus in the same region of the spectrum.

Endocrine Glands of Rabbits Fed Cod Liver Oil or Irradiated Cholesterol.

Studies on the indirect effects of ultra violet light on growth, and its therapeutic action in rickets and tetany in children have received new impetus recently from the independent observations of Hess and Weinstock 1 , and of Steenbock and his collaborators 2 that diets which are deficient in certain respects may be made complete by irradiation with light of short wave lengths, or by the addition of certain irradiated substances, such as cholesterol. The light of the quartz mercury vapor lamp has been found to have definite secondary effects upon some of the endocrine tissues, also, producing a notable hypertrophy of the hypophysis and the (external) parathyroid glands of normal rabbits 3 . The question arises whether the affected glands mediate some of the metabolic effects ascribed to the radiations, and whether irradiated food materials, which appear to have effects similar to those of ultra violet light on growth and in disturbances of calcium and phosphorus metabolism, also act by a specific stimulation of certain endocrine glands. In these experiments we fed Norwegian cod liver oil, or irradiated cholesterol, to adult male rabbits for periods of several weeks, and at autopsy compared the weights of certain of their endocrine organs with those of normal rabbits, and of rabbits irradiated with a quartz mercury vapor lamp over similar periods of time. Experiments I and II. Cod liver oil. In addition to their regular diet, two groups of 6 and of 10 normal adult male rabbits (one group studied in April, the other in December) received 2 to 4 cc. of cod liver oil daily for periods of 3 to 4 weeks. Two groups of 17 and 14 normal rabbits, from the stock of Drs. Brown, Pearce, and Van Allen, examined coincidently with the experimental animals, served as controls.

Collodion sacs for aërobic and anaërobic bacterial cultivation

The collodion sacs demonstrated before this society a year ago, 1 while suitable for intraperitoneal implantation are not so well adapted to microbic cultivation in vitro. We have therefore been making sacs of 5-10 c.c. capacity in test tubes lined with a dried film of gelatin 2 which softens in warm water and permits the easy removal of the collodion membrane. The sac is slipped on to a supporting glass tube, inserted into one limb of a V-shaped tube, open at both ends, and sealed in place with a collar of rubber tubing. Sac and V tube are partly filled with water, plugged with cotton and sterilized in the autoclave. Shrinkage during sterilization may be avoided by maintaining a pressure of 10-12 cm. of water in the sac. The sac may even be expanded by this method, but its permeability apparently is not thereby increased. After sterilization the chosen medium is placed within the sac, and dialysis of nutritive and growth-promoting substances occurs into the surrounding fluid, which is accessible for inoculation through the other limb of the V tube. For anaärobic cultivation both medium and dialysate may be layered with vaseline. The vaseline seal excludes oxygen, but also retains C02 and may therefore tend to a more rapid acidification of the medium. Osmotic pressure adjustments take place automatically by changes in the levels of medium and dialysate. With experience the approximate osmotic pressure of a given medium may be anticipated and the passage of water into the sac may be avoided by filling it with medium to a higher level. These sacs were prepared especially for use with the Smith Noguchi fresh tissue medium. This medium, consisting of ascitic fluid or dilute serum and a fragment of fresh rabbit kidney or testicle is placed within the sac, in the dialysate of which we have grown subplants of T. pallidurn, S. microdentiurn and Bacterium pneumosintes.

Subcutaneous tubes for chemotactic studies and leucocyte collection

For the study of the chemotactic influences of various substances, including bacteria and their products, and for the collection of phagocytes and other wandering cells, small glass test tubes of about 5 c.c. capacity have been placed in the loose subcutaneous tissues of the rabbit. The procedure is as follows. The mouths of test tubes, 1.2 × 5 cm., are covered with drum heads of fine bolting silk, tied on with thread, which permit free interchange of liquids with the subcutaneous tissues and the migration of cells and bacteria. The chemotactic material or the organism to be studied, either in agar (1 c.c.) overlaid with Ringers solution, or in fluid form is then added to the sterilized tubes through a hollow needle. A tube may be placed in each flank through a single dorsal incision, after blunt dissection of the loose subcutaneous tissue. Surgical anesthesia and asepsis are required for the operation. Removal of the tube contents is accomplished by hypodermic puncture through the cloth heads of the tubes, after careful cleansing of the skin with iodine and alcohol. The needles of two 20 C.C. syringes are plunged into the tube. One syringe is already filled with Ringers solution or other liquidto replace the tube contents as it is drawn into the second syringe. Thus the tube is washed out and left full of fresh material. The tubes themselves are practically non-irritating and their contents may be reclovered daily, or at longer intervals, for weeks or months. From such tubes, containing a small amount of aleuronat in agar and filled up with Ringers solution, leucocytes for phagocytic experiments may be collected daily over a long period. During the first day few leucocytes enter the tubes, but after 48 hours, and subsequently, a 15-20 C.C. specimen of ground glass opacity may be recovered from each tube.

Factors influencing anaërobiosis with special reference to the use of fresh tissue

With methylene blue as an indicator, we have studied the influence of certain elements in promoting or in hindering the development of anaerobic conditions in tissue cultures. As a result of our experiments, we have come to the following conclusions: Liquid paraffin oil, used extensively as a seal for anaërobic cultures and in gas analysis, has very little value in inhibiting the access of oxygen. Solid vaseline, on the other hand, forms an effective oxygen-resisting seal. The difference is due to the physical states of the substances at incubator temperature. Fresh kidney tissue is an active reducing agent and quickly decolorizes methylene blue in its vicinity. The reducing effect of fresh kidney tissue is relative to the amount used. As a reducing agent, at least 0.6 gm. per tube is required for the establishment of an adequate oxygen free zone. Culture media may be classified as reducing or non-reducing. Those containing dextrose or peptone in a faintly alkaline solution belong to the former class. Ascitic fluid and dilute serum belong to the latter class, for their content of reducing substances is practically insignificant. For the prompt establishment of strictly anaerobic conditions these media require the addition of reducing substances such as dextrose, peptone, or kidney tissue aided by an effective seal or an anaerobic jar. Semisolid media effectively inhibit the penetration of oxygen to the depths of the tube, but they likewise limit the diffusion of reducing substances and presumably of nutrient substances from imbedded kidney tissue. The length of the column of medium is of minor importance under a vaseline seal.

Preparation of collodion sacs for use in bacteriology

A standardized method was described by which collodion sacs suitable for intraperitoneal incubation and for other bacteriological experiments may be produced in large numbers, sterilized, and handled with convenience and the minimum danger of contamination. Following the procedure of Prudden and McCrae, 1900, as modified by Harris, 1902, the collodion sacs are made on a gelatin capsule foundation which is then dissolved out with hot water. The essentials of the method are the alcohol treatment of the collodion membranes, as recommended by Brown, 1915, and the protection of the sacs in individual glass containers before and after use. Various factors influencing permeability have been subjected to experiment. Quantitative experiments on the dialysis of sodium chloride indicate a uniformly high degree of permeability but the permeability conferred by the alcohol treatment is lost during heat sterilization if the membrane was previously allowed to dry. Simple tests with other substances show that the sacs are permeable to gases in solution, to inorganic salts, to dextrose, to certain protein-split products which are nutritive to bacteria, and to certain toxic products of bacterial metabolism, but they hold back antibodies, unsplit proteins, and formed elements such as bacteria and body cells. The preparation of the sacs is described in detail in the forthcoming (January, 1921) number of the Journal of Experimental Medicine.

A method of standardizing bacterial suspensions

If a wire loop is thrust down into a suspension of bacteria in a test tube, and viewed by looking down into the mouth of the tube, the depth at which the loop disappears will be determined by the opacity of the supervening suspension. If, however, a second suspension of the same organism containing half as many bacteria per cubic centimeter is similarly examined, or if an equal amount of the diluent is added to the original suspension, and the “depth of disappearance” again measured it will be found to be less than twice as great as in the original suspension. In other words, the observed depths of disappearance are not in proportion to the bacterial concentrations or the corresponding volumes. This discrepancy is due to the presence in each reading of a constant which is apparently related to the size and opacity of the individual organisms. It is found that this constant may be eliminated, thus bringing the opacity observations into inverse ratio with the corresponding bacterial concentrations, and a corrected reading (the observed reading minus the constant) for any suspension may be obtained by making two readings at different dilutions of the suspension, and substituting the observed values in the following equation: in which A = the corrected reading for the first volume of the suspension = (a - constant) a = first observed reading. vol a = first volume of the suspension on which reading a is made. b = second observed reading. vol b = second volume of the suspension (diluted), on which reading b is made. A concrete example will illustrate the method. In 4 C.C. (vol a) of a given suspension the loop disappears 1.2 cm. below the meniscus (reading a).