o TORY DfACN^SIS ER AND UNCOLN Class JLS^lZ Book - h . C. count moderately low. or may be normal. Color index verv 24 BLOOD low, often 0.5. W. B. C. count normal or slightly reduced. Stained film: All the red cells show lack of haemoglobin and are slightly undersized. Qualitative changes are absent or inconspicuous. Normoblasts may be found in severe cases. Blood plates are increased. There is a tendency to relative lymphocytosis. Pernicious Anaemia. The etiology is unknown. Haemolysis is the chief change. Whether the haemolytic agent acts in the blood-making marrow or in the circulation is not known. The only lesions constantly found post-mortem are hyperplasia of red bone marrow and increased iron pigment in the liver. The dis- ease is nearly always finally fatal, but three or four remis- sions may occur, lasting from a few days to many months. The blood examination is necessary to a diagnosis, but clin- ical history and symptoms are also important. Blood picture : — Appearance, pale, watery and slow to coagulate. Haemoglobin, low but not so reduced as the red cell count- R. B. C. count very low, the most striking change, (1,000,000 to 2,000,000 common, has been reported as low as 150,000). Color index high, the only condition in which it is high. The higher the color index the worse the prognosis. W. B. C. count low. Leucopenia may be pronounced (3,500 common, has been reported as low as 500). Stained smear: Cells scattered in the field. Qualitative changes marked, poikiloc} r tosis, anisocytosis (more macro- cytes than microcytes), polychromatophilia and basic stip- pling all present in typical cases. Variation in haemoglobin content is shown by dark and pale staining cells. The MANUAL OF LABORATORY DIAGNOSIS 25 more macrocytes with excess of haemoglobin, the higher the color index. Nucleated reds are present in all cases, but vary in number at different times. Typically, megaloblasts exceed normoblasts. The differential leucocyte count shows a relative lymphocytosis proportional to the leucopenia. Eosinophils are decreased when the disease is progressing .and increased when there is improvement. An occasional myelocyte may be found. Blood plates are reduced in number. Leukemia. — Leukemia is a condition characterized by persistent in- crease in leucocytes, and changes in lymph glands, spleen and marrow. There are two types of blood findings: 1st, the myeloid, in which there is an overproduction of those leucocytes formed in the marrow, that is, all kinds except lymphocytes; 2nd, the lymphatic, in which there is an overproduction of lymphocytes. In the myeloid form there is hyperplasia of the myeloid marrow, and myeloid tissue may appear in the spleen, lymph glands, liver and many other places in the body. In the lymphatic form lymph tissue may infiltrate the various organs and tissues of the body. Some pathologists regard leukemias as malignant tumors of the marrow and of lym- phatic tissue. The tumor cells, being- cells belonging to the circulating blood, are carried to all parts of the body and form metastases. Either form of the disease may be acute or chronic in its' course. Remissions sometimes occur in which the leucocyte count may be normal, but examination of a stained smear usually shows the presence of abnormal cells and abnormal percentages of the different varieties of leucocytes. Blood picture in myeloid leukemia. (Synonyms, myelogenous leukemia, splenomyelogenous leukemia, myeloblastoma.) 26 BLOOD Haemoglobin is reduced as the disease progresses. Esti- mation by usual methods is difficult because the large num- ber of leucocytes changes the color and consistency of the blood. . R. B. C. count is little reduced at first, more later. Color index low. W. B. C. count enormously increased (250,000 common, may be 1,000,000). Stained film : The diagnosis is often made at a glance from the great number of granular leucocytes in the field The differential count shows many myelocytes (30% aver- age). All kinds of leucocytes except lymphocytes are in- creased. The percentage of the different varieties varies greatly. Qualitative changes in the reds are usual. Blasts are constantly found and are more numerous than in any other blood disease. Blood picture in lymphatic leukemia. (Synonyms, lymphaemia, lymphoblastoma. ) Haemoglobin low. R. B. C. count low. Color index low. W. B. C. count very high (150,000 common, may be 1,000,000, but is often as low as 30,000). Some acute cases show very moderate counts. Stained film : Although the erythrocyte count is often lower than in myeloid leukemia on account of hemorrhages which are frequent in this condition, the qualitative changes are not so marked and nucleated reds are much fewer. The differential count usually shows 90% or more of lymphocytes. The other varieties of leucocytes are not much changed. MANUAL OF LABORATORY DIAGNOSIS 11 Diagnosis of Malaria from the blood examination. - The plasmodia of malaria arc found in the circulating blood during and shortly before and after febrile paroxysms. Their presence during quiescent periods is not easy to demonstrate. Quinine, even one dose, causes most of the parasites to disappear from the peripheral blood. Methods of Examination : — , 1. Moist preparation. Take a small drop of blood on a slide, cover with cover glass, pressing upon the cover glass to make the layer of blood thin. Examine at once. The parasites appear as hyaline, variously shaped bodies in the red cells. Except in their earliest stages they contain dark, brownish pigment which has a dancing motion. The pigment is usually the first thing to attract the eye. The parasite itself, except in the full-grown asexual and sexual forms, has a slow amoeboid motion. 2- The stained film. This method is preferred when the patient is not convenient to the laboratory. It is less liable to error in the hands of the inexperienced. With Wright's stain the parasite stains blue with a red- dish chromatin body within it. The pigment is unchanged, looking about the same as in the unstained blood. In tertian and quartan fevers the asexual forms are numer- ous in the peripheral blood and the occasionally found sex- ual forms are not easily distinguished from the full-grown asexual. In aestivo-aiitinnnal fever the asexual forms are at first seen in the peripheral blood, but their later development takes place in the spleen. The sexual form of this variety, the crescent, is distinctive and is constantly found in the periph- eral blood after the first few days of the infection. These 28 BLOOD crescents are resistant to quinine and often remain in the blood a long time. The blood in malaria early in the infection may show noth- ing abnormal but the presence of the parasites. Anaemia develops rapidly and in long-standing cases the blood may show a picture much like that of pernicious anaemia. CHAPTER II. SERUM DIAGNOSIS. Widal Reaction. The Typhoid Culture.— Old laboratory cultures which have been many times transplanted are often better, being more motile than fresh- ly-isolated bacteria. For making the test use an 18 to 24- hour bouillon culture which has been inoculated from the stock agar culture and incubated. At this stage the bacilli are actively motile and are about numerous enough to make a good field. Examine a loopful of the culture under the high dry lens to see that the bacilli are motile and are not spontaneously clumped. Obtaining the Blood or Serum. — The blood is obtained from a puncture as for a blood count. AVhole blood may be used. It can be diluted in a white blood counting pipette. The disadvantage is that the corpuscles obscure the field. Blood serum is the best form to use. Obtain the blood in a capillary tube (Wright capsule), centrifuge to separate the serum, or let stand until the serum separates. Dried blood may be used, will keep for some time and is convenient for sending to the labora- tory. Large drops of blood are allowed to drop on glass slides or glazed paper and are dried in the air. 29 30 SERUM DIAGNOSIS Dilution of the Serum. — A dilution of 1 to 40 or higher is required that the test may have any diagnostic value, because normal blood undi- luted or 1 to 20 will sometimes clump typhoid bacilli. For whole blood. — Draw the blood to 1 mark in white blood counter, draw up physiological salt solution to mark 11 and mix. This, makes 1 in 20 dilution (blood is half serum). Blow out into a watch glass. For serum. — Remove the serum with capillary pipette- To one drop of serum add 19 drops of physiological salt solution. This makes 1 in 20 dilution. For dried blood. — Scrape the dried blood from the slide into a watch crystal. Add 9 drops of salt solution. This makes dilution 1 in 20 (dried blood is half serum). Making the Mixture. — Place a loopful of the 1 in 20 dilution of serum on a cover glass. Mix with a loopful of the bouillon culture. The fluid of the culture doubles the dilution, making 1 in 40 dilution of the serum. Ring a hollow ground slide with vaseline and mount the mixture as a hanging drop. Observing the Reaction. — Use the high dry lens and subdued light. Artificial light is sometimes better than daylight. The field should show at first actively motile separate bacilli. If the reaction is positive, in from a few minutes to 1 hour the motion gradu- ally slows and ceases, and the bacilli gather into groups. The reaction is more or less marked according to the amount of agglutinin present. To call a reaction positive the majority of the bacilli must have stopped their motion MANUAL OF LABORATORY DIAGNOSIS 31 and there must be many clumps of 5 or more bacilli. For a 1 in 40 dilution 40 minutes' time of observance is enough ; for higher dilutions. 1 hour. Value of Widal Reaction in Typhoid Fever. In typhoid fever the Widal reaction rarely fails to appear, and is our most certain confirmatory test after the bacilli have left the blood. During the first week of the disease the typhoid bacilli may be found in the blood. The Widal appears first on the seventh or eighth day. usually continues well into convalescence, and may continue an indefinite time after recovery. The Wassermann Test for Syphilis. Haemolysis. — If the erythrocytes of an animal, for example, a sheep, are injected into the blood stream of an animal of another species, for example, a rabbit, the blood of the animal receiv- ing the corpuscles (the rabbit ) gains power to dissolve the erythrocytes of the species from which the corpuscles come (sheep) that is. becomes immunized to these corpuscles. But if the serum of the rabbit is heated to 56" C. for one-half hour (inactivated), or kept at room temperature for twenty- four hours, it loses this power. Haemolysing power may be restored to this serum by adding to it some fresh un- heated serum from any animal. The haemolysis depends upon two substances present in the serum of the immunized animal, one destroyed by heat (complement) and the other not so destroyed (amboceptor). A haemolytic system consists of a suspension of erythro- cytes, the serum of an animal immunized to these erythro- cytes, and complement (contained in fresh unheated serum of any animal). 32 SERUM DIAGNOSIS Complement Fixation. — Complement is present in all sera and will react with different kinds of amboceptors, but amboceptors are devel- oped as the result of the introduction into the blood of some foreign material ; for example, erythrocytes of another spe- cies give rise to haemolytic amboceptors. Bacteria give rise to bacteriolytic amboceptors, which react in a similar manner with complement and the invading bacteria. The substance giving rise to these amboceptors is called antigen ; the corpuscles are the antigen in the haemolytic system, the bacteria are the antigen in the bacteriolytic system. If a bacteriolytic antigen, the corresponding amboceptor and complement are incubated together until the combina- tion has had time to take place, and then the antigen of a haemolytic system, that is, erythrocytes, and haemolytic amboceptor are added to the former combination, no haemol- ysis will take place, because the complement has been used up or bound in the first system. If, however, the first sys- tem contained no amboceptor, then haemolysis would take place, as the complement would be free to unite with the haemolytic system. The Wassermann reaction in syphilis seeks to demon- strate, by this method, the presence or absence of syphilitic amboceptor in a patient's blood. In general terms the proceeding in the Wassermann reac- tion is this : The patient's blood serum, inactivated to remove its own complement, is combined with complement supplied by fresh guinea pig serum, and syphilitic antigen. These three substances are incubated together until com- bination of complement, antigen and amboceptor has had time to take place if the patient's serum contains syphilitic amboceptor. There is no visible change to show whether MANUAL OF LABORATORY DIAGXOSIS 33 combination has taken place or not. If the patient's serum contains syphilitic amboceptor and this has combined with the antigen and complement, then there will be no free complement left in the mixture. Adding another system of antigen and amboceptor, one in which the combination, if it takes place, will cause a visible change, will then show whether there is complement in the mixture free to act in the latter system. A haemolytic system is used, as haemol- ysis is a plainly visible phenomenon. After an hour's incubation haemolytic amboceptor (the serum of a rabbit immunized to sheep's erythrocytes) and washed sheep's corpuscles are added and the mixture again incubated. If there is free complement it will unite with the haemolytic amboceptor and sheep's corpuscles and dis- solve the corpuscles. If the complement has been used in the former combination, the sheep's cells will not be dis- solved. Absence of haemolysis, then, indicates that the patient's blood contains syphilitic amboceptor, that is, the reaction is positive. Haemolysis shows that the comple- ment was not used in the first combination, owing to the absence of the third necessary element, syphilitic ambocep- tor in the patient's blood, but was free to enter into the haemolytic system and bring about haemolysis of the sheep's cells, that is, a negative reaction. Preparation of Materials Used in the Test. — (1) Sheep Corpuscles. — If the sheep's blood is obtained from a slaughter house it must be used within a day or two, as bacterial contamination will soon cause haemolysis. But if obtained in a sterile manner and kept sterile the corpuscles may be used for a week, as a rule. In either case, as soon as the blood is drawn it must be defibrinated by shaking five minutes in a bottle with bits of glass or wire. It is then kept in a refrigerator until time to prepare for use. 34 SERUM DIAGNOSIS (2) Haemolytic Amboceptor. — The inactivated serum of a rabbit which has been immunized to sheep erythrocytes furnishes the haemolytic amboceptor. Two intravenous injections of one to two c.c. of washed sheep corpuscles are given to a rabbit at an interval of four or live days. Five to seven days after the second injection the rabbit's serum will contain the maximum amboceptor. At this time the rabbit is bled from the ear vein, the serum tested, and if found of sufficient strength to use in a dilution as high as 1 to 1,000, the rabbit is anaesthetized and bled from the carotid with aseptic precautions. The serum is separated by the centri- fuge and inactivated at 56° C. for one-half hour, to destroy its complement. It is well to inject two or three rabbits at a time, as not every one injected produces good ambo- ceptor. The amboceptor is stored in sealed glass tubes and kept in the refrigerator. (3) Complement. — Guinea pig serum is used as its com- plement content is high and rather constant. The animal may be bled by puncturing the heart with a hypodermic needle and withdrawing four or live c.c. of blood into the syringe. This requires some skill and many prefer to kill the pig and bleed from the vessels of the neck. The blood should stand until coagulation has taken place. The serum is separated by the centrifuge and pipetted from the clot. It must be used within twenty-four hours unless it is kept frozen. The serum can be placed in small tubes with water- tight stoppers and buried in salt and ice in a thermos bot- tle. By renewing the salt and ice every day or two the complement will be kept frozen and will remain potent for weeks. lust what is needed for a set of tests may be melted at the time the tests are made. (4) Antigen. — The substances used as antigens in the Wassermann reaction are not biologically specific antigens. Various lipoid substances have been found to bind comple- MANUAL OF LABORATORY DIAGNOSIS 35 ment in the presence of syphilitic serum and not to hind it in the presence of non-syphilitic serum. Among the substances found most successful as antigens are, first, alcoholic extracts of syphilitic liver ; second, alco- holic extracts of normal organs, as guinea pig hearts ; third, cholesterinized extracts of normal organs, as guinea pig, beef or human heart muscle. These extracts are not always of the same strength nor enduring quality. (5) The Patient's Blood Serum. — The blood is obtained from a vein at the bend of the elbow. A ten c.c. Luer syringe with a No. 19 needle is convenient for puncturing the vein and obtaining the blood, or the blood may be allowed to drop directly through the needle into a sterile tube. It is well to take as much as 5 c.c. of blood, although less may be sufficient. The blood is allowed to stand until coagulated. The serum is separated by the centrifuge and pipetted from the clot. The serum is inactivated in a water bath at 56° C. for one-half hour to destroy its complement. If the serum is not used within a few hours it should be kept in a refrigerator. If handled in a manner to prevent contamination the serum will be good for the test for a few days, but more reliable results are obtained if the test is made within forty-eight hours after the blood is taken. The glassware should be clean and dry. All tubes and pipettes should be thoroughly washed in hot running water. A supply of test tubes ^x4 inches is required. This size allows the contents to be well mixed by shaking. Racks to tit the tubes with holes arranged in parallel rows are useful- Five rows of a dozen in a row form a good rack. A water bath with the same arrangement for tubes is desirable. One c.c. outflow pipettes marked in tenths are used and a good supply of them is needed. Sterile centrifuge tubes, and a good supply of capillary pipettes with rubber nipples, should be on hand. 36 SERUM DIAGNOSIS 0.85% sodium chloride solution is used for diluting all reagents. Diluting and Titrating Reagents Ready for Test. 1. Corpuscles. — A few c.c. of the defibrinated sheep's blood are put into a centrifuge tube, the tube filled with 0.85% sodium chloride solution and centrifuged until the corpuscles are settled. The fluid is pipetted off, and the washing repeated twice, mixing the corpuscles well with the salt solution each time. After the last washing all the salt solution is carefully pipetted off. A 2.5% suspension of the sheep cells is made by taking 1 c.c. of the cells and 39 c.c. of salt solution ; 0.5 c.c. of this suspension is used in the test. 2. Amboceptor. — The amboceptor may be diluted 1-1000 for the first trial. In order not to waste amboceptor it is well to dilute one drop with nine drops of salt solution, and make the higher dilutions from this. 0.1 c.c. of this 1-10 dilution added to 9.9 c.c. salt solution will make the 1-1000 dilution. To titrate the amboceptor when the strength of the com- plement is still unknown it is necessary to use a probable excess of complement; 0.5 c.c. of a 1-10 dilution of comple- ment will usually be enough. A series of tubes is prepared, each containing 1 c.c. salt solution, 0.5 c.c. corpuscle suspension, and 0.5 c.c. comple- ment dilution 1-10. To these add increasing doses of 1-1,000 dilution of amboceptor, to the first 0.1 c.c, to the second 0.2 c.c, and so on. Incubate one-half hour. The first tube which shows complete haemolysis contains one unit of amboceptor. Two units are used in the test. 3. Complement. — The amount of guinea pig serum needed for the set of tests is diluted 1-10 with salt solution. To titrate the complement a series of tubes is prepared, MANUAL OF LABORATORY DIAGNOSIS 37 each containing 1 c.c. salt solution, 0.5 c.c. corpuscle sus- pension and one unit of amboceptor. To these are added increasing amounts of complement, to the first 0.1 c.c, to the second 0.2 c.c, and so on. The tubes are incubated one-half hour. The first tube which shows complete hae- molysis contains one unit of complement. One and one- half or two units are used in the test- 4. Antigen. — Antigen must be titrated to obtain its anti- complementary unit and also its antigenic unit. When these units are once obtained they usually remain the same for some time, and these titrations need not be repeated every time the test is made. The anticomplementary unit. — Antigens alone absorb some complement, and if used in large enough amount would prevent haemolysis in a haemolytic system without the presence of any serum or in the presence of a normal serum. The amount of antigen which will absorb or bind the two units of complement used in the test is the anticomplementary unit. To ascer- tain this a series of tubes is prepared, each containing 1 c.c. of salt solution, two units of complement, 0.1 c.c. of known normal serum- To these increasing amounts of antigen are added. The first tube may have 0.1 c.c. of a 1-10 dilution of antigen, the second 0.3 c.c, etc- These are incubated one- half hour in a water bath, then to each is added two units of amboceptor and 0.5 c.c. of corpuscle suspension. Incubate again for one-half hour- The first tube which shows im- perfect haemolysis contains the anticomplementary unit of antigen. Not more than one-fourth of this amount should be used in the test. The antigenic unit. — This is the amount of antigen necessary to inhibit haemol- ysis with a syphilitic serum. To obtain this unit a series of tubes is prepared, each containing 1 c.c salt solution, two 3$ SERUM DIAGNOSIS units of complement and 0.1 c.c. of known syphilitic, serum. To these are added decreasing amounts of antigen, begin- ning with J /\ the anticomplementary unit. These are incu- bated one-half hour in a water bath and then to each tube two units of amboceptor and 0.5 c.c. of corpuscle suspen- sion are added. The tubes are again incubated for one hour. The first tube which shows inhibition of haemolysis contains the antigenic unit. This should be much less than one-fourth the anticomplementary unit in a good antigen. An abundance of antigen should be used in the test. One- fourth the anticomplementary unit is often used rather than smaller amounts. A fresh dilution of the antigen is pre- pared for each set of tests, according to the strength of the antigen. It is convenient to dilute the antigen enough so that the amount used in the test is contained in 0.5 c.c. of the dilution. The Patient's and Control Sera. 0.1 c.c. of inactivated serum is used in the tests. Making the Test. These titrated and diluted reagents and the inactivated sera are arranged in convenient order. Tubes ^x4 inches are numbered in blue pencil as indicated in the chart. The chart indicates the order of the tubes in the rack and the contents of each tube. The rows are lengthened for other sera to be tested and other controls. More rows are added for other antigens, if more than one is used. At the end of two hours the reaction is usually complete, but it is well to place the tubes in the refrigerator and make a final read- ing next morning. The front row of tubes, 1, 3 and 5, con- taining no antigen' should show complete haemolysis. The second row, 2, 4 and 6, will show no haemolysis in 2, haemolysis in 4, and haemolysis in 6 if the unknown serum is negative and none or little of it is positive. MANUAL OF LABOR* i ORY DIAGNOSIS 3<) ■o ■D u o 05 10 10 m -M u T3 *; o r ~ c E c o 3 re 3 e o in E CM c t_ CM 3 e t_ +J o O u JB a e o w O T3 ■D o (0 m o T3 +-> o r- c E c O 3 re 3 E o CO E 3 C\J C o £_ 3 O CM S_ o (A E u n a> c E a 3 co E o CO - 0) o 3 (A a +- 1 Z o O co < (A 3 a i_ o c v " O ■a (J ■a O o . 1 re o«2 ° w ^ o T3 ±! o r- E * E E d = © re = E O E CM E 1 ° c o 3 O , E J2 — a w h E -o o - 57 = o — a c « E O) re 0) re 3 O E 1- E O Ol o — Z < CO <5 a s_ o >" O E <0 T3 6 ■a o U re o to CO o ■a ■M o ,_ E o E C o 3 ,_ re 3 E O V E 3 eg C o S- 3 O a u a 3 h to ai 0) E 0) 3 o CO - n 3 B9 > a -H Q. a>-4-> a E £_ E re re E E O E +J F a) (A E O -m ■H O to X E CO E 6 o a L. o 01 E E O o TJ re •a c re re o E +■> re re m o E o t. a o o o re L. re E re X) o O CO F *- E !_ -a V o re 3 E a) — en -a O O ™ +J CO re u a ■a L. ■a m 0) ^ 0) E h < re * 40 SERUM DIAGNOSIS The test as described provides for the use of one anti- gen, but it is desirable to use two or more as in this way the tests are repeated. Sera which contain small amounts of antibody may give a clearer positive with one antigen than with another. The antibody in a positive serum may be titrated by using decreasing amounts of the serum. A strongly posi-' tive serum will give a positive reaction with a fraction of the standard amount used in the test. By substituting drops for tenths c.c. throughout, the Was- sermann reaction may be made with smaller amounts of material. While this method is less accurate, it can be used with good results and when the serum is too small in amount for the c.c. method, it proves very convenient. The Wassermann Test on spinal fluid is made just as the test on blood serum is made except that the spinal fluid is not inactivated, and that five to ten times the amount is used, that is 0.5 c.c. to 1 c.c. of undiluted fluid. Diagnostic Value of the Wassermann Test. A positive Wassermann may be obtained as early as four or five weeks after infection with syphilis, that is, a few days after the appearance of the chancre, but as a rule the reaction first becomes positive seven or eight weeks after infection and remains so throughout all stages of the dis- ease if the case is untreated. In hereditary syphilis the reaction is positive in an equally high percentage of cases. There are occasional exceptions to this rule, a negative Wassermann being found in undoubted cases of active syphilis. Treatment renders the reaction negative in many cases, but a positive reaction recurs if the treatment has been insufficient to cure the disease. Three weeks should elapse after the cessation of treatment before the test is made. In syphilitic diseases of the nervous system the blood Wassermann may be negative and the spinal fluid Wassermann positive. MANUAL OF LABORATORY DIAGNOSIS 41 Positive Wassermann reactions have been found in some cases of jaundice, leprosy, relapsing fever, frambesia, trypanosomiasis and malaria in the febrile stage. The in- gestion of alcohol may change a positive reaction to a negative. Complement Fixation Test for Gonorrhoea. The complement fixation test for gonorrhoea is per- formed in the same manner as the Wassermann test for syphilis, substituting gonococcus antigen for syphilitic antigen. Gonococcus antigen made from many strains of gonococci can be purchased from Parke, Davis & Co. The diagnostic value of the complement fixation test for gonorrhoea is considerable, a positive reaction being more valuable than a negative. The test is seldom positive dur- ing the first few weeks of the infection but later in the course of the disease a large percentage of the cases show positive reactions. CHAPTER III. URINE. Obtaining Specimens. A twenty-four hour specimen is usually desirable, be- cause the total output of both solids and water is learned in this way. A single specimen is sometimes useful, as sugar or albumin may be present in the urine at one time of the day, and not at other times. Under ordinary circumstances no preservative is needed. When the urine must be transported a long distance or kept more than a day or two a preservative may be re- quired. Formalin, one drop to four ounces of urine, will prevent decomposition. If more is added the formalin may reduce copper solutions used in sugar tests. Physical Properties. I. Amount. The normal quantity for an adult is from 1000 to 1500 ex. in twenty-four hours. Children pass more in proportion to body weight than adults. A new born infant passes 150 to 200 c.c. A child of 5 years about 700 c.c. In health more is passed during the day than during the night. This may be reversed in disease, notably in chronic interstitial nephritis. The amount varies physiologically according to the 43 44 URIN E amount of fluids and watery foods taken and the activity of other organs of elimination. Pathological factors influencing the amount are : 1. The condition of the renal parenchyma. For ex- ample there is oliguria or temporary anuria in acute ne- phritis. 2. The circulation in the kidney. The amount of urine is influenced by the rapidity of the blood flow, as well as by blood pressure. For example, there is oliguria in chronic passive congestion. Weak heart from any cause produces oliguria. Most diuretics increase the amount of urine by improving the circulation. 3. Abnormal quantity or quality of substances excreted. The sugar in diabetes causes polyuria. At the beginning of convalescence from acute fevers, accumulated waste products stimulate the kidney to increased output of urine. 4. Nervous causes. In hysteria there may be anuria, oliguria or polyuria. In various other nervous diseases variations in amount occur. The cause is thought to be vasomotor. 5. The unknown cause of diabetes insipidus produces polyuria. II. Appearance. Normal urine is transparent, though a slight cloud of mucus may appear on standing. Cloudiness is generally due to pus, blood, bacteria, urates or phosphates. Albu- min itself even in large amounts causes no change in the appearance of the urine. The consistency of normal urine is watery, pathologically it may be frothy from the pres- ence of albumin, syrupy from the presence of sugar, or ropy from mucus or pus in alkaline urine. MANUAL OF LABORATORY DIAGNOSIS 45 III. Color. Normal urine is amber or straw colored. It may vary a great deal within normal limits. Pathological Changes of Color : 1. Blue or green. The administration of methylene blue usually accounts for a blue or greenish color. Rarely a bluish urine appears from putrefactive changes in the in- testines in cholera. 2. Dark yellow or greenish brown color is usually due to bile 3. Black. Melanin from a melanotic sarcoma may color the urine black. In salol, carbolic acid or iodoform pois- oning, the urine may turn black on standing. 4. Red, brownish or smoky color may be due to blood. 5. Red color in alkaline urine may be due to phenol- phthalein, rhubarb, senna or cascara. 6. Milky color is found when there is an admixture of chyle. IV. Reaction. Normal urine is usually acid in reaction but may be temporarily neutral or alkaline. An amphoteric reaction, the urine turning red litmus paper blue, and blue paper red is not unusual, and has no pathological significance. Alkaline urine may be due to decomposition in the bladder as in cystitis with retention of the urine, or to admistration of alkaline drugs. A meat diet has the tendency to increase the acidity, and fruit and vegetable diet to diminish acidity. The reaction is determined by litmus paper, acid urine turning blue litmus red, and alkaline urine turning red litmus blue. 46 URINE Total Acidity. — The total acidity of urine varies considerably normally. The average normal acidity is 400° to 600°. In estimating the total acidity the specimen of urine should be a mixture of the twenty-four hour output and should be as fresh as possible. Folin's Method of Estimation. — Measure 10 c.c. of urine into a 100 c.c. flask, add about 25 c.c. of distilled water, 10 drops of 1% alcoholic solution of phenolphthalein and about 2 grams of potassium oxalate. Add slowly N/10 NaOH until a permanent faint pink color appears. The flask should be shaken after each addition of alkali and the alkali added drop by drop after the first one or two c.c. has been allowed to run in. The number of c.c. of N/10 NaOH required to neutralize 1 c.c. of urine multiplied by the number of c.c. of urine passed in 24 hours gives the degree of total acidity. V. Specific Gravity. The normal specific gravity of a 24 hour specimen of urine is between 1.012 and 1.020. Normally a great vari- ation occurs in separate passages of urine, the night urine having a high specific gravity. Physiologically the specific gravity varies inversely as the quantity. It is increased by nitrogenous diet. Patholog- ically the specific gravity is high in the following diseases : 1. Diabetes mellitus. 2. Acute nephritis. 3. Acute fevers. The specific gravity is low in : 1. Diabetes insipidus. MANUAL OF LABORATORY DIAGNOSIS 47 1. Chronic interstitial nephritis. 'The specific gravity is markedly fixed and low, due to inability of the kidney to excrete a concentrated urine. The specific gravity is estimated 1))' means of the Squibbs urinometer. Dr. W. S. Harpole's Practical Urinometer en- ables one to take the specific gravity of a very small amount of urine. Total Solids. — By the total solids is meant the solids excreted in 24 hours. The chief value of the estimation of the specific gravity is to give an idea of the amount of solids excreted. The normal total solids of an adult on ordinary diet is about 60 grams. About 4% of the weight of the urine is that of the solids and of this urea forms a little less than half. The total solids are estimated by the use of Haeser's coefficient. The last two figures of the specific gravity multiplied by 2.33 (Haeser's coefficient) equals the grams per litre. This multiplied by the number of litres passed in 24 hours gives the total solids. Example : 1400 c.c. of urine is the 24 hour amount. The specific gravity is 1.016. 16x2.33x1.4=52.19. The total solids are 52.19 grams. Chemical Composition of Normal Urine. The 24 hour output consists of: 1. Water, 1000 to 1500 c.c. 2. Solids, 60 grams. a. urea, 30 grams. b. chlorides, 15 grams. c. sulphates, 2.5 grams. d. phosphates, 2.5 grams. 48 URINE e. ammonia, 0.7 gram. f. uric acid, 0.7 gram. g. traces of numerous other substances. The amount of water and the various solids varies within wide limits in health, the above figures only serving to indi- cate the average output. Abnormal Substances Found in Solution in Urine. 1. Proteids. Serum albumin, serum globulin, albumo- ses, nucleo-albumin, Bence-Jones proteid, mucin. 2. Carbo-hydrates. Glucose, lactose, rarely other sugars. 3. Acetone bodies. Acetone, diacetic acid and oxy-bu- tyric acid. 4. Bile. 5. Indican. Albuminuria. True albuminuria is the presence in the urine of albumin which has escaped through the cortex of the kidney. The conditions in which true albuminuria occurs are classified as follows : I. Albuminuria with definite renal lesions (usually large amounts of albumin). 1. Nephritis in all its forms. 2. Chronic passive congestion. 3. Acute congestion of the kidney. II. Albuminuria without definite renal lesions (usually small amounts of albumin). 1. Functional. After severe exercise, after prolonged cold baths, in the new born, cylic albuminuria. MANUAL OF LABORATORY DIAGXOSIS 49 2. Febrile. 3. Haematogenous. In severe anaemias and cachexias, and in chronic diseases as lues. 4. Nervous. Usually transitory. False or accidental albuminuria is the presence in the urine of albumin from the admixture of pus or blood out- side the kidney substance, as in cystitis or injuries to the ureter, bladder or urethra. Serum albumin and serum globulin are usually both present in albuminuria, sometimes also nucleo-albumin and albumoses. Most ordinary tests respond to all these sub- stances. The appearance of proteoses or globulin except in association with serum albumin is rare. The Bence- Jones proteid appears in the urine in multiple myeloma. When present there is usually a large amount. This proteid coagulates on heating at about 60° C, clears up on further heating and reappears on cooling. For all albumin tests the urine must be clear. If cloudy, filter. If still cloudy, mix the urine with light magnesia (magnesium oxide) and filter. Qualitative Tests for Albumin. — Heller's Test. Take an inch of urine in a test tube, un- derlay this with half an inch of nitric acid. If the urine is alkaline, acidify it with a few drops of dilute acetic acid before making the test. Interpretation of the test : Precipitates. 1. Albumin shows a white ring at junction of acid and urine. 2. Nucleo albumin or mucin shows white ring above the contact with clear zone between. 50 URINE 3. Urates show a cloud above the contact line which disappears on heating or on using diluted urine. 4. Oleo-resins show a milkiness at the contact. They are not coagulated by heat. Colors. 1. Normal — varies from faint pink to brownish ring at contact. 2. Biliary pigment — dark green or blue. 3. Indican — violet or bluish when great excess is present 4. Iodides — intense brownish red. 5. Anilin compounds — red to purple. Robert's Modification of Heller's Test. Use in place of nitric acid a reagent consisting of saturated aqueous solu- tion of magnesium sulphate 100 c.c. and 20 c.c. of nitric acid. The interpretation is the same as that of Heller's test so far as the precipitates are concerned and the test is more delicate. Purdy's Test for Serum Albumin. — Take 1/2 test tube full of clear urine, add 1/6 its volume of saturated solution of sodium chloride and 5 drops of 50% acetic acid. Boil the upper portion. A cloud in the boiled portion indicates serum albumin. Quantitative Tests for Albumin. Purdy's Centrifuge Method. — Place 10 c.c. of urine in a graduated centrifuge tube. Add 3 c.c. of a 10% solution of potassium ferrocyanide, and 2 c.c. of 50% acetic acid. Mix by turning, let stand 5 minutes, then centrifuge three minutes at 1500 revolutions per minute. Read the percentage of the precipitate. Each mark represents 1%. The percentage by weight is approximately 1/50 of the bulk percentage as estimated by this method. MANUAL OF LABORATORY DIAGNOSIS 51 Tsuchiyas modification of the Esbach method. — Use Esbach's albuminometer. Fill the tube to the mark U with urine. Add the reagent to the mark R. Mix by turning and let stand 24 hours. The scale reads in grams of albumin per litre of urine. To find the percentage move the decimal point one place to the left. If the precipitate comes up to the mark 4 the percentage is 0.4. Tsuchiva's Reagent : Phosphotungstic acid, 1.5 grams. Concentrated HO, 5 c.c. 95% alcohol q.s. 100 c.c. Glycosuria. Glycosuria is the presence of glucose in the urine. The assimilation limit for glucose is the minimum amount of sugar the ingestion of which is followed by sugar in the urine. The assimilation limit may be tested by giving 100 grams of glucose dissolved in )A pint of water two hours after a breakfast of bread and coffee. Two hours later test the urine. If sugar is present the assimilation limit is pathologically lowered. The assimilation limit is lowered in the following condi- tions : diabetes mellitus, pregnancy, starvation, acute dis- eases, exophthalmic goitre and destructive lesions of the liver and pancreas. Spontaneous permanent glycosuria is nearly always due to diabetes mellitus. Lactose is the only other sugar often found in the urine. This may appear when the function of lactation is inter- rupted, sometimes during lactation. 52 URINE Qualitative Test for Glucose. — Haines' Test. — Boil an inch of Haines' solution in a test tube, add 5 drops of urine, bring to a boil ; if no reaction occurs add 5 more drops of urine and bring to a boil again. If sugar is present a yellow or reddish yellow precipitate is thrown down. Avoid doubtful results by not using too much urine nor boiling too long. A slight greenish precipitate often appears on standing in urine containing no sugar. Haines' Solution : Cupric sulphate, 12 grams. Potassium hydroxide, 45 grams. Glycerine, 90 c.c. Water q.s. 1000 c.c. Quantitative Test for Glucose. Haines' Method. — Measure 10 c.c. of Haines' quantitative sugar test solu- tion into a 100 c.c. flask and add 50 c.c. of water. Dilute the urine by adding 4 parts of water to 1 part of urine and fill graduated burette with diluted urine. Bring the Haines' solution in the flask to a boil. Allow the diluted urine to run from the burette into the boiling solution very slowly and finally drop by drop until the blue color dis- appears. 10 c.c. of Haines' solution is decolorized by 0.01 gram of glucose, therefore, whatever amount of urine was used to decolorize the Haines' solution in the flask contained 0.01 g. of glucose. 0.01 divided by the number of c.c. of undiluted urine used would give the grams of sugar in each c.c. of urine. 100 times this would give the amount in 100 c.c. or the per cent of sugar. Example : 1 c.c. of urine decolorized the fluid in the flask. 0.01-1-1X100=1=% of sugar. If the 24 hour amount MANUAL OF LABORATORY DIAGNOSIS 53 of urine was 3000 c.c. the grams of sugar excreted in that time would be 1% of 3000 or 30 grams. Haines' Solution for Quantitative Sugar Determination : Copper sulphate 8.314 grams Potassium hydroxide 25 grams Ammonia 350 c.c. Glycerine 40 c.c. Distilled water q.s 1000 c.c. The Acetone Bodies. B-Oxybutyric acid, diacetic acid and acetone are spoken of as the acetone bodies. One or more of them will ap- pear in the urine when they are in excess in the blood. Poisoning by these bodies is called acidosis. This acidosis occurs when there is carbohydrate starvation. The source of the acetone bodies is the fats. The ammonia out- put in the urine is increased in proportion to the output of acetone bodies. The system is protected against the in- creased acidity due to the acetone bodies by neutraliza- tion with ammonia. This sort of acid poisoning may occur, 1st, when car- bohydrates are not ingested, as in starvation ; 2nd, when carbohydrates cannot be retained or digested, as in can- cer of the stomach and some intestinal disorders ; 3rd, when carbohydrates cannot be assimilated, as in diabetes mellitus. Test for acetone. (Gunning's) — To Y* test tube of urine add about 1 c.c. each of tincture of iodine and ammonia. A black precipitate of nitrogen iodide forms and gradually disappears. If acetone is present iodoform is formed, and is recognized by its odor, yellow color and the microscopic appearance of its crystals. 54 U R I N E Test for diacetic acid. ( Gerhardt's ) — To y 2 test tube of urine add a few drops of tincture of ferric chloride. If a precipitate (phosphates) occurs filter and add more ferric chloride to the filtrate. A dark red color which fades somewhat on boiling or on standing sev- eral hours, indicates diacetic acid. Salicylates, phenol and antipyrine cause a dark red color which does not fade on boiling. Urea. Normally about 85% of the nitrogen excreted in the urine is in the form of urea. This proportion is altered in acidosis because the nitrogen is taken up in forming the ammonia which combines with the acid. Urea is increased when there is, 1. Increased intake of nitrogen, as with meat diet. 2. Increased tissue destruction, as in fevers or after excessive exercise. Urea is decreased when there is, 1. Decreased intake of nitrogen in the food. 2. Destructive disease of the liver, as acute yellow atrophy. 3. Renal insufficiency as in acute nephritis and chronic interstitial nephritis. Test for Urea. — Use Doremus' ureometer. Remove albumin from urine if more than a trace is present, by acidifying with acetic acid, boiling and filtering. Fill the long arm of the ure- ometer with freshly made sodium hypobromite. Add slowly through the curved pipette exactly 1 c.c. of the urine. The gas evolved is nitrogen and bears a constant relation to MANUAL OF LABORATORY DIAGNOSIS 55 the amount of urea present. The percentage of urea is read on the scale. Sodium hypobromite is made by adding 1 c.c. of bromine to 30 c.c. of a 20'/ solution of sodium hydroxide. The Doremus ureometer is filled with the 20^ sodium hydrox- ide and the bromine introduced with a curved 1 c.c. pipette. Ammonia. The normal excretion of ammonia in the urine is less than one gram in twenty-four hours. Notable increase in ammonia occurs in acidosis, the acid bodies being combined with ammonia to protect the tissues against the acids. In pernicious vomiting of pregnancy ammonia is much increased. The estimation of it may help to distinguish between pernicious vomiting and ner- vous vomiting. When the urea forming function of the liver is interfered with more of the nitrogenous waste appears as ammonia and correspondingly less as urea. Formalin Method of Estimation. To 10 c.c. of urine in a 100 c.c. flask add 25 c.c. of dis- tilled water and 5 drops of a 1% alcoholic solution of phe- nolphthalein. Neutralize by the addition of N/10 NaOH. In a second flask measure 5 c.c. of formalin, add 25 c.c. of water and neutralize with N/10 NaOH, using phenol- phthalein as an indicator. Add the neutralized formalin to the above neutralized urine. The resulting mixture will become colorless because the formalin breaks up the am- monium salts and liberates the acids. Titrate this acid in the mixture with N/10 NaOH. The number of c.c. N 10 NaOH used in this last titration X0.0017 will give the number of grams of NH :J in 10 c.c. of urine. From this the amount in 24 hours can be calculated. Example: Suppose 4 c.c. N/10 NaOH was used to 56 URINE neutralize the acidity after adding- the formalin. 4X0.0017= 0.0068 grams of ammonia in 10 c.c. of urine. If 1000 c.c. is the amount of urine passed in 24 hours 0.0068X100=0.68 grams ammonia in 24 hours. Uric Acid. The source of the uric acid is the nucleins. It is present normally as urates of sodium, potassium and ammonium. It crystallizes in the urine when in excess, or when the urine is very concentrated and acid. The variations in amount are considerable in health. Uric acid is increased : 1. After ingestion of foods rich in nucleins, as sweet- breads or liver. 2. In leukaemia, from the breaking down of many nu- cleated cells. 3. During and after the paroxysm in gout. 4. In fevers, corresponding to the increase in urea. Uric acid is decreased : 1. In advanced kidney disease. 2. Preceding the paroxysm in gout. Test for uric acid. — Use Ruhemann's uricometer. Introduce through a pipette carbon disulphid up to the mark S. Add the iodine mixture to the mark J. Mix well. The carbon disulphid becomes a deep red color. Add urine up to the lowest calibration, mix by turning. Add more urine, a few drops at a time, turning after each addition, until the carbon disuJphid i.3 white. The reaction is then complete. Read the mark on the scale even with the surface of the urine. The scale reads in grams of uric acid per litre of urine. MANUAL OF LABORATORY DIAGNOSIS 57 Ruhemann's reagent : Iodine, 0.5 g. Potassium iodide, 1.25 g. Absolute alcohol, 7.5 c.c. Glycerine, 5 c.c. Distilled water q.s., 100 c.c. Indican. Indol is a product of decomposition of proteid in the in- testine. It is oxidized in the blood to indoxyl, combines with sulphuric acid to form indoxyl sulphate of potassium (indican), in which form it is excreted in the urine. An excess appears in 1. Intestinal putrefaction, a. Due to intestinal indigestion, diarrhoea, intes- tinal tuberculosis, constipation, etc. b. When HC1 is lacking in the stomach as in chronic gastritis or cancer. c. When there is a lack of peristalsis as in peri- tonitis or ileus. 2. Putrefactive processes elsewhere in the body, as em- pyema, lung abscesses, advanced pulmonary tuberculosis. Obermayer's Test. — Pour y 2 inch of urine into a test tube, add an equal amount of Obermayer's reagent and about 1 c.c. of chloroform. Mix well. If an excess of indican is present the chloroform will be colored blue, the depth of the color depending upon the amount of indican present. Obermayer's Reagent : Hydrochloric acid, 500 c.c. Ferric chloride, 1 gram. 58 • URINE The HC1 breaks up the potassium indoxyl sulphate and frees indoxyl. The ferric chloride oxidizes the indoxyl to indigo, which colors the chloroform blue. In urine containing bile or in any highly colored urine a doubtful color may appear in this test. This is avoided by removing disturbing substances with lead acetate. To half a test tube of urine add 1/5 its volume of a saturate solution of lead acetate. Let stand a few minutes, filter and test filtrate. Other oxidizing agents can be used with HC1 in detect- ing indican. The following test is more delicate than Obermayers and shows a faint color with normal urine : Pour 5 ex. of HC1 into a test tube, add 1 drop of HNO' and 15 drops of urine. Mix well. If indican is present an amethyst color appears, reaches its maximum in from 5 to 10 minutes and then changes to yellow. Bile. Bile in the urine is always pathological. It appears whenever bile is present in the blood, that is, in jaundice from any cause. Bile may appear in the urine before the jaundice is apparent in the skin. Bile salts and bile pig- ments usually appear together and tests for bile pigments usually suffice for both. Tests.— Urine containing bile has a very yellow or greenish color, it foams when shaken and the foam is distinctly yellow. It stains filter paper yellow. The formed elements in the sediment are stained vellow. MANUAL OF LABORATORY DIAGNOSIS 50 Gmeliris Test. — Layer the urine with nitric acid. If bile is present a dark green color will be seen at the contact. Smith-Rosen Test. — Layer 2 c.c. of Smith's reagent upon an inch of urine. Emerald green ring appearing at the contact indicates bile. Smith's reagent : Tincture of iodine, 10 c.c. 95% alcohol, 90 c.c. The Diazo Reaction of Ehrlich. This reaction never occurs in health, and rarely in non- febrile diseases. It is nearly always present in typhoid fever and measles, occasionally in pneumonia, scarlet fever, diphtheria, ery- sipelas and tuberculosis. This reaction occurs in about 80% of the cases of ty- phoid, appearing early, often during the first week, and disappearing with the subsidence of the fever. Its reap- pearance during a relapse may serve to distinguish relapse from complication. The reaction is more constant and more marked in severe cases. A positive Diazo assists in distinguishing measles from rotheln. The appearance of a positive Diazo in tubercu- losis indicates a bad prognosis. Test : To 40 parts of solution I and one part of solution II (two fingers of solution 1+4 drops of solution 2) in a test tube add an equal amount of urine. Add quickly an excess of ammonia and shake. A deep red color and a pink foam constitute a positive reaction. A dark greenish pre- cipitate appears within 24 hours. 60 URINE Solution I : Sulphanilic acid, 1 gram. Hydrochloric acid, 5 c.c. Distilled water, 100 ex. Solution II : Sodium nitrite, 0.5 grams. Distilled water, 100 c.c. The solution of sodium nitrite should be freshly made. Sediments in Urine. Sediment in urine is never strictly normal but may not be of serious significance. Methods of examination. A few facts may be obtained by macroscopic examination. Urates and phosphates may be recognized by appearance and chemical reactions. Uric acid in large amount is visible to the naked eye and char- acteristic in appearance. For microscopic examination the urine is centrifuged, the liquid poured off, the last drop in the centrifuge tube is shaken, then poured upon a slide, covered with a cover glass, and examined first with low power, afterwards with high, if it contains small bodies not easily identified with the low power. The light should be considerably cut down with the iris diaphragm as hyaline casts and other transparent bodies will be entirely invisible if there is too much light. Unorganized or Chemical Sediments. Those which have no cellular form and no connection with the cellular ele- ments of the body. 1. In acid urine. A. Amorphous. Urates, a heavy cloud usually pinkish or brick dust color, soluble on warming. B. Crystalline. a. Uric acid. Amber, rarely colorless, large crystals of various shapes, rhombic plates, diamonds, "whetstones" and "butcher's block" or short MANUAL OF LABORATORY DIAGNOSIS 61 cylinders. They tend to form in groups and masses or rosettes. They are soluble in caustic soda and insoluble in hydrochloric or acetic acid. They appear when uric acid is in excess and the urine is very acid and concentrated. They are only significant when found in a freshly voided specimen. They may form renal or vesical cal- culi, and may be found in the urine together with blood cells in this case. b. Oxalate of calcium. Small, colorless, refractile. Common form is the envelope. Sheaf, hour glass and oval forms may appear. They vary much in size. They dissolve in strong hydro- chloric acid. They appear after a diet rich in oxalates, as strawberries, rhubarb, tomatoes or spinach. They often accompany intestinal in- digestion and muscular pains. They may form calculi and masses of them with blood cells may be found in this case. c. Cystin. Colorless, six sided plates. They are rare. They are due to abnormal proteid metab- olism and individuals sometimes have them in the urine throughout life. They form calculi. d. Lencin and tyrosin. They appear together, the leucin in yellowish balls, the tyrosin as needle crystals in sheaves They are rare, being found in acute yellow atrophy of the liver and phos- phorous poisoning. 2. In alkaline urine. A. Amorphous. a. Phosphates. Granular whitish cloud. Dissolves on the addition of acids. b. Carbonates. Indistinguishable from phosphates in appearance. Dissolves on addition of acids 62 U R I N E with «erTervescence. They indicate decompo- sition of the urine. B. Crystalline. a. Ammonium-magnesium phosphate (triple phos- phate). Colorless, usually large, prism forms. The "coffin lid/ 5 "boot jack'' and occasionally feathery forms are seen. They are soluble in acetic acid. They usually signify nothing but decomposing urine, but when found in fresh specimens they point to alkaline fermentation in the bladder. b. Ammonium urate. The only urate which appears in alkaline urine. Brown prickly balls, "thorn apple" crystals, often grouped. They are soluble in acetic acid with formation of uric acid crys- tals. They appear only in decomposed urine containing free ammonia and have no pathologi- cal significance. c. Calcium carbonate. Small indefinite "dumb bells" associated with amorphous phosphates. Organized or Anatomical Sediments. — 1. Epithelial cells. Some are always present. The por- tion of the urinary tract from which the cells come can- not be definitely located. They are of three general types. a. Squamous. Large, thin, flat, leaf like cells, often in sheets. Rather small round nucleus. From bladder, urethra or vagina. b. Irregular cells. Caudate, pyramidal, cylindrical. May come from deep layers of epithelium any- where in the tract. c. Round cells. Small round cells with compara- tively large nuclei, often slightly granular. These include the renal cells, but cells similar in appearance may come from other portions of the tract. Such cells are not common in normal urine. MANUAL OF L4B0RAT0RY DIAGNOSIS 63 In nephritis they are often seen to be in a state of fatty degeneration. 2. Red blood cells. Small round, homogeneous, non- nucleated cells. 'They may retain their normal disc shape and pale yellow color or they may be crenated, or swollen and colorless (shadow cells). Blood cells are always path- ological. Their source cannot be determined by appear- ance but may sometimes be suggested. Sources of blood : a. Acute nephritis and exacerbations of chronic nephritis. From an occasional cell to large amounts. Urine usually smoky colored. b. Malignant disease of the kidney or bladder. Hem- orrhage almost constant and may be profuse. c. Renal tuberculosis. Blood is not constant but is often found. d. Calculus, after the passage of a stone from the kidney or bladder. • e. Acute cystitis. Urine usually red. f. Polypoid tumor of bladder. Often causes profuse hemorrhage. g. Acute urethritis. h. Traumatism from catheter. i. Poisons, as turpentine, carbolic acid, cantharides and urotropine (occasionally after long administra- tion). j. Acute infectious diseases, as yellow fever, malaria and small pox. k. Hemorrhagic diseases, as scurvy, leukemia, purpura and haemophilia. 3. Pus cells. About twice as large as red blood cells, round, granular, with irregular nuclei indistinct or invisible. They may be much or little degenerated. The addition of acetic acid to the slide will clear I'p the nuclei and make 64 URINE their characteristics more distinct. An occasional leuco- cyte may be found in normal urine. Sources of pus : Urethritis, cystitis, pyelitis, from various bacterial causes, including gonococci and tubercle bacilli. In nephritis a few pus cells may be present. 4. Casts. They are molds of kidney tubules. They are cylindrical, finger shaped bodies, of varying length. They appear when the kidney is affected by nephritis, circu- latory changes or toxic irritants. The same causes which produce albuminuria cause casts. They are usually found together, though either may be found without the other. The following varieties are recognized : a. Hyaline. These are homogeneous and so transpar- ent that the light must be considerably cut down in order to see them. They vary much in size, some being very small. They appear with less provocation than other varieties of casts, being found in temporary irritation of the kidneys, after anaesthetics, etc. They appear also in connection with other varieties in all forms of nephritis, and may be the only cast found in advanced interstitial nephritis. b. Granular. The substance of this cast appears finely or coarsely granular and more or less dark look- ing. Sometimes the base of the cast seems to be hyaline with granules partly filling the clear sub- stance. Granular casts indicate a definite lesion of the kidney and seldom appear except in nephritis. The coarsely granular cast has a more serious sig- nificance than the finely granular. c. Waxy. Homogeneous, but less transparent and more refractile than hyaline. They are usually large, sometimes very long. Their color is some- times slightly yellowish. They are of stiffer con- sistency than hyaline casts as shown by their ten- MANUAL OF LABORATORY DIAGNOSIS 65 dency to break off square at the end, and the fact that they are not seen to bend. They are some- times twisted and often show transverse cracks. They appear in advanced nephritis and are of seri- ous significance. In acute nephritis a cast called by many writers "fibrinous" is occasionally found which is difficult to distinguish from waxy. It is apt to be yellowish or brownish in color. d. Epithelial. The cells of the tubules compose or cover these casts. Sometimes a hyaline or granu- lar base appears. Sometimes the cast seems en- tirely formed of epithelial cells. They are found in acute and chronic nephritis and point to a seri- ous lesion of the kidney. e. Blood cell casts. These casts are covered by or formed of red blood cells. They appear whenever there is exudation of blood into the kidney tubules, oftenest in acute nephritis. f. Pus casts. A few leucocytes may be attached to casts of any variety but true pus casts are formed entirely of pus cells. They are found in pyelo- nephritis, and are always of grave significance. g. Fatty casts. These consist of masses of fat globules. They are the product of fatty degenera- tion of epithelial cells composing epithelial casts. If the degeneration is only partial and the cells can still be made out they usually are called epi- thelial. They appear in both acute and chronic parenchymatous nephritis. Pus, blood and epithelial cells may all be found in the same cast. It will take its name from the predominating cells or be called a mixed cell cast. Cylindroids are bodies of the apparent composition of hya- line casts, only less solid. One end appears to be molded 66 URINE in a kidney tubule and the other end trails off into a nar- row thread. They have but little significance, appearing often in normal urine as well as in company with hyaline casts. Narrow, long, transparent, microscopic threads of mucus are a frequent finding in urine. Their source is the bladder and their significance nothing. These should not be con- fused with mucous shreds (tripperfaden) which are opaque flecks or strings seen by the naked eye, often a centimeter long, and when viewed under the microscope are seen to be thickly studded with leucocytes. 5. Bacteria. A few of the varieties which cause infection of the urinary tract can be identified without cultural meth- ods, as the tubercle bacillus and the gonococcus. Normal urine is sterile but is a good culture medium, therefore all specimens not obtained and kept in an aseptic manner rap- idly grow bacteria which have no significance but which cause cloudiness of the urine. 6. Spermatozoa. Spermatozoa may appear in the urine of adult males after intercourse or nocturnal emissions, as well as in cases of spermatorrhea and after epileptic or other convulsive attacks. 7. Yeasts and moulds are usually contaminations, but have been known to infect the bladder in cases of dia- betes, the saccharine urine furnishing a favorable medium for their growth. MANUAL OF LABORATORY DIAGNOSIS 67 _l < o E o o CO O cc o 2 Many casts of all varieties es- pecially blood and epithelial blood cells few or many. A few leucocytes. Many casts of all varieties, especially granular, fatty and waxy. Occasional blood cells. A few leucocytes. Hyaline and granular casts. May be but few. Occasional red blood cells. Pus in small or large amounts. Blood may be present. Tubercle bacilli. Red blood cells, few or many. Pus cells if there is pyelitis. Crystals of calcium oxalate. Uric acid or cystin. _J < o i UJ I o Large amount of albu- min. Normal solids diminished. Very large amount of al- bumin. Normal solids diminished. Albumin present, often in small amounts. May be temporarily absent. Sugar present. Acetone bodies in severe cases. No albumin nor sugar. Normal solids in normal amounts in 24 hr. output. Albumin present, a trace or more. >> re 3 CO 3 *i a « E E 3 < -I < o CO > I Q. Small amount, highly colored. Cloudy or smoky. Specific gravity high. Small or normal amount. Normal or dark in color, cloudy. Specific gravity high or normal. Large amount, light color, clear. Specific gravity low. Very large amount. Color very light, may be greenish, clear. Specific gravity very high. Very large amount. Color very light, clear. Specific gravity very low. Amount normal or large. Cloudy, acid reaction. re £ c o C 4» o t- c o re c. * 2 3 o ° m i. c >,- o E CO CO 4> 'p !* < D z Chronic Parenchymatous Nephritis Chronic Interstitial Nephritis Diabetes Mellitus CO w V 3 n a. ■ * « o£ CO io _ O re 3 c o 4) t- 3 h Renal Calculus CHAPTER IV. STOMACH CONTENTS. Test Meal.— A specified test meal removed at a definite time is nec- essary so that the findings may be comparable. The Ewald breakfast is usually used in this country and con- sists of 35 grams of bread (2 slices) and 400 c.c. (2 cups) of water or clear weak tea. The test meal is given on an empty stomach and the stomach contents are then removed in 1 hour. Stomach contents after a test meal normally consist of 1. acids; hydrochloric acid, free and combined, 2. ferments; pepsin or pepsinogen, rennin or rennin zy- mogen ; 3. food products; starch and products of starch diges- tion, i. e., erythrodextrin and achroodextrin; proteids and products of proteid digestion, i. e., acid albumin, albumose and peptone ; and acid salts. Pathological substances which may be present are lactic acid (more than a trace), mucus (excess), blood, bile, pus, bacteria (large numbers), yeasts and moulds. Examination of Stomach Contents After Ewald Test Breakfast. I. Macroscopical Examination. — Observe amount, color, odor, character of food particles 69 70 STOMACH CONTENTS whether coarse or finely divided, consistency whether watery or viscid, mucus whether mixed with food or float- ing*. If mucus is in excess and mixed with the food the stomach contents will string out when poured from one container into another. II. Microscopical Examination. — Place a drop of unfiltered stomach contents on a slide, add a drop of Lugol's solution, mix, cover with cover glass and examine with high dry lens. Starch granules are stained blue or violet, epithelial cells, pus cells, yeast cells and bacteria, yellow, and fat droplets are unstained. III. Chemical Examination. — Filter and use the filtrate for the chemical examinations. 1. Titration. Measure 5 c.c. of filtrate into each of two flasks. Dilute each to 25 c.c. with distilled water. To the first flask add 3 drops of phenol phthalein (1 % alcoholic solution) and 1 drop of dimethylamido-azo-benzol (0.5 % alcoholic solution). If free HC1 is present, solution turns red; if free HC1 is absent solution turns yellow. To the second flask add 1 drop of alizarin (1 % aqueous solu- tion). If free acid is present solution turns bright yellow, if absent solution turns violet. Titrate mixture in flask No. 1 with N/10 NaOH from burette until red color changes to orange. Take burette reading. Number of c.c. N/10 NaOH used X20 indicates degree of acidity due to free HC1. Continue titration until first permanent pink color appears. Take burette read- ing. Total number of c.c N/10 NaOH used, including the amount used in first determining the free HC1X20 indi- cates the degree of total acidity. MANUAL OF LABORATORY DIAGNOSIS 71 Titrate solution in flask No. 2 until yellow color changes to violet. Take burette reading-. Number of c.c. N/10 NaOH used X20 subtracted from degree of total acidity indicates degree of combined HC1. The sum of the free and combined HC1 subtracted from the total acidity, gives the degree of acidity due to acid salts and organic acids. The acidity of stomach contents is usually expressed in degrees, degree of acidity meaning the number of c.c. of N/10 NaOH required to neutralize the acidity of 100 c.c. of stomach contents. Phenol phthalein indicates total acidity. Dimethyl- amido-azo-benzol (Topfer's Reagent) indicates free HC1. Alizarin indicates all acidity except that due to combined HC1. 2. Test for Ferments.— If hydrochloric acid is absent test for ferments. Pepsin and rennin run parallel, therefore the test for rennin being simpler is sufficient for both. Pour 5 c.c. of milk into each of 3 t. t. and add 2 c.c. of 1 % solution calcium chloride to each. Dilute 1 c.c. of filtrate from stomach contents with 9 c.c. of water, and add 5 c.c. of this dilution to tube 1. To the remainder of the di- luted filtrate add 5 c.c. of water, and add 5 c.c. of this sec- ond dilution to tube 2. Incubate the three tubes J / 2 hour. If rennin is present in normal amount the milk in tubes 1 and 2 will be coagulated. If the milk is not coagulated in either tube rennin is absent. If the milk is coagulated in tube 1 and not in tube 2. rennin is deficient. 3. Test for Lactic Acid. — Lactic acid is never found in the presence of free HO, hence test for it only when free HO is absent. Kelling's Test. To a t. t. full of distilled water add ferric chloride solution to just color the water. Divide into 2 t: t. 72 STOMACH CONTENTS Add filtrate from stomach contents drop by drop to one of the tubes, the second tube being used as a control. If lactic acid is present a canary yellow color will appear as drops are added. 4. Test for Blood.— Weber's Test. Add 1/3 volume glacial acetic acid to tin- filtered stomach contents (about 10 c.c.) and shake. Add about 5 c.c. ether and gently invert tube a few times. Add 1 c.c. of fresh tincture of guaiac (alcoholic solution of gum guaiac), and excess of hydrogen peroxide (about 2 c.c). If blood is present a blue ring will appear at the line of con- tact of ether and stomach contents spreading upward throughout the ether. The depth of color indicates roughly the amount of blood present. 5. Test for Peptone. — Add a few drops of Haines' solution to about 5 c.c. of filtered stomach contents. A violet color will appear if peptone is present. Peptone is present if combined HC1 is present. 6. Test for Starch.— Add Lugol's solution drop by drop to about 5 c.c. of fil- tered stomach contents. A blue color will appear if un- changed starch is present, a brownish color if erythrodex- trin is present and no change in color or an absorption of the color of the Lugol's solution if achroodextrin is pres- s ent. Normally after the addition of the first drops of Lu- gol's solution there is some absorption of color due to the presence of achroodextrin followed later as more Lugol's is added by the appearance of the brownish color due to erythrodextrin. MAX UAL OF LABORATORY DIAGXOSIS 73 Normal Stomach Contents After Ewald Test Breakfast. Macroscopic: Amount, less than 150 c.c. Color, grayish white to yellow. Character of food particles, finely divided. Odor, sour. Consistency, semi fluid, separates into two layers. Mucus, little or none mixed with food. Chemical : Total acidity. 40 : -60 . Free HC1. 20'-40\ Combined HC1, 10 : -20'. Organic acids and acid salts, less than 10''. Pepsin and rennin present. Peptone present. Erythrodextrin and achroodextrin present. Lactic acid absent. Blood absent. Microscopic : Few bacteria. Xo Boaz-Oppler bacilli, sarcinae or yeasts. Few epithelial cells. Xo pus or blood cells. Mucus from the throat is often found in the stomach contents and shows numerous leucocytes and epithelial cells. Numerous starch granules stained blue or violet with Lugol's solution. Pathological Variations. Amount. — Is increased in hypersecretion and motor in- sufficiency'. Color. — Blood may give a red, brown or black color. Green color may be due to bile or green algae. 74 STOMACH CONTENTS Character of Food Particles. — Coarse when HC1 is de- ficient or absent. Remnants of former meals found in stasis. Consistency. — Watery in hypersecretion, thick and viscid when excess of mucus is present. Mucus. — If intimately mixed with food is pathogno- monic of mucous gastritis. If in lumps or floating in liquid portion, it is swallowed mucus. Free HC1. — Increased in hyperacidity and most cases of gastric ulcer. Decreased in acute gastritis, chronic gas- tritis, and general systemic depression. Absent in most cases of carcinoma, advanced chronic gastritis and perni- cious anemia. Lactic Acid. — Present only in absence of free HC1. Its presence is the most valuable single diagnostic symptom of gastric cancer. Ferments. — If ferments are absent it is due to actual de- struction of the secreting glands. They are seldom absent except in atrophic gastritis and carcinoma. Starch Digestion. — Hyperacidity inhibits starch diges- tion, therefore in hyperacidity unchanged starch is present. If HC1 is diminished or absent, starch digestion proceeds to achroodextrin. Proteid Digestion. — Proteid digestion is poor when there is no free HC1 and little combined HC1. Little or no pro- teid digestion takes place if total acidity is very low. Pro- teid digestion is rapid in hyperacidity. Blood. — Usually present in carcinoma (coffee ground). Hemorrhages occur in many cases of ulcer. Occult blood (chemical, not visible) at times in ulcer, as a rule in car- cinoma. Gastric hemorrhages may occur (a) in primary disease of the stomach as ulcer or cancer, (b) secondary MANUAL OF LABORATORY DIAGNOSIS 75 disease of other organs as in chronic passive congestion and cirrhosis of the liver. Bacteria. — Large numbers of bacilli, micrococci and yeast cells are present when there is stasis. Yeasts and sarcinae are present in gastric dilatation of benign origin. In gas- tric carcinoma large numbers of large, long bacilli, the Boaz-Oppler bacilli, are present. 76 STOMACH CONTENTS < O CO H W. H O O K o < S o CO to O CO O H co i— i W H O < < o o I— I H CO O o<2 (0 << o — 'T3 E cog S © 0) O o — H- O o i°"-HO oo : &-f (0 o o Oil to co £. <» S- E <» E "O +J *; U- E O •m co cd pa < w CO > t- « o a «i. Q) o W ■E 0"J w re™ E -W E C 0) o E f > J.O < E m w* _]- a. 0. E o~ < 0) O w S°fe"5 -§£E = 3 E ° £ g h "4- ELUg Geo" ^5 , or ;?o >» <^ re O h Q HI < DC + O°o 25 ■do o «2o5 re •*- o w ^? ■5 o-m TS~ E « c > CO fi- re 5, o° -Eg CO r- 3 Ol re £.E u >-a O ? < O re Q LJ 5* Z co — .o. "Ere Q^ o i §1 E° re o CI -- O a> D* 2E T3 oia> CO E-E E O Dark reddish brown shell with rough sur- face. Thin colorless shell. One sur- face flattened. Protoplasm in stages of em- bryonic devel- opment. Colorless, thin shell. Proto- plasm granular and segmenting into 2, 4, 8 or more rounded segments. Brown thick shell. Button projection at each end. c/5 i o 2 Ova in feces. May find worm in feces. Worms in feces. Ova about the anus. Ova in feces. Ova in feces. Worm rarely in feces. Rhabditiform or filariform embryos in feces. Ova and adult forms rare. z O h o iu u. z re > O Ova. Larvae enter intestine through food or dirt or burrow through skin. Ova. Little known. Probably through em- bryo. DC UJ h o < DC < ai o > o LU Ll _l E s_ o IU E re (0 E (0 a. o ■D Ee O 3 *o 3£ ■D O Embryo set free from ovum in small intestine, becomes sexually mature as it passes along the in- testine. Pregnant females in colon. Ova develop in dirt or feces out- side the intestine to larval form. Larval form enters intestine, con- tinues development in small in- testine and attaches itself to mu- cous membrane. Adult worm develops in same form from ovum. Life history complicated. From ova develop the rhabditiform embyro which generally passes into filari- orm embryo which develops intc pathenogenetic females (strongy- loides intestinals) and sexually ma- ure (Rhabditis stercoralis.) < h m < (0 ■h re s - E c »E el- O a, -e" C0 = o ox 0) +* *' D)'- ■Jj ■« ■•-' +J w MO 5 >, -i! DC E oI^S -E — 'it- O^iio" 5 " > +'.2 5 MM coo 0)OC +* t> 3 ' a ■a >s E >E o a5 - g «o °£?5 +j *- 3 3 O — C re 1r! - c a § O > a, E a> E s-*> o > (BE TJ S- <-> SJ = °t ""E * OEre.5.. ±! c re ^> — o E ai X « O*" O.E £E*2 J; re ra* Q.L. o m o o E ">a> . o c re °^ a> e-o ° re^ - - .e re •a ■ . re-* o g£S c §/? ■ X C--OE CO o re • ■M 0) re a c re^-s VE c a) 5 a> a; re CO CHAPTER VI. HUMAN MILK. Composition of Normal Human Milk. Reaction — slightly alkaline or neutral. Sp. Gr.— 1028-1032. Fat— 3 to 5%. Lactose — 6 to 7%. Protein— 1 to 2.25%. Microscopically, fat droplets are present and during the first few days of lactation, colostrum corpuscles (large granular cells). Variations in the amount of fat, proteid and sugar are the chief changes which have a practical bearing on infant feeding. Fat. Use Holt's cream gauge. Fill cream gauge to zero mark with fresh milk. Let stand 24 hours. Read % of cream. The ratio of cream to fat is as 5 to 3. Thus 5% cream indi- cates 3% fat. The fat content may be determined immediately with the small Babcock tube which fits the cup of the ordinary centrifuge. 5 c.c. of milk is pipetted into the tube, 5 c.c. of concentrated H 2 SO is added a little at a time, mixing with each addition, and finally enough of a mixture of equal parts of concentrated HC1 and amyl alcohol is added to fill 85 $6 HI 'MAX MILK the tube. Centrifuge the tube 5 minutes and read the per- centage of fat directly on the tube. Protein. Boggs' Modification of the Esbach Method. Dilute the milk ten times and rill Esbach albuminometer to mark U with diluted milk. Add the reagent to the mark R. Mix by turning. Let stand 24 hours. With this dilu- tion the marks on the tube give the percentage of the pro- tein. Boggs' Reagent. Phosphotungstic Acid 25 grams. Concentrated Hydrochloric Acid 25 c.c. Distilled water q. s. ad 250 c.c. Lactose. Mix equal parts of milk and Boggs' Reagent, dilute with equal volume of water, filter and estimate lactose in the fil- trate by the titration method as in the quantitative esti- timation of sugar in urine. 0.014 grams of lactose re- duces 10 c.c. of Haines' quantitative solution. To calculate : 0.014 divided by the number of c.c. of undiluted milk used multiplied by 100 gives the % of lac- tose. CHAPTER VII. CEREBRO-SPINAL FLUID. Normal cerebrospinal fluid is limpid and colorless and contains very few cells. It contains enough sugar to re- duce copper solutions and a very slight trace of proteid material. Bacteriological Examination. Make smears from centrifuged sediment of spinal fluid. Stain with Gram, also with . carbol fuchsin if tubercle ba- cillus is suspected. Make cultures of centrifuged sediment on blood agar and blood serum, using culture tubes and Petri dishes in which blood agar has been poured. In smears and cultures examine for meningococcus intracel- lularis, tubercle bacillus, pneumococcus. streptococcus, ty- phoid bacillus, colon bacillus and influenza bacillus. Other bacteria have been reported but rarely. In examining for tubercle bacilli in spinal fluid it is best to allow the fluid to stand 24 to 48 hours to allow a pellicle to form. This pellicle may contain the bacilli when they cannot be found in the fluid proper. Cytology. Differential Cell Count. The fluid is centrifuged, the sediment smeared, dried and fixed on a slide and stained with Wright's blood stain. A differential count of the cells is then made as a differential 87 88 CEREBROSPINAL FLUID leucocyte count is made. In the acute infections the poly- morphonuclear cells predominate and in the chronic infec- tions as tuberculosis and syphilis the lymphocytes predom- inate. Total Cell Count. In fluids containing few cells (clear fluids) the number of cells per cu.mm. is estimated as follows : The leucocyte pipette is filled to the 1 mark with 10% acetic acid, the fresh spinal fluid, well shaken, is then drawn up to the mark 11. The mixture is shaken and a drop mounted in the counting chamber as in counting blood. The Tiirck counting chamber is used and the cells in the whole ruled space, 9 sq. mm., are counted. This space contains 9/10 cu. mm., of fluid. The mixture is 9/10 spinal fluid and 1/10 diluting fluid. Therefore the number of cells counted X10/9X10/9= the number of cells per cu. mm. For ex- ample the 9 sq. mm., contains 40 cells. 40X10/9X10/9= 49+. If cells are so numerous as to cause clouding, the spinal fluid must be diluted as for a leucocyte count of the blood. The number of cells per cu. mm. of normal spinal fluid is less than 10. Chemical. Test for Globulin.— The Ross-Jones modification of the Nonne test for ex- cess of globulin is as follows : 1 c.c. of spinal fluid is care- fully layered upon 2 c.c. of saturated solution of ammonium sulphate in a small test tube. A grayish white ring at the contact, appearing within a few minutes, is a positive re- action and indicates a pathological amount of globulin in the spinal fluid. The test is positive in all infections of the nervous system and negative in normal spinal fluid. MANUAL OF LABORATORY DIAGNOSIS 89 The Lange Colloidal Gold Reaction. Technique of the Test. Ten test tubes (6 in. x Y\ in.) are placed in a row in a test tube rack. To the first add 1.8 c.c. of a freshly made 0.4% sodium chloride solution. To each of the other tubes add 1 c.c. of the same solution. Draw up into a 1 c.c. pipette 0.2 c.c. of the spinal fluid to be tested and add it to the first tube. This makes a dilution of 1-10 in tube 1. From the first tube remove with the same pipette 1 c.c. of the mixture and place it in tube 2. This will make a dilu- tion of 1 in 20 in tube 2. Remove 1 c.c. from tube 2 and place it in tube 3, making a dilution in tube 3 of 1 in 40. Continue in this way to the end of the series, discarding the 1 c.c. removed from tube 10. The dilution in each tube is double that in the tube before it. the dilution in tube 10 being 5120. Add to each tube in the series 5 c.c. of the colloidal gold solution. At the end of twenty-four hours a final reading is made, although strong reactions will show in a much shorter time. The reaction consists in color changes due to more or less precipitation of the colloidal gold. The solution itself is red and clear. Slight precipitation gives a bluish tint to the fluid. Increasing amounts of precipitation change it to violet blue, grayish and when precipitation is com- plete, colorless. The amount of change is expressed by numbers, 5 representing complete precipitation and the least observable change 1. A negative reaction would be written 0000000000. One in which precipitation is com- plete in the first four tubes and less in the fifth, sixth and seventh would be written 5555431000. Preparation of the colloidal gold solution used as indicator requires great care and is often unsuccessful in inexperienced hands. Specific directions for its preparation with discussion of its difficul- ties may be found in Bull. Johns Hopkins Hospital xxvi, 90 CEREBROSPINAL FLUID 1915, p. 391. The colloidal gold solution can be purchased from E. H. Sargent. Diagnostic Value. The diagnostic value of the test is considerable as it dis- tinguishes several types of cerebro-spinal disease. In general paresis complete precipitation occurs in the lower dilutions, the typical reading being 5555431000. Com- plete precipitation may occur in even higher dilutions. In cerebro-spinal syphilis and tabes the greatest color change occurs in the third and fourth tubes, a typical read- ing being 1133200000. In non-syphilitic meningitis the greatest precipitation oc- curs in the higher dilutions, that is, beyond the fourth tube. In normal spinal fluid the color of the whole series re- mains unchanged. MANUAL O/-' LABORATORY DIAGNOSIS < DC III h O < CO Meningococcus Pneumococcus Streptococcus Influenza B, etc. Tubercle bacillus. Spirochaeta Pallida Absent. DIFFEREN. TIAL CELL COUNT Polynuclears predominate Mononuclears predominate (0 « «1 4) .E 1 E O 0) o a 2 &3 . _J O LU zo .e CI S- V > ■o (0 re 0) o c ■a minute. 3. Wash in water and dry between filter paper. Loeffler's Methylene Blue. (Wright's formula.). Methylene blue 0.5 gram. Sodium carbonate 0.5 gram. Water 100 c.c. 102 BACTERIOLOGY Gram's Method. — 1. Fix smear in flame. 2. Stain ]/ 2 minute in anilin gentian violet. 3. Wash in water. 4. Cover with Gram's iodine y 2 minute. 5. Drain and drop on 95% alcohol until alcohol runs off clear. 6. Wash in water. 7. Counterstain y 2 minute with pyronin (0.5% aqueous solution). 8. Wash in water and dry between filter paper. Anilin- gentian-violet. Anilin water 75 c.c. Sat. ale. sol. gentian violet 25 c.c. Anilin water is prepared by adding 2 c.c. of anilin oil to 98 c.c. distilled water, shaking the mixture vigorously for 1 minute and filtering through filter paper until filtrate runs clear. Grains Iodine. Iodine 1 gram. Potassium iodide 2 grams. Water 300 c.c. The object of the iodine is not to stain and not to decol- orize, but to act as a mordant which sets the stain in some bacteria so that the alcohol will not decolorize them. Those bacteria in which the stain is set are Gram positive, i. e., they keep the gentian violet stain. Those bacteria which are decolorized by the alcohol are Gram negative and take the counterstain. In pus smears Gram positive bacteria appear dark purple. Gram negative bacteria and leucocytes stain pink. MANUAL OF LABORATORY DIAGNOSIS 103 Ziehl-Neelsen Method. (Stain for tubercle bacillus.) 1. In making smear from sputum, select purulent por- tion, pick up with wooden toothpick, smear on slide, fix in flame. Cover with carbol fuchsin. Keep steaming hot three minutes. 2. Wash in water. 3. Decolorize in 10% sulphuric acid in 95% alcohol until well spread portions are decolorized. 4. Wash in water. 5. Counterstain Yi minute in Loeffler's Methylene Blue. 6. Wash in water and dry between filter paper. 7. Examine with oil immersion lens. The tubercle bacilli show as slender red rods. Everything else in the sputum stains blue. Carbol fuchsin. Phenol crystals, melted 25 c.c. Absolute alcohol 50 c.c. Fuchsin (basic) 2 grams. Allow to remain over night in an incubator to insure complete solution, cool and filter. This stock solution is permanent and does not require further filtering. For use, add 1 part of this stock solution to 4 parts of distilled water. Diagnostic Characters of Pathogenic Bacteria. Staphylococci. Morphological and Cultural Characteristics. The varieties of staphylococci are differentiated on the basis of pathogenicity, pigment formation, liquefaction of gelatin and other cultural properties. They are named from their distinguishing characteristics, as Staphylococcus 104 BACTERIOLOGY pyogenes aureus, Staphylococcus pyogenes albus, Staphy- lococcus citreus, Staphylococcus epidermidis albus, etc. Typically the pathogenic staphylococci are Gram posi- tive, appear in smears as spheres in grape-like clusters and as diplococci. Culturally they grow luxuriantly on all media, form moist elevated colonies, white, yellow or orange, depending upon the variety, liquefy gelatin freely, acidify and coagulate milk, and ferment sugars. Atypically staphylococci may vary in size from small to very large cocci, and may appear as flattened diplococci with a narrow space between the cocci. They may be Gram negative. They may grow in tiny colonies, or in the form of flat colonies with concentric markings, or they may form a film like adherent growth. They may liquefy gel- atin very slowly or not at all. Strains which liquefy gelatin freely are in general more virulent. Staphylococcus pyogenes aureus is more pyo- genic than the albus or citreus. Occurrence. The staphylococcus is the most common pus producer. It is found most frequently in skin infections as acne pus- tules, skin abscesses, furuncles, carbuncles. It may cause primary infection of sinuses and may cause septicemia. It is the most common cause of osteomyelitis. Streptococci and Pneumococci. Varieties. No absolute method has been found of differentiating the different varieties of streptococci from one another or from the pneumococcus, but a fairly satisfactory differentiation can be made on the basis of the capsule formation, the be- havior on blood agar, the ability to ferment the various sugars, the serum reactions (agglutination and complement fixation), and the pathogenicity towards animals. MANUAL OF LABORATORY DIAGNOSIS 105 Q Z < CO O u o o o 5^ a u u co CO CO hW <> 3s <° l-H H W M H = ° c o 0> Q.— c ;: w -o re t- ro s- re III! .EfU'OO sill E o F a> J: c > a, w re m W TO O ^aE 5 p« o 3 a> re 1) s_ 0) O) Q,-t-« W O 3~ .c o — ■* <- < c £o .2 >, re '43 oj l.Ere CO 4) * s ENTA- SUGA Raffin + + + 1° Z UJZ J + O O + U-O D h E ZDC o< cj si *E iJ o « « o o ° c re . I 85 1 — re c «j iJ J2 (U OCO i < < L. -a> >» ■ o ' . ■ . 4) > o o J o I Q. DC o 2 o ^ a.- q. .55 T3 re c t. <•- o o 11 re oi o w i§5g 5E°S 1? 5 ra Q. re E ° «- f +J C T3 > re «?3iic ^ ■- o 3 a> O >, s_ WD o o o HI O 05 +J o ° .- s o O r- — O W O c O 3 < E «> & li~ a 5 •u w z ^o Q. O 0) u £ " LlflODB ^ w^ 2. CO 3 5 o ■^ 3 >^ 3 *> 3.- co o E Q. o 1 co u a-^ o 1 CO O > 106 BACTERIOLOGY There are many strains of streptococci found in the hu- man body and its secretions which cannot be readily classi- fied among the streptococci or pneumococci mentioned in the table, partly because of their morphology, partly because of their appearance on blood agar, but chiefly because of their lack of pathogenicity. Some are Gram negative. Some produce dry, brown adherent colonies on blood agar with very slight or no haemolysis or green col- or ization of the agar and are non-pathogenic towards ani- mals, or at least produce no visible lesion in heart or joints even in enormous doses. Pathogenicity. The virulence shown by the streptococci and pneumo- cocci varies considerably. The lesions produced by animal inoculations depend upon the virulence of the strain, the method of inoculation and the number of bacteria inocu- lated. The most virulent streptococci will cause fatal septicaemia, less virulent strains may cause localized ab- scesses, erysipelatoid inflammations or endocardial or joint involvement. Streptococcus hemolyticus tends to locate in the joints, while streptococcus viridans tends to locate in the endocardium. The pneumococcus and streptococcus viridans often show little virulence toward animals. Occurrence. Found in erysipelas, lymphangitis, cellulitis and puer- peral septicaemia, in suppurative inflammatory conditions in joints (as arthritis and acute rheumatism) and serous membranes (as peridicarditis and pleurisy), in otitis media and the throat affections of tonsillitis, diphtheria, scarlatina and measles, in enteritis in infants, and in pneumonia (pneumococcus in lobar pneumonia, streptococcus in the lobular pneumonia usually). MANUAL OF LABORATORY DIAGNOSIS 107 Gram Negative Diplococcus Group. Gonococcus. (Diplococcus of Neisser.) Morphological and Cultural Characteristics. The gonococcus is a coffee bean shaped large diplococcus with flat sides adjacent. It is Gram negative. In pus smears the diplococci appear grouped in the cytoplasm of the pus cells. In freshly infected cases they as a rule are the only bacteria found. In chronic gonorrhea many other bacteria may be present and the microscopic diagnosis is not so simple. The gonococcus grows best on blood agar or ascitic agar in the form of delicate, fine, grayish white colonies. After continued cultivaton it may grow on ordinary media. Isolation of the gonococcus is only pos- sible from acute cases and even then it is very difficult. Occurrence. The gonococcus is found in the pus of acute gonorrhea and gonorrheal ophthalmia. Arthritis and endocarditis occur as metastatic complications. Micrococcus Catarrhalis. — Morphological and Cultural Characteristics. The micrococcus catarrhalis has the same morphology in general as the gonococcus, but is often slightly larger and tends to show a sharper outline. In pus smears it gen- erally stains more deeply than the gonococcus and is less often seen within the leucocytes. It grows well on ordi- nary media forming grayish white or yellowish white often adherent colonies of mortar like consistency. Occurrence. Found commonly in secretions of normal and diseased mucous membranes. 108 BACTERIOLOGY Meningococcus. (Micrococcus intracellularis meningitidis.) Morphological and Cultural Characteristics. The meningococcus is a biscuit shaped micrococcus oc- curring usually in pairs, but may be seen also in fours or masses. Involution forms are common. It is Gram nega- tive and shows some irregularity in staining. In pus smears it is chiefly intracellular. It grows best on neutral ascitic glucose agar, forming flat, grayish white disk like colonies tending to become confluent. It may grow on agar after prolonged cultivation. It tends to die out in a few days after isolation. The different strains vary in the ease with which they can be grown on artificial media, in their fermentative action on sugars, and in their virulence. Occurrence. The meningococcus is the most frequent cause of puru- lent meningitis either sporadic or epidemic. In meningo- coccus meningitis the spinal fluid is cloudy and contains a large number of polynuclear leucocytes. The meningo- coccus is found chiefly within the cells. Bacillus of Tuberculosis. Morphological Characteristics. The tubercle bacilli are slender, straight or slightly curved rods often occurring in small clumps, the bacilli lying at an acute angle with one another. They vary in length and often show small nodules or swellings. They are acid fast, that is, they retain the fuchsin stain and are not decolorized by acids or alcohol as the non-acid fast bac- teria are. They commonly stain uniformly but may show a beaded appearance due to the deep staining of the nodules and the pale staining of the areas between. MANUAL OF LABORATORY DIAGNOSIS 109 Diagnosis. Examination of. sputum for tubercle bacilli is commonly made. The usual and most satisfactory method is directly by stained smears. Care must be taken to obtain the sputum from the lungs. Purulent portions are smeared on slides, fixed in the flame and stained with carbol fuchsin. (See stains.) Thicker smears may be made in examining for tubercle bacilli than for the usual bacteriological exam- ination, thus permitting one to examine easily more ma- terial. One has no difficulty in distinguishing the red stained bacillus even in a thick smear. A careful exami- nation of more than one slide prepared from suspicious purulent portions will show the bacilli if they are present. The tubercle bacilli show as bright red rods against a blue background and show a characteristic arrangement and morphology. Tubercle bacilli in the urine are much more difficult to find than in sputum. Large amounts of urine must be centrifuged and the sediment smeared on slides, dried, fixed in the flame and stained as sputum smears. The pos- sible presence of other acid fast bacilli as the smegma bacil- lus rarely makes the diagnosis of tubercle bacilli in the urine uncertain. The examination of a catheterized speci- men will usually exclude the smegma bacillus. The only certain method is to inject a guinea pig with the sediment from a large volume of urine. Diphtheria Group. (Bacillus diphtheriae and the diphtheroid bacilli.) Morphological and Cultural Characteristics. The typical diphtheria bacillus produces a powerful toxin, grows rapidly and grows best on serum media, and shows a characteristic morphology in stained smears. The. 110 BACTERIOLOGY diphtheria bacilli are slender rods which vary in length, are often slightly curved, often clubbed or swollen at the end or middle. They may lie at an acute angle with one another or they may lie in palisade arrangement. They are Gram positive. They do not stain uniformly with Loefller's methylene blue but show granules or barred staining. The pseudo-diphtheria bacilli tend to be short plump rods more uniform in size and shape, and generally do not show polar granules. The true and pseudo-diph- theria bacilli, however, cannot always be differentiated morphologically. Culturally the diphtheroid bacillus like the diphtheria bacillus grows best on serum media, but may grow well on all media. A valuable means of differentiating the Hof- mann bacillus or pseudo-diphtheria bacillus from the diph- theria bacillus is by growing the bacilli in glucose bouillon. The Hofmann bacillus does not produce acid by the fer- mentation of the glucose while the diphtheria bacillus does. It is not possible, however, to classify the numerous strains of diphtheria and diptheroid bacilli on the basis of their fer- mentative action on the various sugars. Diphtheroid bacilli of acne and those found in enlarged glands can be isolated anaerobically, but after cultivation may grow aerobically. The acne bacillus is easily and constantly isolated from acne pustules in anaerobic cultures on blood serum. Pathogenicity. The virulence of diphtheria bacilli varies considerably, but the majority are of nearly equal virulence. Diphtheria like bacilli are commonly found which are pathogenic to guinea pigs especially in enormous doses, but which pro- duce no diphtheria toxin. Animal inoculation is used as a test of virulence and as a test of toxin production. True diphtheria bacilli cause death in 72 hours of a guinea pig injected subcutaneously with a broth culture in amount MANUAL OF LABORATORY DIAGNOSIS 111 less than 1/5% of the body weight. If a guinea pig in- jected with antitoxin lives after the injection of about 2 c.c. of the broth culture of a bacillus and the control pig which received only the broth culture dies, one is deal- ing with a virulent diphtheria bacillus. Occurrence. Virulent diptheria bacilli are found in diphtheritic mem- branes, also occasionally on normal mucous membranes. Diphtheroid bacilli are commonly present on normal and inflamed mucous membranes as of the pharynx, nose, ear, eye (B Xerosis), urethra, vagina, etc., and on the skin as the acne bacillus in acne pustules. They are found in en- larged glands in Hodgkins disease, lymphatic leukemia, leprosy, etc. Diagnosis of Diphtheria. The diagnosis of diphtheria often rests upon the bacteri- ological findings and the early diagnosis is very important. The bacteriological diagnosis can often be made from a smear directly from the throat membrane. This should always be made as it may permit the administration of antitoxin many hours earlier. If the throat smear is doubt- ful or negative, wait for the examination of the culture. The culture is best made on blood serum and should be examined after 8 to 10 hours incubation as the diphtheria bacillus grows rapidly. Smears from the throat or smears from the culture should be stained with methylene blue. If the diphtheria bacilli are present they will show their characteristic clubbed form or their barred or orranule Hemoglobinophilic Bacillus Group. Occurrence. In this group belong the influenza like bacilli found in 112 BACTERIOLOGY some epidemics of influenza, the pertussus bacillus found in whooping cough, the Koch-Weeks bacillus found in acute contagious conjunctivitis, the bacillus of Ducrey found in soft chancre, the influenza like bacilli found in diseases of the respiratory tract, in some cases of acute meningitis, measles and scarlet fever. The bacilli found in these various diseases belong to the group of hemoglo- binophilic bacilli and are identical morphologically and culturally, differing only in the matter of virulence and varying somewhat in that. Morphological and Cultural Characteristics. All the bacilli of this group require hemoglobin in the culture media for growth. They grow in tiny transparent colonies and die out quickly. They are very small, non- motile, Gram negative, bipolar staining bacilli. Bacterial Diagnosis. Differentiation between non-pathogenic influenza like bacilli found in sputum, etc., and this group of hemoglo- binophilic bacilli cannot be made in smears. Streak spu- tum on blood agar. Examine small shining colonies in stained smear to see if there are any influenza like bacilli. Transfer suspicious colonies to plain agar and blood agar. No growth will take place on plain agar if it is a true hemoglobinophilic bacillus. For further identification make agglutination test. Mucosus Capsulatus Group. This group includes B. Mucosus capsulatus (Bacillus ol Friedlander), B. lactis aerogenes, B. ozenae (Abel bacillus) and probably the Perez bacillus. MANUAL OP LABORATORY DIAGNOSIS 113 Morphological and Cultural Characteristics. The bacilli of this group are non-motile, Gram negative, generally short broad bacilli but varying in their propor- tions and usually with prominent capsule. They grow well on all media, and form a viscid mucoid growth, the viscid- ity depending upon the degree of capsule formation. They generally ferment sugars with acid and gas production. There is a wide variation within the group from the bacilli of the colon type with no capsule to those with prominent capsule formation, from the bacilli growing like B. coli to the bacilli growing in mucus like masses and from the bacilli producing slight or no carbohydrate fermentation to those which ferment all carbohydrates with a large amount of gas production. Occurrence. The bacilli of this group are found constantly in the crusts and secretion of atrophic rhinitis and ozena, may be found in diseases of the middle ear and accessory sinuses of the nose, in lobular pneumonia, occasionally in cystitis, pyelitis, pericarditis, pleuritis and meningitis (secondary). B. lactis aerogenes is a normal inhabitant of the intestine es- pecially of children. Colon-Typhoid Group. This group includes the colon and paracolon bacilli, the typhoid and paratyphoid bacilli and the group of dysentery bacilli. Diagnostic Methods. These consist of (1) the isolation of the bacilli from the blood, feces, urine or other sites of infection, (2) the morph- ological and cultural study of the bacilli, and (3) for final identification the agglutination test, using immune sera against the various types. o 1— 1 H cfl ►— 1 ti H H U < K < B u < 1— 1 ID o H < J P ID to U O Q < Ph ID o hJ to < o o W 1 Q CD i— i o o J ffi O Ph ffi t* Ph H P4 o O o o o HH to < t-H 2 to w O to O to pq < o = c . ns « cb re +j w D. E *§ w a; re w c ■"= o c£± o>£ ■E c a re s.«) "' ■°.E=£_ £■£ * re re w c c . a>o „•= L 4 c w « £ — o r *> = JJO+' CQ O £ •o £ o c re £ **■ ~ 3 01.Q < core *^ re a) *a * 3 o a> £ re 4>~ CO £ 0) TJ s| •E-K- £x a a 22" 2 re£"Ore 0. a> £ a W 3 a> o (TLl 3s o o .2 « re o o J. a v> o >>rejc hOO o o z "2 E 4) a) «> ° S w £ «- re .2 >>re «> -o ■o ■? » — 0) — M- E -G +J (0 O 5 ct e O ■o-o"£ 0) E o II £-o a a>'o Ll re t re oi a> tj 3 re-H .-wore E w o f6~ Olo Z o S « o « "SIS' Si X"«- a> • '■o re •— 1L O £ -H o X 1 *- a> • "O c "WO re-- «S ra w Se E £^ * 5 CT.2 E E o = re « 4J W 0) i «- OT o ai 4> 4)—, s_ o cbtj a' 3 oi > a> E E o~ *;« UJh o a>"o O 4> g .2J ^3 _tt> o _l ■ t- . t- . O 8."-2«|« ? *£ E o,E E Ol c E O 4>o>> >, c E 01 C s O 4>0 >» >^ Egg o I O 0, O * a. DC O t- •- ^ 5i E 5= ££2 5 « £e re a> CO ^ re ao w z s < < < UJ < z 1° E w ~ OE ciilus raty- osus. pe A. ciilus raty- osus. pe B« a* C = Q O OO re a re re e >, « «e >, DB>, m aa|T CD aa|_ MANUAL OF LABORATORY DIAGNOSIS 115 Methods of Isolation of the Bacilli from Blood, Urine and Feces. From blood. Inoculate blood from the vein into broth in the proportion of 1 part of blood to 50 of broth, or into bile media in the proportion of 1 part of blood to 3 of bile. Identify by cultural characteristics and agglutination test with known immune sera. Front urine and feces. To isolate typhoid bacilli, streak plates of Endo medium with centrifuged urine sediment or a suspension of feces made by rubbing up feces with sterile water. By streaking several plates the inoculation can be sufficiently thin to have isolated colonies. Fish translu- cent dewdrop like colonies and make agglutination test with known typhoid serum. A probable diagnosis of colon bacilli in urine can easily be made by centrifuging catheterized urine in sterile centri- fuge tubes, and adding a few drops of sediment to a fer- mentation tube of dextrose bouillon. Colon bacilli will ferment the dextrose w r ith gas production Bacillus Proteus. (Putrefying bacillus.) Morphological and Cultural Characteristics. The proteus bacillus is an actively motile Gram nega- tive bacillus varying greatly in size, and ranging from short to long filaments. It grows on all media and best at room temperature, producing a putrefactive odor on blood serum and gelatin with liquefaction of the gelatin. The growth on gelatin plates is characteristic, consisting of an irregular radiating mass of colonies with liquefying center. Occurrence. The proteus bacillus is not uncommonly found in pye- 116 BACTERIOLOGY lonephritis and cystitis. Meat poisoning may be due to bacilli of this group. Sometimes associated with other bac- teria as pus cocci or alone, the proteus bacillus has been found in purulent peritonitis and phlegmonous inflamma- tions. It may be found in the nasal secretion. Bacillus Pyocyaneus. (Bacillus of blue and green pus. ) Morphological and Cultural Characteristics. The pyocyaneus bacillus is very small, slender, actively motile and Gram negative. It grows well on all media, liquefies gelatin, and colors all media bright green in the presence of oxygen. Occurrence. The pyocyaneus bacillus may be found in inflammations of the serous membranes as of the pericardial sac and joints, of the mucous membranes as of the ear and sinuses of the nose. It has been found in broncho pneumonia and has been known to cause general infection. Bacillus Tetani. Morphological and Cultural Characteristics. Tetanus bacilli are motile, long slender rods usually single but often in long threads, form large round terminal spores and are Gram positive. They grow on ordinary media only under anaerobic conditions, producing an arbo- rescent growth on solid media. They liquefy gelatin and ferment sugars with gas production. They produce a powerful toxin. Occurrence. The tetanus bacillus is found in tetanus through wound infection. The bacilli multiply but little in the animal body and are usually associated with other bacteria, hence it is difficult to isolate them in pure culture. They produce MANUAL OF LABORATORY DIAGNOSIS 117 a powerful toxin at the point of infection, and this spreads to the motor nerves by various channels, causing death. Diagnostic Methods. These consist of (1) the examination of smears from the pus of the wound, (2) the study of the cultures made from the pus or infecting" material and (3) animal inoculation. 1. Make smears from the pus of the wound, stain with Gram, and examine for spored bacilli ; the tetanus bacillus is seldom found in the pus. 2. Inoculate pus or bits of tissue or foreign bodies found in the wound into glucose bouillon and make anaerobic. Incubate the cultures 24 to 48 hours, then heat them j/ 2 hour at 80° C. to kill all vegetative forms of bacteria. In- oculate the heated culture into glucose bouillon or milk, make anaerobic, incubate 24 hours and examine for the tetanus bacillus. 3. To make certain test, inoculate mice or guinea pig subcutaneously with salt solution emulsion of material from the wound. Tetanus will result in 1 to 4 days if the tetanus bacilli or spores are present. Bacillus Anthracis. Morphological and Cultural Characteristics. Anthrax bacilli are large, non-motile rods with square cut ends. They grow in long chains, often in twisted bundles and show a prominent capsule. They are Gram positive. They form spores only in the presence of oxy- gen, hence not in the animal body. They grow on all media and liquefy gelatin. Their colonies are very characteristic and consist of a tangled mass of filaments. Occurrence. The anthrax bacillus is the causative agent in malignant pustule and intestinal and pulmonary anthrax. They may 118 BACTERIOLOGY be recognized in- smears from the fluid of the malignant pustule, in the feces from intestinal anthrax and in the sputum from pulmonary anthrax. The diagnosis may be established by cultural stud}' and animal inoculation, the anthrax bacillus causing death of guinea pig by septicaemia. Bacillus. Aerogenes Capsulatus. (B. Welchi.) Morphological and Cultural Characteristics. The B. Welchi is a large non-motile bacillus growing singly and in chains. It forms a capsule when growing in the body and spores occasionally in cultures. It is Gram positive. Culturally it is a strict anaerobe and grows well on all media. It ferments the sugars with energetic gas production. It produces a characteristic growth (stormy fermentation) in milk, that is, it forms a much riddled clot because of the abundant gas formation. The milk is acidified by the formation of butyric acid which gives its characteristic odor to the culture. Occurrence. B. Welchi is common in the intestine and soil. It is the cause of emphysematous gangrene. Pathogenic Trichomycetes. Microorganisms of this group of higher bacteria grow in the form of long threads varying in length and thick- ness, some showing club like terminations, some branching. Among them there are Gram positive and Gram negative varieties, acid fast and non-acid fast varieties, aerobic and anaerobic varieties, some growing readily in artificial media, some grown with great difficulty. The growth on solid media is generally granular, adherent, dry like a mould, and in liquid media white, thistledown like tufts of inter- lacing filaments, or they may form a pellicle. They are widely distributed and not infrequently met with. They are of a low grade of pathogenicity, the usual reaction to MANUAL ()} ; LABORATORY DIAGNOSIS 119 infection in man and animals being chronic granulation tumors with or without suppurative foci. Varieties. — Leptothrix. This grows in long, straight, thread like form and shows no branching. It is usually Gram negative. It is found frequently in the human mouth about the teeth and tonsils. It is of doubtful pathogenicity. Nocardia (Streptothrix). This grows in branching threacj like form and in smears appears as a tangled mass of threads. There are Gram positive and Gram negative strains. The growth is usually like that of a mould, but the different strains vary much in the ease with which they grow on artificial media. The anaerobic varieties generally show a scanty growth as compared with the aerobic. It may be found in skin abscesses, alveolar abscesses, brain abscesses, cerebrospinal meningitis, pneumonic areas, pseudotuberculosis of the lungs, etc. Actinomyces. This grows as branching threads. It is typically anaerobic. In the tissues the actinomyces grows in colonies in the form of yellow granules of about pin- head size. For diagnosis examine the pus or granulation tissue for these yellow granules. Crush the granules be- tween slides and examine unstained with high dry lens, also stain with Gram and examine with the oil immersion lens. The granules will be seen to be made up of a tangled mass of filaments tending to radiate from a center and showing characteristic club like terminations. The center of the colony may have a mass of coccus like bodies or conidia. The filaments and spores are Gram positive and the clubs usually Gram negative. Actinomyces causes the disease actinomycosis, which is rare in man. It may occur primarily in the mouth, head or neck, on the skin, in the lungs and in the intestine. 120 BACTERIOLOGY Bacteriology of Conjunctival Secretions. The diagnosis of the bacteria in conjunctivitis can often be made by the examination of a Gram stained smear from the conjunctival secretion. In making a smear the dis- charge should be taken from the conjunctival surface avoiding" contaminations from the lid margins, etc. If the discharge is slight it is best to obtain what has collected at the inner canthus of the eye. Sometimes gentle pressure upward along the lachrymal duct will bring forth some discharge into the inner canthus of the eye. For collection of the discharge it is con- venient to use wooden applicators wound at the tip with a bit of cotton and sterilized. A platinum loop may also be used. Smears are made on clean slides by rubbing the pus collected on the cotton tip of the applicator or plat- inum loop into a thin layer on the slide. The smear is then stained with Gram's stain and examined with oil im- mersion lens. Koch-Weeks Bacillus. This may be found in acute con- tagious conjunctivitis. The bacilli are Gram negative slender rods of varying length. Morphologically they .cannot be distinguished from the influenza bacillus. Bacillus Influenzae. This may be found in catarrhal con- junctivitis. The bacilli are Gram negative very short rods, often resembling elongated diplococci. Morax-Axenfeld Bacillus. This may be found in subacute conjunctivitis. The bacilli are large Gram negative diplo- bacilli, some appearing in short chains. Bacillus Xerosis. This may be found on the normal con- junctiva. The bacilli are Gram positive diphtheroid bacilli. Pneinnococcus. This may be found in acute catarrhal con- junctivitis. The pneumococci are Gram positive, elongated often lancet shaped diplococci. The capsules are not seen in eye smears. MANUAL OF LABORATORY DIAGNOSIS 121 Gonococcus. This may be found in purulent conjunctivitis. The gonococcus is a Gram negative biscuit shaped diplo- coccus, chiefly intracellular. Micrococcus Catarrhalis. This may be found occasionally on the normal conjunctiva and in simple catarrh. It is a Gram negative biscuit shaped diplococcus, generally extra- cellular and often in clusters. Generally the secretion is scanty and poor in cellular elements in proportion to the number of cocci. Meningococcus. This may be found in conjunctivitis ac- companying meningococcal meningitis. The meningo- coccus is a Gram negative biscuit shaped diplococcus, chiefly intracellular. Streptococcus. This may be found in severe membranous conjunctivitis. The streptococci are Gram positive spheri- cal diplococci and in chains. Bacteriology of Vincent's Angina. The diagnosis is easily and quickly made by examination of a smear from the exudate or membrane in the throat. The smear may be stained with methylene blue or Gram's stain. Spirilla and fusiform bacilli in large numbers, and a moderate number of leucocytes will be found in the smears. The spirilla are Gram negative, small and show shallow spirals. The bacilli are Gram negative, irregularly stained with methylene blue, are long, slender and spindle shaped. Cultures from the exudate on blood serum under anaero- bic conditions may yield a growth of the fusiform bacillus mixed with other bacteria. Such cultures have a foul odor. Preparation of Bacterial Vaccines. The preparation of an autovaccine consists in, (1) the obtaining of a bacterial culture, (2) the making of an emul- sion in salt solution of this culture, (3) the sterilizing of the emulsion, (4) the counting of the bacteria in the 122 BACTERIOLOGY emulsion, and the diluting and bottling of the vaccine ready for use. Cultures. — The most difficult and most important step in the mak- ing of vaccines is the obtaining of satisfactory cultures. It is always advisable to first examine the smears made directly from the specimen, and to use this information about the varieties and numbers of bacteria present as a basis in selecting proper media and determining the amount of the material to be used in inoculation. To obtain cultures from abscesses, pustules or other pus containing a single organism, platinum loopfuls of the pus are smeared over the surface of 4 or 5 tubes of Loeffler's blood serum or plain agar, and incubated 24 hours. If there is a mixture of bac- teria in the specimen of pus, sputum, urine, feces, tissue, etc., from which the vaccine is to be prepared it will be necessary to separate the varieties of bacteria and get them in pure culture. To do this the material should be streaked over the surface of the media in Petri dishes of blood agar, over slant surface of a series of tubes of blood serum, and inoculated into liquid media as ascitic glucose bouillon, some of the inoculations being rendered anaero- bic. Pure cultures from the different bacteria can then be obtained from the single colonies and subcultures used in the preparation of the vaccine. Solid media, especially blood serum and blood agar, (5 to 10 tubes) is usually most practical for making the cultures for the vaccine, but in the case of streptococci and pneumococci more growth can be obtained in inoculation of about 50 c.c. of glucose bouillon or ascitic glucose bouillon. Preparation of the Emulsion. — If the cultures are on solid media add a few c.c. of sterile salt solution to each tube and gently scrape the growth from the surface with a platinum loop mixing with the solution. Pour the bacterial emulsion from each tube MANUAL OF LABORATORY DIAGNOSIS 123 into a sterile centrifuge tube, centrifuge 2 or 3 minutes and pour off turbid supernatant fluid into sterile 25 c.c. bottle. Add a few c.c. of sterile salt solution to sediment in centrifuge tube, mix thoroughly by shaking, repeat cen- trifuging and again pour off supernatant cloudy fluid into same bottle with previous emulsion. This process can be repeated if necessary in order to get into an emulsion nearly all of the sediment, but usually twice is sufficient. This method gives a uniformly cloudy fluid, removes any clumps of bacteria and avoids the labor of prolonged shaking to break up the clumps. If the culture is in bouillon, this must be poured into sterile centrifuge tubes and centrifuged. The supernatant bouillon is then poured off and discarded, and sterile salt solution added in its place. The bacterial sediment is mixed well with the salt solution, the tube centrifuged and the supernatant fluid poured into a sterile bottle, the pro- cess being repeated if necessary until most of the bacterial sediment is in the form of a uniformly turbid emulsion. Sterilizing the Vaccine. — Sterilize the bacterial emulsion by keeping it at a tem- perature of 60° C. for one hour. This is done by placing the bottle containing the emulsion in a water bath at 60° C. allowing the water to come well up on the neck of the bottle. Any bacteria on the inside of the neck of the bottle are first killed by thoroughly flaming the neck of the bottle with Bunsen flame. Counting the Bacteria in the Emulsion. — Pour a few drops of the sterilized bacterial emulsion into a watch crystal. Have ready a clean dry watch crystal, a watch crystal with normal salt solution, three clean slides which have been passed slowly through the Bunsen flame 4 or 5 times to burn off grease, one capillary glass pipette with rubber teat fitted over the large end, and a few strips of cigarette paper cut the width of a slide. 124 BACTERIOLOGY Mark the glass pipette with a glass pencil one inch from the tip. Stab finger with a needle and press out drop of blood. By suction with rubber teat draw up blood to pencil mark, draw in small measure of air then same measure of bacterial emulsion as blood, followed by 8 measures of salt solution each separated by air. Press all out in dry watch crystal, mix by drawing back and forth through pipette, then place a drop of the mixture near the end of each of the three slides. Make a thin smear by touching cigarette paper to the drop and pulling paper gently along the slide. Dry smear in the air, fix in 7% mercuric chloride for one minute, wash in water and stain in alkaline methylene blue 5 minutes. To count the bacteria examine the smears with oil im- mersion lens, counting the red blood cells and the bac- teria in about 25 fields on each slide, usually a total of about 300 or 400 red cells. Put the number of red cells counted in each field in one column and the number of bacteria counted in each corresponding field in another column, and sum up each separately. A small circle about Yz inch in diameter drawn on the lower glass, of the eye piece of the microscope with a pen or glass pencil forms a convenient small field in which all the cells can be easily and quickly counted. Well and evenly spread portions of the smears should be used in counting. To calculate the number of bacteria per c.c. in the emul- sion the following proportion is determined, it being known there are 5 million red cells per cmm. of blood. Number of red cells counted 5 million Number of bacteria counted x x= number of bacteria per cmm. multiplied by 1000= number of bacteria per c.c. Diluting and Bottling the Vaccine. — Bacterial vaccines of staphylococci are usually diluted to contain 1000 million bacteria per c.c, streptococci 200 mil- MANUAL OF LABORATORY DIAGNOSIS 125 lion per c.c, and most bacilli 500 million per c.c. In dilut- ing calculate the total number of bacteria which will be present in the vaccine; for example there will be 25,000 million staphylococci in a vaccine bottle containing 25 c.c. of vaccine with 1000 million bacteria per c.c. If the bac- terial emulsion as prepared contains 5000 million bac- teria per c.c, 5 c.c. of this emulsion must be added to 20 c.c. of salt solution in order to have 25 c.c. of vaccine with 1000 million bacteria per c.c. It is convenient to have on hand amber bottles contain- ing 25 c.c. of normal salt solution cotton plugged and ster- ilized in the autoclav. Use a glass Luer syringe (10 c.c.) in making the dilution, sterilizing the syringe by drawing ether back and forth through the needle and the syringe a few times. Having calculated the number of c.c. of the bacterial emulsion required in the complete vaccine, that number of c.c. of salt solution is withdrawn from a bottle containing 25 c.c. with the sterile syringe and replaced by a corresponding number of c.c. of the emulsion. Add as antiseptic J / 2 c.c. of a 5% solution of carbolic acid to the 25 c.c. of vaccine. Fit rubber top previously immersed in 7% mercuric chloride for 5 minutes on bottle and place rubber band tightly just below rim of bottle. It is prac- tical to use heavy surgeon's rubber finger cots, cutting off the finger portion so as to leave about 1 inch of tip. To determine the sterility of the vaccine, shake the bottle, touch rubber top with lysol, withdraw from inverted bottle about y 2 c.c. of vaccine into sterile Luer syringe. Inoculate this into a tube of agar or blood serum and incubate 24 hours. If the vaccine has been prepared from an anaero- bic organism requiring special media for growth, use the same conditions in planting out vaccine as were necessary in growing the bacteria in the first place. INDEX Abel bacillus, 112 Abscess of lung, sputum in, 95 Acchroodextrin, test for, 72 in stomach contents, 69 Acetonuria, Gunning's test, 53 Acidosis, 53 Acid, diacetic in urine, 53, 54 hydrochloric, combined, 69. 70, 73 free, 69, 70. 73. 74 lactic, 71, 74 Acidity, total of gastric con- tents, 70, 73 of urine, 45, 46 Aestivo-autumnal parasite, 27 Agglutination. See Widal re- action Albumiuria, 48 Esbach's test, 51 Heller's test. 49 nitric acid test. 49 occurrence, 48 Purdy's qualitative test, 50 Purdy's quantitative test, 50 Robert's test, 50 Tsuchiya's test, 51 Alizarin 70, 71 Amboceptor, 34 titration of, 36 Anaerobic cultures, 101 making of, 101 Anemia, blood in secondary, 22 pernicious, 24 chlorosis, 23 Anilin-gentian violet, 102 Anisocytosis, 19 Antigen, 34 titration of, 37 Ascaris lumbricoides, 82 Ascitic glucose agar, 100 Asthma, sputum in, 95 B Bacillus, Abel, 112 anthracis, 117 aerogenes capsulatus, 118 coli, 113, 114, 115 diphtheriae, 109 diphtheroid, 109 Ducrey, 112 dysenteriae, 113, 114 Friedlanders, 112 influenzae, 111 Koch-Weeks, 112, 120 Morax-Axenfeld, 120 mucosus capsulatus, 112 ozenae, 112 paratyphosus, 113, 114 Perez, 112 pertussus, 112 proteus, 115 pyocyaneus, 116 tetani, 116 tuberculosis, 108 typhosus, 113. 114, 115 Welchi, 118 Xerosis, 111, 120 Bacterial examination of blood, 98, 115 conjunctival secretion, 120 feces, 98, 115 glands, 98 sputum, 98 tonsils, 98 urine, 98, 115 Bacterial smears, making of, 99 Bacterial vaccines, 121 Basic stippling, 19 Basophiles, 16, 22 Bence Jones proteid, 48, 49 Bile, Gmelin's test, 59 Smith-Rosen test, 59 Bile medium for typhoid cul- tures, 100 127 128 INDEX Blood, agar, 100 casts, 65 coagulation time, 17 color index, 14 culture, 9$ eosinophilia, occurrence of, 21 erythrocyte, counting of, 10 variation in, being nucle- ated, 19 number, 18 shape, 19 size, 19 staining, 19 film, making of, 14 staining, 15 guaiac test for, 72, 79 hemoglobin, 9 in chlorosis, 23 feces, 79, 80 leukemia, 25 lymphatic leukemia, 25 myeloid leukemia, 25 pernicious anemia, 24 - secondary anemia, 22 sputum, 93 stomach contents, 72, 74 urine, 63 leucocytes, counting of, 13 differential count, 17 percentage, 16 varieties, 16 leucocytosis, occurrence, 21 pathologically, 21 physiologically, 21 lymphocytosis, occurrence, 21 pathologically, 21 physiologically, 21 malarial parasites, methods of examination for, 27 obtaining of, 9 pathology, 18 polynucleosis, 21 serum, Loefflers, 100 stain, 15 typhoid bacillus in, 115 Wassermann reaction, 31 Weber's test for, 72, 79 Widal reaction, 29 Wright's stain, 15 Blood agar, 100 Blood cast, 65 Blood cells. See Erythrocyte. Blood culture, 98 Boaz Oppler bacillus, 75, 76 Bronchiectasis, sputum in, 95 Bronchitis, sputum in, 94 Calcium oxalate crystals in urine, 61 Carbol iuchsin, 103 Carbonates in urine, 61 Casts, 64 blood, 65 epithelial, 6 C fatty, 65 granular, 64 hyalin, 64 pus, 65 waxy, 64 Cerebrospinal fluid. See Spinal fluid. Chlorosis, blood in, 23 Coagulation time of blood, 17 determination of, 17 Colon-typhoid group of bacilli, 113 Colostrum corpuscles, 85 Complement, 34 fixation, 32 fixation for Gonorrhea, 41 titration of, 36 Conjunctival secretion, bacteriology of, 120 INDEX 129 Culture media, 99 Cystin in urine, 61 Cytology of spinal fluid, 87 Degree of acidity of gastric contents, 71 Diacetic acid in urine, 53 Gerhardt's test, 54 Diazo-reaction, 59 Dimethylamido-azobenzol indi- cator, 71 Diphtheria, diagnosis, 111 Doremus' ureometer, 54 Ehrlich's diazo reaction, 59 Endo-medium, 100 Eosinophile leucocyte, 16 Eosinophilia, occurrence, 21 Epithelial cast, 65 Erythrocyte, counting of 10 variation in being nucleated. 19 number, 18 shape. 19 size, 19 staining, 19 in urine. 63 Erythrodextrin. 69. 72 Ewald test breakfast, 69 Fat in feces, 81 in milk, 85 Fatty cast. 65 Feces, blood in. 80. 78, 79 color of, 77 examination of, 77 bacterial, 115 chemical, 79 macroscopical, 77 microscopical, 77 fat in, 81 food remnants in, 77 uncus in, 78, 79 ova in, 78, 82 parasites of, 78, 82 pathological findings and sig- nificance of, 79 stones in, 81 vegetable detritus of, 78 Friedlanders bacillus, 112 Grangrene of the lung, sputum in, 95 Gastric contents, blood in, 72, 74 Boaz oppler bacillus in. 75, 76 combined hydrochloric acid in, 71, 73. degree of acidity of, 71 examination of, 69 chemical, 70 macroscopical, 69 microscopical, 70 ferments of, 69, 71, 74 free hydrochloric acid of, 70, 73, 74 in chronic gastritis, 76 gastric carcinoma, 76 gastric ulcer, 76 hyperacidity, 76 lactic acid in, 71, 74 normal after Ewald test breakfast, 73 mucus in, 70, 74 pathological variation from normal, 73 proteid digestion, 69, 72, 74 sarcinae in, 75 test meals, 69 titration of, 70 total acidity of. 70. 73 yeasts in. 75 130 IXDEX Gastritis, gastric contents in, 76 Globulin in cerebrospinal fluid, 88 Ross-Jones test for, 88 Glycosuria, 51 Haines' test, 52 Haines' quantitative test, 52 Gmelin's test for bile, 59 Gonococcus, 107 Gonorrhea Complement fixation test for, 41 Gram negative diplococcus group, 107 Granular cast, 64 H Hayem's fluid, 10 Hemoglobin, estimation of, 9 Hemoglobinophilic group of bacilli, 111 Hemolysis, 31 Hemolytic system, 31 Hook worm, 82 Hyalin cast, 64 Hydrochloric acid, free, 70. 71, 73, 74 combined, 71, 73 titration of, 70 Hyperacidity, gastric contents in, 76 Hypobromite method of esti- mation of urea, 54 Indican, excess, 57 Obermayer's test, 57 K Kelling's test for lactic acid, 71 Lactic acid in gastric contents, 74, 76 Lactose in milk, 86 in urine, 51 Lange colloidal gold reaction, 89 diagnostic value, 90 Leucin and tyrosin in urine, 61 Leucocytes, counting of, 13 differential count, 17 percentage of each variety, 16 varieties of, 16 Leucocytosis, 20 Leukemia, blood in, 25 Loeffler's methylene blue, 101 blood serum, 100 Lymphatic leukemia, blood in, 26 Lymphocytes, small, 16 Lymphocytosis, 21 M Macrocyte, 19 Malarial parasites, 27 methods of examination, 27 aestivo autumnal, 27 quartan, 27 tertian, 27 Mast cell, 16 Megaloblast, 19 Meningococcus, 108 Meningitis, spinal fluid in, 91 Microblast, 19 Milk, human, fat in, 85 colostrum corpuscles of, 85 composition of, 85 lactose of, 86 proteid of, 86 reaction of, 85 specific gravity of, 85 INDEX 131 Morax-Axenfeld bacillus, 120 Mucin in urine, 49 Mucosus capsulatus group of bacilli. 112 Mucus, in feces, 78. 79 in gastric contents. 70, 74 Myelocyte, 20 Myeloid leukemia, blood in. 25 N Normoblast. 19 Rennin in gastric contents, test for, 71 Ross-Jones test for globulin, 88 Rudolpf's method, modification of, for determina- tion of coagulation time of blood. 17 Ruhemann's method of estima- tion of uric acid. 56 Ova in feces, 78, 82 Oxyuris vermicularis. 82 Oligocythaemia, 18 Obermaver's test for indican, 57 Parasites in feces. 78. 82 Perez bacillus, 112 Phenolphthalein, 70, 71 Phosphates in urine, 61 Pneumococcus, 104 Pneumonia, sputum in, 94 Poikilocytes. 19 Polychromatophilia. 19 Polycythemia, 19 Polynucleosis, 20 Proteid digestion in gastric contents, 74 Proteid in milk, 86 Pulmonary tuberculosis, sputum in, 94 Pulmonary edema, sputum in. 95 Pus casts in urine, 65 Pus cells in urine, 63 Pus in feces, 80 urine. 63 Quartan malarial parasite, 27 Sarciriae in gastric contents, 75 Seat worm, 82 Smears, making of, 99 Smith-Rosen test for bile, 59 Specific gravity of urine, 46 of breast milk, 85 Spinal fluid, 87 bacteriological examination of, 87 chemical examination of, 88 cytology of, 87 differential cell count of, 87 globulin of, 87 in acute meningitis, 91 tubercular meningitis. 91 syphilitic disease of nerv- ous system, 91 Lange colloidal gold reac- tion. 89 normal. 87 Ross-Jones test for globulin. 88 total cell count of, 88 Wassermann. 40 Sputum, bacteria of, 94 character of. 93 color of, 93 consistency of, 93 examination of. macroscopic, 93 132 IXDEX microscopic, 93 in abcess of lung, 95 acute bronchitis, 94 acute lobar pneumonia. 94 asthma, 95 bronchiectasis, 95 chronic bronchitis, 95 gangrene, 95 pulmonary edema, 95 pulmonary tuberculosis, 94 tubercle bacillus in, 109 unstained, 93 Staines and methods of stain- ing, 101 Gram, 102 Methylene blue, 101 Wrights, 15 Ziehl-Neelsen, 103 Staphylococci, 103 Starch digestion in stomach contents, 74 Stomach contents. See Gastric contents. Stomach worm, 82 Stones in feces, 81 Stronglyoides intestinalis, 82 Streptococci, 104 Tallquist-hemoglo'bin scale. 10 Test meals, 69 Tertian malarial parasite. 27 Tonsils, bacterial examination of, 98 Topfer's reagent, 71 Transitional leucocytes, 16 Trichina spiralis, 82 Trichomycetes, 118 Trichocephalus trichiuris, 82 Tubercle bacillus, in sputum, 109 in urine, 109 staining of, 103 Typhoid bacillus, in blood, 115 feces, 115 urine, 115 Typhoid fever, diazo reaction in, 59 Widal reaction in, 31 U Ulcer, gastric, gastric contents in, 76 Uncinaria duodenalis, 82 Urates in urine, 60 Urea, decreased, 54 increased, 54 estimation of by hypobro- mite method, 54 Uric acid, decreased, 56 increased, 56 estimation, Ruhemann's method, 56 crystals, 60 Urine, acidity, 46 acidosis, 53 albuminuria, 48 appearance, 44 amount, 43 bacterial examination of, 98 bile, 58 blood cells, 63 blood cast, 65 calcium oxalate, 61 carbonates, 61 casts, 64 chemical composition, normal, 47 color, 45 cylindroids, 65 cystin, 61 diazo reaction, 59 epithelial casts, 65 epithelial cells, 62 fatty casts, 65 INDEX 133 glycosuria, 51 granular casts, 64 hyalin casts, 64 in acute nephritis, 67 chronic parenchymatous nephritis, 67 diabetes mellitus, 67 diabetes insipidus, 67 renal calculus, 67 tuberculosis of kidney, 67 indican, 57 leucin and tyrosin. 61 obtaining, 43 phosphates. 61 pus casts, 65 pus cells, 63 reaction. 45 red cells, 63 sediment, unorganized in acid acid urine, 60 in alkaline urine, 61 organized, 62 specific gravity, 46 total solids, 47 urates, 60 urea, 54 uric acid, 56 waxy casts. 64 yeast. 66 V Vaccines, preparation of. 121 \ incents angina. 121 W Wassermann test, 31 amboceptor, 34. 36 antigen, 34. 37 complement, 34, 36 complement fixation, 32 diagnostic value, 40 hemolysis, 31 hemolytic system, 31 patients serum. 35 sheeps corpuscles, 33 technic, 38 titration of reagent, 36 with cerebrospinal fluid, 40 Waxy cast, 64 Weber's test for blood, 72, 79 Whip worm, 82 Widal reaction for typhoid, 29 Wright's blood stain, 15 Yeast in stomach contents, 75 Ziehl-Neelsen method for stain- ing tubercle bacil- lus, 103 FORM FOR REPORT BLANKS NO. Patient Date Physician Haemoglobin BLOOD EXAMINATION Erythrocyte Count Leucocyte Count Color Index STAINED FILM DIFFERENTIAL LEUCOCYTE COUNT Polymorphonuclear Neutrophiles Poikilocytosis ERYTHROCYTES Small Mononuclears Anisocytosis *_arge Mononuclears Polychromatophilia Transitionals Nucleated Reds Per 100 Leucocytes Eosinophiles Normoblasts Basophiles Megaloblasts Myelocytes Microblasts Parasites Coagulation Time Widal Reaction Opsonic Index Serum Tests Wassermann Complement Fixation Test for Gonorrhoea FORM FOR REPORT BLANKS Date Physician URINALYSIS QUALITATIVE Color QUANTITATIVE Quantity in 24 Hours Reaction Total Acidity Specific Gravity Total Solids Albumin Quantity Sugar Quantity Indican Urea Bile Uric Acid Rlnod Chlorides Acetone Sulohates Diacetic Acid Phosphates Diazo Reaction Ammonia Functional Kidney Test MICROSCOPIC: Casts Cylindroids Blood Pus Crystals AmorDhous Deposits EDithelial Cells FORM FOR REPORT BLANKS Patient Date Physician Test Meal EXAMINATION OF STOMACH CONTENTS Withdrawn in CHEMICAL Hrs. Quantity Rel. Amt. Liquid Odor Food Particles Color Mucus Total Acidity Starch Free HCL Erythrodextrin Combined HCL Acchroodextrin Free Acids and Salts Peptone Organic Acids and Salts Bile Lactic Acid Blood Ferments Pus Bacteria MICROSCOPIC Pus Yeast Blood Sarcinae Epithelium Oppler Boas B Mucosa Food Remnants From Former Meals FORM FOR REPORT CLANKS No. Patient Date Physician EXAMINATION OF FECES Color Consistency Reaction Undigested Food Connective Tissue Stones Fat Blood Bile MICROSCOPIC: Muscle Fibre Free Starch Granules Neutral Fat Crystal: Pus Parasites FORM FOR REPORT BLANKS No. Patient Date Physician Amount CEREBROSP NAL FLUID Appearance Total Cell Count Differential Cell Count Globulin Tests Fehling's Solution Reduction Lange's Colloidal Gold Test Wassermann Bacteriological Examination Stained Smears from Sediment Cultures LIBRARY OF CONGRESS 007 635 065 6