Macroscopic Agglutination.—For the macroscopical or sedimentation test, take a series of small tubes (⅜ × 3 inches) and deposit 1 cc. of salt solution in each of the series. Now, having taken an empty test-tube, drop 4 drops of serum in it and then add 12 drops of salt solution. This approximately gives 1 cc. of a 1 to 4 dilution of the serum. It is more exact to make the 1 to 4 dilution with a graduated pipette. With a rubber-bulb capillary pipette, which has been graduated to hold 16 drops or 1 cc., draw up the contents of the tube containing the 1 to 4 serum and add it to the next tube containing 1 cc. of salt solution. This gives 2 cc. of a dilution of 1 to 8. Now mix thoroughly by drawing up and forcing out with the bulb pipette, and then withdraw 1 cc. and add to the next tube containing 1 cc. of salt solution. This gives a dilution of 1 to 16. Having mixed as before, again withdraw 1 cc. of the mixture and add it to the 1 cc. in the next tube. We now have a dilution of 1 to 32. Again withdrawing 1 cc. and adding it to the fourth tube containing 1 cc. of salt solution we have a dilution of 1 to 64. In tube 1 there is now 1 cc. of a dilution of the serum of 1 to 8; in tube 2, there is 1 cc. of a dilution of 1 to 16; in tube 3 of 1 to 32. Tube 4 contains 2 cc. of 1 to 64. The dilutions can be carried on in the same manner to any extent that may be desirable. In cholera agglutinations we may run up to 1 to 5000 or thereabouts. Of course, where such dilutions are employed, we generally start with 2 cc. of 1 to 50 in the first tube. When we have completed the series, each tube having 1 cc. of diluted serum, and the last 2 cc., we remove with the pipette 1 cc. from the last tube and discard it by ejection from the pipette leaving 1 cc. in the last tube. Now adding 1 cc. of a culture of typhoid or any other organism, we have the dilution of the serum in each tube doubled. Tube 1 now contains a serum in dilution of 1 to 16, acting on the bacteria; tube 2 of a 1 to 32; tube 3 of a 1 to 64. Now place these tubes in the incubator and, after two to five hours or overnight, we examine for the clearing up of the supernatant fluid. If the serum in a certain dilution agglutinates, the clumps gravitate to the bottom and the upper part becomes clear. If so desired, these dilutions may be carried on to 1 to several hundred in the same way. It is safer to work with dead cultures instead of living ones. To prepare, take a twenty-four-hour agar slant culture of typhoid or paratyphoid and emulsify in salt solution (about 6 cc. to a slant).
By adding 0.1 of 1% of formalin to the typhoid emulsion and placing in the ice-box the cultures will be found sterile in about three days. The emulsion should be shaken twice daily while undergoing sterilization in the ice-box. Such cultures are not easily contaminated and appear to retain their agglutinable qualities for several months. The macroscopic methods are preferable with such dead cultures. For our Dreyer emulsions we use a two-billion suspension of typhoid or para-typhoid organisms in 1 cc. of the formalinized culture.
Combination of Microscopical and Macroscopical Methods.—Microscopic: Prepare dilutions of serum as above described and take from each or several of the series, a loopful of the diluted serum. For control use a loopful of salt solution. Place on a cover-glass and add loopful of bouillon culture of the living organisms. Make hanging drop preparation, report after one hour at room temperature. Use ⅔ inch lens for examination.
Macroscopic: Add to each of the series, including the control, an equal amount of an emulsion of killed organisms.
The method of using a slide with two vaselined rings, one containing an emulsion in the specific serum and the other in salt solution, is of great practical value. This method is described under cholera.
Complement Fixation.—Complement fixation tests have been employed in the diagnosis of several tropical diseases but do not seem to be at present sufficiently reliable or practical with the exception of that for yaws and tularaemia. The chief difficulty with complement fixation tests for suspected sera is to obtain a reliable antigen. Should we later on be able to prepare bacterial antigens as satisfactory as Noguchi’s acetone-insoluble antigen is for the Wassermann test there may be a field for such tests in tropical pathology.
Other Practical Methods of Haematological Study
Haemoglobin Estimation
The standard method now is the estimation of the oxygen capacity of the blood, using some gas apparatus, such as Van Slyke’s. Otherwise, the most accurate instrument for this purpose is the Miescher modification of the v. Fleischl haemoglobinometer.