Clot Cultures.—A very simple method is to take blood with a Wright U-tube. Then centrifuge and use the serum for agglutination tests and the clot, emulsified in some liquid medium, for the blood culturing. For paratyphoid culturing bile media are preferable, just as for typhoid.
Lyon Blood Tube.—Quite recently I have been using the blood tube recommended by Lyon. To make it, heat a 5- or 6-inch section of ¼ inch tubing in the centre and draw out as for making 2 bacteriological pipettes. Divide and seal off the large end in the flame. Next seal off the capillary end. Then apply a very small flame to a point on the large end just before it begins to taper to the capillary part. The heat causes the heated sealed-off air inside to force out a blow hole. To use: Break off the sealed capillary end and allow the capillary end to suck up blood from a drop just as with the Wright tube. I consider this tube superior to the Wright one.
N. N. N. Medium.—In culturing blood for protozoa the N. N. N. medium is usually employed. Novy and MacNeal originally used a 12½% meat infusion containing 2½% agar, 2% peptone, 1% normal sodium carbonate solution and ½% salt. To one part of this agar, melted and cooled to 60°C., they added twice the amount of defibrinated rabbit’s blood. In the N. N. N. medium, as modified by Nicolle, there is beside the blood only salt and agar—no peptone or meat extractives.
Citrated salt solution was the medium used by Rogers in the cultivation of splenic juice from kala-azar patients.
The Taking of Blood for Serological Tests
This can be done with the Wright tube, pipetting off the clear serum after centrifuging. We usually draw blood from a vein by use of the system of stopper and tubing described under blood culturing but employing an empty, sterile centrifuge tube.
Agglutination Tests
There are two methods of testing the agglutinating powers of a serum—the microscopical and the macroscopical or sedimentation method.
For the microscopical method draw up serum to the mark 0.5 of the white pipette. Then draw up salt solution to the mark 11. This when mixed gives a dilution of 1 to 20. One loopful of the diluted serum and one loopful of a bouillon culture or salt solution suspension of the organism to be tested gives a dilution of 1 to 40. One loopful of the 1-20 diluted serum and 3 loopfuls of the bacterial suspension give a dilution of 1-80. These two dilutions answer in ordinary diagnostic tests. The red pipette with a 1-100 or 1-200 dilution may be used where dilutions approaching 1-1000 are desired. Having mixed the diluted serum and the bacterial suspension on a cover-glass, we invert it over a vaselined concave slide and examine with a high power dry objective (⅙ inch). It is simpler to make a ring of vaseline to fit the cover-glass and make the mixture of diluted serum and culture in the centre of this ring or square. Then apply the cover-glass, press it down on the vaseline ring and examine as with the ordinary hanging drop. In making dilutions it is preferable to use salt solution, as the phenomenon of agglutination requires the presence of salts. Ordinarily, thirty minutes is a sufficient time to wait before reporting the absence of agglutination. Agglutination is more rapid at body temperature than at room temperature. In reporting agglutination, always give time and dilution. It is absolutely necessary that a control preparation be prepared in every instance; that is, one with the bacterial culture alone or with a normal serum of the same dilution as the lowest used. Some normal sera will agglutinate in 1 to 10 dilution, and group agglutinations (as paratyphoid with typhoid serum) may occur in 1 to 40 or possibly higher. It is very unusual for sera to agglutinate any other bacteria then the specific one in dilutions as high as 1-80.