J. Franklin Yeager

Micromethod for Determining Insect Hemolymph Specific Gravity (Periplaneta americana Linn).

This is a preliminary report of a falling drop method (based on Stokess law) that permits the determination of the specific gravity (S.G.) of a single drop of insect hemolymph (4.85 mm.3 or less) within the temperature range of 15°C.-40°C., with an average accuracy of ±0.0021 (1 determination) or ±0.0008 (mean of 10 determinations), and with an average determination time of about 8 minutes. The diameter of the hemolymph drop at a beeswax-mineral oil surface is measured with a calibrated ocular micrometer scale. The hemolymph drop, surrounded by oil, is sucked into a glass tube and thus transferred to the sedimentation cylinder. The time required for the drop to sediment through a given falling distance in mineral oil is measured and converted to falling time at 25°C. by means of conversion factors calculated from the equation in which n is the viscosity of the oil medium, ps is the S.G. of the drop, pm is the S.G. of the oil medium, t is the falling time and the subscripts 1 and 2 refer respectively to the experimental temperature and to 25°C. The correction factor, k, for fluidity of the drop, was defined by Hadamard 1 as k = (2n + 3n′)/(3n + 3n′), where n is the viscosity of the medium and n′is that of the drop. This variable factor was found to be k2 = 0.6692 at 25°C. By means of curves that show falling time-specific gravity difference relationships for drops of various sizes at 25°C., the drop-medium S.G. difference is found and is added to the S.G. of the medium to obtain that of the hemolymph at 25°C. The curves were calculated by means of the equation which is derived from Stokess law In these equations, d is drop diameter, D is diameter of the container of the medium, h is the height of the column of the medium, g is the gravitational constant, a is the radius of the drop, s is the falling distance, V is the falling velocity and n, k, ps, pm and t2 have the meanings already given them. The factors (1 + 2.4 d/D) and (1 + 1.65 d/h) correct for side wall and end effects of the container of the medium. 2

On Counting Mitotically Dividing Cells in the Blood of the Cockroach, Periplaneta orientalis (Linn.)

Although cell divisions in insect blood have been described by Hollande 1 and others, few quantitative counts apparently have been reported. This is a preliminary report of blood cell counts in P. orientalis, especially mitotically dividing cell (M.D.C.) counts. Large nymphs were used. Method. Antennal blood is taken into a pipette, made to handle 1.22 mm.3 blood, and rapidly diluted; a total count is obtained with a hemocytometer. A small drop of the antennal blood is placed also in a larger drop of diluting fluid on a slide, immediately stirred to prevent cell coagulation, covered with a coverslip, and rimmed with oil to prevent evaporation. The cells are randomly counted (oil immersion) and the % M.D.C. determined. The diluting fluid consists of 0.081 M NaCl, 0.002 M KCl, 0.001 M CaCl2, 0.005% Gentian Violet, 0.125% glacial acetic acid. Results. Some of the counts obtained are in Table I. Groups I and II include normal animals. In Group I only anaphases and telophases, while in Group II prophases, metaphases, anaphases, and telophases were counted. Each animal was bled only once, to make the count. Group III contains 3 series of 3 animals each: 1 control, bled once for the count, and 2 experimental animals bled extensively at previous times; for example, No. A-2 was bled at 4 and again at 2 days before the count. Group IV includes 1 control, bled once for each count, and 1 experimental animal, also bled extensively after each count. This experiment lasted 14 days. Discussion and Conclusions. The average total cell count of Groups I and II is 31,672 cells/mm.3 blood, or about the same as the count for P. fuliginosa obtained previously. 2 The average. % M.D.C. is 0.07 in Group I and 0.51 in Group II. Assuming this, difference to be due (1) to the counting of all phases in Group II and only anaphases and telophases in Group I and (2) to a longer time duration of pro- plus meta- as compared to ana- plus telophase, then the observed difference would indicate that the average mitotically dividing cell remains in all mitotic phases about 7 times as long as in the last 2 phases.

Reducing Power of Hemolymph from the Roach, Periplaneta americana Linn., with Special Reference to Coagulation

Hemolymph (blood) coagulation of the roach, P. americana, involves cytolysis of the hemolymph (blood) cells. This paper reports experimental results bearing on the question: do reducing substances enter the plasma from the coagulating cells? The Hage-dorn-Jensen blood sugar micromethod 1 was used to determine total reducing power, expressed as mg. glucose per 100 cc. hemolymph. Hemolymph samples were obtained from severed antennae, a single sample coming from a single animal. Ten samples were from animals submerged in water at 60° for 10 minutes to fix the cells, 10 from animals subjected to glacial acetic acid vapor until cell fixation had occurred and 10 samples of “serum” were obtained from untreated animals by collecting the normal hemolymph under oil to prevent drying, letting stand 10–15 minutes to allow cell coagulation to occur and then removing the uncoagulated fluid with a fine glass capillary tube. The “serum” was transferred from the capillary tube to a 0.1 cc. micropipette, graduated to 0.001 cc., used also to measure the volumes of samples of uncoagulated hemolymph. All of the samples were introduced from this pipette into the tube for precipitation of cells and proteins, as required by the method. All of the animals treated with heat and acid to inhibit coagulation and half of the untreated animals were imagos; the others were large nymphs. The mean total reducing powers and their standard deviations are 63.2 ± 10.2 for the heat-treated imagos (whole blood), 57.3 ± 15.7 for the acid-treated imagos (whole blood), 65.5 ± 20.5 for the untreated imagos (“serum”), 65.3 ± 9.1 for the untreated nymphs (“serum”), 65.4 ± 16.3 for the untreated animals and 62.0 ± 14.4 mg. “glucose” per 100 cc. hemolymph for the whole group of 30 animals.

Physiological Effects of Injections of Various Benzene and Furan Derivatives into the Cockroach

Summary Various benzene and furan derivatives, dissolved or emulsified in Hobsons solution, have been injected quantitatirely into the cockroach, Periplaneta orientalis (Linn.). The effects of the injections upon various body activities are given in condensed and tabular form. No clear-cut relationship between chemical structuse and physiological effect has been detected, although the methyl groups appear to be associated with lesser toxic effects. Several observations of physiological interest are described.

The Polynuclear Count in the Rat, with Special Reference to Vaginal Smears

The presence of polynuclear leucocytes in vaginal smears of the rat has been noted by Long and Evans, 1 who observed the presence of such cells during certain phases of the oestrous cycle, no quantitative measurements being made. The present report deals with polynuclear counts made from blood and from vaginal smears taken simultaneously from the same animal. All data were obtained from healthy rats kept under ordinary laboratory conditions. Blood smears were prepared in the usual manner. The smears, upon drying, were fixed with methyl alcohol for one minute and were stained subsequently with Wrights blood stain. A few smears were stained with “Fadicit”, only Solution I being used. Either stain permits an easy identification of the polymorphonuclear neutrophiles and counts may be made without difficulty. Vaginal smears, obtained by means of a wire loop and clean cotton, were stained with Delafields hemotoxylin. In making a polynuclear count, cells showing fragmentation or obvious distortion were omitted, the count including only cells possessing a distinct cytoplasmic outline. It will be noted from Table I, a representative cycle, that the blood count is decidedly left-handed as compared to that of normal man, and that the vaginal smear count, while also decidedly left-handed as compared to man, is also somewhat right-handed when compared to the animals own blood count. The average weighted mean of a group of 35 blood polynuclear counts is 1.09, with a standard error of ±0.0016, while the average weighted mean of a group of 20 vaginal polynuclear counts is 1.69, with a standard error of ±0.039. The polynuclear count of the rat is similar to the polynuclear counts obtained by Simpson 2 , 3 from the cow, the sheep and the horse.

Changes Produced by Sugar Solutions in Hypotonic Hemolytic Systems Containing Red Cells of Man

The inhibitory effect of sucrose solutions upon saponin and taurocholate hemolysis has been studied in a previous paper. 1 The present report deals with the changes produced by solutions of sucrose and other sugars in the resistance of the hypotonic hemolytic system: The quantities of (a), (b) and (c) are made such that the resulting system is of the hypotonicity desired. “Resistance” is expressed as that tonicity of the system which will just produce complete hemolysis in one hour. 2 A typical experiment is presented. In Fig. 1, Curve A shows the change in resistance of the system when increasing quantities of 0.80% NaCl are replaced by equal quantities of M/3.6 sucrose, the cell suspension being a citrate-NaCl-NaCl suspension; similarly, Curve B shows the effect of increasing quantities of M/3.6 sucrose, when the suspension is a citrate-sucrose-sucrose suspension. These curves indicate that the replacement, in this way, of NaCl solution by sugar solution in the system produces at least two effects: (1) an immediate effect, resulting in an increased resistance of the system and (2) a more prolonged effect, resulting in a comparative decrease of resistance. Curve A represents the first effect only. In Curve B, the first effect is superimposed upon the second; here the immediately produced increase in resistance occurs in the case of red cells in which a comparative decrease of resistance has already been brought about by a more prolonged contact with the solution of sugar. Essentially the same results are obtained with M/3.6 maltose and M/3.6 lactose, which increase resistance, and M/3.6 dextrose, which decreases the resistance of the system. These experiments, together with observations of the following type, indicate that the destruction of red cells by a hypotonic hemolytic system involves to a considerable extent factors other than those of pure osmosis: (a) red cells suspended for 3 hours in M/3.6 lactose show a resistance of less than 0.20%; (b) red cells suspended in 0.80% NaCl and kept for 36 hours at 10° C. show a resistance of less than 0.20%; (c) in both cases, microscopic observation of a hypotonic NaCl system that just fails to completely hemolyse shows that the few remaining cells are not swollen into spheres, as might be expected from an hypothesis involving the occurrence of only osmotic changes, but are mostly crenated, cup-shaped and disc-shaped; (d) the resistance (as defined above) is altered by altering the rate at which the suspension is added to the system.

The Use of Equations of the n-th Order to Describe the Action of Simple Haemolysins:

In all recent work concerned with the fitting of formulae to curves obtained for the action of the simple haemolysins it has been assumed that the “fundamental reaction” between the cells and the lysin is one in which the latter combines with some component (probably protein) in the membrane of the former, thus forming a new compound as the result of the formation of which the integrity of the cell is destroyed. Thus, the quantity of the cell component, S, destroyed, is proportional to the quantity of lysin, x, used up in the system, and the velocity of the reaction is given by whence where c is the initial quantity of lysin (in milligrams), where t is the time required to produce lysis of an arbitrary number of red cells, and where S is large compared to c. Since it is assumed that the complete lysis of n cells corresponds to the utilization of a constant quantity of lysin, we obtain, by putting × = const., and varying c in (2), a relation between the time for complete lysis of n cells and c, the initial concentration of lysin; when plotted, this relation gives the “time-dilution curve” for any particular lysin. If we are concerned with the number of cells, N, haemolysed from moment to moment by a particular concentration of lysin, from the beginning of the reaction until its completion, we solve (2) simultaneously with and obtain the S-shaped “percentage haemolysis curves”. For certain haemolysins under certain conditions, these expressions describe the experimental results excellently. Recently, however, we have examined the action of several simple lysins over very much longer periods than previously, observing the time-dilution and percentage haemolysis curves over periods as long as 300 minutes and as short as 6 seconds.