io aa ri qa (iiece ma eon ma sein ih entionyslay as if es oy iy ‘ Maen Hine ety ¢ ceteris bhi Aue aH : iN us) Gm hea peers AN ae en RCE ae aie or ie eas i hs igs Wed ty mut mh teide a ne ay ne a ste tee See tee batiata rat tet ary vagha hs sprites tet yarren eta’ shia ib she CN aes ate ha cee se lcensiatoeen — ropa sae Mgt aie ba Pot rine ie ee tet Ge oo pon ie ie vs ia ype, meh at : ‘ a Hr Mast ony nite bth, 8 44 ny) ibebeted PAA Ae Rinne soa i en r esa Wa EM AH 4 ae Se inh ia ora ope ay re ah ae sins in a me D a ‘ ehdantat p, i ea he ; ree et erie a sal easier rae rl a Ae cena a : er Neopet: jrecies paint tec ane ete ee iERbe jig hiale ing nite Petes pia tec tp Sohaa “ip ea i ioe isesencik pagan bn eee peptone Phebe an nese toy ie ng Jala inelis leatl eitec ae ete me mt titel ne ies eto Brean ce ons bkenretira a ae Me rei tenagthaeatie apne Penge brie cen cies i Bh a ite pa Bedi Weis Batons Vike Weert bb Uy iy Hi ge ne Pe isan rca neat es mopman ebtrrcontant Hi es Seaman as irele nee Fp fy pig “ fear, rete Yi eS dul IPaaia silos hse Seana Ns pain { bf if fs i sa iene Nt ak Haiti eels Sh h if tide AACE cata tetel ferent ae gente det tet ie eae b eau Tah fini Ricca dat cait t ea a UT erie ia Pa at Eeaollyn ce ie aut a aoged TL gabemeneeeten iets a edited na ite tap ALBERT R. MANN LIBRARY NEw YorRK STATE COLLEGES OF AGRICULTURE AND HOME ECONOMICS AT CoRNELL UNIVERSITY ornell University Library laboratory course in bacteriology, for Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003200791 A LABORATORY COURSE IN BACTERIOLOGY For the Use of Medical, Agricultural, and Industrial Students BY FREDERIC P. GORHAM, A.M. Associate Professor of Biology, Brown University ; Bacteriologist, Health Department, Providence, R. I. WITH 97 ILLUSTRATIONS PHILADELPHIA AND LONDON W. B. SAUNDERS & COMPANY 1903 CopyrIGHT, 1g01, By W. B. SAUNDERS & COMPANY. ELECTROTYPED BY PRESS OF WESTOOTT & THOMSON, PHILADA, W. &. SAUNDERS & COMPANY. PREFACE. BACTERIOLOGY is essentially a laboratory study. It is only by actual laboratory work that it can be taught in such a manner as to serve any useful pur- pose. It is also a subject of very general scientific interest. Courses in bacteriology are no longer con- fined to the medical schools, but are being intro- duced into colleges and agricultural and industrial schools. ‘This volume has been prepared as a guide to the practical details of laboratory work. It is intended to present the subject in such a general way as to lay a broad foundation for later specializa- tion in any branch of bacteriology. By a judicious selection the course can be made to conform to the requirements of medical, agricultural, or industrial students. Brown UNIVERSITY, August, 1901. CONTENTS. CHAPTER I. _ Microscopic EXAMINATION OF BACTERIA .........4, II Manipulation of the Microscope, 11.—Measurement of Bac- teria, 11.—Examination of Living Bacteria, 13.—Ordinary Examination, 13.—Hanging Drop, 15.—Examination of Stained Bacteria, 16.—Ordinary Stains, 16.—Gram’s Stain, 19.—Staining Bacteria in Tissues, 20. CHAPTER II. MorPHOLOGY OF BACTERIA . Ce ee ee 22 Demonstration of Form, 22, Benenden of Motion, 24.— Staining Flagella, 25.—Demonstration of Capsules, 31. CHAPTER III. REPRODUCTION OF BACTERIA . = 2 ......4.4. 34 Division, 34.—Spores, 35.—Staining Spores, 36.—Germina- tion of Spores, 38. CHAPTER IV. CLASSIFICATION OF BACTERIA .... «2... ses 39 CHAPTER V. STERILIZATION © we te ee 43 Steam, 46.—Autoclave, 46.—Hot-air, es CHAPTER VI. PREPARATION OF CULTURE-MEDIA . ... ean 50 Bouillon, 50.—Gelatin, 50.—Agar, 50.—Media from Meat- extracts, 55.—Potato, 55.—Dextrose, Lactose, and Saccharose Bouillon, 56.—Milk and Litmus Milk, 57.—Blood serum, 59. 7 8 CONTENTS. CHAPTER VII. PAGE CULTURES OF BACTERIA . . Bouillott Cultures, 60.—Gelatin or these Gultares, 6 1.—Potato and Blood-serum Cultures, 66.—Plate Cultures, 66.—Im- pression or Adhesive Preparations of Colonies, 75.—Cultures in the Fermentation-tube, 76.—Anaérobic Cultures, 78.— Demonstration of Liquefying Ferment, 80,—Isolation of Species, 81. ~ CHAPTER VIII. DETERMINATION OF SPECIES . Morphology and Life-history of a eurdes 83. ay ieraina: tion of the Name of a Species, 95.—Classification of Bacteria by Groups, 107.—Classification of Water Bacteria by Groups, 112. CHAPTER IX. BACTERIAL ANALYSIS OF WATER, MILK, AIR, AND SOIL Water Analysis, 114.—Milk Analysis, 120.—Bacteria in the Air, 121.—Bacteria in the Soil, 122. CHAPTER X. PATHOGENIC BACTERIA Pyogenic Organisms, 125. a ieanbisuere, mn imine iach —Glanders, 133.—Diphtheria, 135.—Influenza, 141.—Ty- phoid and Colon Bacilli, 144.—Pneumonia, 150.—Tubercu- losis, 151.—Actinomycosis, 158.—Malaria, 159. APPENDIX Bacterial Measarements iy Plicteaphy, 163. __Mivalds and Yeasts, 165.—Stains and Reagents used in the Study of Bacteria, 170.—Table of Synonyms, 175. TER wn, we ie ee SR ie ue 60 83 . 114 . 124 . 163 183 A LABORATORY COURSE IN BACTERIOLOGY A LABORATORY COURSE BACTERIOLOGY. CHAPTER I. MICROSCOPIC EXAMINATION OF BACTERIA. 5 I. MANIPULATION OF THE MICROSCOPE. 1. Examine mounted slides of bacteria with the low-power (# inch) and high-power (4 inch) objec- tives and with different eyepieces. 2. Manipulate the condenser, diaphragm, and mirror, in order to ascertain what combination gives the best result. 3. (a) Affix the oil-immersion lens (7; inch) to the microscope. (4) Place a drop of immersion oil on the cover- glass. (c) Bring the lens to a focus in the drop of oil. (2) Again manipulate condenser, diaphragm, and mirror to determine what combination now is best. il. MEASUREMENT OF BACTERIA. The most accurate method of measurement is by photography ;' but as this requires special appa- 1 See Appendix, page 163. ll I2 MICROSCOPIC EXAMINATION OF BACTERIA. ratus, the following fairly accurate methods are recommended : I. First Method. (a) Place a micrometer eyepiece in the micro- scope. (6) Examine the bacteria to be measured, and record their lengths in divisions of the eyepiece micrometer. (c) Remove the slide of bacteria. (d) Place a stage micrometer on the stage of the microscope, and determine the relation of the divisions of the eyepiece micrometer to the. divis- ions of the stage micrometer. (e) The length of the divisions of the stage mi- crometer is a fixed quantity (usually 737 mm.) ; therefore we have the equations : Length of bacteria = x divisions of eyepiece micrometer ; x divisions of eyepiece micrometer = y di- visions of stage micrometer ; I division of stage micrometer = 745 mm. From these equations we can readily determine the length of the bacteria in millimeters. The unit of length for microscopic measurements is the thou- sandth part of a millimeter ; it is called a micron or micromillimeter, and is designated by the Greek letter u. Therefore in the above equations we can substitute for zi; mm. Io #, and write the result in so many #. 2. Second Method. (a) Adjust the ‘‘camera lucida’’ to the micro- scope. EXAMINATION OF LIVING BACTERIA. 13 (4) Draw by means of the camera the exact size of the bacteria to be measured. (c) Remove the slide of bacteria, and replace it with the stage micrometer. - (d) Over the figure of the bacteria now draw the scale of the stage micrometer, and read off directly the size of the bacteria in divisions of the stage micrometer. UI. EXAMINATION OF LIVING BACTERIA. 1. Ordinary Examination. (a) Place a drop of water on a clean slide. (4) Pass the cotton plug of the tube containing the culture to be examined through the flame ; ex- Fic. 1.—Method of holding tubes during inoculation (McFarland). tinguish the ignited cotton by blowing or pinching out the flame. (c) Hold the test-tube containing the culture be- 14. MICROSCOPIC EXAMINATION OF BACTERIA. tween the thumb and finger of the left hand, allow- ing the lower end of the tube to rest on the back of the hand. (d) Hol& a straight platinum needle between the thumb and forefinger of the right hand, and ster- ilize it by heating red-hot. Allow it to cool. £ | | X Fic. 2.—Platinum needles for transferring bacteria ; made from No. 27 platinum wire inserted in glass rods. (e) Grasp the cotton plug between the third and fourth finger of the right hand, and remove it ; in- sert the platinum needle, and transfer an exceed- ingly minute portion of the culture of bacteria to the drop of water. (/) Return the cotton plug to the tube, and ster- ilize the needle.’ (g) Place a clean” cover-glass over the drop of water, and examine with the 4 inch objective. 1 The needle should invariably be heated before and after _using. If this practice is not carefully followed, cultures will be contaminated, and perhaps pathogenic organisms spread about the room. It is well also to pass the handle of the needle through the flame before beginning work each day. ? Cover-glasses and slides may be cleaned in the follow- ing solution : Potassium bichromate, 6 gm. ; Concentrated sulphuric acid, 6 c.c. ; Water, 100 ¢c.c. Wash in water and store in alcohol. Or boil the cover-glasses in sulphuric acid, wash in EXAMINATION OF LIVING BACTERIA. 15 (2) Manipulate condenser, diaphragm, and mir- ror to determine the best adjustment for the exam- ination of unstained bacteria. 2. Examination in the ‘“‘Hanging Drop.” Fic. 3.—A ‘concave slide’ with ‘hanging drop” (McFarland). Fic. 4.—A slide with cell for hanging drop. The ring may be made of glass or of zylonite, and is cemented on the slide with Canada balsam. (a) Paint a ring of vaselin around the hollow in water, and keep in alcohol; or wash in strong nitric acid for some time, rinse in water, and store in alcohol. Very often simply passing them several times through a Bunsen flame will clean them sufficiently. 16 MICROSCOPIC EXAMINATION OF BACTERIA. a ‘concave slide ;’ or use a slide on which is cemented a small glass ring, and vaselin the top of the ring. (6) On “the centre of a clean cover-glass place a small drop of water. (c) With a sterile platinum needle add to the drop of water a very small portion of the culture to be examined. (d) Invert the slide over the cover-glass, so that the drop of water is covered by the concavity or is inside the glass ring, but does not touch the sides of either ; press down so that the chamber is sealed tight by the vaselin. (e) Invert carefully and examine. The hanging-drop examination is for the purpose of determining the motility of bacteria or for watch- ing their reproduction. The preparation may be kept for examination from day to day without loss by evaporation. IV. EXAMINATION OF STAINED BACTERIA. 1. Ordinary Stains. (a) Prepare a clean cover-glass.' (4) Place a drop of water on the glass.’ (ce) With a sterile platinum needle transfer a minute portion of a culture to the drop of water 1 See footnote, page 14, for directions for cleaning covers. 2 Tf the cultures are in bouillon or other fluid, it is often unnecessary to use the drop of water in spreading them on the cover-glass. EXAMINATION OF STAINED BACTERIA, 17 and spread uniformly over the surface of the cover- glass.' (2) Allow the film to dry. (e) When dry pass the cover-glass, snieared sur- face upward, three times through a Bunsen or alco- hol flame at about the rate of the pendulum of a clock. The heat coagulates the albuminous material 1The cover may be held, while staining, in one of the special forceps devised for the purpose. 2 18 MICROSCOPIC EXAMINATION OF BACTERIA. around the bacteria and fixes them firmly to the glass. (/) Place a drop of stain ' on the cover-glass large enough td! cover the film. Allow it to stain for from two to ten minutes; the length of time de- pends on the stain, the strength of the staining solution, and the kind of bacteria.” Fic. 8.—Bottles for stains. (g) Wash in water, dry the unsmeared side on filter-paper, mount, film side down, in a drop of water on a clean slide, and examine with the } inch objective. ‘Almost any of the anilin stains may be employed. Gentian-violet, basic fuchsin, and methylene-blue are those most commonly used. For methods of preparing these staining solutions, see Appendix, page 170. - ? It very rarely happens that bacteria are over-stained., But if such is the case, either a new film must be prepared or the stain drawn with weak acetic acid (1: 1000), or the cover-glass swept through 1 per cent. sulphuric acid. If the film is not sufficiently stained, repeat the staining process, EXAMINATION OF STAINED BACTERIA. 19 (A) If the bacteria are evenly distributed’ and properly stained, dry both sides thoroughly between filter-paper and mount, film side down..in a drop of Canada balsam. Label and preserve. ~ 2. Gram’s Stain. The value of this method depends on the fact that the mycoprotein of certain bacteria forms with an anilin dye and an iodid a compound insoluble in alcohol. There are many bacteria in which such an insoluble compound is not formed, and this method consequently has considerable diagnostic value. (a) To a drop of water on a clean cover-glass add a very small amount of a culture of Bacillus subtilis. (4) To the same drop add a very small amount of a culture of Bacillus vulgaris, or any culture that does not stain by this method. (c) Dry,. fix, and stain for five minutes in anilin gentian-violet.’ (2) Wash in water. (e) Treat with Gram’s solution * for one minute. 1 In a successful preparation the bacteria are evenly and not too thickly distributed over the surface. If too many bacteria are present, either a smaller amount of the culture must be used, or a little from the first cover-glass must be added to another drop of water on a second cover-glass, and so on until the proper dilution is reached. 2 See Appendix, page 171. 3 Gram’s solution : Todin, I part ; Potassium iodid, 2 parts ; Distilled water, 300 parts. 20 MICROSCOPIC EXAMINATION OF BACTERIA. (f) Wash in 95 per cent. alcohol until no more color comes away. (g) Dryeand contrast-stain in safranin' (thirty seconds), or Bismarck-brown? (two to three min- utes), or eosin® (one minute). (2) Wash, dry, and mount. 3. Staining Bacteria in Tissues. (1) First Method. (a) Harden the tissue in absolute alcohol or Zen- ker’s fluid.* (6) Dehydrate, embed, and section by the usual methods. (c) Stain as directed for cover-glass preparations, but a little more deeply. (d@) Draw the color with dilute acetic acid (1 : 1000) until the bacteria alone are stained. (e) Contrast-stain in eosin or any stain not re- quiring acid for differentiation. (f) Clear and mount. (2) Second Method. Stain the sections by Gram’s method as follows : (2) Stain in anilin gentian-violet for five minutes. (4) Wash in water. (c) Treat with Gram’s solution for one minute. (2) Wash in 95 per cent. alcohol until no more color comes away. 1 Stock solution is ar per cent. solution of safranin in equal parts of methylated spirit and water. For use, dilute with 5 parts of water. * Saturated solution in equal parts of alcohol and water. 5 Aqueous solution, I : 1000. 4 See Appendix, page 173. EXAMINATION OF STAINED BACTERIA. 21 (e) Wash in water. (/) Contrast-stain in an aqueous solution of eosin (1: 1000) for one-half to one minute. (g) Wash in 60 per cent. alcohol "for thirty seconds. (2) Wash in absolute alcohol for thirty seconds. (z) Clear in xylol and mount in balsam. (3) Third Method, (2) Stain in Kuhne’s methylene-blue! for one- half to one hour, or in carbol-thionin-blue? for five minutes. (4) Wash in water. (c) Treat with 0.5 per cent. acetic acid till pale green. (7) Wash in water, 60 per cent. alcohol, and absolute alcohol, each for thirty seconds. (e) Contrast-stain as in above methods, clear, and mount. (4) Fourth Method. (a) Stain the dried preparations in a dilute aque- ous solution of methylene-blue. (4) Wash in water and dry. (c) Stain in aqueous eosin solution (1 : 1000) for one to one and a half minutes. (2) Dehydrate, clear, and mount. 1 See Appendix, page 171. 2 See Appendix, page 171. CHAPTER II, MORPHOLOGY OF BACTERIA. BACTERIA ate minute, unicellular, vegetable or- ganisms. ‘They consist of a sharply defined mass of protoplasm which reacts to anilin stains very much like the nuclei of other cells, and outside of this a more or less well-developed envelope. They are classified according to their form into three main groups, the spherical cocci, the rod-shaped bacilli, and the curved or spiral spirilla. a b t) eo g h CO) O ® oO Fic. 9.—Diagram illustrating the morphology of the cocci : @, coccus or micrococcus ; 4, diplococcus ; ¢, d, strep- tococci ; ¢, f, tetragenococci or merismopedia ; g, 2, modes of division of cocci; z, sarcina; 7, coceus with flagella; 4, staphylococci (McFarland). I. Demonstration of Form. (a) Make hanging-drop and stained preparations from cultures of cocci, bacilli, and spirilla. (6) Examine with the } inch or with the oil- immersion lens. 22 DEMONSTRATION OF FORM. “23 In the hanging-drop preparations are the indi- vidual bacteria spherical, rod-shaped, or spiral? If rod-shaped, are the ends pointed, sounded, or square? Are the bacteria motile?’ Are they single a & ce é ; te ule ale oe L/D Pr a Fic. 10.—Diagram illustrating the morphology of the bacilli: a, 6, ¢, various forms of bacilli; d, e, bacilli with flagella; f, chain of bacilli, individuals distinct; g, chain of bacilli, individuals not separated (McFarland). c a b oo ne Fic. 11.—Diagram illustrating the morphology of the spirilla: @, 5, ¢, spirilla ; d, e, spirocheeta. or united in pairs, fours, irregular masses, or chains? Preserve the hanging-drop preparations for further study. (c) Repeat these observations on the stained prep- arations. 1 See 2 II., page 24. 24 MORPHOLOGY OF BACTERIA. Are the individuals stained uniformly or irregu- larly, deeply or faintly? In the rods are the ends more deeply stained than the centers (polar stain- ing)? (@) Measure the various kinds and make draw- ings of them. II. Demonstration of Motion. (a) To a small drop of water on a cover-glass add a very little carmine or Bismarck-brown. Fic. 12.—Bacillus suipestifer, showing flagella. (4) Mount in the same manner as a hanging-drop preparation. (c) Examine with the } inch objective. Are the bits of pigment in motion? Do they change their position relative to one another, or do they dance about in one place? This is the so- called Brownian movement. STAINING FLAGELLA. 25 (7) Examine the hanging-drop preparations of the last section in reference to this movement. Do they all show the Brownian movement? Are some actively swimming about, changing their position in relation to one another ? All bacteria exhibit the Brownian movement, but certain ones are motile of themselves. They possess organs of locomotion or flagella, lash-like appendages, by the movements of which they propel themselves along. The flagella may be very numerous, extending from all sides of the cell, or they may be collected in a tuft at one end, or Hieie may be a single one or a pair (periecichous, lophotrichous, monotrichous, amphitrichous). The flagella may be demonstrated by appropriate methods of staining. Ill. Staining Flagella. 1. Pittield’s Method. (2) Prepare a mordant as follows: Tannic acid (10 per cent. solu- tion, filtered), 10 C.c.3 Corrosive sublimate (saturated aqueous solution), 5¢¢. 3 Alum (saturated aqueous solu- tion), 5 ee, 5 Carbol-fuchsin ' 5 Gc. Allow to stand and draw off the clear fluid. The mordant will keep one or two weeks. 1 See Appendix, page 172. 26 MORPHOLOGY OF BACTERIA. (6) Prepare a stain as follows : Alum (saturated aqueous solu- tiow), IO C.C.5 Gentian-violet (saturated alco- holic solution), 2.0. The stain will keep two or three days. (c) On an absolutely clean cover-glass' place a drop of distilled water. (Z) With a sterile platinum needle add to the drop of water, without stirring, a very small por- tion of an actively motile culture. (e) Stand on a water-bath at 60° C. for one-half hour. (f) Treat the film with the cold mordant for twenty-four hours, or with hot mordant (steaming, but not boiling) for three minutes. (g) Wash thoroughly in running water and dry. (2) Treat with the stain the same as directed for the mordant. (¢) Wash, dry, mount, and examine with the oil- immersion lens. 2. Modification of Pitfield’s Method. (a) Prepare solution A as follows : Alum (saturated aqueous solu- tion), TO Gc, * Gentian-violet (saturated alco- holic solution), -" Tee ’ See footnote, page 14. STAINING FLAGELLA. 27 (4) Prepare solution B as follows : Tannic acid, I gm.; Distilled water, 19, ¢.c. (c) Filter when cold, mix the two solutions, and use immediately. (@) Prepare films as before. (e) Treat with the above mixture, gently heating until it almost boils, then set aside for a minute. (/) Wash, dry, and mount. Fic.13.—Microspira comma, showing the flagella; x 1000 (Ginther). 3. Loffler’s Method. (a) Prepare a mordant as follows : Tannic acid (20 per cent. solu- tion), filtered, IO C.c.3 Ferrous sulphate (cold, satu- rated solution), filtered, ones Fuchsin (saturated alcoholic so- lution), 1 c.c. 28 MORPHOLOGY OF BACTERIA. Filter each time before using. This mordant will keep two or three days. (4) Prepare films as before. (¢) Dry and pass once through the flame. (@7) Cover the film with the mordant, and warm over the flame for a few seconds only. Fic. 14.—Bacillus typhosus, from an agar culture six hours old, showing the flagella stained by Léffler’s method; x 1000 (Frankel and Pfeiffer). (e) Wash in water, dip in absolute alcohol, and again wash in water. (f) Cover the film with anilin-water fuchsin,! and warm over the flame from three to four min- utes, taking care that the solution steams, but does not boil. 1 See Appendix, page 171. STAINING FLAGELLA. 29 (g) Wash in water, dry, mount, and examine with oil-immersion lens. 4. Modification of Loffler’s Method. - (a) Prepare a mordant as follows : Tannic acid (20 per cent. solu- tion), TO €.6, 5 Ferrous sulphate (saturated so- lution), 10 GC; Logwood solution (x gram boiled in 8 cc. of water and filtered), 3-4 CC. (4) Prepare a stain as follows : Anilin-water,' IOO C.C.; Sodium hydrate (1 per cent.), tee; Methylene-violet or methylene- blue or fuchsin, 4-5 gm. Filter. (c) Prepare films as before. (2) Heat with the mordant until steam rises, and then move over flame for one minute. (e) Wash in water, and if mordant does not dis- appear, in absolute alcohol. (/) Dry and heat with the stain until steam rises, then leave in the warm stain one minute. (g) Wash, dry, and mount. 1 See Appendix, page 171. 30 MORPHOLOGY OF BACTERIA. 5. Van Ermengem’s Method. | (a) Prepare solution A as follows : Osmic «acid (2 per cent. solu- tion), I part ; ‘Tannic acid (10 to 25 per cent. solution), 2 parts. To each 100 c.c. of the tannic acid add 4 or 5 drops of glacial acetic acid. Fic. 15.—Bacterium Faschingii in blood; x tooo (Frankel and Pfeiffer). "Various modifications of Van Ermengem’s method have been suggested. See Centralbl. f, Bakt., Erste Abteilung, Bd. xxvii., Nos. 16-17, p. 597; and Lancet, vol. ii., No. 14, p. 874, 1898. DEMONSTRATION OF CAPSULES. 31 (6) Prepare solution B as follows : Gallic acid, 5 gm. ; Tannic acid, Bon oe Fused acetate of sodium or ‘* potassium, Io ‘ Distilled water, 350 Cc. (c) Prepare films as before. (d) Place in solution A for one-half hour ; or, if the solution is warmed to 60° C., five minutes are sufficient. (e) Wash thoroughly in water, then in alcohol, then again in water. (f) Place for two minutes in solution of nitrate of silver (0.25-0.5 per cent.). (g) Without washing, place in solution B for one and one-half to two minutes, using fresh solu- tion for each preparation. (A) Wash thoroughly in water, and examine in water. If the flagella are not sufficiently stained, begin again at (/). Always change the silver nitrate solution as soon as any precipitate appears. IV. Demonstration of Capsules. In certain forms the envelopes surrounding the cells can be stained as follows : 1. Welch’s Method.' (a) Prepare films without using water. (4) Place in glacial acetic acid for a few seconds. (c) Remove the acid with filter-paper. 1 Bulletin of the Johns Hopkins Hospital, p. 128, Decem- ber, 1892. 32 MORPHOLOGY OF BACTERIA. (2) Wash in anilin-water gentian-violet repeat- edly until all the acid is removed. (ec) Wash in a } per cent. solution of sodium chlorid and’examine in the same solution.. 2. Johne’s Method. (a) Prepare films as usual. (6) Warm in a 2’per cent. solution of gentian- violet until steam arises. Fic. 16.—Bacterium pneumoniz of Friedlander, from the expectoration of a pneumonia patient; x 1000 (Frankel and Pfeiffer). (c) Wash in water. (@) Place in 2 per cent. acetic acid for six to ten seconds. (€) Wash in water. (/) Dry and mount in balsam. DEMONSTRATION OF CAPSULES. 33 3. Boni’s Method.! (a) Prepare a solution as follows : White of one egg ; Glycerin, 50 '¢.c. 3 Formalin, 2 drops. Shake together and filter. (6) Prepare films of the organism to be studied, using the above solution in place of water. (c) Spread very thin, and heat over flame until the glycerin is evaporated, (d@) Stain in Ziehl’s carbol-fuchsin’ for twenty to thirty seconds. (e) Wash in water and dry with filter-paper. (/) Contrast-stain in Léffler’s methylene-blue* for four to six minutes. - (g) Wash, dry, and mount in balsam. 1 Centralbl. fiir Bakteriologie, Erste Ab., Band xxviii., No. 20, p. 705. 2 See Appendix, page 172. 5 See Appendix, page 171. 3 CHAPTER III. REPRODUCTION OF BACTERIA. THE common method of reproduction among the bacteria is by binary division. In some forms this is the only mode of reproduction known. Under favorable conditions an individual cell grows in length, a transverse constriction appears in the middle and gradually becomes deeper until two new cells are formed. Separation may be com- plete or there may be the formation of chains. In the spherical forms division may take place in one, two, or three planes, forming chains (streptococci), or groups of two (diplococci), irregular groups (staphylococci or micrococci), or cubical packets (sarcina). In the rod-shaped forms division takes place in but one plane, forming chains, pairs, or single individuals (Figs. 9 and 10). Some bacteria, besides having the power of re- production by division, form endogenous spores. These spores are developed from the plasma of the cell, and have a dense wall that protects them from injury by drying, enables them to withstand high temperatures, and also causes them to resist the action of stains. I. Reproduction by Division. (a) On a clean cover-glass place a large drop of bouillon.' 1 See page 50. = 34 REPRODUCTION BY SPORES. 35 (4) Add to the bouillon a very small portion of a culture of Bactllus subtilis, or any chain-forming and spore-producing species. (c) Mount as a hanging-drop preparation and place in the incubator. (d7) Examine at intervals of one-half hour. Notice the elongation of the bacilli and the for- mation of chains. (e) Inoculate a bouillon-tube as directed on page 60. (/) Place in the incubator and make stained preparations at half-hour intervals. “ Preserve both hanging-drop preparation and bouillon-tube for further examination. II. Reproduction by Spores. 1. After the formation of chains has taken place in the above cultures, continue the examination at intervals. The formation of spores within the rods will be observed ; finally, the rods will disappear and nothing but spores remain. a 4 é ad e ft ce> <2 ' oe eS @€ -Fic. 17.—Diagram illustrating sporulation: a, bacillus enclosing a small oval spore; 6, drumstick bacillus, with the spore at the end ; ¢, clostridium ; d, free spores ; é, and J, bacilli escaping from spores. 2. Make stained preparations from the bouillon culture when rods and spores within the rods and spores alone are all present. Stain as follows: 36 REPRODUCTION OF BACTERIA. III. Staining Spores. (1) First Method. (a) Prepare films in the usual manner. (6) Stain in warm Ziehl’s carbol-fuchsin ' for twenty to thirty minutes, or in the same solution steaming, but not boiling, for five minutes. (c) Wash in water. (a2) Dip in acid alcohol (70 per cent. alcohol, 97 Fic. 18.—Bacterium anthracis, stained to show spores; x 1000 (Frankel and Pfeiffer). c.c. ; hydrochloric acid, 3 ¢.c.) or in I per cent. sulphuric acid a few seconds and wash in water. (e) Examine in water. Spores should be red and rods unstained or faintly eee If the spores are not sufficiently 2 # See Appendix, page 172. STAINING SPORES. 37 stained, place again in the fuchsin. If the rods are still stained, wash longer in the acid alcohol. (/) Dry and pass.three times through the flame. (g) Stain in Léffler’s methylene-blue! for three to four minutes, or in gentian-violet? one minute, or in saturated aqueous solution of methylene-blue for half a minute. (2) Wash, dry, and mount. Spores should be red, rods blue. (2) Second Method. (a) Prepare films in the usual manner. (4) Place in chloroform for two minutes. (c) Treat with 5 per cent. chromic acid for one minute. The acid acts on the membranes of the spores and permits the entrance of the stain. (2) Wash thoroughly in water. (e) Stain in hot Ziehl’s carbol-fuchsin for three minutes. (f) Without washing decolorize in 5 per cent. sulphuric acid. (g) Wash in water and stain in Loffler’s methy- lene-blue' for two minutes or longer. (2) Wash, dry, and mount. (3) Third Method. (a) Suspend the spore-bearing bacteria in a nor- mal salt solution® in a test-tube. (6) Add an equal volume of Ziehl’s carbol-fuchsin. (c) Place the test-tube in boiling water for fifteen minutes. 1 See Appendix, page 171. 2 See Appendix, page 171. 3 See Appendix, page 173. 38 REPRODUCTION OF BACTERIA. (2) Spread a loopful on a cover-glass; dry; fix. (e) Decolorize in a 14 per cent. (by volume) solu- tion of hydrochloric acid in alcohol. Fic. 19.—Bacillus chauveei, showing spores ; x 1000 (Frankel and Pfeiffer). (/) Wash in water and contrast-stain in Léffler’s methylene-blue. (g) Wash, dry, and mount. (4) To Observe the Germination of Spores. (a) Make a hanging-drop preparation, using ster- ile bouillon and some of the spores from the above culture. (4) Place on a warm stage and watch the growth of rods from the spores. (c) Inoculate a sterile bouillon-tube with a large number of spores from the above culture. (2) Make stained cover-glass preparations from this at intervals of one-half hour. CHAPTER TY. CLASSIFICATION OF BACTERIA. THE position occupied by the bacteria in the vegetable world is shown by the table on page qo. The Bacteria or Schizomycetes are classified into families according to their form, and again into genera according to certain other characters. The following classification is that proposed by Migula in his System der Bakterien, 1900: BACTERIA, SCHIZOMYCETES. EUBACTERIA, cells contain no sulphur granules or bacterio-purpurin. 1. Family CoccacE#, spherical forms. Genus : 1. Streptococcus, non-motile ; cells divide in one plane. 2. Micrococcus, non-motile; cells divide in two planes. 3. Sarcina, non-motile; cells divide in three planes. 4. Planococcus, motile; cells divide in two planes. 5. Planosarcina, motile; cells divide in three planes. 2. Family BACTERIACE#, straight, rod-shaped forms without envelope. Genus: 1. Bacterium, non-motile. 2. Bacillus, motile ; flagella over whole surface. 3. Pseudomonas, motile; flagella polar. 39 CLASSIFICATION OF BACTERIA. 40 “BLIa}0eq Jo 18unj-uorssty ‘sej}aAwI0ZzIYOS *xB[e-UOISSI] “suoqory ‘sauoqory ) “run gq ‘sajaoAuroyd Ap *SJJOM3U0}S ‘eaoBleysy ‘eae pay ‘eao) ‘srejas1o py ‘sui3aq ‘eyAydoyeqy, “eydydokag ‘eyhqdopuaig ‘spaas Suruioy syueid Suramoy ss ‘viuesorsueyg ‘sorods Surunsoy \ sjuvid ssajiomog ‘ermesojyddi5 L “syuelg CLASSIFICATION OF BACTERIA. 41 3. Family SPIRILLACE#, curved, rod-shaped forms without envelope. . Genus > 1. Spivosoma, non-motile; cells rigid. 2. Microspira, motile; one, rarely two or three, polar flagella. 3. Spirvillum, motile ; polar tufts of flagella. 4. Spirocheta, cells flexile. 4. Family CHLAMYDOBACTERIACE®, cells with envelopes. Genus: 1. Chlamydothrix, unbranched threads ; cell-division in one plane. 2. Crenothrix, unbranched threads ; cell-division in three planes ; sheath visible. 3. Phragmidiothrix, unbranched threads ; cell-division in three planes ; sheath scarcely visible. 4. Spherotilus, branched threads. THIOBACTERIA, Cells contain sulphur granules or bacterio-purpurin ; red or violet color, never green. 1. Family BEGGIATOACE&, thread-forming, with- out bacterio-purpurin. Genus: 1. Thiotrix, attached threads ; non-motile. 2. Beggiatoa, unattached threads; motile. 2. Family RHODOBACTERIACE, cells contain bacterio-pupurin and sulphur granules; red or violet. 42 CLASSIFICATION OF BACTERIA. I. Subfamily THrocapsacr#, cells divide in three planes. Genus: 1. Thiocystis. 2. Thiocapsa. 3. Thiosarcina. II. Subfamily LAMPROCYSTACEA, cells divide first in three, then in two planes. Genus : 1. Lamprocystis. III. Subfamily TH1opEprace&, cells divide in two planes. Genus: 1. Thiopedia. IV. Subfamily AMEBOBACTERIACEA, cells divide in one plane. Genus : 1. Amebabacter. 2. Thiothece. 3. Thiodictyon. 4. Thiopolycoccus. V. Subfamily CHROMATIACEA. Genus: 1. Chromatium. 2. Rhabdochromatium. 3. Thiospirillum. CHAPTER 'V. STERILIZATION. STERILIZATION is the process of killing micro- organisms. A body is said to be sterile when all the bacteria in or upon it have been killed or re- moved. Sterilization is effected by the application of heat, by treating with certain chemicals, or, in the case of fluids, by filtration. When spores are not present, bacteria are killed by exposure for twenty minutes to boiling water or steam. ‘To destroy spores, however, a much longer exposure is necessary, in some cases several hours. Steam under a pressure of thirty pounds will give a temperature of 120° C.; this will kill all organ- isms and spores in fifteen minutes. Dry heat is not so fatal to either bacteria or their spores as is moist heat or steam ; therefore to steril- ize by dry heat a higher temperature, sustained for a longer time, is necessary ; 150° C. continued for one hour will kill all ordinary bacteria and their spores. Liquids and objects liable to be injured by dry heat can be sterilized only by steam. The method of dzscontinuous sterilization is em- ployed in sterilizing objects which would be injured by long exposure to a high temperature. The ob- jects to be sterilized are first subjected to a temper- ature sufficiently high to destroy all bacteria not 43 44 STERILIZATION. in the spore condition, say 100° C. for twenty minutes. They are then allowed to cool for twenty-four hours, and again sterilized as before. This is repeated several times. ‘The intermissions allow such spores as are present to develop into rods, and these are killed by the subsequent heating. Pasteurization is the term applied to the partial sterilization of milk, effected by subjecting it to a temperature sufficiently high to kill all pathogenic and most of the souring and spore-forming bacteria, but not high enough to produce any physical changes, such as are brought about when the milk is sterilized. A temperature of 60° C. continued for fifteen or twenty minutes is usually sufficient, though temperatures as high as 85° C. are fre- quently employed. Enhanced keeping qualities and the destruction of pathogenic organisms are the results of pasteurization. The chemical substances most frequently em- ployed for sterilization or disinfection ' are solutions of corrosive sublimate (1 : 1000), carbolic acid (1:20), or formalin (1 : 20). A substance that prevents the development of bacteria but does not destroy them is an antiseptic. One that destroys all germs and spores is a germicide or disinfectant. There are many gases, acids, salts, etc., that have anti- septic or germicidal properties. Bacteria may be remnoved from a liquid by pass- ing it through a properly constructed filter of un- 1 Disinfection is the term applied to sterilization by means of chemicals. FILTRATION. 45 glazed porcelain. The most satisfactory filters in use at present are the Chamberland and the Berke- feld. Fic. 20.—Filter: a, porcelain bougie; 4, at- tachment for suction- pump; ¢, reservoir; d, sterile receiver ; ¢, rubber tube wired to bougie and reservoir. Fic. 21.—Filter: a, porcelain bougie; 4, at- tachment for suction- pump; ¢, reservoir; d, sterile receiver; ¢, perfo- rated rubber collar; 7 glass chimney for draw- ing the fluid around the bougie (Page). Bacteria in the air are unable to pass through a cotton-wool filter, and consequently sterile flasks or 46 STERILIZATION. test-tubes stopped with a cotton-wool plug remain sterile indefinitely. I. Steam Sterilization. Ber objects liable to be injured by dry heat, such Fic. 22.—Kitasato’s fil- ter: a, porcelain bougie ; 6, attachment for suction- pump; ¢, reservoir; d, sterile receiver. as culture-media, fluids, in- struments, etc.: (a) Fill the water-tank of the sterilizer’ and start the flame. (6) When the chamber is filled with steam place the objects ,to be sterilized within and close the door. (c) If the objects will not be injured by prolonged heating, allow them to re- main for one hour. (2) If prolonged heating is injurious, allow them to remain twenty minutes, and repeat the process three times at intervals of twenty-four hours. II. Steam Sterilization under Pressure. For rapid and effective sterilization. In this method an ‘‘ autoclave’’ is used. (a) Adjust the safety-valve at the desired press- ure, say thirty pounds. See that an abundance of water is present, so that the steam will not be superheated. ? An “Arnold”? sterilizer is the form usually employed. HOT-AIR STERILIZATION. 47 (6) Place the objects to be sterilized in the chamber. (c) Close the door and turn on the steam, first allowing the air present in the chamber to escape. (d@) Bring the temperature to, say 120° C. ; allow it to remain there fifteen minutes. Fic. 23.—Arnold steam sterilizer, Boston Board of Health pattern. (e) Shut off the steam and allow the apparatus to cool well below 100° C. before opening the door or allowing the steam to blow off. III. Hot-air Sterilization. For glassware and other objects not liable to be 48 STERILIZATION. injured by dry heat. The oven of an ordinary gas- stove into which a thermometer can be inserted makes an excellent hot-air sterilizer. (a) Wash thoroughly, using, if necessary, the cleaning mixture recommended on page 14, all FIGs. 24, 25.—Autoclaves for rapid sterilization by steam under pressure. flasks, test-tubes, Petri dishes, etc., to be used in the preparation of culture-media. (4) Plug the flasks and test-tubes with cotton.! 1“ Sliver’? obtained from cotton-mills is excellent for plugging test-tubes, etc. HOT-AIR STERILIZATION. 49 In the case of the test-tubes, the plugs should be tight enough so that the tubes can be lifted by them. (c) Place in the hot-air sterilizer, close the door, and bring the temperature to 150° C.; keep it there Fic. 26.—Hot-air sterilizer. | for three-quarters of an hour, or until the cotton plugs begin to turn brown. (2) Allow the oven to cool before opening the door. Place the glassware in a clean place free from dust until used, 4 CHAPTER VI. PREPARATION OF CULTURE-MEDIA.' I. Bouillon. Il. Gelatin. Il. Agar. 1. Infuse finely chopped lean beef for twenty hours with ¢wice its weight of distilled water in the re- frigerator, say Io0o grams of meat, 2000 grams of water. 2. Make up weight of meat-infusion (and meat) to original weight by add- ing water—z.e., to 3000 grams. 3. Filterinfusion through cloth to remove meat. 4. Weigh infusion, say 1800 grams. 5. Set infusion on water- ’ bath, keeping temperature below 60° C. 6. Add peptone, 1 per cent., 18 grams; salt, 0.5 per cent., 9 grams. 7. After ingredients are dissolved, titrate ;?_ reac- tion probably + 2.3 to + 2.5 per cent. 8. Neutralize (Fuller's method).3 Ditto. Ditto. Ditto. Ditto. Ditto. Ditto, and sheet gelatin, 10 per cent., 180 grams. Ditto, probably + 4.0 to + 5.0 per cent. Ditto. Boil 30 grams of thread agar in 1 liter of water for half an hour. Make up loss by evaporation to a weight of 1000 grams. 1. Infuse finely chopped lean beef for twenty hours with its own weight of distilled water in the re- frigerator, say Io0o grams of meat, 1000 grams of water. 2. Make up weight of meat-infusion (and meat) to original weight by add- ing water—z.e., to 2000 grams. 3. Ditto. 4. Ditto, say 900 grams, 5. Ditto. 6. Add peptone, 2 per cent., 18 grams; salt, 1 per cent., 9 grams. 7. Ditto, probably + 4.5 to + 4.7 per cent. 8. Ditto. To the 900 grams of meat-infusion (containing now peptone and salt) add goo grams of the 3 per cent. agar-jelly de- scribed at the head of this column, For notes see page 51. 50 BOUILLON—GELATIN~—AGAR. 5I 1 Substantially as recommended by the Bacteriologic Committee of the American Public Health Association. Some minor changes, suggested by Dr. H. W. Hill, in the Report of the Health Department of Boston for 1898, have been’ incorporated without detracting from their value as standard media. ? Acid media are denoted by the f/us sign, and alkaline media by the minus sign; the degree of acidity or alka- linity is denoted by parts per hundred. Thus, a medium marked + 1.5 indicates that the medium is acid, and that 1.5 per cent. of = sodium hydroxid is required to make it neutral to phenclphthalein. ® Following is Fuller’s method of obtaining the degree of reaction of culture-me‘ia : (a) Prepare a = solution of sodium hydroxid. (6) Prepare a . solution of hydrochloric acid. (c) Transfer 5 c.c. of the medium to be tested to a porce- lain evaporating-dish. (d2) Add 45 c.c. of distilled water. (e) Boil for three minutes. (/) Add 1 cc. of a 0.5 per cent. solution of commercial phenolphthalein in 50 per cent. alcohol. (g) Titrate while still hot with the acid or alkali as re- quired, and determine the reaction. To determine exactly when the neutral point is reached, notice that in bright daylight the first change that can be seen on the addition of alkali is a very faint darkening of the fluid, which on the addition of more alkali becomes a more evident color and develops into what may be de- scribed as an Italian pink. A still further addition of alkali suddenly develops a clear and bright pink color, and this is the reaction always to be obtained. (2) When the reaction has been obtained, calculate the amount necessary to neutralize the bulk of the medium or to produce the required reaction, and add the proper amount of a normal solution of the acid or alkali. 52 PREPARATION OF CULTURE-MEDIA. 9. Heat over boiling water (or steam) bath thirty minutes. 10. Restore weight lost by evaporation to origi- nal weight of filtered meat-infusion, for bouillon and gelatin, and to éwce that weight for agar— 1800 grams in each case. 11. Titrate, reaction probably + 0.3 to + 0.5 per cent. 12. Adjust reaction to final point desired, + 1.5 per cent. 9... Graniteware funnel. f---. Layer of absorbent cotton. Seserananstebenseceecelier Coil of wire to pre- vent cotton from entering tube of funnel Fic. 27.—Filter for culture-media. 13. Boil five minutes over free flame, with stir- ring. 14. Add water if necessary to make up loss by evaporation to 1800 grams. 15. Filter through absorbent cotton, passing the filtrate through the filter repeatedly until clear. 16. Titrate to determine whether or not the de- sired reaction has been maintained. BOUILLON—GELATIN—AGAR. 53 17. Tube and sterilize for fifteen minutes in the steam sterilizer on three successive days. Some of the gelatin- and agar-tubes after the Fic. 28.—Funnel for filling tubes with culture-media : a, funnel containing the culture-media in liquid condition ; 6, pinch-cock by which the flow of fluid into the test-tube is regulated ; c, rubber tubing (Warren). 1 Allow the chamber to fill with steam before placing the media within. Do not leave the media in the sterilizer to cool. Plunge gelatin-tubes into ice-water immediately after each sterilization, in order to maintain a high melting-point. 54 PREPARATION OF CULTURE-MEDIA. VILLA Ruy Fic. 29.—Providence Health Fic. 30.—Potato-tube Department tube of heavy (Mallory and Wright). glass, with etched surface for writing data. These tubes are etched by dipping for thirty seconds in ‘‘ white acid.” last sterilization should be allowed to solidify with slanting surfaces. POTATO. 5g IV. Media from Meat-extracts. (2) Mix thoroughly the white of 1 egg with 1000 c.c. of water for bouillon or gelatin ; with 500 c.c. of water for agar. (4) Proceed then as directed in the above table, beginning with No. 4, substituting ‘‘ white of egg solution ’’ for ‘‘infusion.”” At No. 6 add 0.5 per cent. of Liebig’s extract of beef for bouillon or gel- atin, I.0 per cent. for agar. V. Potato. (a2) Select large sound potatoes and wash thor- oughly. (4) Cut off the ends, and with a sterile cork-borer cut out cylinders of the potato a little smaller than the tubes in which they are to be placed. Handle the potatoes under water as much as possible, to prevent darkening of the surface. (c) Cut the cylinders into two equal parts by a diagonal cut. (2) Place in cold running water for twelve to eighteen hours. This will usually render the potato neutral. (If necessary to change the reaction of the potato, steam in a measured quantity of distilled water for one-half hour. ‘Titrate and add the required amount of : sodium hydroxid, and repeat the boil- ing for thirty minutes.) (e) Distribute in tubes in the bottom of which a small amount of non-absorbent cotton or a short piece of glass rod has been placed, and sterilize in 56 PREPARATION OF CULTURE-MEDIA. the steam sterilizer for thirty minutes on three suc- cessive days. VI. Dextrose, Lactose, and Saccharose Bouillon. . (a) After filtration of the meat-infusion, prepared as above described, place in an Erlenmeyer flask, and inoculate with a fluid culture of the Bacillus coli or an allied gas-producer. (4) Place in the incubator at 37.5° for twenty- four hours. Fic. 31.—The Smith fer- Fic. 32.—A graduated fer- mentation-tube. mentation-tube. This removes the ineat-sugar. (c) Fiom this infusion prepare bouillon in the ordinary way. (2) To the completed broth add 1 per cent. of the required sugar. (e) Distribute in test-tubes or in fermentation- tubes,' and sterilize in the steam sterilizer on three successive days. 1A small test-tube inverted inside a large one will answer for a fermentation-tube. MILK AND LITMUS MILK. 57 VII. Milk and Litmus Milk. (2) Heat fresh milk for fifteen minutes in the steam sterilizer. (4) Place in the ice-box over night. (c) Siphon off the milk, without cream or sedi- ment. \ Hollow Upirable stopper Surfaces in contact "grou. wo Fic. 33.—The Hill fermentation-tube. (dz) Titrate. (e) If less than 2 per cent. acid to phenolphtha- lein, place in tubes and sterilize for twenty minutes on four successive days in the steam sterilizer. If more than 2 per cent. acid, adjust to + 1.5 per cent. by the addition of 7 sodium hydroxid. 58 PREPARATION OF CULTURE-MEDIA. (f) A solution of litmus may be added just _ It f rubber = glass go cm. ¥ Fic. 34.—Siphon, with one-way valve for starting the flow of serum. The end of the glass tube is turned over to prevent the clot from entering. Thermometer. Glass and Asbestos cover 7 Fa SF EF SE SS iF fre gauge Adjustable legs for regulating slant. ia = Fic. 35.—Coagulator for blood-serum tubes. Providence Health Department pattern, with wooden rack for holding tubes away from sides. When in use, the four sides and the space below are covered with asbestos boards. previous to its distribution in tubes, sufficient to give the milk a pale-blue color. BLOOD-SERUM. 59 VIII. Blood-serum (Zéffer’s). (a) Receive freshly drawn beef blood in sterile jars. (2) Allow twenty minutes for coagulation to begin, then with a sterile glass-rod break up any adhesions between the coagulum and the jar. (c) Allow the jars to stand twenty-four hours in the ice-box. (2) Siphon off the clear serum.' (e) To 3 parts of serum add 1 part of 1 per cent. dextrose bouillon.” (/) Adjust to + 0.8 per cent. (g) Distribute in tubes and solidify with a slant- ing surface by heating for three hours in a blood- serum coagulator. (2) Sterilize in the steam sterilizer for twenty minutes on three successive days. 1If the serum is not clear, filter through the coagulum left after the filtration of bouillon, as suggested by Hill. 2 If 1.25 per cent. glycerin is added also, it seems to pre- vent the tubes from drying. CHAPTER Vt. CULTURES OF BACTERIA. OWING to their small size and to their similarity of form, the different species of bacteria cannot be recognized by microscopic examination alone. Re- course must be had to a study of their biologic peculiarities. For this purpose it is necessary to grow them on artificial culture-media. If a single germ is planted on a suitable nutrient medium, and is protected from contamination, it multiplies rapidly and forms a colony. Such a colony is com- posed of but one species, since all its members are the descendants of a single germ. Cultures made from such a colony are known as ‘‘ pure cultures,”’ since they contain but one species. From a study of the behavior of these pure cultures, under differ- ent conditions, the diagnostic characters of the species are determined. I. BOUILLON CULTURES. 1. Inoculate a tube of bouillon by touching the culture to be studied with a sterile platinum needle, and then dipping the needle in the bouillon. 2. Place at the room-temperature or in the incu- bator, and examine from day to day, and note any changes as follows : 60 “STAB” CULTURES. 61 (a) Does the bouillon become uniformly clouded, or only at the surface or bottom ? (4) Does a pellicle form on the surface or a de- posit at the bottom ? Fic. 36—Method of holding tubes during inoculation. (c) Is the color, odor, or reaction of the bouillon changed ? Il. GELATIN OR AGAR CULTURES. 1. “Stab”? Cultures.! (1) Sterilize a straight platinum needle and touch it to the culture to be studied ; hold the gelatin- or agar-tube upside down, remove the cotton plug, and stab the needle carefully up through the cen- ter of the medium nearly to the bottom of the 1“Stab” cultures are usually made in gelatin-tubes ; “stroke” cultures, on agar-tubes. 62 CULTURES OF BACTERIA. tube ; withdraw the needle carefully and replace the cotton plug. (2) Allow to growat the room-temperature, if gela- Fic. 37.—a,‘‘ Stab ”’ cult- ure; 5, ‘‘stroke’’ cult- ure. tin is used ; at either room- or incubator-temperature, if agar is used; examine from day to day. (3) Note in stab cult- ures: (a) Does the growth ap- pear along the line of puncture, at the surface, or in both places? (4) Is the surface growth abundant or scanty; does it spread over the whole surface or is it confined to the point of puncture ; is its margin regular or in- dented ; is it flat or raised, dry or moist; what is its color, luster ? (c) Is the medium changed in color, odor, or condition ? (2) In gelatin cultures, if liquefaction takes place, is it at the surface, deep, or throughout the line of puncture? What is the form of the liquefied area. The following terms have been suggested by “STAB” CULTURES. 63 Fic. 38.—Various forms of gelatin ‘‘stab’’ cultures: a, Bacillus typhosus; 6, Bacterium anthracis; c, Bacillus mycoides ; d, Bacillus mesentericus ; ¢, Bacillus cedematis ; J, Bacillus radiatus. Chester’ for describing the characters of gelatin stab cultures : L Non-liquefying Cultures. Line of puncture may be— filiform, euifema growth without special charac- ers. Nodose, consisting of closely aggregated colonies. Beaded, consisting of loosely placed, disjointed. colonies. Papillate, beset with papillate extensions. Echinate, beset with acicular extensions. Villous, beset with short, undivided, hair-like ex- tensions. Plumose, a delicate feathery growth. Arborescent, branched or tree-like, beset with branched hair-like extensions. 1 Eleventh Annual Report of the Delaware College Agri- cultural Experiment Station, Newark, Delaware, 1898-1899. 64 CULTURES OF BACTERIA. Il, Liquefying Cultures. Line of liquefaction may be— Crateriform, a saucer-shaped liquefaction. Saccate, shape of an elongated sac, tubular, cylindrical. Infundibuliform, shape of a funnel, conical. Napiform, shape of a turnip. Fusiform, shape of a parsnip, narrow at either end, broadest below the surface. Stratiform, liquefaction extending to walls of the tube and downward horizontally. Fic. 39.—Microspira comma (Asiatic cholera): gelatin puncture cultures aged forty-eight and sixty hours (Shakespeare). 2. “Stroke”? Cultures. (1) Sterilize the platinum needle, touch it to the Fic. 40.—Microspira Finkleri: gelatin puncture cultures aged forty-eight and sixty hours (Shakespeare). Fic. 41.—Microspira Metschnikovi: puncture culture in gelatin forty-eight hours old (Frankel and Pfeiffer). 5 65 66 CULTURES OF BACTERIA. culture to be studied, and draw it over the surface of an agar- or gelatin-tube that has been solidified with a slanting surface. (2) Allow to grow as before, and examine from day to day. (3) Note: (2) What are the size and shape of the streak ? (6) What is the character of the margin ? (c) Is the growth abundant or scanty, flat or raised, opaque or transparent?! (2) What is the color, luster ? : (e) Has the color, consistence, or odor of the medium changed ? II. POTATO AND BLOOD-SERUM CULTURES. Stroke cultures are employed in using potato or blood-serum tubes. The methods of study are essentially the same as described for gelatin or agar cultures. IV. PLATE CULTURES. In making plate cultures the nutrient medium is liquefied and a very small portion of the culture to be studied mixed with it; it is then poured into sterile Petri dishes, solidified, and the colonies allowed to develop. By this method the bacteria are scattered through the medium, and the colonies that develop are the descendants of a single germ. This permits different species to be separated from a mixture in pure culture, and the peculiarities of 1 For descriptive terms, see p. 70, ef seg. PLATE CULTURES. 67 ‘colonies’? in distinction from ‘cultures’ to be determined. 1. Liquefy three gelatin- or agar-tubes! by placing in a water-bath. Gelatin-tubes must not be heated above 40° C. Agar-tubes may be heated to 100° C., but-must be cooled to 40° C. before the inocu- lations are made. 2. With a sterile platinum needle transfer a Fic. 42.—Petri dish with colonies. minute portion of a culture to the liquefied medium in the first tube. 3. Shake the tube thoroughly without producing bubbles in the medium, and transfer a loopful to the second liquefied tube. 4. Shake the second tube, and transfer a loopful to the third tube. 1 Bouillon-tubes or tubes of sterile water may be substi- tuted for the first two gelatin- or agar-tubes when it is desired to make but one plate, 68 CULTURES OF BACTERIA. Fic. 43.—Brown University electric water or paraffin bath.! 1A porcelain-lined dish fitted with a tin cover, provided with four doors. In the figure the cover is raised from the dish to show the thirty candle-power electric lamp which projects through the cover into the water or paraffin below. The lamp is provided with a regulating socket giving PLATE CULTURES. 69 5. Shake the third tube, and pour the contents of the three tubes into sterile Petri dishes, having first passed the lip of each tube through the flame. 6. On the surface of the first Petri dish, before 4 Fic. 44.—The various appearances of colonies of bacteria under the microscope: a, colony of Bacterium parvum ; 4, colony of Bacillus polypiformis ; c, colony of Bacillus radiatus. the medium has entirely solidified, press a sterile’ cover-glass or small piece of mica. candle-powers from five to thirty. By adjusting the socket to the different candle-powers the requisite temperature of water or paraffin is secured and indicated by the thermom- eter. When used as a water-bath, the dish is provided with a wire rack to support the tubes to be melted. When used for paraffin, little baskets of wire gauze, containing the specimens to be embedded, are hung about the sides. It is convenient to have the water-bath kept at a temperature of 40° C. In this gelatin-tubes may be melted, or agar- tubes, after being melted at a higher temperature, may be cooled down and kept melted until used. 1Sterilize by heating in the flame. Allow it to cool before placing on the plate. 7O CULTURES OF BACTERIA. 7, Allow to develop as directed for tube cultures, and examine from day to day, or oftener as necessary. 8. In plate cultures note : (a) Is there a difference in the number of colonies in the three dishes? ‘The use of the three tubes and plates is for the purpose of reducing the num- ber of colonies, so that only a few will be present in the third plate. (4) Is there more than one kind of colony present ? Notice that colonies on the surface differ from those below the surface, though of the same species. If the culture from which the inoculations were made is a pure culture, and if no germs have been allowed to enter during the process of making the plates, there should be but one kind of colony present. (c) What are the size, shape, texture, and color of the colonies, both surface and deep? What is the character of their margins? The following terms are those suggested by Chester for the description of colonies : I. Form. Punctiform, dimensions too small for defini- tion. Round, of a more or less circular outline. Lrregular. Elliptical. Fustform, spindle-shaped. Cochleate, spiral or twisted like a shell. Amebord, very irregular, streaming. Mycelioid, a filamentous colony with the radiate character of a mould. SURFACE ELEVATION. : 71 Filamentous, an irregular mass of loosely woven filaments. floccose, of a dense woolly structure. khizozd, of an irregular, branched root-like character. Conglomerate, an aggregate of colonies of similar size and form. Torulotd, an aggregate of colonies like the budding of the yeast plant. Rosulate, like a rosette. II. Surface Elevation. 1. General character of a surface as a whole. Flat, thin, leafy, spreading over the sur- face. Liffused, spread over the surface as a thin, veily layer more delicate than the pire ceding. Raised, growth thick with abrupt terraced edie. Convex, surface the segment of a circle, but very flatly convex. Pulvinate, surface the segment of a circle, but decidedly convex. Capitate, surface semi-spherical. 2. Detailed characters of surface. Smooth, surface even, without any of the following distinctive characters. Alveolate, honeycombed. Punctate, dotted with punctures. Bullate, biistered. Vesicular, more or less covered with mi- 72 CULTURES OF BACTERIA. nute vesicles due to gas formation; more minute than bullate. Verrucous, bearing wart-like prominences. Sguamose, scaly. Echinate, beset with pointed prominences. Papillate, beset with nipple-like processes. Rugose, short irregular folds due to shrink- age. Corrugated, in long folds due to shrinkage. Contoured, a smoothly undulating surface. Rimose, abounding in clefts or cracks. III. Microscopic Structure. 1. Refraction weak. Outline and surface of relief not strongly defined. 2. Refraction strong. Outline and surface of relief strongly marked. (a) Dense, not filamentous colonies. 1. General. Amorphous, without definite structure. fTyaline, clear and colorless. Homogeneous, structure uniform through- out all parts of the colony. Homochromous, colony of uniform color throughout. 2. Granulations or blotchings. Finely granular. Coarsely granular. Grumous, coarser than preceding. Morulord, segmented. Clouded, having a pale ground with ill- defined patches of a deeper tint. EDGES OF COLONIES. 73 3. Colony marking or striping. Retzculate, in the form of a network. Areolate, divided into rather irregular or angular spaces. Gyrose, marked by wavy lines. Marmorated, traversed by vein-like mark- ings, Rivulose, marked by lines like the rivers on a map. Rimose, showing cracks or clefts. (4) Filamentous colonies. Filamentous, as already defined. Floccose, composed of filaments densely packed. Curled, filaments in parallel strands. IV. Edges of Colonies. Entire, without toothing or division. Undulate, wavy. Repand, like the border of an open um- brella. Erose, as if gnawed, irregularly toothed. Lobate, divided into lobes. Lobulate, minutely lobate. Auriculate, with ear-like lobes. Lacerate, irregularly cleft, as if torn. Fimbriate, fringed. Ciliate, hair-like extensions. Tufted, Filamentous, Floccose, Curled, as already defined. 74 CULTURES OF BACTERIA. V Optical Characters. Transparent. Vitreous, transparent and colorless. Oleaginous, transparent and yellow. Resinous, transparent and brown. Translucent. Fics. 45, 46.—The various appearances of colonies of bacteria under the microscope: a, colony of Bacillus muscoides (Liborius); 6, colony of Bacterium anthracis (Fliigge). Porcelaneous, translucent and white. Opalescent, translucent, grayish white by reflected light, smoky brown by trans- mitted light. IMPRESSION OR ADHESIVE PREPARATIONS. 75 Nacreous, translucent, grayish white with pearly luster. Sebaceous, translucent, yellowish or gray- ish white. Butyrous, translucent and yellow. Ceraceous, translucent and wax-colored. Opaque. Cretaceous, opaque and white ; chalky. Dull, without luster. Glistening. Fluorescent. Tridescent. Phosphorescent. (d) Is there growth beneath the cover-glass or mica plate? ‘This determines, roughly, whether they require oxygen for their growth or not (aérobic or ana€érobic). (e) In the gelatin plates is the gelatin liquefied, and what is the nature of the liquefaction ? V. IMPRESSION OR ADHESIVE PREPARATIONS OF COLONIES. An entire colony of bacteria may be preserved as a microscopic specimen. (a) Slightly warm a clean cover-glass. (6) Lay it upon the surface of the gelatin or agar containing the colonies. Apply sufficient pressure to remove all air-bubbles, but not enough to disturb the colony. (c) Remove the cover, gently lifting it from one side. ‘The colonies will adhere to the glass. 76 CULTURES OF BACTERIA. (dz) Dry, fix, stain, and mount as for ordinary preparations. Museum preparations of gelatin or agar tube or plate cultures may be made by exposing the cultures to formaldehyd vapor until the growth is killed, and then sealing the tubes or plates tightly with sealing-wax or paraffin. Fic. 47.—Bacterium tuberculosis: adhesive preparation from a fourteen-day blood-serum culture; x I0o (Frinkel and Pfeiffer). VI. CULTURES IN THE FERMENTATION-TUBE. This method is for the purpose of studying gas formation, and for the study of the aérobic or anaé- tobic properties of organisms. (a) Prepare fermentation-tubes with dextrose, lac- tose, or saccharos¢ bouillon. (4) Inoculate the tubes by floating a little of the CULTURES IN THE FERMENTATION-TUBE, 77 culture to be studied on the fluid in the bulb with a sterile needle. (c) In the case of gas formation, at the end of every twenty-four hours, for several days, mark the level of the fluid in the closed branch upon the tube or measure it with a millimeter scale. (d@) Record the result in percentages of the length Fic. 48.—Bacterium anthracis: colony three days old upon a gelatin plate ; adhesive preparation ; x 1000 (Frankel and Pfeiffer). of the closed branch. If I cm. of gas forms in a 10 cm. tube, 10 per cent. of gas is said to have formed. (e) To test the relative amount of carbon dioxid and hydrogen present. Fill the bulb completely with a 2 per cent. solution of sodium hydroxid. Place the thumb over the mouth of the bulb, and 78 CULTURES OF BACTERIA. run the mixture six or eight times through the length of the tube, returning the remaining gas to the closed branch before removing the thumb, Measure the amount of gas remaining ; the differ- ence between this and the former measurement shows in millimeters the amount of carbon dioxid absorbed by the alkali. The remaining gas, mostly hydrogen, may be transferred to the bulb and exploded by a flame. ‘The proportion of hydrogen to carbon dioxid is usually expressed in the form of a fraction called the gas formula, as ; 2 The fermentation-tube affords a ready method of determining the oxygen requirement of bacteria. Growth, indicated by cloudiness, in the bulb only, is to be found only among obligatory aérobes ; in the closed branch only, among obligatory anaé- robes; while growth in both, only among the facultative anaérobes. VII. ANAEROBIC CULTURES. Growth under the mica plate or cover-glass’ and in the fermentation-tube are methods for the deter- mination of the aérobic properties of organisms. For the growth of strictly anaérobic forms special methods have been devised : I. Place the cultures in a vessel from which the air can be withdrawn and hydrogen substituted. 2. Buchner’s Method.—Use two test-tubes, one inside the other. ‘The outer one is partially filled 1 See page 75. ANAEROBIC CULTURES. 79 with pyrogallic acid made alkaline with sodium hydrate, and is sealed tightly with a rubber stopper. The inner tube contains the culture. The oxygen is absorbed by the mixture in the outer tube. 3- Wright’s Method.—Make an ordinary cult- ure in a test-tube. Clip off any superfluous cotton from the plug, and push the plug into the tube so Fic. 49.—Novy’s jars for anaérobic cultures. that it lies 1 centimeter below the mouth. For test-tubes 6 X 34 inches, run into the cotton plug, from a pipet, approximately % c.c. of a freshly prepared solution of pyrogallic acid (1 part of acid, I part of water), and then approximately 1 c.c. of a solution of sodium hydrate (1 part of sodium hydrate, 2 parts of water). Quickly insert a rubber stopper into the tube. 4. Make a hanging-drop culture. On one side of the cover-glass introduce a little pyrogallic acid, and on the other side a little sodium hydrate, so that it runs around and unites with the acid. Seal with vaselin, 80 CULTURES OF BACTERIA. 5. Distribute the germs to be studied in bouillon or in liquefied gelatin or agar, and draw some of the solution into sterile pieces of glass tubing of small caliber. When the tube is full seal the ends in a flame. 6. Put large quantities of culture-medium in the tubes and puncture deeply. The surface of the medium is then covered with sterile oil. 7. Park’s Method.—Cover the culture-medium with melted paraffin.’ Sterilize by the ordinary methods, and when cool enough for inoculation, but before the paraffin solidifies, inoculate through the paraffin into the medium below. Wright recommends? the following precautions in growing anaérobic bacteria : 1. ‘The medium should contain 1 per cent. of glu- cose, and should be boiled and cooled immediately before inoculation. 2. The medium should be freshly prepared. 3. The reaction should not be more acid to phenolphthalein than +1.5. With 1 per cent. glucose bouillon, growth is better if the reaction is nearer the neutral point of phenolphthalein than +15. VHI. DEMONSTRATION OF LIQUEFYING FERMENT. (a) Inoculate several gelatin-tubes with Bacellus prodigiosus. (4) Allow them to grow until all the gelatin is liquefied. 1 Melting point, 42° C. 2 Jour. Boston. Soc. Med. Sci., vol. v., No. 4, p. 114. ISOLATION OF SPECIES. 8% (c) Add ¥%4 c.c. of chloroform, or 5 per cent. car- bolic acid, to each tube, shake thoroughly, and filter. (d) Add the filtrate, now containing no living bacteria, to tubes of sterile gelatin. Note the liquefaction that takes place, caused by the fer- ment produced by the bacteria in the first set of tubes, IX. ISOLATION OF SPECIES. Given a bouillon culture, containing three species of bacteria, to isolate the species.' (a) Liquefy three gelatin or agar tubes and number 1, 2, and 3 respectively.’ (2) Transfer a minute loopful of the bouillon cult- ure to tube No. I. (c) Shake thoroughly, and transfer 2 loopfuls from tube No. 1 to tube No. 2. (a) Shake and transfer 3 loopfuls from tube No. 2 to tube No. 3. (e) Flame the lips of the tubes and pour their contents into sterile Petri dishes. — (/) Examine in twelve to twenty-four hours. (g) Select the dish in which the colonies are well developed, and in which they have not run together ; look for three kinds of colonies. If more than three kinds are present, it shows 1 This method is applicable to the separation of species from any fluid. ? Tubes of bouillon or sterile water may be substituted for tubes Nos. 1 and 2, in which case, of course, but one plate can be made—z. ¢., from tube No. 3. 6 82 CULTURES OF BACTERIA. that others have been allowed to enter through carelessness in manipulation. (h) Record the appearance of the different colo- nies, and inoctilate them as pure cultures in tubes of culture-media. Study them as directed in the following chapter. CHAPTER: VIIL. DETERMINATION OF SPECIES. I. MORPHOLOGY AND LIFE-HISTORY OF A SPECIES. THE following points in the morphology and life- history of any form of bacterium must be known before it can be fully described or assigned a place in any particular species.' I. SouRcE AND HapsiratT. II. MoRPHOLOGIC CHARACTERS. 1. Form. 2. Dimensions. 3. Manner of grouping’ and arrangement in the growths. 4. Staining powers: (a) with aqueous stains; (6) by Gram’s method. . Presence or absence of capsule. . Presence or absence of flagella (motility). . Spore-formation. . Tendency to pleomorphism. . Involution and degeneration forms. III. BroLoGic CHARACTERS. A. Cultural characteristics, mode of growth in and upon— Oo ON AN - Bouillon. : . Gelatin plates (single colonies, surface and deep), . Gelatin-tubes. . Agar plates (single colonies, surface and deep). PwNH 1 These are the points recommended by the Committee of Bacteriologists of the American Public Health Associa- tion. 83 84 DETERMINATION OF SPECIES. 5. Agar-tubes. 6. Potato. 7. Milk. 8. Blood-serum. B. Biochemic features. 1. Temperature relationship (activity of growth at 18°-22° C. and at 36°-38° C. and thermal death-point). | 2. Relation to free oxygen (aérobic and andérobic growth). 3. Relation of growth to acidity and alkalinity of media. 4. Action upon gelatin (presence or absence of lique- faction). 5. Action upon proteids (milk and serum). 6. Action upon carbohydrates (fermentation and gas formation). 7. Action upon nitrates. 8. Production of indol. , g. Production of acid or alkali. 10. Pigment formation. 11. Development of odor. C. Pathogenesis. The following tests are of value in certain cases : I. MORPHOLOGIC. 1. Staining reactions with special stains. 2. Study of flagella by special stains. 3. Permanency of morphologic characters after long- continued growth and successive transplantation upon artificial media. 4. Photographic reproductions of isolated bacteria. 5. Cover-glass impressions. II. PHysIOoLoGic. A. Cultural characteristics, mode of growth in or upon— 1. Litmus gelatin. 2. Léffler’s blood-serum. 3. Synthesized media. 4. Photographic reproduction of characteristic cultures. STANDARD CHART FOR BACTERIAL ANALYSIS. INTAWME cance: cesecsccssnscseacth POUR CE cccre: steers asus HaBITAT.. TAI i execencisdoncehdcrnases REPORTED BY wrcsccccescccsssctsenet Form and arrangement in bouillon, grown ............. hours at 18°-20° C.; ditto, grown ............. hours at 36°-38° C. Size, length ............. Hy breadth ............- #; extreme lengths from ............. PERO recsccreier: B. Capsules, none observed, easily observed or demonstrated. Conditions under which they are present, agar, serum, milk, or Spores, none observed within ............. hours at Germinate within ............. hours at ... When present are polar, central, cells ............. swollen. °C. Stain by .method. Are killed at 100° C. in .. .° C., or when stained with 2.000.000... Vacuoles, observed when grown on Motility, sluggish or active, rotary or direct, more pronounced in -.......-e cultures grown at ............ OCS FOr cassie hours. Flagella stain by ......................... method ; are monotrichous, lophotrichous, amphitrichous, peritrichous. ~- Pleomorphism, observed in 0.2.1.0: sees cultures grown at ............. ONG AOR cccacees days. Stain, easily or with difficulty with ..........-...+ , uniformly or irregularly. Stained or decolorized by Gram’s method. Gainers Boar Surface colonies. Deep colonies. BouiLion. SkeTcH of Germ AND COLony. PLaTEs. = é Opacity begins after .... hrs. at ....°C. si cane Gelatin. Agar. Gelatin. Agar. Pellicle forms in .... hrs. at ....° C. | os Color .... appears in’... hrs. at ....°C. ae —, i Deposit forms in .... hrs. at ....9 C. Under mica plate. ma Stas CULTURES. ; ; Character, compact, flocculent, granular, GELATIN OR AGAR-TUBE. flaky, or viscid on agitation. Puncture. Color. é Form Odor. . rT °° | Surareipiowels « .* Reaction, wee sahesnat joa Ce Size. Shape. pone Potato or BLoop-sERuM. Color. | ee Consistence. M ape Deep growth. Surce veliee anes Laas ap 4 St Se te =e 3 J g fs ve - : eeat re ee SHEE bbles. ae ee ne ats cn PES RCULTURESS OT Size. Texture. » <= ss Pa aaa Shape. Consistence, 1S) iS) Margin. Liquefaction. ° 3 Surface relief. Gas production. : : Light transmission. fe Color. 4 4 Luster. a Consistence. os aa . 3 : : ; eduction of nitrates. 2 — in medium. Dyers ices bouillon. Pa - ‘olor. ndol production. é A Consistence. ¥ > YZ : eZ Odor. MILK. Curd, curdles or does not curdle in .... days at 18°-20° C. or aes Ler ee Ge eS en ‘boiling after .... daysat ....°C. hard or soft, in one mass or in fragments, gas-bubbles. changed or unchanged by boiling. W hey, separates from curd or not, amount, transparent or turbid. Reaction, w..e.206 AD: scacaaasa days at. 32 fey Color. Gas production. Digestion becomes gradually transparent without forming curds. complete in .... daysat. effects of boiling, odor, clear or cloudy, watery or viscid. reaction at end of digestion sievenicravonaeiae 2S Saas i a : = apeere z aoe ae Sucar BovuiLion IN ~ Amount of gas in ae at Reaction of fluid in. A Pelli- | Opac- Col FERMENTATION-TUBES 18°-20° C, 60-389 C. after....days at. h cle. ity. OOK: Micrococcus, single, pairs, chains, tetrads, or cubical packets ; Bacil/us, single, pairs, chains, or filaments; Spzrzd/m, comma, spiral. inzd. as BS iy eT inrd,, 2ds. a tegen ENR DDOREHOSE eacigesicsl| ides Sees WES OAH MERE | YE FT Seg GEES VT EpCtosecinscmdscee | Goan, satus Sens HPS KEN || Re Soe Skee SHES Gees Saccharose.. #3 PIGMENT, PATHOGENESIS. developed in presence or absence of oxygen. es TE, onensemant cultures at ...... °C.in hours. color 5 by acid or alkali. soluble in Optimum Temperature ....0C. Growrn Limits ....°C.to....°C. THermat Dearnu-point ....°C., time of exposure .... minutes. PropucTion oF Acips oR ALKALIES. RELATION TO FREE OXYGEN. RetaTion oF GrowTH To AcipiTy or ALKALINITY OF MEDIUM. Carbohydrates absent or present. Obligatory aérobe. -... @acid to.... % alkaline. Facultative anaérobe. Obligatory anaérobe. aia STUDY OF FORM AND GROUPING. 89 B. Biochemie. 1. Minimum, optimum, and maximum temperatures of growth. 2. Growth in atmospheres of various inert gases (when anaérobic power of growth has been determined). 3. Optimum reaction of media and reaction limits, acid and alkaline (indicated by phenolphthalein). 4. Chemic properties and solubility of pigments pro- duced, and spectroscopic observations upon the pigment. C. Pathogenesis. 1. Inoculation of various species of animals, with minute study of the pathologic changes produced. 2. Immunity-producing properties. 3. Agglutinating properties of specific sera. 4. Determination and isolation of toxic substances (from non-pathogenic, as well as from pathogenic, bacteria). A suitable blank should be prepared, on which are recorded the observations made on each species. The appended form (pages 86, 87) is substantially the one recommended by the Bacteriologic Com- mittee of the American Public Health Associa- tion. A further explanation in regard to some of these points is required. (a) Study of Form and Grouping.—Determine and describe the morphology from the growth obtained upon at least one solid medium and in at least one liquid medium. Growth at 36°-38° C. should in general be not older than from twenty- four to forty-eight hours, while growth at room- temperature (18°-22° C.) should be not older than from forty-eight to seventy-two hours. Growth on solid media may be studied from cover-glass prep- arations ; in liquid media growth is best observed go DETERMINATION OF SPECIES. in the hanging-drop, preferably in a fresh medium inoculated with a very small amount of the culture to be examined. It is desirable that the form and grouping be determined in bouillon, gelatin, and on agar, and that any variation found upon the examination of the growth on other media be accu- rately noted. (b) Test for Motility.—For the study of motility the hanging-drop preparations should be made from young cultures grown at or near the optimum temperature for only a few (six to eighteen) hours. (c) Tests for Spores.—The tests for the presence of spores are : ' (a) Do colonies develop from cultures which have been subjected to a temperature of 80° C. for ten minutes ? (6) Are there highly refracting bodies within the bacteria in unstained preparations, and can they be demonstrated by the spore-staining methods?. ~ Cultures to be tested should be grown for forty- eight hours in bouillon, and when possible at 36°-38° C. Three loopfuls of this culture’ after agitation are transferred to tubes containing 10 c.c. of bouillon. This is exposed to a temperature of 80° C. for ten minutes, and then placed under con- ditions favorable for the development of any of the organisms which may have survived. . (d) Pleomorphism.—In regard to pleomorphism, attention is called to the variations in size and shape brought about by the following conditions of growth : (a) At: different temperatures, ACIDITY AND ALKALINITY OF MEDIA. gl (6) Upon or in media of different composition: (c) Upon or in media of different degrees: of acidity and alkalinity. 5 (2) In cultures of different ages. (e) As well as to the variations in the size and shape of different individual bacteria obtained from one culture and appearing often in the same field of view—z. e., subjected to exactly the same condi- tions of growth. (e) Determination of the Thermal Death- point.—In determining the thermal death-point, the facts required to be known are (1) the time of exposure to heat, (2) the presence or absence of moisture, (3) the presence or absence of spores, (4) the age of the culture, (5) the amount: of the culture used for the tests, and (6) the character of the con- taining vessel. The temperature required to destroy the species under consideration is to be determined within 2 degrees C.; thus, if samples are exposed to temper- atures of 50°, 52°, 54°, 56°, 58°, and 60° C., and it is found that development in a suitable medium occurs after exposure to 56° C., but not after expos- ure to 58° and 60° C., the thermal death- -point | is to be given as 58° ., although further study might show it to be domewhat less than this. (f). Relation to Free Oxygen.—For the methods of determining the aérobic properties of organisms, see under Anaérobic Cultures, on page 3. : (g) Relation to Acidity and Alkalinity of Media.—In-determining the relation of growth to g2 DETERMINATION OF SPECIES. acidity and alkalinity of media, all that is necessary is to add to tubes containing equal quantities of any of the usual media a calculated amount of a standardized solution of hydrochloric acid or of sodium hydroxid to obtain the desired reaction. A record should be made of cultures upon at least one medium reacting +3 per cent., +1.5 per cent., neutral, and —1.5 per cent. to phenolphthalein. (h) Action upon Carbohydrates.—For the methods of determining the action upon carbo- hydrates and for measuring the gas production, see under Cultures in the Fermentation-tube, page 76. (i) Action upon Nitrates.—To determine the power of certain bacteria to reduce nitrates to nitrites and ammonia, incubate them for seven days at 20° C. in the following: NITRATE BROTH. Peptone, I gm.; Potassium nitrate, 0.2 gM. ; Tap water,’ I000 ¢.¢c. Submit the inoculated tubes and also several un- inoculated control-tubes to the following tests for nitrites : Prepare two solutions : I, Naphthylamin, O.I gm. ; Distilled water, 20 c.c. Boil until the naphthylamin is dissolved ; cool, 1 Better than distilled water because of the other salts present, which favor the growth of the bacteria. ACTION UPON NITRATES. 93 filter, and add the filtrate to 150 c.c. of dilute (1 to 16) hydric acetate. II. Sulphanilic acid, OF Ce 5 Dilute (1 to 16) hydric acetate, 150 c.c. Keep these solutions in separate glass bottles, tightly stoppered, and mix in equal parts before use. To 3 c.c. of the solution to be tested, in a per- fectly clean test-tube, add gradually 2 c.c. of the test-solution. A red color develops of an intensity in proportion to the amount of nitrites present. The appearance of the color may be hastened by heating. If this test shows the presence of nitrites, test one-half of the remaining solution for ammonia with Nessler’s solution.' The presence of ammonia is shown by the im- mediate development of a yellow color or precipi- tate on the addition of a few drops of the test-solu- tion. When these tests are positive our inquiry has been answered. When negative the nitrates may have remained unchanged or may have been re- duced to free nitrogen. It is therefore necessary to determine whether the nitrates are still present or not, as follows: (2) Invert the tube containing the culture and evaporate to dryness the small amount of culture remaining on the inside of the tube. 1 See Appendix, page 174. 94 DETERMINATION OF SPECIES. (6) Add, in order, phenol-sulphonic acid,’ water, and sodium hydrate. A yellow. color shows the presence of nitrates. (j) Acid or Alkali Production. —For the deter- mination of acid or alkali production, cultures are made on solid media to which have been added. 1 per cent. of dextrose, saccharose, or lactose, and a sufficient quantity of litmus solution to produce a blue tinge. A pink coloration of the colony or a reddening of the surrounding medium indicates acid production. (k) Test for Indol Production.—For the deter. mination of indol production, incubate the species: to be tested in the dextrose-free bouillon described on page 56. Add to the tube of culture 1 c.c. of a 0.0I-0.02 per cent. solution of sodium or potassium nitrite. T’o each 10 c.c. of medium add one drop of concentrated sulphuric acid. A red coloration indicates indol. If no result is obtained at once, it is well to allow the tube to stand for one hour. Pathogenesis.—In Chapter VIII. are described the various methods of determining the pathogenic properties of bacteria. 1. When a given form grows only at or below 18° to 20° C. inoculation should be made into the dorsal lymph-sac of a frog, using about 1 per cent. of the body-weight of a bouillon culture seven. days old. _ 2. When a given species grows at 35” C. or up- ward, inoculation should be. made into the peri-. 1 See Appendix, page 174. DETERMINATION OF SPECIES. 95 toneal cavity of a white or ordinary ‘house mouse, using I per cent. of the body-weight of a forty-eight hour bouillon culture. If the mouse is killed, it is well to try the culture on guinea-pigs or rabbits. A careful autopsy should be made in all cases, cult- ures taken from the organs, and all pathogenic changes noted. Il. DETERMINATION OF THE NAME OF A SPECIES. When the points mentioned at the beginning of this chapter have been determined, the name of the species, if it has been described and named, can be found by referring to the bacterial analysis tables in Migula’s System der “Bakterien, Stern- berg’s Manual of Bacteriology, Fligge’s Dre Mikroorganismen, or Chester’s “Studies in Sys- tematic Bacteriology’’ in the ninth, tenth, and eleventh Reports of the Delaware College Agrt- cultural Experiment Station, 1897, 1898, and 1899. The following table, adapted from Migula, may assist in the determination of the name of a species : BACTERIA. ' I. Family. Coccaceze. 1. Genus. Streptococcus. I. Grow on gelatin. 1. Colonies white. A. Do not liquefy gelatin. (a2) No growth on the surface in gelatin stab cultures. S. pyogenes. S. equtti. S. cystitidis. DETERMINATION OF SPECIES. S. urine. S. acidi lactici. (6) Growth on the surface and along the puncture in gelatin stab cultures. S. mastitidis. S. mirabilis. B. Liquefy gelatin. S. septicus. S. morbi brightii. S. gracilis. S. vermiformis. S. albus. 2. Colonies colored. S. cerasinus. S. citreus. II. Do not grow on gelatin. S. giganteus. 2. Genus. Micrococcus. I. Grow on gelatin. 1. White. A. Do not liquefy gelatin. . candicans. . tardissimus. . tardus. . plumosus. viticulosus. stellatus. . tenuissimus. albocereus (cereus albus). ureze. coryze. . salivarius. SSSSS5555555 DETERMINATION OF SPECIES. Gx REE ERE ESC ESE ESE . lacticus. . acidi lactici. . phosphoreus. . catarrhalis. . bovis. . similis. iquefy gelatin. pyogenes. ovis. ’ foetidus. conoideus. liquefaciens. radiatus. . faviformis. . amplus. . dissimilis. 2. Yellow. A. Do not liquefy gelatin. eEESEe . aurantiacus. . tardigradus. luteus. cereus. versicolor. . vatians. B. Biguely gelatin. SSSSSS8E . aureus (S. pyogenes aureus). . beri-beri. . fuscus. . coronatus. . conjunctivitidis. . tardus. . flavus. 97 98 98 DETERMINATION OF SPECIES. . desidens. . conglomeratus. ' . citreus (S. pyogenes. citreus). . citrinus. ; . corrugatus. . mollis. 3. Red. A. Do not liquefy gelatin. M. cinnabareus. M. carneus. B. Liquefy gelatin. M. roseus. M. rosaceus. M. carnicolor. M. fragilis. 4. Blue and violet. M. violaceous. M. cyaneus. II. Do not grow on gelatin. . gonorrheeee. . intracellularis. . subflavidus. . rugatus. . cuniculorum. . progrediens. . tenuis. . nlitrosus. . foetidus. 3. Genus. Sarcina. I. Grow on gelatin. 1. White. Sarcina alba. SEEEES SSSSSE55585 DETERMINATION OF SPECIES. 99 a. Yellow. A. Do nat liquefy gelatin. Sarcina lutea. Sarcina ventriculi. B. Liquefy gelatin. Sarcina liquefaciens. Sarcina aurantiaca. II. Family. Bacteriacez. 1. Genus. Bacterium. I. Form spores. I. Spores polar. Bact. anthracis. Bact. anthracoides. Bact. subtile. 2. Spores central. Bact. carotarum. Bact. angulans. 3. Position of spores undetermined. A. Grow on gelatin at the room-temperature. (a) Colonies white. 1. Do not liquefy gelatin. Bact. acidi lactici. Bact. coprogenes. 2. Liquefy gelatin. (1) Center of colonies plainly floc- cose. Bact. vermiculare. (2) Colonies not floccose. Bact. tricomii. Bact. nephritidis. Bact. sempervivum. Bact. lacteum. 100 DETERMINATION OF SPECIES. (4) Colonies colored. Bact. brunneum. B. Do not grow on gelatin at the room- temperature. Bact. termophilum. II. Spore formation not observed. 1. Grow well on gelatin. A. Form no pigment. (a) Do not liquefy gelatin. I. Stained by Gram’s method. Bact. pneumonie. Bact. proteus. Bact. rhusiopathiee. Bact. murisepticum. Bact. lacticum. Bact. parvum. 2. Do not stain by Gram’s method. Bact. pneumonicum. Bact. rhinoscleromatis. Bact. faschingil. Bact. endocarditidis. Bact. cuniculicidi. Bact. suicida. Bact. palumbarium. Bact. ribberti. Bact. cuniculi. Bact. pseudotuberculosis. Bact. columbarum. Bact. a€rogenes. Bact. aceti. Bact. salivee. 3. Gram’s stain undetermined. Bact. capsulatum. DETERMINATION OF SPECIES. Io! Bact. keratomalaciz. Bact. felis. Bact. pyogenes. Bact. welchii (aérogenes capsu- latus). Bact. bienstockii. Bact. laerii. Bact. ubiquitum. Bact. candicans. Bact. phosphorescens. Bact. giardi. (4) Liquefy gelatin. Bact. bovis. Bact. vignali. Bact. varicosum. Bact. buccale. B. Colonies yellow. (2) Do not liquefy gelatin. Bact. erythrogenes. Bact. citreum. Bact. aurescens. (4) Liquefy gelatin. Bact. arborescens. Bact. aquatile. Bact. chlorinum. Bact. aureum. C. Colonies red. Bact. mycoides. Bact. pyocinnabareum. D. Colonies blue or violet. Bact. coeruleum. Bact. amethystinum. 102 DETERMINATION OF SPECIES. 2. Do not grow well on gelatin at room- temperature. Bact. tuberculosis. Bact. tuberculosis avium. Bact. leprze. Bact. syphilidis. Bact. smegmatis. Bact. mallei. Bact. diphtherie. Bact. xerosis. Bact. pseudodiphtheriticum. Bact. vaginee. Bact. influenze. Bact. pseudoinfluenze. 2. Genus. Bacillus. I. Form spores. 1. Spores polar. B. ramosus. 2. Spores central or oblique. B. subtilis. B. megaterium. 3. Position of spores undetermined. A. White or dirty white on gelatin. (a) Do not liquefy gelatin. B. spermophilinus. B. polypiformis. B. muscoides. (4) Liquefy gelatin. I. Stain by Gram’s method. B. mycoides. B. gracilis. B. granulosus. DETERMINATION OF SPECIES. 103 B. tetani (anaérobic). 2. Gram’s stain undetermined. . cereus. . laevis. . vermicularis. . thalossophilus. . intricatus. . limophilus. . circulans. . pseudanthracis. . globigii. mesentericus. . vulgatus. . Sporogenies. . liodermos. . albolactus. . butyricus. . chauveei (anaérobic). . tadiatus (anaérobic). . cedematis. B. Form a black pigment on gelatin. B. aterrimus. B. niger. II. Spore formation not observed. 1. Grow on gelatin. Not phosphorescent. A. Colonies white. (2) Do not liquefy gelatin. 1. Stain by Gram’s method. B. zenkeri. B. muripestifer. 2. Do not stain by Gram’s method. B. typhosus. lcoMccMccMcoMcvMcomccMccMs McoMerRicoMcomcoMcoes ics ie:) 104 DETERMINATION OF SPECIES. coli. . pestis. . avium. . suipestifer. Zee. . glacialis. DW do , Crait s stain undetermined. B. murium. B. solanacearum. B. phaseoli. B. amylovorus. B. sorghi. B. zopfii. (4) Liquefy gelatin. Is 2. Stain by Gram’s method. B. dysenteriz. Do not stain by Gram’s method. B. pseudotuberculosis. B. ozeenee. B. vulgaris. B. halophilus. . Gram’s stain undetermined. . arthuri. . sulfureus. . liquidus. . diffusus. . nubilus. . reticularis. diaphanus. . mirabilis. gasoformans, . delicatulus. es MocMesMesMe-Me-MocMecMe Me DETERMINATION OF SPECIES. 105 B. cloacz. B. hyalinus. B. superficialis, B. Colonies yellow. B. arborescens. C. Colonies red. B. prodigiosus. B. indicus. B. plymouthensis. B. rubescens. 2. Grow on gelatin. Phosphorescent. B. phosphorescens. B. fischeri. B. phosphoricus. 3. Do not grow on gelatin. B. equi. 3. Genus. Pseudomonas. I. Grow on gelatin. 1. Colonies white. Form no pigment. P. litoralis. 2. Form fluorescent pigment. A. Do not liquefy gelatin. P. alba. P. tenuis. P. eisenbergii. P. stewarti. B. Liquefy gelatin. P. zruginosa (B. pyocyaneus). P. fluorescens. P. minutissima. 3. Colonies blue or violet. P. ianthina. 106 IIT. DETERMINATION OF SPECIES. P. pseudianthina. P. laurentia. 4. Phosphorescent species. P. javanica. II. Do not grow on gelatin. P. europea. P. javaniensis. Family Spirillacee. . Genus. Spirosoma. Spirosoma nasale. Spirosoma linguale. Spirosoma aureum. Spirosoma flavum. Spirosoma flavescens. Spirosoma attenuatum. Spirosoma gregarium. . Genus. Microspira. I. Not phosphorescent. 1. Do not liquefy gelatin. Microspira canalis. Microspira saprophiles. Microspira tonsillaris. 2. Liquefy gelatin. Microspira comma. Microspira metschnikovi. Microspira finkleri. Microspira sputigena. Microspira marina. II. Phosphorescent. Microspira dunbari. Microspira coronata. Microspira annularis. CLASSIFICATION BY GROUPS. 107 Microspira glutinosa. Microspira delgadensis. Microspira tuberosa. Microspira degenerans. Microspira luminosa. Microspira caraibica. Microspira papillaris. Ill, CLASSIFICATION OF BACTERIA BY GROUPS. Systematic bacteriology is at present in a state of chaos. Many times the most that can be done in attempting to classify a new species, is to refer it to some group, the members of which have certain characters in common, and are probably the descendants of one ancestral type. Chester has proposed’ a synopsis of several groups of bacteria which, slightly modified, is given below. BACTERIUM. I. Spore-formers. 1. No growth at room-temperature or below 22°-25° C. TTHERMOPHILIC GROUP. Bact. termophilum type. 2. Grow at room-temperature. A. Do not liquefy gelatin. BACT. FAICALIS GROUP. Bact. subtile type. B. Liquefy gelatin. 1 Eleventh Annual Report of the Delaware College Agri- cultural Experiment Station for 1898-99. 108 DETERMINATION OF SPECIES. ANTHRAX GROUP. ; Bact. anthracis type. II. Spore formation not observed. facultative anaérobic. 1. Do not liquefy gelatin. A. Do not stain by Gram’s method. (a) Obligate aérobic. ACETIC FERMENT GROUP. Bact. aceti type. (4) Aérobic and facultative anaérobic. 1. Gas generated in glucose bouillon. a. Gas generated in lactose bouil- lon. BACT. AEROGENES GROUP. Bact. aérogenes type. 6. Little or no gas generated in lactose bouillon. FRIEDLANDER GROUP. Bact. pneumonicum type. 2. No gas generated in glucose bouillon. a. Coagulate milk. FOWL-CHOLERA GROUP. Bact. cuniculcida type. 5. Do not coagulate milk. SWINE-PLAGUE GROUP. Bact. suicida type. B. Stain by Gram’s method. Gas generated in glucose bouillon. Lactic FERMENT Group. Bact. lacticum type. 2. Liquefy gelatin. Aérobic and CLASSIFICATION BY GROUPS. 109 GLANDERS GROUP. Bact. mallei type. 3. Do not grow well on gelatin at room-tem- perature. A. Stain with basic aniline dyes, but are easily decolorized by mineral acids when stained with carbol-fuchsin. (a) Grow well in bouillon at body-temper- ature and stain by Gram’s method. DIPHTHERIA GROUP. Bact. diphtheriz type. (4) Do not grow in bouillon or on ordinary media. 1. Rods slender. a. Stain by Gram’s method. LEPROSY GROUP. Bact. lepree type. 5. Do not stain by Gram’s method. INFLUENZA GROUP. . .Bact. influenzze type. 2. Rods variable. ROOT-TUBERCLE GROUP. B. Do not stain with aqueous solutions of basic anilins and are not easily decolorized by acids. TUBERCLE GROUP. Bact. tuberculosis type. BACILLUS. I. Spore formers. 1. Aérobic and facultative anaérobic. Rods not swollen at sporulation. IIO DETERMINATION OF SPECIES. A. Liquefy gelatin slowly. UROBACILLUS GROUP OF MIQUEL, B. Liquefy gelatin quickly. (a) Potato cultures rugose. POTATO-BACILLUS GROUP. B. mesentericus type. (4) Potato cultures smooth. B. SUBTILIS GROUP. B. subtilis type. 2. Obligate anaérobic. A. Rods not swollen at sporulation. MALIGNANT EDEMA GROUP. B. chatveei type. B. Rods clavate at sporulation. TETANUS GROUP. B. tetani type. II. Spore formation not observed. 1. Aérobic and facultative anaérobic. A. Gelatin colonies roundish, not distinctly ameboid. (a) Do not liquefy gelatin. 1. Do not stain by Gram’s method. a. Generate gas in glucose bouillon. (1) Coagulate milk. CoLON GROUP. B. coli type. (2) Do not coagulate milk. HOG-CHOLERA GROUP. B. suipestifer type. CLASSIFICATION BY GROUPS. Tir 5. Do not generate gas in glucose bouil- lon. TYPHOID GROUP. B. typhosus type. 2. Stain by Gram’s method. . B. MURIPESTIFER GROUP. B. muripestifer type. (4) Liquefy gelatin and generate gas in glu- cose bouillon. B. CLOACH GROUP. B. cloacee type. B. Gelatin colonies ameboid or irregular. (a) Do not liquefy gelatin. B. ZOPFI GROUP. B. zopfi type. (4) Liquefy gelatin. PROTEUS GROUP. B. vulgaris type. MICROSPIRA. I. Not phosphorescent. 1. Do not liquefy gelatin, or only slightly. Msp. SAPROPHILES GROUP. Msp. saprophiles type. 2. Liquefy gelatin. A. Produce indol. (a) Very pathogenic to pigeons. Msp. METSCHNIKOVI GROUP. Msp. metschnikovi type. 112 DETERMINATION OF SPECIES. (4) Not distinctly pathogenic to pigeons. CHOLERA GROUP. Msp. comma type. B. Do not produce indol, or very little, at least after twenty-four hours. CHOLERA-NOSTRAS GROUP. Msp. finkleri type. IV. CLASSIFICATION OF WATER BACTERIA BY GROUPS. Fuller and Johnson have applied the group method of classification to the bacteria of water as follows :! WATER BACTERIA. I. Fluorescent. Group I. II. Non-fluorescent. 1. Chromogenic. A. Red. Group II. B. Orange. Group III. C. Yellow. Group IV. D. Violet. Group V. 2. Non-chromogenic. A. Gelatin liquefied. (a) Characteristic colo- nies on gelatin plates. 1 Jour. of Exp. Med., vol. iv., Nos. 5-6, p. 609. Conn has adopted a somewhat similar classification into groups for the dairy bacteria. See Report of the Storrs (Connecti- cut) Agricultural Experiment Station for 1899. CLASSIFICATION BY GROUPS. 113 1. Proteus forms. Group VI. 2. Subtilis forms. Group VII. (4) Non-characteristic colonies on gelatin plates. 1. Fermentation of carbohydrates. a. Gas production. Group VIII. 6. Nogas production. Group IX. 2. Non-fermentation of carbohydrates. Group X. B. Gelatin not liquefied. (a) Fermentation of car- bohydrates. 1. Gas production. Group XI. 2. No gas production. Group XII. (4) Non-fermentation of carbohydrates. Group XIII. CHAPTER 1%; BACTERIAL ANALYSIS OF WATER, MILK, AIR, AND SOIL. WATER ANALYSIS. THE biologic examination of water is for the purpose of determining the number and kinds of organisms present. It serves to supplement chem- ical analysis, and both are necessary to arrive at a sound conclusion as to potability. I. Quantitative Analysis of Water.—The num- ber of bacteria present in water varies within wide limits without affecting the value of the water; consequently no fixed standard can be of much value in determining the quality. The proper study of a water-supply should include the deter- mination of its normal mean number of bacteria at every season of the year. Any variation from the mean can then readily be determined, and its cause investigated. If simultaneous analyses of waters from various sources are made, the preferable water for drinking-purposes can easily be selected. Beyond this the value of the water can be deter- mined by quantitative analysis alone only within wide limits. Water containing less than 100 bac- teria per cubic centimeter is presumably from a deep source and uncontaminated by surface drain- age. It can usually be recommended for drinking- 114 WATER ANALYSIS. II5 purposes. Water containing more than 500 per cubic centimeter should be looked upon with sus- picion. 2. Qualitative Analysis of Water.—The quali- tative examination of water requires not only the iso- lation of the several species present, but also their cultivation and the determination of their patho- genic or non-pathogenic properties. Such an examination takes a long time, and under most favorable circumstances it is very difficult to recognize the presence of pathogenic forms. The principal value of the qualitative analysis of water is in the detection of contamination by sew- age. Sewage is always liable to contain the evacu- ations of patients sick with typhoid fever or other transmissible diseases. The germs of typhoid fever are not easily identified, but there are certain bacteria common in human and other animal evacuations and in sewage (B. coli, B. vulgaris, B. cloace, B. sporogenes, Bact. aérogenes) whose presence is easily detected. Consequently the presence of such forms, though harmless in themselves, always indi- cates contamination. 3. Laboratory Work in Water Analysis.—All samples should be collected in sterile flasks, and cultures should be made immediately to secure ac- curate results. If transportation is necessary, the samples should be packed in ice. Tap water should be allowed to run a few minutes before the sample is taken ; if spring or well water is to be examined, the sample should be collected from about a foot below the surface. 116 BACTERIAL ANALYSIS. (a) Transfer 1 c.c. of the water to be examined, by means of a sterile graduated pipet, to each of three tubes of liquefied gelatin.’ Use a sterile pipet for each transfer. (4) Shake the tubes, flame the lips, and pour into sterile Petri dishes. Place these in the dark at 20° C. (c) Examine the plates from day to day, and count the colonies that appear. Fic. 50.—Simple microscope for counting colonies, It is best to count all the colonies if possible; but when they are very numerous, some one of the various methods devised for counting colonies must be employed. (1) Wolfhiigel’s counting-plate consists of a glass plate on which are ruled square centimeters. The Petri dish is ‘If the water is suspected of containing large numbers of bacteria, smaller quantities than 1 c.c. should be added to each tube, or, better, dilute the water by the addition of a known quantity of sterile water. Many workers prefer to mix the water and the gelatin in the Petri dish instead of in the tube. WATER ANALYSIS. 117 placed thereon and the number of colonies in several of the square divisions counted, the average taken, and the Fic, 51.—Wolfhiigel’s apparatus for counting colonies of bacteria upon plates, number in the whole dish estimated. A lens is used in counting the colonies. Fic. 52.—Jeffer’s plate for counting colonies in circular dishes. The area of each division is 1 square centimeter. 8 118 BACTERIAL ANALYSIS. (2) Colonies in circular dishes may be counted by means of Jeffer’s plate. In this each circle is marked with its area in square centimeters, and each division equals 1 square centimeter. (3) The colonies appearing under the microscope in the field of a low-power objective may be counted, in several parts of the dish, the average taken, and the number in the whole dish estimated by the equation : Number of colonies in the field _ (4 diam. of field)? Whole number of colonies (4 diam. of dish)? * (ad) If a qualitative analysis is required, isolate and cultivate the different kinds of colonies.’ 4. Test for the Presence of Bacillus coli.— (a) Prepare 10 fermentation-tubes of sterile bouil- lon containing I per cent. glucose. (6) ‘To each tube add 1 cc. of the water to be tested. (c) Place the tubes in the incubator at 37.5° C. for three days. (2d) Note the amount of gas which forms on each of the three days. If gas-forming bacteria are present, gas will col- lect in the closed tube. The number of tubes showing the presence of gas gives a rough idea of the number of gas-producing bacilli present. Bacillus coli, if present, will fill the closed tube by the second day. ‘Too little or too much gas does not point to the presence of Bacillus coli. Bacillus coli forms most of its gas during the first twenty-four hours. The liquid in the bulb must 1See p. 112 for Fuller and Johnson’s groups of water bacteria, WATER ANALYSIS. 1I9g be distinctly acid to indicate the presence of Bacil- lus coli. 5. Isolation of Bacillus coli.—First Method.— (a) Add 50 c.c. of the water to be tested to 50 c.c. of sterile bouillon in a sterile flask. (6) Place in the incubator at 37.5° C. for two days. The high temperature will destroy the common water bacteria, but will encourage the growth of the coli group. (c) Test part of this culture for indol. Its pres- ence indicates the presence of Bacillus coli. (2) Make plates from this culture. (e) If any non-liquefying colonies are whitish with irregular leafy outlines and show lines more or less radial, they are probably colonies of Bacillus coli, and must be carefully studied in cultures.! Second Method.—(a) Add 70 c.c. of the water to be tested to 30 c.c. of sterile bouillon. (4) To the mixture add 1 c.c. of 5 per cent. car- bolic acid. (c) Incubate at 37.5° C. for twenty-four hours. Carbolic acid restrains the growth of the ordinary water bacteria, while the coli group and other intestinal forms grow unhindered. (7) From the growth that results inoculate fer- mentation-tubes containing 1 per cent. glucose bouillon. Note the amount of gas that forms as before. (e) Make plates from the growth in the tubes, 1See page 60. 120 BACTERIAL ANALYSIS. and study the colonies that resemble those of Bacillus coli. 6. Milk Analysis.—The analysis of milk is conducted in the same manner as is that of water, but on account of the great number of bacteria in Fic. 53.—Hesse’s apparatus for collecting bacteria from the air. milk the samples must be diluted with sterile water. Bacillus typhosus and Bacillus coli are detected in milk by the same methods as in water analysis. Tuberculosis bacilli may be detected in milk by the method described in the next chapter.' 1 See page 157. AIR ANALYSIS. 121 For the classification of bacteria in milk, see H. W. Conn, Report of the Storrs (Connecticut) Agrz- cultural Experiment Station for 1899. iN 2 eS HAS cD . Fic. 55.—Sedgwick’s ex- panded tube for air exam- ination. Fic. 54.—Petri’s sand-fil- ter for air examination. 7. Bacteria in the Air.—(a) Prepare 3 gelatin plates and expose them to the air for four or five 122 BACTERIAL ANALYSIS. minutes in different places. (In recitation-rooms before and after class, out of doors, etc.) (4) Cover and allow to grow. (c) Examine from day to day, and make cultures from the different colonies. This is a rough method of determining the rela- tive number of bacteria in the air. For more exact results recourse must be had to special apparatus for aspirating through bouillon or through sugar, as described in the text-books. 8. Bacteria in the Soil.—Numerous species of Fic. 56.—Frankel’s instrument for obtaining earth from various depths for bacteriologic examination. bacteria occur in the soil; some are of special interest on account of their pathogenic properties. Many are anaérobic, and this fact must be kept in mind while studying them. To determine the number of bacteria in a sample of soil: (a) Collect the soil without contamination from bacteria from other sources. (4) Introduce a measured quantity into a tube of liquefied gelatin. Crush with a platinum needle, and mix thoroughly with the medium. (c) Make plates, count, isolate, and study in the usual way. SOIL ANALYSIS. 123 Another method, and one which does away with the presence of particles of soil in the medium, but which perhaps does not give such accurate results, is to mix the sample thoroughly with sterile water and then make plates from the water. CHAPTER X. PATHOGENIC BACTERIA. THE methods for the study of pathogenic bacteria are exactly the same as those already described. In certain cases only are special culture-media necessary for their growth. Most pathogenic forms grow better in the incubator at body-temperature, 37.5° C. In all cases animal inoculations are necessary for the determination of pathogenicity. Fic. 57.—Micrococcus aureus, from an agar-agar culture (Ginther). Great care must be used in handling pathogenic cultures in order to avoid accidents. Have at hand a solution of corrosive sublimate (1 : 1000) or carbolic acid (1: 20), with which to flood any material that 124 PYOGENIC ORGANISMS. 125 may by accident be spilled on floor or table. Care- fully sterilize everything that the pathogenic material may have contaminated. Thoroughly dis- infect the hands, instruments, and table with the corrosive sublimate solution after completing the work. Fic. 58.—Streptococcus pyogenes (from a bouillon culture). I. PYOGENIC ORGANISMS. (a) Study, according to the schedule in Chapter VIII., the morphology and biology of : Micrococcus aureus (Staphylococcus pyogenes aureus). Micrococcus citreus (Staphylococcus pyogenes citreus). Micrococcus pyogenes (Staphylococcus pyogenes albus). Streptococcus pyogenes. Sarcina tetragena (Micrococcus tetragenus). (4) Select and weigh a well-grown rabbit. In- oculate into the ear vein by means of a hypodermic syringe’ I cc. of a twenty-four or forty-eight hour bouillon culture of one of the above species, or of pus taken directly from an abscess. 1 Keep the syringe for some time before the operation in a 2 per cent. solution of carbolic acid. Wash it out five or six times with sterile water or bouillon before the inocula- tion; or the syringe and needle may be boiled for five minutes before using. 126 PATHOGENIC BACTERIA. ——<< es es By Pa PE Fic. 59.—Koch’s syringe. (c) Record the daily weight of the animal until death. (d) Perform an autopsy on the dead rabbit, note Fic. 60.—Method of making an intravenous injection into arabbit. Observe that the needle enters the posterior vein from the hairy surface (McFarland). the various pathologic changes, and make cultures and cover-glass smears from all the organs and from the blood.’ 1Sear the surface of the body-wall and of each viscus with a red-hot spatula or old scalpel before cutting into PYOGENIC ORGANISMS. 127 (e) Preserve some of tissues in absolute alcohol or in Zenker’s fluid.! (/) Stain the smear preparations and examine for the inoculated organism. (g) Make pure cultures from whatever growth is Fic. 61.—Streptococcus pyogenes ; cover-glass preparation from the pus of an abscess ; x 1000 (Frankel and Pfeiffer), obtained in the cultures from the blood and organs, and try to recover the original organism. them ; sterilize the blades of the knives, scissors, and for- ceps used in making the incisions by dipping them in methyl alcohol and passing them quickly near enough to a Bunsen flame to ignite the alcohol. Smears and cultures from the blood are best made from the blood in the heart. For the method of preparation of blood-smears, see page 59. 1 See Appendix, page 173. 128 PATHOGENIC BACTERIA. Fic. 62.—Streptococcus pyogenes, seen in a section through human skin; x 500 (Frankel and Pfeiffer). Fic, 63.—Sarcina tetragena in pus from a white mouse; x 615 (Heim). GONOCOCCUS. 129 (Z) Section the preserved tissue and stain for bacteria with carbol-thionin-blue, Kuhne’s methy- lene-blue, or by Gram’s inethod. Fic. 64.—Mouse-holder, with mouse in position for inocu- lation. Other animals—guinea-pigs, rats, or mice—may be used for inoculations. Inoculations may be sub- cutaneous, peritoneal, or intravenous, according to the organisms used or the nature of the experi- ment. Il. GONOCOCCUS. 1. Examine cultures of Micrococcus gonorrhee, and stain the organism with Loffler’s blue and by Gram’s méthod. 2. Examine gonorrheal pus as follows : (a) Prepare films on cover-glasses. (4) Pass three times through flame. (c) Stain with Léffler’s methylene-blue or with any aqueous anilin stain one minute. (d) Wash, dry, and mount. (e) Examine with the oil-immersion lens. 130 PATHOGENIC BACTERIA. Gonococci are of medium size, composed usually of two hemispheres separated by a narrow un- stained interval. Occasionally two pairs of cocci form a ‘‘tetrad.’? The cocci are usually within the leukocytes. (/) Stain another film by Gram’s method. The gonococci are decolorized. Il. ANTHRAX. 1. Study the morphology and biology of cultures of Bacterium anthracis. 2. Inoculate a guinea-pig as follows : Fic. 65.—Gonococcus in urethral puS; x 1000 (Frankel and Pfeiffer). (a) Remove the hair from a small area on the abdomen. ANTHRAX. 131 (4) With a snip of a pair of sterile scissors make a little subcutaneous pocket. (c) Introduce into this pocket spores and bacteria from a pure culture by means of a platinum loop. (a) At the autopsy prepare cultures, smears, and sections from the organs. Fic. 66.—Bacterium anthracis; colony three days old upon a gelatin plate ; adhesive preparation ; x 1000 (Fran- kel and Pfeiffer), (e) Make cover-glass preparations from the blood as follows : 1. Place a small drop of blood between two abso- lutely clean cover-glasses, draw them apart and allow the smears to dry. 2. Fix in equal parts of ether and alcohol for thirty minutes, or in absolute alcohol five minutes, 132 ; PATHOGENIC BACTERIA. or in vapor of formaldehyd two and a half minutes; or heat in the thermostat at 110°-120° C. for twelve hours ; or heat on a brass plate for one hour at the point where water boils. Fic. 67.—Bacterium anthracis; cover-glass preparation from the spleen of a mouse (Mallory and Wright). | 3. Stain in eosin (4 per cent. in 60 per cent. alcohol) from one to five minutes. 4. Wash in water and dry. 5. Contrast-stain in aqueous methylene-blue from one-half to one minute. 6. Wash, dry, and mount, GLANDERS, 133 IV. GLANDERS. 1. Study the morphology and biology of the glanders bacterium. 2. Inoculate a male guinea-pig intraperitoneally with 1 c.c. of a bouillon culture. 3. Note in two or three days the great swelling Fic. 68.—Bacterium anthracis, stained to show spores; x 1000 (Frinkel and Pfeiffer). and redness of the testicles, caused by a semipuru- lent affection of the tunica vaginalis. This is a diagnostic test for the glanders bacterium. 4. At the autopsy prepare smears, cultures, and sections from all the organs and from the peritoneal and scrotal nodules. 5. Stain sections as follows : 9 134 PATHOGENIC BACTERIA. (2) Carbol-thionin-blue for ten to fifteen minutes. (4) Wash thoroughly in water. (c) Dehydrate in anilin oil. (2) Treat with equal parts of anilin oil and xylol. (e) Pass through pure xylol to balsam. Fic. 69.—Bacterium mallei, from a culture upon glycerin agar; x 1000 (Frankel and Pfeiffer). 6. For the diagnosis of a suspected case of glan- ders proceed as follows : (a) Rub a large swab made of absorbent cotton in the discharge from the nose or in the suspected ulcer. (4) Transfer the swab to 5 c.c. of sterile water and shake thoroughly. (c) Inoculate the resulting suspension intraperi- toneally into a well-grown male guinea-pig. DIPHTHERIA, 135 (7) In two to seven days scrotal inflammation will develop if the glanders bacterium was present. If the glanders bacterium was not present, after a few hours or days of depression the guinea-pig recovers completely. It may happen that some acute septic organisms were present in the material injected. In such cases the guinea-pig usually dies within twenty-four hours, and the test is evidently of no value and must be repeated. (e) If the scrotal lesions appear, perform an autopsy. Look particularly for. nodular deposits in the peritoneum and visceral layer of the tunica vaginalis, infiltration of the scrotal tissue, and edema extending into the groin and suprapubic region. (f) Transfer aseptically a portion of a nodule to potato, and place at 37.5° C. In twenty-four to forty-eight hours siall, smooth, glistening, amber- colored colonies of the glanders bacterium should develop. The bacterium is a short, thick rod, with rounded ends, usually slightly curved or bent, soinetimes elongated into threads. It stains faintly in Loffler’s methylene-blue. (g) A positive diagnosis can usually be made from the gross lesions, but the isolation of the organism makes the diagnosis certain. Vv. DIPHTHERIA. 1. Make swabbings from the throats of healthy individuals and from several diphtheria patients in hospital. Depress the tongue and rub a sterile 136 PATHOGENIC BACTERIA. FIG. 70. Fie. 71. Fic. 70.—Providence Health Department outfit for diph- theria diagnosis. A pasteboard box containing a swab- tube and a serum-tube, both with etched surface on which to write the name and address of patients, etc. Fic. 71.—Bacterium diphtheriz; agar culture (photo- graph by Dr. Henry Koplik). swab made of non-absorbent cotton over the back of the throat, tonsils, diphtheritic membrane, ete. 2. Rub the swab over the surface of a Loffler blood-serum tube. DIPHTHERIA, 137 3. Place the tube in an incubator at 37.5" C. for sixteen hours or longer. 4. Examine the growth for the small grayish, slightly elevated diphtheria colonies. 5. Prepare films from a suspected colony. 6. Pass three times through the flame and stain in Loffler’s methylene-blue for one minute. Fic. 72,—Bacterium diphtheria, from culture upon blood- serum ; x 1000 (Frankel and Pfeiffer). 7. Wash, dry, mount, and examine with the 7, inch oil-immersion lens. The characteristic diph- theria bacteria can easily be detected. 8. Isolate the diphtheria organisms in pure cult- ure by inoculating tubes from a single colony which on examination proves to be diphtheria. If a single colony cannot be found, touch the needle 138. PATHOGENIC BACTERIA. once to the growth on the serum-tube and make a series of strokes on 3 or 4 tubes. When the colonies: develop, those in the last tube will be sufficiently distinct so that pure cultures may be made from them. Another method is to inoculate a bouillon- tube or a tube of sterile water or normal salt solu- tion, and immediately from this make stroke cult- Fic. 73.—Bacterium diphtheriz, colony twenty-four hours old upon agar; x 100 (Frankel and Pfeiffer). ures on serum-tubes. The growth on these will probably be in individual colonies. Either of these methods obviates the necessity of making plates. g. Study the morphology and biology of the diphtheria bacterium obtained above. to. The following stains are diagnostic for the diphtheria bacterium : DIPHTHE RTA. 139 Hunts Stain, (a) Prepare films as usual. (6) Stain in aqueous methylene-blue for one minute. (c) Wash in water and dry. (d) Treat with a Io per cent. solution of tannic acid for one minute. (e) Wash in water and dry. (/) Stain in aqueous methyl-orange for one minute. (g) Wash, dry, and mount. Neisser’s Stain. (a) Prepare solution A as follows: Methylene-blue, I gm.; Alcohol (95 per cent.), 20 €.C. Dissolve and add Acetic acid, 50 ¢.C.3 Water, 950 c.c. (4) Prepare solution B as follows: Bismarck-brown, 2gm.; Boiling water, IOOO ¢.¢. (c) Treat films with solution A for one to three seconds. (@) Wash in water. (e) ‘Treat with solution B for three to five seconds. (f) Wash, dry, and mount. 11. Test the virulence of the diphtheria organism isolated as follows : 140 PATHOGENIC BACTERIA. (a) Prepare a twenty-four to forty-eight hour bouillon culture. (4) Sterilize a hypodermic syringe and needle by soaking in 2 per cent. carbolic acid and washing out in sterile water or bouillon, or by boiling for five minutes. (c) Select and weigh a full-grown guinea-pig. (d) While the pig is held on its back on the table @| 3] oF] 6) Ey F rr) 37) ow 42) 43} 40, #5) 47 46) 9) 50) 46 4o| Ez) Je E? Jo} i “34 33] J2| cz 2 22] a 24 25 Zé 27 Ey) 29 Jol 20] 79) 78 a 76 75 74 23| 7) 7 ‘ 2 oI + 5 6 7 6 F] i) Fic. 74.—Slide, 7 x 2} inches, for the routine examina- tion of diphtheria cultures. Each square can be placed under the lens of the microscope without disturbing the equilibrium of the slide. by an assistant, the hair is removed from a small area on the ventral abdominal wall. (e) With the thumb and forefinger of the left hand pinch up a fold of the skin, and with the right hand insert the hypodermic needle between the skin and the muscular body-wall. (/) Inoculate an amount of the culture equal to I per cent. of the weight of the pig. (g) Record the daily weight of the pig until death. INFLUENZA. I4I (2) Perform an autopsy on the dead pig; note the pathologic changes, and prepare cultures, smears, and sections from the organs and from the point of inoculation. VI. INFLUENZA. 1. Prepare blood-agar tubes by smearing the sur- face of a ordinary agar-tube with a drop of blood senate fe Poy adr Fic. 75.—Bacterium influenze, from a gelatin culture; x tooo (Itzerott and Niemann). obtained aseptically from man, rabbit, guinea-pig, pigeon, or frog. 2. Break up a distinctly purulent portion of influeuza sputum in x or 2 c.c. of bouillon, and spread a loopful of the suspension over the surface of the blood-agar tube. 142 PATHOGENIC BACTERIA. 3. Place in the incubator and examine at the end of from eighteen to twenty-four hours. 4. The influenza colonies appear as minute color- less, glassy, transparent points, resembling drops of dew. ‘They are barely visible to the unpractised eye, and require a low magnifying power to be seen clearly. Fic. 76.—Bacterium influenzz: colonies on blood-agar ; low magnifying power (Pfeiffer). 5. Study the morphology and biology of the organisms in one of these colonies. "They should not grow on ordinary media, and should have the morphology of the influenza bacteria. 6. Prepare smears from one of the purulent INFLUENZA. 143 masses in the sputum. Stain in very dilute carbol- fuchsin for five to ten minutes, or in L6ffler’s methylene-blue heated to the steaming-point. The influenza bacteria are very small, short, with Fic. 77.—Bacterium influenzz: cover-glass preparation of sputum from a case of influenza, showing the bacteria within the leucocytes ; highly magnified (Pfeiffer). round ends, are often present in large numbers, and are frequently within the pus-cells. They may occur in pairs, and then resemble cocci. The ends are usually more deeply stained than the central portions. bo SUSAN 144 PA mPUT Aung, fo tdeaq VII. TYPHOID AND COLON BACILLI. Fic. 78.—Bacillus typhosus, from a twenty-four hour agar culture; x 650 (Heim). Fic. 79.—Bacillus coli, from an agar culture; x 1000 (Itzerott and Niemann). 1. Study the morphology and biology of cultures TYPHOID AND COLON BACILLI. of Baciltus typhosus and Bacillus colt. 145 Some of the differences between them are indicated below : B. TyPHOsus. Rods usually slender. Flagella more numerous, longer, more wavy (10-20). In artificial media growth gener- ally slower and not so vigorous. Growth on fresh acid potato a nearly transparent film. Very slight acid production in ordinary media, followed sometimes by a production of alkali, Litmus milk—no change. Milk not coagulated. Fermentation of lactose slight if any. Litmus lactose agar—no change very in color. Glucose media—no gas forma- tion. No production of indol in ordi- nary bouillon. Does not grow in Maassen’s as- paragin-glycerin solution. Agglutination-test positive. B. cout. Rod inclined to be a little thicker. Flagella | fewer and _ shorter (8-10). Growth faster and more vigor- ous. Growth on potato a brownish pellicle. Well-marked acid production. Litmus milk—pink color, Milk coagulated. Fermentation of lactose pro- nounced. Litmus tion of red color. Abundant gas formation, lactose agar—produc- Well-marked indol production in ordinary bouillon. Grows in Maassen’s solution. Agglutination-test usually nega- tive. 2. Study sections of spleen, intestinal lesions, mesenteric lymph-glands, Stain with carbol-thionin-blue typhoid autopsy. or carbol-fuchsin. ete., from a human 3. Widal Reaction in Typhoid Fever.— If the blood-serum of a person suffering with typhoid fever or of one who has recently recovered from it 146 PATHOGENIC BACTERIA, be added to a bouillon culture of actively motile typhoid bacilli, the bacilli lose their motility and Fic. 80.—Bacillus typhosus: superficial colony two days old on a gelatin plate; x 20 (Heim). Fic. 81.—Bacillus coli: superficial colony two days old, on a gelatin plate; x 21 (Heim). soon aggregate in clumps. Dilutions of serum 1;10 and 1:30 with a half-hour time-limit are TYPHOID AND COLON BACILLI. 147 those most commonly employed. In doubtful cases a dilution of 1: 100 with a one-hour time-limit is recommended. (a) Place 9 drops of a twenty-four-hour bouillon culture of actively motile typhoid bacilli on separate Fic. 82.—Bacillus typhosus, from an agar culture six hours old, showing the flagella stained by Lé6ffler’s method ; x 1000 (Frankel and Pfeiffer). spots on a clean cover-glass. Add 1 drop of serum from the blood of the suspected typhoid case,’ mix 1 The blood may be collected in a capillary tube and after coagulation the serum removed; or dried blood may be moistened with water and the resulting solution used as serum. The latter method, however, does not permit accu- rate dilution. Chester and Robin have recently devised a pipet for delivering a measured drop of blood, so that dilu- 148 PATHOGENIC BACTERIA. all together, and mount as a hanging-drop prepara- tion.! (6) Examine with the 4 inch or with the oil- Fic. 83.—The Chester and Robin pipet for delivering uniform drops of blood for the Widal test. A glass and rubber pipet, the bulb of which is enclosed between two strips of metal held in place by a Hoffman clamp. & immersion lens. If the case is one of typhoid, after some time, varying between a few seconds and a tions may be made with a fair degree of accuracy from dried blood. It consists of an ordinary medicine-dropper of a given size, the bulb of which is enclosed on either side by two narrow strips of metal (Fig. 83, ¢, ¢.), and both placed in a medium-sized Hoffman clamp. The inward movement of the clamp by means of the screw a compresses the bulb, while a slight turn in the opposite direction dilates it a little and permits a small drop of blood to enter. In expel- ling the blood the dropper is held vertically over a strip of thick filter-paper, and the clamp is slowly compressed until a single drop falls of its own weight. This drop is then dried, and when the test is to be applied the blood-spot is cut from the paper and drops of the diluting fluid are added from an exactly similar pipet until the required dilution is reached. ‘It is convenient to use a slide on which two glass rings have been cemented, so that one may be used as a control, containing the culture of bacilli alone. TYPHOID AND COLON BACILLI. 149 half-hour, the bacilli will be seen to become less motile, and finally to cease all movement and Fic, 84.—Slide with two cells for observing Widal reaction with a control. appear in clumps here and there throughout the field. a@ Fic, 85.—Widal reaction: @, bouillon culture of Ba- cillus typhosus ; 4, the same after the addition of typhoid serum. This reaction can be reversed and made to serve as a means of identifying the typhoid bacillus if the serum of a person known to have typhoid fever is at hand. 10 I50 PATHOGENIC BACTERIA. VII. PNEUMONIA. BACTERIUM PNEUMONIA (FRANKEL’S PNEUMOCOCCUS) AND BACTERIUM PNEUMONICUM (FRIEDLANDER'S PNEUMOBACILLUS). Fic. 86.—Bacterium pneumonize, from the heart’s blood of arabbit ; x 1000 (Frankel and Pfeiffer), 1. Study the morphology and biology of cultures of B. pueumonie and B. pneumonicum. 2. Make cover-glass preparations from pneu- monia sputum. Stain in Loffler’s methylene-blue or carbol-fuchsin, diluted one-half. 3. Prepare sections of lung-tissue from cases of TUBERCULOSIS. I5I lobar pneumonia, general infection, etc. Stain in carbol-thionin-blue and by Gram’s method. 4. Inoculate a guinea-pig or rabbit subcutaneously or intravenously with virulent cultures or with Fic. 87,—Bacterium pneumonicum, ‘from the expectoration of a pneumonia patients x 1000 (Frinkel and Pfeiffer). pneumonia sputum ; also with sputum from healthy persons. 5. At the autopsy note the pathologic changes, prepare smears from the blood and organs, make cultures, and prepare sections. IX. TUBERCULOSIS. I. Examination of Cultures. Study the morphology and biology of Bac- tertum tuberculosts. 152 PATHOGENIC BACTERIA. (1) For cultures use blood-serum, agar, and bouil- lon to which from 4 to 8 per cent. of glycerin has been added. (2) Stain films, prepared from cultures, with ordi- nary stains’ and by the Ziehl-Neelson method as follows : (a) Prepare films as usual. (4) Stain in Ziehl’s carbol-fuchsin, steaming but not boiling, five minutes ; cold, twenty minutes. (c) Wash in 20 per cent. sulphutic acid for three to five seconds. (2) Wash in 60 per cent. alcohol until no red color is left. (e) Wash in water. (f) Stain in aqueous solution of methylene-blue for one minute. (g) Wash in water, dry, and mount. The bacteria of leprosy, syphilis, and smegma stain by this method. Bacterium lepre stains much more quickly than B. tuberculosis, B. syphil- idis is decolorized more quickly, especially in sul- phuric acid, and B. smegmatis is decolorized by the alcohol. 2. Examination of Tuberculous Sputum. (1) Ziehl-Neelson Method. (a) Place the sputum in a shallow glass dish on a black surface. (4) Select several of the characteristic yellowish 1The tubercle bacillus takes the ordinary stains very slowly and faintly. TUBERCULOSIS. 53 particles, place between two cover-glasses, and spread evenly. (c) Draw the covers apart and allow the films to dry. ‘(d) Pass three times through flame. ayy ¢ J Fic. 88.—Tubercle bacteria in sputum (carbol-fuchsin and methylene-blue). (e) Stain in hot (steaming but not boiling) Ziehl’s carbol-fuchsin for five minutes or in the cold solution for twenty minutes. (7) Wash rapidly in water. (g) Decolorize in 20 per cent. sulphuric, hydro- chloric, or nitric acid, for three to five seconds, 154 PATHOGENIC BACTERIA. (2) Wash in water. (z) Contrast-stain in saturated aqueous solution of methylene-blue. or in Loffler’s blue for one-half minute. (7) Wash in water, dry, and mount. (2) Examine with the 7, inch oil-immersion lens. The bacteria should be red in a blue field.! (2) Koch-Ehrlich Method. (a) Stain cover-glass preparations in anilin fuch- sin or anilin gentian-violet for twelve to twenty- four hours. (4) Decolorize for three to five seconds in 20 per cent. nitric acid. (c) Wash in water, in 60 per cent. alcohol for five to ten seconds, and again in water. (2) Contrast-stain in aqueous methylene-blue for one minute. (e) Wash, dry, and mount. (3) Gabbet’s Method. (a) Stain in steaming Ziehl’s carbol-fuchsin for one minute. (2) Wash in water for two to three seconds. (c) Stain in Gabbet’s blue? for thirty seconds or longer. (2) Wash in water, dry, and mount. (4) Rosenberger’s Method. (a) Stain in carbol-fuchsin (cold) for five to ten minutes. (4) Without washing stain for one to two minutes 1For the detection of tubercle bacteria in sputum when present in very small numbers see page 157. 2 See Appendix, page 172. TUBERCULOSIS. 155 in sweet spirits of nitre to which has been added enough alcoholic solution of malachite-green, Bis- marck-brown, or methiylene-blue to give a deep- colored fluid. (c) Wash in water, dry, and mount. 3. Inoculations. (a) Inoculate a guinea-pig subcutaneously in the abdoniinal wall with tuberculous material. Fic. 89.—Tubercle bacteria in sputum (Frankel and Pfeiffer). (6) After four to six weeks,' when the inguinal lymphatic glands have become enlarged, kill the animal, (c) With proper aseptic precautions make three or four cultures on blood-serum from two or three 1 For class-work such an animal must be previously inoc- ulated. 156 PATHOGENIC BACTERIA, glands, spreading a large quantity of the material on the surface of the tubes. (d@) Seal air-tight, place in the incubator, and examine the growth that occurs. 4. Sections. (a) Prepare sections of tubercular lesions from the inoculated animals or from the human lung in acute phthisis. (6) Stain by the Ziehl-Neelson, Koch-Ehrlich, or Rosenberger methods. 5. Detection of Tubercle Bacteria in Urine. (a) Make smears from the deepest layer of sedi- ment thrown down by the centrifuge, or in the sedimenting glass. (4) Stain in hot (steaming but not boiling) Ziehl’s carbol-fuchsin for one minute. (¢) Wash in water. (2) Decolorize in 20 per cent. sulphuric acid until pink. (e) Wash in water, 95 per cent. alcohol for thirty seconds, and again in water. (f) Stain in Loffler’s methylene-blue for twenty seconds. (g) Wash in water, dry, and mount. The smegma bacillus, which frequently occurs in urine, does not stain by this method, as it is decolorized by alcohol. Rosenberger’s method also gives excellent results with urine sediment, TUBERCULOSIS. 157 6. Detection of Tubercle Bacteria in Milk. (1) First Method.’ (2) To 50 c.c. of suspected milk add 10 cc. of carbolic acid. (6) Shake vigorously for two to five minutes. Pour into a sedimenting glass, cover, and allow to stand for twenty-four hours ; or use a centrifuge. (c) With a pipet remove the deepest layer of sedi- ment and prepare films. (2) Dry and pass three times through flamie. (e) Pass through equal parts of ether and alcohol. (/) Dry and pass three times through the flame, and stain as directed for sputum. (g) Mount and examine with the 5 inch oil- immersion lens. (2) Second Method. (2) To 20c.c. of the milk add 1 c.c. of a 50 per cent. potash solution. (6) Heat in boiling water until the mixture turns yellowish brown. (c) Add 20 c.c. of acetic acid, shake, heat again for three minutes, and centrifuge. (d) Wash the sediment with hot water, again centrifuge, and make cover-glass preparations from the second sediment. (e) Stain as directed for sputum. 1This method may also be applied to the detection of tubercle bacteria in sputum when present in very small numbers, using 10-15 c.c. of sputum, Io c.c, of water, and 6 c.c. of carbolic acid. 158 PATHOGENIC BACTERIA. X. ACTINOMYCOSIS. 1. Study the morphology and biology of cultures of Actinomyces bovis. 2. Prepare sections from tissue containing colo- nies of the parasite. 3. Stain by Gram’s method or as follows : Fic. 90.—Actinomyces (von Jaksch). (a) Stain deeply in saturated aqueous eosin solu- tion for ten minutes. (4) Wash in water. (c) Stain in anilin-gentian-violet for two to five minutes. (2) Wash in normal salt solution.’ (e) Treat with iodin solution (iodine, 1 part; potassium iodid, 2 parts ; distilled water, 100 parts), for one minute. (f) Wash in water and dry slightly with filter- paper. 1 See Appendix, page 173. MALARIA. 159 (g) Clear in anilin oil, then in several changes of xylol. (2) Mount in balsam. XI. MALARIA. ie I. Examination of Fresh Blood for the Ma- larial Hematozoon. (a) Clean a slide and cover-glass. (4) Place a small drop of the blood to be ex- amined on the slide, cover, and seal with vaselin, (c) Examine with the oil-immersion lens. The ameboid movements of the Hematozoon malariz are visible at the room-temperature, but become more active if the slide is warmed. 2. Stained Preparations. (1) First Method. (a) Place a small drop of blood between two cover-glasses, slide the covers apart, and allow the films to dry. (4) Fix by placing in absolute alcohol for five minutes or in the vapor of formaldehyd for two and a half minutes, or by one of the methods given under Anthrax (page 131). (c) Stain in a I per cent. aqueous solution of eosin for five minutes or, if formaldehyd is used for fixa- tion, in an alcoholic (60-75 per cent.) solution of eosin for three to ten minutes. (2) Contrast-stain in saturated aqueous solution of methylene-blue for five minutes for the alcoholic preparations, for one minute for the formaldehyd 160 PATHOGENIC BACTERIA. ? 2 a ¥ (eS) ‘ é 7 & 7 ; @ gh 2) CEI? (0) © 2p colt ro 4

, i t aN | + + #-\-- mY y saat | fans ff E- fen y sum E fa +t : t ‘ t Tit t way i t | \ ‘is \ wf ‘ | \ 7 . — \ tt YY + X T Fe 4 XX ¥ ‘ te te is gies anges saan ms At Cie Tarr + a PTT im, rr ml fot i as ; EN if ay Ht tf th aa Ae ae aaaee y oa. H perry E . Ha rH ee or {J Ze Let ‘ane aut a tH £ caaen a eee 5 rt i A ade + eise> es H . aa f +H HEE a = Ley G ioanues i Fic. 93.—Wilson and Randolph’s method of measuring bacteria by photography. MOULDS AND YEASTS. 165 Il. MOULDS AND YEASTS. Moulds and yeasts frequently contaminate plate and tube cultures, and inasmuch as these growths are occasionally the cause of pathologic conditions, Fic. 94.—Saccharomyces cerevisiz. it is advisable that the bacteriologist be acquainted with certain typical forms. Fic. 95.—Mucor racemosus: a, spore-bearing head; 8, spores; ¢, branch; d, resting spores (after Jelliffe). 11 166 APPENDIX. 1. Saccharomyces cerevisiz. Examine cultures of this yeast, mount, and stain exactly as directed for bacteria. 2. Mucor racemosus. (1) Cultures of this mould may be made on the surface of gelatin- or agar-tubes, but preferably in plates. The developing colonies may be examined directly with the low-power lens of the microscope. Fic. 96.—Aspergillus repens: a, conidia-bearing head; 4, conidia ; ¢, peritheca ; d, sterigmata (after Jelliffe). (2) For examination with the high power, re- move a small portion of the growth to a mixture of glycerin and water on a slide; spread out as thin as possible, cover, and examine. (3) The preparation may be stained by adding a little eosin solution to the glycerin and water. (4) Permanent preparations may be made as follows : (a) Transfer a small amount of the mould to a slide, MOULDS AND YEASTS. 167 (4) Drop a little alcohol upon it to remove the air from the hyphe. Fic. 97.—Penicillium crustaceum : a, conidia with myce- lium; 6, conidia cluster; ¢, sterigma; d@, conidia (after Jelliffe), (c) Treat again with water. (2) Stain in methylene-blue. (e) Mount in glycerin or glycerin-jelly. 168 APPENDIX. 3. Aspergillus repens and Penicillium crus- taceum. These moulds may be examined exactly as directed for Mucor. 4. Key for the Identification of the Yeasts and Moulds that most frequently contaminate Cultures. Mycelium growth absent: I. YEASTS, SACCHAROMYCETACE. 1. Growth white to dirty white, S. cerevista. : S. albicans. 2. Growth reddish, S. glutints. 3. Growth brownish to black, S. niger. Mycelium growth present: II. MOULDS. 1. Spores inside of sporangia. PHYCOMYCETES. Family, MucorInl1. (2) Mycelium of one kind, 1. Spore-bearing bodies on unbranched hyphe, Mucor. 2. Spore-bearing bodies on branched hyphe, Circinelia. (2) Mycelium of two kinds, Rhizopus. ‘Adapted from ‘‘Some Laboratory Moulds,” by S. E. Jelliffe, in Journal of Pharmacology, Nov., 1897. MOULDS AND YEASTS. 169 2. Spores free, at the ends of modified hyphe, HYPHOMYCETES. (2) Hyphe pallid, loose, not collected into fascicles, MUCEDINE. 1. Conidia undivided, a. Hyphe short, (1) Hyphee unbranched, Oospora. (2) Hyphe branched, Monilia. b. Hyphe elongated, (1) Conidia aggregated, * Fertile hyphze enlarged at apex, + Conidia on simple sterigmata. Aspergillus. tt Conidia on compound sterigmata, Sterigmatocystis. ** Fertile hyphz not enlarged at apex, Pentcillium. (2) Conidia separated or loosely aggre- gated, Botrytis. 2. Conidia once septate, Cephalothecium. (4) Hyphe brownish or black, not collected into fascicles, DEMATIER. 1. Conidia non-septate, a. Hyphz short, only slightly different from conidia, Torula. 170 APPENDIX. b. Hyphee distinct from conidia, fHormodendron. 2. Conidia septate, a. Conidia in chains, Alternarta. 6. Conidia single, Macrosportum. (c) Hyphe pallid or dark, collected into fasci- cles, STILBE. (2) Hyphee pallid or reddish, collected in wart- like masses, TUBERCULARIES. Conidia elongated, septate, Fusarium. Il. STAINS AND REAGENTS USED IN THE STUDY OF BACTERIA. I. Simple Anilin Stains. Prepare saturated alcoholic solutions of gentian- violet, methylene-blue, thionin-blue, basic fuchsin, saffranin, or Bismarck-brown,’ by adding sufficient stain to absolute alcohol to make a saturated solu- tion, and leave some undissolved stain at the bot- tom of the vessel. T’o these stock solutions alcohol may be added from time to time, taking care that some undissolved stain always remains. When re- quired for use add 5 c.c. of the saturated alcoholic solution to 95 c.c. of distilled water, and filter. 1Use the anilins prepared especially for microscopic work by Griibler. -STAINS AND REAGENTS. 171 The watery solutions soon decompose, and must be made only as required for use. Methylene-blue, however, may be made up as a saturated aqueous solution, as it is permanent. Always filter a stain before use. 2. Kuhne’s Methylene-blue. Methylene-blue, 1.5 gm.; Absolute alcohol, IO CC} Carbolic acid (x : 20), 100 ¢.c. Stain films five minutes. 3. Loffler’s Methylene-blue. Saturated alcoholic solution of methylene-blue, 20 G8} Solution of potassium hydrate in water (1: 10,000), 100 ¢.¢. - 4, Carbol-thionin-blue. Thionin-blue, I gm.; Carbolic acid (1 : 40), 100 ¢.c. Dilute 1 volume of the stain with 3 of water, when required for use. Stain from three to five minutes. 5. Anilin-water Solution. Anilin oil, 5 .¢.c.5 Water, IOO ¢c.c. Shake together, allow to stand for five minutes, and filter through a moistened filter. 6. Anilin-gentian-violet and Anilin-fuchsin. Anilin-water, Io parts; Saturated alcoholic solution of gentian-violet or fuchsin, I part. 172 APPENDIX. 7. Gram’s Iodin Solution. Todin, I part; Potassium iodid, 2 parts; Distilled water, 300° 8. Ziehl’s Carbol-fuchsin. Basic fuchsin, I part; Absolute alcohol, 10 parts; Carbolic acid (1 : 20), Ioo ‘ 9g. Gabbet’s Blue. Sulphuric acid (25 per cent. solution), 100 ¢.¢.} Methylene-blue, 2 emi. Allow the diluted acid to stand twenty-four hours or until it is cold before adding the methylene-blue. ro. Unna’s Polychrome Methylene-blue. Methylene-blue, I part ; Potassium carbonate, Go? Water, 100 parts. Must be ripened for months. The ripéned solution may be procured from Griibler. 1. Ehrlich’s Triacid Stain. Saturated aqueous solution of orange G, 120 parts. Acid fuchsin, 80“ Methyl-green, too (‘ Distilled water, 300. «‘S Absolute alcohol, 180‘ Glycerin, 50 ‘ STAINS AND REAGENTS. 173 Never shake the solution. Pipet from the top what is needed for use. 12. Ehrlich-Biondi Stain. Is best procured ready made from Griibler: 13. Chenzinsky-Plein Stain. Saturated aqueous solution of methylene-blue, 40 CC. 5 Alcoholic (70 per cent.) solu- tion of eosin (1 : 200), 2G cue: 2 Distilled -water, 40 c.c. The best results are obtained by staining’ for twenty-four hours. Fairly good results may be obtained by using warmed stains for fifteen minutes. 14. Normal Salt Solution. Distilled water, 100 ¢.C.; Sodium chlorid, 0.75 gm. 15. Acid Alcohol. Alcohol (7o per cent.), 97 C.C. 5 Hydrochloric acid, oe 16. Zenker’s Fluid. Potassium bichromate, 2.5 gm.; Sodium sulphate, I gm.; Corrosive sublimate, 5 gm.; Glacial acetic acid, 5c.c.3 Water, ad 100 ¢.c¢. Do not add the acetic acid until ready for use. Fix tissues from one to twenty-four hours. 174 APPENDIX. Wash in running water from twelve to twenty-four hours. Preserve in 80 per cent. alcohol. 17. Cleaning Mixture, for Slides, Cover- glasses, and Glassware. Potassium bichromate, 6 gm.; Sulphuric acid, 6c¢.3 Water, 100 ¢.c. Wash in water and alcohol. 18. Nessler’s Solution. A. Potassium iodid, 35 gm.; Water, 200 €.¢. B. Mercuric chlorid, 16 gm. ; Water, 500 ¢.c. Add B to A until faint show of excess is indicated, then add 160 grams of solid potassium hydrate. Dilute to x liter, and add strong solution of mercuric chlorid, little by little, until the red mercuric iodid just begins to be permanent. Do not filter. Solution should be pale straw-color. It is improved by age. 19. Phenol-sulphonic Acid. Sulphuric acid (pure and concentrated), 148 c.c.; Distilled water, 12 Ges Pure carbolic acid, 24 gm. TABLE OF SYNONYMS. 175 IV. TABLE OF SYNONYMS. The scientific names of species used throughout this work are those adopted by Migula. They are preferable because they conform to the laws of scientific nomenclature and priority. The follow- ing table gives the synonyms of many of the common species in alphabetic order : BaciLLus— albolactus, Mig.; B. lactis albus, Léffler. amylovorus (Burrill), De Toni ; M. amylovorus, Burrill. aterrimus, L. et N.; B. mesentericus niger, Lunt. avium, Mig.; B. diphtheriz avium, Kruse. chauvei, Arloing, Cornevin et Thomas; B. carbonis, Mig.; B. anthracis symptomatici, Kruse. coli, Mig.; Bact. coli commune, Escherich. diaphanus, Mig.; Halibacterium pellucidum, Fischer. dysenteriz (Kruse), Mig.; B. dysenteriz liquefaciens, Kruse. equi, Mig.; B. equi intestinalis, Dyar et Keith. fischeri, Mig.; Photobacterium fischeri, Beyerinck; B. phosphorescens indigenus, Kruse. globigii, Mig.; B. mesentericus ruber, Globig. indicus, Koch; B. indicus ruber, Mig. intricatus, Mig.; Cladothrix intricata, Russell. limnophilus, Mig.; B. limosus, Russell. mesentericus, Mig.; B. mesentericus fuscus, Fliigge. mirabilis, Mig.; Proteus mirabilis, Hauser. murium, Mig.; B. typhi murium, Léffler. niger, Mig.; B. lactis niger, Gorini. ozene, Mig.; B. foetidus ozeene, Hajek. phosphorescens, Fischer ; Photobacterium indicum, Bey- erinck ; B. phosphorescens indicus, Kruse. 176 APPENDIX. BaciLLus— phosphoricus, Mig.; B. argenteo-phosphorescens III., Katz. prodigiosus, Fliigge ; Monas prodigiosa, Ehrenberg ; Bact. prodigiosum, Schréter; M. prodigiosus, Cohn. pseudotuberculosis, Mig.; B. pseudotuberculosis lique- faciens, Kruse. suipestifer, Kruse; B. of hog-cholera, Salmon-Smith ; B. cholerze suum, Mig. sulfureus, Mig.; Proteus sulfureus, Lindenborn. typhosus, Mig.; B. typhi abdominalis, Mig. vulgaris, Mig.; Proteus vulgaris, Hauser. vulgatus, Mig.; B. mesentericus vulgatus, Fliigge. zenkeri, Hauser ; Proteus zenkeri, Hauser. zopfii, Mig.; Bact. zopfii, Kurth. BacTERIUM— aceti, Zopf; Ulvina aceti, Kiitzing ; Mycoderma aceti, Thomsen. acidi lactici, Mig.; B. acidi lactici I., Hiippe. aérogenes, Mig.; Bact. lactis aérogenes, Escherich. amethystinum, Mig.; B. membranaceus amethystinus, Eisenberg. anthracis, Mig. ; B. anthracis, Koch. anthracoides, Mig.; B. anthracoides, Hiippe et Wood. aquatile, Mig.; B. aquatile, Frankland. arborescens, Mig.; B. arborescens, Frankland. aurescens, Mig.; B. aurescens, Frankland. bienstockii, Schréter ; B. coprogenes parvus, Bienstock. bovis, Mig.; Pneumobacillus liquefaciens bovis, Arloing ; B. pneumonicus liquefaciens, Kruse. brunneum, Mig.; B. brunneus, Adametz-Wichmann, buccale, Mig.; Leptothrix buccalis, Robin. candicans, Mig.; B. candicans, Frankland. TABLE OF SYNONYMS. 177 BacTrERIUM— capsulatum, Pfeiffer; B. capsulatus, Pfeiffer, Koch. carotarum, Mig.; B. carotarum, Koch. chlorinum, Mig.; B. chlorinus, Frankland. citreum, Mig.; B. citreus, Frankland. ceeruleum, Mig.; B. cceruleus, Smith. columbarum, Mig.; B. diphtheriz columbarum, Léffler. coprogenes, Mig.; B. coprogenes fcetidus, Fliigge. cuniculicida, Koch; B. cholere gallinarum, Fliigge ; Bact. septichzemiz, Schréter. cuniculi, Mig.; B. cuniculi pneumonicus, Kruse. endocarditidis, Mig.; B. endocarditidis capsulatus, Weich- selbaum. felis, Mig.; B. felis septicus, Kruse. giardi, Mig.; B. phosphorescens giardi, Kruse. keratomalacie, Mig.; B. septicus keratomalacie, Babes. lacteum, Mig.; B. lactis III., Kruse. lacticum, Mig.; B. lacticus, Kruse ; B. lactis acidi, Lieb- mann. laerii, Mig.; B. viscosus I., van Laer; B. viscosus cere- visize, Kruse. lepree, Mig.; B. leprae, Hansen. mallei, Mig.; B. mallei, Léffler. murisepticum, Mig.; B. murisepticus, Fliigge ; B. muri- nus, Schroter. mycoides, Mig.; B. mycoides roseum, Scholl-Holschew- nikoff. nephritidis, Mig.; B. nephritidis interstitialis, Letzerick. palumbarium, Mig.; B. choleree columbarum, Kruse. phosphorescens, Fischer; Photobacterium phosphores- cens, Beyerinck. proteus, Mig.; Proteus capsulatus septicus, Banti; B. capsulatus septicus, Kruse. 178 APPENDIX. BaCTERIUM— pseudodiphthereticum, Mig.; Corynebacterium pseudo- diphthereticum, L. et N. pseudoinfluenze, Mig.; B. pseudoinfluenze, Kruse. pseudotuberculosis, Mig.; B. pseudotuberculosis, Pfeiffer ; Streptobacillus pseudotuberculosis rodentium, Preisz. pyocinnabarium, Mig.; B. pyocinnabarius, Kruse. pyogenes, Passet ; B. pyogenes foetidus, Passet. rhusiopathie, Mig.; B. rhusiopathize suis, Kitt ; Bact. ery- sipelatus suis, Mig. salive, Mig.; B. salivee minutissimus, Kruse. sempervivum, Mig.; B. lactis XII., Kruse. smegmatis, Mig.; B. smegmatis, Kruse. subtile, Mig.; B. subtilis simulans I., Bienstock; B. fe- calis I., Kruse. suicida, Mig.; B. suisepticus, Kruse. termophilum, Mig.; B. termophilus, Miquel. tuberculosis avium, Mig.; B. tuberculosis avium, Maf- fucci; Mykobacterium tuberculosis avium, L. et N. tuberculosis, Mig.; B. tuberculosis, Koch; Mykobacte- rium tuberculosis, L. et N. ubiquitum, Mig.; B. ubiquitus, Jordan. varicosum, Mig.; B. varicosus conjunctive, Hombert. vermiculare, Mig.; B. vermicularis, Frankland. vignali, Mig.; B. g, Vignal; B. buccalis minutus, Stern- berg. welchii, Mig.; B. aérogenes capsulatus, Welch. xerosis, Mig.; B. xerosis, Neisser et Kuschbert. Micrococcus— albocereus, Mig.; Staphylococcus cereus albus, Passet. amplus, Mig.; M. albicans amplus, Fliigge. aurantiacus, Cohn; Staphylococcus cereus aureus, Mig. TABLE OF SYNONYMS. 179 Micrococcus— aureus, Mig.; Staphylococcus pyogenes aureus, Rosen- bach ; M. pyogenes aureus, Mig ; M. pyogenes, L. et N. cereus, Mig.; Staphylococcus cereus flavus, Passet. citreus, Mig.; Staphylococcus pyogenes citreus, Passet. citrinus, Mig.; Diplococcus citreus liquefaciens, Unna. conglomeratus, Fliigge; M. citreus conglomeratus, Fliigge; Diplococcus citreus conglomeratus, Eisen- berg. conjunctivitidis, Mig.; M. flavus conjunctive, Gombert. conoideus, Mig.; Staphylococcus salivarius pyogenes, Biondi. corrugatus, Mig.; Merismopedia mesenterica corrugata, Dyar. coryze, Mig.; Diplococcus coryze, Hajek. cuniculorum, Mig.; M. pyemiz cuniculorum, Schroter. cyaneus, Cohn; Bacteridium cyaneum, Schréter. desidens, Mig.; M. flavus desidens, Fliigge. faviformis, Mig.; M. lacteus faviformis, Fliigge. fragilis, Mig.; Merismopedia fragilis, Dyar. gonorrheee, Fliigge ; Gonococcus, Neisser. intracellularis, Mig.; Diplococcus intracellularis menin- gitidis, Weichselbaum ; Streptococcus intracellularis, L. et N. lacticus, Mig.; Sphzerococcus acidi lactici, Marpmann. liquefaciens, Mig.; M. urez liquefaciens, Fliigge. luteus, Cohn ; Bacteridium luteum, Schroter. mollis, Mig.; Merismopedia mollis, Dyar. phosphoreus, Cohn ; M. lucens, v. Tieghem ; M. pfliigeri, Ludwig, ex parte; Photobacterium phosphorescens, Beyerinck. pyogenes, Mig.; Staphylococcus pyogenes albus, Rosen- bach ; M. pyogenes albus, L. et N. roseus, Fliigge ; Diplococcus roseus, Eisenberg. 180 APPENDIX. Micrococcus— rugatus, Mig.; M. endocarditidis rugatus, Weichsel- baum. salivarius, Mig.; Coccus salivarius septicus, Biondi. stellatus, Frankland ; Coccus stellatus, Lustig. subflavidus, Mig.; M. tetragenus subflavus, v. Besser. tardigradus, Mig.; M. flavus tardigradus, Fliigge. tardior, Mig.; Diplococcus flavus liquefaciens tardus, Unna et Tommasoli. tardissimus, Mig.; Diplococcus albicans tardissimus, Fliigge. tardus, Mig.; Diplococcus albicans tardus, Unna et Tom- masoli. tenuis, Mig.; M. pyogenes tenuis, Rosenbach. tenuissimus, Mig.; M. cumulatus tenuis, v. Besser. varians, Mig.; Merismopedia flava varians, Dyar. MIcROSPIRA— annularis, Mig.; Photobacterium annulare, Fischer. canalis, Mig.; Vibrio saprophiles 7, Weibel. caraibica, Mig.; Photobacterium caraibicum, Fischer. comma, Schréter; Spirillum cholerz asiatice, Fliigge ; Vibrio cholere asiatice, Mig.; Vibrio comma, Mig. coronata, Mig.; Photobacterium coronatum, Fischer. degenerans, Mig.; Photobacterium degenerans, Fischer. delgadensis, Mig.; Photobacterium delgadense, Fischer. dunbari, Mig.; Vibrio dunbari, Mig. finkleri, Schréter ; Spirillum finkleri, Mig.; Vibrio fink- leri, Mig.; Vibrio proteus, Mig. glutinosa, Mig.; Photobacterium glutinosum, Fischer. luminosa, Mig.; Photobacterium luminosum, Fischer. marina, Mig.; Spirillum marinum, Russell. metschnikovi, Mig.; Vibrio metschnikovi, Gamaleia. papillaris, Mig.; Photobacterium papillare, Fischer. TABLE OF SYNONYMS. 181 MIcRosPIRA— saprophiles, Mig.; Vibrio saprophiles, 8, Weibel. tonsillaris, Mig.; Vibrio tonsillaris, Klein. tuberosa, Mig.; Photobacterium tuberosum, Fischer. PsEUDOMONAS— eruginosa, Mig.; Bact. eruginosum, Schréter; B. zru- ginosus, Schréter; B. pyocyaneus, Gessard. alba, Mig.; B. fluorescens albus, Zimmermann; B. fluor- escens non-liquefaciens (?), Eisenberg. eisenbergii, Mig.; B. fluorescens non-liquefaciens, Eisen- berg europea, Mig.; Nitrosomonas curopza, Winogradsky. fluorescens, Mig.; B. fluorescens liquefaciens, Fliigge. ianthina, Mig.; Bact. ianthinum, Zopf; B. janthinus, Zimmermann. javanica, Mig.; Photobacterium javanense, Eijkmann. javaniensis, Mig.; Nitrosomonas javaniensis, Winograd- sky. laurentia, Mig.; B. violaceus laurentius, Jordan. litoralis, Russell; B. litoralis, Russell. minutissima, Mig.; B. fluorescens liquefaciens minutissi- mus, Unna et Tommasoli. pseudianthina, Mig.; B. violaceus, Frankland. tenuis, Mig.; B. fluorescens tenuis, Zimmermann. SaRcINA— ventriculi, Goodsir; Merismopedia goodsiri, Husem ; Merismopedia ventriculi, Robin ; Sarcina fuscescens, de Bary. SPIROSOMA— attenuatum, Mig.; Spirillum attenuatum, Warming. flavescens, Mig.; Vibrio flavescens, Weibel. 12 182 APPENDIX. SPIROSOMA— flavum, Mig.; Vibrio flavus, Weibel. gregarium, Mig.; Myconostoc gregarium, Cohn. STREPTOCOCCUS— cerasinus, Mig.; .M. cerasinus siccus, List. citreus, Mig.; M. citreus, List-Eisenberg. cystitidis, Mig.; Diplococcus ure pyogenes, Rovsing. equi, Schiitz; S. coryzz contagiose equorum, Eisen- berg. ; giganteus, Mig.; S. giganteus urethre, Lustgarten et Mannaberg, gracilis, Mig.; S. coli gracilis, Escherich. mastitidis, Guillebeau ; S. mastitidis sporadice,, Guille- beau ; S. agalactize contagiose, Kitt. pyogenes, Rosenbach; S. erysipelatos, Rosenbach; S. conglomeratus, Kurth; S, brevis, v. Lingelsheim; S. longus, v. Lingelsheim; S. murisepticus, v. Lingels- heim; S. septo-pyzemicus, Biondi. septicus, Mig.; S. septicus liquefians, Babes ; S. septicus liquefaciens, Babes. INDEX. ACID alcohol, 173 carbolic, 44, 124 phenol-sulphonic, 94, 174 production, 94. white, 55 Acidity, determination of, 52 relation to, of medium, 91 Actinomyces bovis, 158 Actinomycosis, 158 Adhesive preparations of colonies, 75 Aérobic organisms, 75, 76, 78 Agar culture-medium, 51 cultures, 61 Air, analysis of, 121 bacteria in, 121 Algze, brown, 40 green, 40 ted, 40 Alkalinity, determination of, 52 relation to, of medium, 91 Alkali production, 94 Amebabacteriacez, 42 Amebacter, 42 American Public Health Associa- tion, 52, 83, 85 Ammonia, production of, 92 test for, 93 Amphitrichous, 25 Anaérobic cultures, 78 organisms, 75, 76, 78, 80 Analysis of air, 121 of milk, 120 of soil, 122 of water, qualitative, 115 quantitative, 114 Anilin fuchsin, 171 gentian-violet, 171 stains, 18 water, 171 Anthrax, 130 Antiseptics, 44 Arnold's steam sterilizer, 46, 47, 48 Arrangement of bacteria, 85 Asiatic cholera, 27, 64 Aspergillus repens, 166, 168 Autoclave, 46, 49 ! Autopsy, making, 126 BACILLI, form of, 22 groups of, 109 reproduction of, 34 species of, 102 Bacillus, 39, 102, 109 acidi lactici I., 176 aérogenes capsulatus, 178 aéruginosus, 180 anthracis, 176 symptomatici, 175 anthracoides, 176 aquatile, 176, arborescens, 176 argenteo-phosphorescens, III., 176 aurescens, 176 brunneus, 176 buccalis minutus, 178 candicans, 176 capsulatus, 176 septicus, 177 carbonis, I75 carotarum, 177 chauveei, 38 chlorinus, 177 cholerze columbarum, 177 gallinarum, 177 suum, 176 citreus, 177 cloacze, 115 coeruleus, 117 coli, colony of, 146 for removing sugars, 56 in water, I15 isolation of, 119, 120 study of, 144 test for, 118 coli communis, 175 coprogenes foetidus, 177 183 184 Bacillus coprogenes parvus, 176 cuniculi pneumonicus, 177 diphtheriz, 136, 137, 138 avium, 175 columbarum, 177 dysenterize liquefaciens, 175 endocarditidis capsulatus, 177 equi intestinalis, 175 feecalis I., 178 felis septicus, 177 fluorescens albus, 181 liquefaciens, 181 minutissimus, 181 non-liquefaciens, 181 tenuis, 181 foetidus ozzenz, 175 g, Vignal, 178 indicus ruber, 175 janthinus, 181 lacticus, 177 lactis III., 177 XII., 178 acidi, 177 albus, 175 niger, 175 leprze, 177 limosus, 175 litoralis, 181 mallei, 177 membranaceus amethystinus, 176 mesentericus, 63 fuscus, 175 niger, 175 ruber, 175 vulgatus, 176 murinus, 177 murisepticus, 177 muscoides, 74 mycoides, 63 roseum, 177 nephritidis interstitialis, 177 cedematis, 63 of hog cholera, 176 phosphorescens giardi, 177 indicus, 175 indigenus, 175 pneumonicus liquefaciens, 176 polypiformis, 69 : prodigiosus, 80 pseudoinfluenzze, 177 pseudotuberculosis, 178 liquefaciens, 176 pyocinnabarius, 178 pyocyaneus, 180 pyogenes foetidus, 178 INDEX. Bacillus radiatus, 63, 69 rhusiopathize suis, 178 salivz minutissimus, 178 septicus keratomalaciz, 177 smegmatis, 152, 156, 178 sporogenes, II5 subtilis, I9, 35 simulans I. 178 suipestifer, 24 suisepticus, 178 syphilidis, 152 termophilus, 178 tuberculosis, 178 avium, 178 typhi abdominalis, 176 murium, 175 typhosus, colony of, 146 gelatin culture, 63 in milk, 120 in water, II5 isolation of, 115 showing flagella, 28, 147 study of, 144 Widal reaction, 145 ubiquitus, 178 varicosus conjunctive, 178 vermicularis, 178 violaceus, 181 laurentius, 181 viscosus I., 177 cerevisize, 177 vulgaris, I9, 115 xerosis, 178 Bacteriaceze, 39, 99 Bacteria, classification of, 39, 99 by groups, 107 cultures of, 60 form of, 22 groups of, 107 in air, 121 in milk, 120 classification of, by groups, 112 I2I in soil, 122 in tissues, 20 in water, 114 classification of, by groups, 112 measurement of, 11, 163 morphology of, 22 pathogenic, 124 reproduction of, 34 species of, 99 Bacteridium cyaneum, 179 luteum, 179 Bacteriologic committee, 52, 83, 85 INDEX. Bacterio-purpurin, 39, 41 Bacterium, 39, 99, 107 aérogenes, 115 aéruginosum, 180 anthracis, colony of, 74, 77, 131 gelatin culture, 63 showing spores, 36 study of, 130, 132, 133 coli commune, 175 diphtheriz, 135, 136, 137, 138 _ diagnosis of, 135, 138 isolation of, 137 slide for examination of, 140 test for virulence, 139 erysipelatos suis, 178 faschingii, 30 ianthinum, 181 influenzze, 141, 142, 143 lactis aérogenes, 176 leprae, 152 mallei, 133, 134 parvum, 69 pneumoniz, 32, 150 pheumonicum, 150, 151 prodigiosum, 176 septiceemize, 177 smegmatis, 152, 156, 178 tuberculosis, 76, 151 zopfii, 176 Basic fuchsin, 18, 170 Bath, paraffin and water, electric, 68, Beef, Liebig's extract of, 55 Beggiatoa, 41 Beggiatoaceze, 41 Berkefeld filter, 45 Binary division, 34 Biologic characters of a species, 83, 84 examination of water, 114 Bismarck brown, 20, 170 Blood agar, 141 examination in anthrax, 131 in malaria, 159, 161 Whitney's method, 161 preparations, 131, 159, 161 Blood-serum cultures, 66 L6ffler’s, 58 tubes, 58 Boni’s method for capsules, 33 _ Boston Board of Health sterilizer, 47 Bouillon, 51 cultures, 60 dextrose, 56 185 Bouillon, lactose, 56 saccharose, 56 Brown algze, 40 Brownian movement, 24, 25 Brown University paraffin and water bath, 68 Bryophyta, 40 Buchner’s method for anaérobes, 78 CAMERA lucida, 12 Capsules, demonstration of, 31 Carbohydrates, 92 Carbol-fuchsin, Ziehl's, 172 Carbolic acid, 44, 124 Carbol-thionin blue, 171 Cell, 15 Chamberland filter, 45 Characeze, 40 Chenzinsky-Plein stain, 173 Chester, 95, 107 Chester and Robin pipet, 147, 148 Chlamydobacteriacez, 41 Chlamydothrix, 41 Chlorophyceze, 40 Cholera, Asiatic, 27, 64, 180 Chromatiaceze, 42 Chromatium, 42 . Cladothrix intricata, 175 Classification of bacteria, 39 by groups, 107 of dairy bacteria by groups, 112, I2I of water bacteria by groups, 112 Cleaning mixture, 14, 174 Clostridium, 35 Club mosses, 40 Coccaceze, 39, 95 Cocci, 22 Coccus salivarius septicus, 179 stellatus, 179 Colon bacillus, 144 Colonies, counting, 116, 117, 118 impression, or adhesive prepara- tions of, 75 Colony, 60 Concave slide, 15 Condenser, 11 Conjugatze, 40 Conjugates, 40 ‘| Conn, 112 Contamination of water by sewage, 115 Cornet’s forceps, 17 Corrosive sublimate solution, 44, 124 186 Corynebacterium pseudodiphtheriti- cum, 177 Cotton, 48 Cotton-wool filter, 45 Counting-plate, Jeffer's, 117, 118 Wolfhiigel’s, 116, 117 Cover-glass forceps, 17 Crenothrix, 41 Cryptogamia, 40 Culture-media, 51 Cultures, agar, 61 anaérobic, 78 blood-serum, 66 bouillon, 60 gelatin, 61 in the fermentation-tube, 76 of bacteria, 60 plate, 66 potato, 66 pure, 60 stab, 61 stroke, 64 DAIRY bacteria, classification of, Il2, 121 Demonstration of arrangement, 85 of capsules, 31 of division, 34 of flagella, 25 of form, 22, 85 of liquefying ferment, 80 of motion, 24, 90 of pleomorphism, go of spores, 35, 9° Determination of acid production, 94 of acidity of media, 52 of alkalinity of media, 52 of alkali production, 94 of arrangement, 85 of form, 22, 85 of motility, 24, go of name, 95 of pleomorphism, 90 of species, 83 of thermal death-point, 91 Dextrose, 56 Diagnosis of diphtheria, 135 of glanders, 134 of influenza, 141 slide for diphtheria, 140 Diaphragm, 11 Diatomeze, 40 Diatoms, 40 Dinoflagellates, 40 INDEX. Diphtheria, 135 diagnosis, 135 slide, 140 diagnostic stains, 138 inoculations of, 140 isolation of bacterium, 137 outfit for diagnosis, 136 test for virulence, 139 Diplococcus, 22, 34 albicans tardissimus, 180 tardus, 180 citreus conglomeratus, 179 liquefaciens, 178 coryz, 179 flavus liquefaciens tardus, 180 intracellularis meningitidis, 179 roseus, 179 urez pyogenes, 181 Discontinuous sterilization, 43 Disinfectant, 44 Disinfection, 44 Division, reproduction by, 34 Drumstick bacillus, 33 EHRLICH-BIONDI stain, 173 Ehrlich’s triacid stain, 172 Electric paraffin and water bath, 63 Endogenous spores, 34 Eosin, 20 Estivo-autumnal fever, 160 Etched tubes, 55 Eubacteria, 39 Examination of gonorrheal pus, 129 of tuberculous sputum, 152 Extract of beef, Liebig’s, 55 Eyepiece, 11 FACULTATIVE anaérobes, 78 Fermentation-tubes, 57 cultures in, 76 Ferment, liquefying, 80 Ferns, 40 Fever, estivo-autumnal, 160 malarial, 159 quartan, 160 tertian, 160 Filter, 45 cotton-wool, 45 for culture-media, 53 Kitasato’s, 46 porcelain, 45 Filtration, 45 of blood-serum, 59 of culture-media, 53 Fission-algze, 40 INDEX, Fission-fungi, 40 Fission-plants, 40 Fixing blood smears, 131, 159, 161 Flagella, 25 Fligge, 95 Forceps, cover-glass, 17 Form, 22, 85 Formalin, 44 Frankel’s instrument for collecting soil, 122 pneumococcus, 150 Friedlander's pneumobacillus, 150 Fuchsin, anilin, 171 basic, 18, 170 Fuller and Johnson’s groups of water bacteria, 112 Fuller's method of titration, 52 Fungi, 40 GABBET’S blue, 172 method for sputum, 154 Gas, 77 formula, 78 production, 77 tests for, 77 Gelatin culture-medium, 51 cultures, 61 Gentian-violet, 18, 170. anilin, 171 Germicide, 44 Germination of spores, 38 Glanders, 133 diagnosis of, 134 inoculation of, 133 isolation of bacterium, 135 Gonococcus, I29, 130, 179 Gonorrheal pus, examination of, 129 Graduated fermentation-tube; 57 Gram’s iodin solution, 19, 172 method, 19 stain, 19 Granules, sulphur, 39, 41 Green algze, 40 Grouping, study of, 85 Groups, classification of bacteria by, 107 of milk bacteria, 112, 121 of water bacteria, 112 HALIBACTERIUM pellucidum, 175 Hanging drop, 15 | Hematozoon malariz, 159 187 Hill fermentation-tube, 57 -| Horsetails, 40 Hot-air sterilization, 47 Sterilizer, 50 Hunt's stain for diphtheria, 139 Hyphomycetes, 40 IDENTIFICATION of species, 83 of yeasts and moulds, 168 Immersion lens, 112 oil, 11 Immunity, production of, 85 Impression preparation of colonies, 75 Indol production, 94 Influenza, 141 diagnosis of, 141 Inoculation of anthrax, 130 of diphtheria, 140 of glanders, 133 of plates, 66 of pneumonia, 151 of pus, 125 of sputum, I51 of tuberculosis, 155 of tubes, 61 Inoculations, 129 intraperitoneal, 133 intravenous, 125, 126, 151 subcutaneous, 130, 140, 151, 155 Instruments, sterilization of, 127 Intraperitoneal inoculations, 133 Intravenous inoculations, 125, 126, I5t Involution forms, 90 Iodin solution, Gram’s, 19, 172 Isolation of Bacillus coli, 119, 120 typhosus, 115 of Bacterium diphtherize, 137 influenzze, 142 mallei, 135 tuberculosis, 155 of species, 81 JEFFER’S counting plate, 117, 118 Johne’s method for capsules, 32 KALTEYER’sS cover-glass_ forceps, 17 Kitasato’s filter, 46 Koch-Ehrlich method for sections, 156 for sputum, 154 Hesse’s apparatus for collecting bac- | Koch’s syringe, 126 teria in air, 120 Ktihne’s methylene-blue, 172 188 INDEX. LACTOSE, 56 Lamprocystaceze, 42 Lamprocystis, 42 Leprosy, 152 Leptothrix buccalis, 176 Lichenes, 40 Lichens, 40 Liebig’s extract of beef, 55 Life-history of a species, 83 Liquefying ferment, 80 Litmus milk, 58 Liverworts, 40 L6ffler's blood-serum, 58 method for flagella, 27 modification of, 29 methylene-blue, 171 Lophotrichous, 25 MALARIA, 159 Malarial hematozoon, 159 Measurement of bacteria, 11, 163 Meat-extracts, 55 Meat-sugar, 56 Media, culture-, 51 Merismopedia, 22 flava varians, 180 fragilis, 179 goodsiri, 181 mesenterica corrugata, 179 mollis, 179 ventriculi, 181 Methylene-blue, 18, 170 Kiihne’s, 171 Léffler’s, 171 Unna's polychrome, 172 Mica, 69 Micrococci, form of, 22 reproduction of, 34 species of, 96 Micrococcus, 39, 96 albicans amplus, 178 amylovorus, 175 aureus, 124, 125 cerasinus siccus, 181 citreus, 125, 181 conglomeratus, 179 cumulatus tenuis, 180 endocarditidis rugatus, 179 flavus conjunctive, 179 desidens, 179 tardigradus, 179 gonorrhoeze, 129 lacteus faviformis, 179 lucens, 179 pfliigeri, 179 Micrococcus prodigiosus, 176 pyzmiz cuniculorum, 179 pyogenes, 125, 178 albus, 179 aureus, 178 tenuis, 180 tetragenus, 125 subflavus, 179 ureze liquefaciens, 179 Micrometer, eyepiece, 12 stage, I2 Micromillimeter, 12 Micron, 12 Microscope, Ir simple, for counting colonies, 116 Microspira, 41, 106, 111 comma, 27, 64, 180 finkleri, 65 metschnikovi, 65 Migula, 39, 95 Milk, analysis, 120 classification of bacteria in, 112, I2I culture-medium, 58 litmus, 58 tubercle bacteria in, 157 Mirror, I1 Monas prodigiosa, 176 Monotrichous, 25 Mordant, 25, 27, 29 Morphologic characters of a species, 83, 84 Morphology of a species, 83 of bacteria, 22 Mosses, 40 Motility, test for, go Motion, 24, 90 Moulds, 165 cultures of, 166 examination of, 166 identification of, 168 staining, 166 Mouse-holder, 129 Movement, Brownian, 24, 25 Mucor racemosus, 165, 166 Museum preparations, 76 Mycoderma aceti, 176 Myconostoc gregarium, 181 Mycoprotein, 19 Mykobacterium tuberculosis, 178 avium, 178 Myxomycetes, 40 NAME, determination of, 95 Needle, platinum, 14 INDEX. 189 Neisser’s stain for diphtheria, 139 Plate, counting, Wolfhiigel’s, 116, Nessler's solution, 174 117 Neutralization, 52 cultures, 66 Nitrates, 92 Platinum needles, 14 test for, 93 wire, 14 Nitrites, production of, 92 Pleomorphism, 90 test for, 92 Pneumobacillus, 150 Nitrosomonas europzea, 181 liquefaciens bovis, 176 javaniensis, 181 Pneumococcus, 150 Normal salt solution, 173 Pneumonia, 150 Novy's jars, 79 inoculation of, 151 Polar staining, 24 OBJECTIVES, Ir Polychrome methylene-blue, Unna's, Obligatory aérobes, 78 172 anaérobes, 78 Porcelain filter, 45 Potato, 55 PARAFFIN bath, 68 cultures, 66 Park's method for anaérobes, 80 Potato-tubes, 56 Pasteurization, 44 Production of acid, 94 Pathogenesis, 85, 94 of alkali, 94 Pathogenic bacteria, 124 of ammonia, 92 Penicillium crustaceum, 167, 168 of gas, 77 Peridineze, 40 of immunity, 85 E Peritrichous, 25 of indol, 94 Petri dish, 67 of liquefying ferment, 80 Petri’s sand filter, 121 of nitrites, 92 Phzeophyceze, 40 of pigment, 85 Phanerogamia, 40 of spores, 35, 90 Phenolphthalein, 52 of toxin, 85 Phenol-sulphonic acid, 174 Proteus capsulatus septicus, 177 Photobacterium. annulare, 180 mirabilis, 175, caraibicum, 180 sulphureus, 176 coronatum, 180 vulgaris, 176 degenerans, 180 zenkeri, 176 delgadense, 180 Pseudomonas, 39, 105 fischeri, 175 Pteridophyta, 40 glutinosum, 180 Pure cultures, 60 indicum, 175 Pus, gonorrheal, staining of, 129 javanense, 181 inoculation of, 125 luminosum, 180 Pyogenic organisms, 125 papillare, 180 phosphorescens, 177, 179 QUALITATIVE analysis of water, tuberosum, 180 II5, 118 Quantitative analysis of water, 114 Quartan fever, 160 Photography, measuring bacteria by, 163 Phragmidiothrix, 41 Pigment, production of, 85 Pipet, Chester and Robin, for ma- | REAGENTS, 170 larial blood, 147, 148 Red algze, 40 Pitfield’s method for flagella, 25 Reproduction by division, 34 modification of, 26 by spores, 35 Planococcus, 39 of bacteria, 34 Planosarcina, 39 Rhabdochromatium, 42 Plants, 40 Rhodobacteriacez, 41 Plate, counting, Jeffer's, 117,118 { Rhodophyceze, 40 190 Rosenberger’s method for sections, 156 for sputum, 154 SACCHAROMYCES cerevisiz, 165, 166 Saccharomycetaceze, 168 Saccharose, 56 Safranin, 20, 170 Salt solution, normal, 173 Sand filter, Petri’s, r2t Sarcina, 34, 39, 98 fuscescens, 181 tetragena, 125, 128 Schizomycetes, 39, 40 Schizophyceze, 40 Schizophyta, 4o Sections, staining, 20, 129 of actinomycosis, 158 of glanders, 133 of tuberculosis, 156 Sedgwick’s tube for air analysis, I2r Sewage, II5 bacteria, 115 contamination, detection of, 115 Slide for diphtheria, diagnosis, 140 for Widal reaction, 148, 149 Slime-fungi, 40 Sliver, 48 Smegma bacterium, 152, 156, 178 Smith fermentation-tube, 57 Soil analysis, 122 bacteria, 122 Species, determination of, 83 isolation of, 81 life-history of, 83 morphology of, 83 names of, 95 Sphzerococcus acidi lactici, 179 Spheerotilus, 41 Spirilla, 22 Spirillaceze, 41, 106 Spirillum, 41 attenuatum, 181 cholerz Asiaticze, 180 finkleri, 180 marinum, 180 Spirocheeta; 23, 41 Spirosoma, 41, 106 Spores, 34 endogenous, 34 germination of, 38 production of, 35, 90 reproduction by, 35 INDEX. Spores, staining, 36 test for, 90 Sputum, examination of influenza, 43 of tuberculous, 152 inoculation of, 151 tubercle bacteria in, 153, 155, 157 Stab cultures, 61 Stain bottles, 18 Gram's, 19 Hunt's, 139 Neisser's, 139 Staining actinomycosis, 158 anthrax blood, 131 bacteria in tissues, 20, 129 blood for malarial organism, 159, 161 preparations, 131, 161 diphtheria bacteria, 137, 138 flagella, 25 gonorrheal pus, 129 influenza sputum, 143 malarial blood, 159 pneumonia bacteria, 150 sections, 20, 129 for. actinomycosis, 158 for glanders, 133 for tubercle bacteria, 156 spores, 36 tubercle bacteria, 152 Stains, 18, 170 anilin, 170 ordinary, 16 Staphylococcus, 22, 34 cereus albus, 78 aureus, 178 flavus, 178 pyogenes albus, 125, 179 aureus, 125, 178 citreus, 125, 178 salivarius pyogenes, 179 Steam sterilization, 46 under pressure, 46 sterilizers, 46, 47, 48, 49 Sterilization, 43 by hot air, 47 by steam, 46 under pressure, 46 discontinuous, 43 intermittent, 43 of instruments, 127 of syringes, 125 Sterilizer, 46 Arnold, 46, 47, 48 hot-air, 50 INDEX. Sterilizer, steam, 46, 47, 48, 49 Sternberg, 95 Stewart's cover-glass forceps, 17 Stonewort, 40 Streptobacillus pseudotuberculosis rodentium, 178 - Streptococci, 22, 34 species of, 95 Streptococcus, 39, 95 agalactize contagiosee, 182 brevis, 182 coli gracilis, 182 conglomeratus, 182 coryzz contagiosae equorum, 182 erysipelatos, 182 giganteus urethra, 182 intracellularis, 179 longus, 182 mastitidis sporadice, 182 murisepticus, 182 pyogenes, 125, 127, 128 septicus liquefaciens, 182 liquefians, 182 septo-pyzemicus, 182 Stroke cultures, 64 Subcutaneous inoculations, 130, 14c, 151, 155 Synonyms, table of, 175 Syphilis, 152 Syringe, Koch’s, 126 Syringes, sterilization of, 125 TERTIAN fever, 160 Test for ammonia, 93 for Bacillus coli, 118 for motility, 90 for nitrates, 93 for nitrites, g2 for spores, go for virulence, Bact. diphtheriz, 139 Tetragenococci, 22 Thallophyta, 40 Thermal death-point, determination of, 91 Thiobacteria, 41 ‘Thiocapsa, 42 Thiocapsacee, 42 _ Thiocystitis, 42 Thiodictyon, 42 Thionin blue, 170 Thiopedia, 42 ‘Thiopediacez, 42 Thiopolycoccus, 42 Thiosarcina, 42 1g! Thiospirillum, 42 Thiothece, 42 Thiothrix, 41 Tissues, staining bacteria in, 20, 129 in actinomycosis, 158 in glanders, 133 in tuberculosis, 156 Titration, 52 Toxin, production of, 85 Triacid stain, Ehrlich's, 172 Tubercle bacteria, cultures of, 152 in milk, 157 in sputum, 152, 153, 155, 157 in urine, 156 staining, Gabbet’s method, 154 in sections, 156 Koch-Ehrlich method, 154 Rosenberger’s method, 154 Ziehl-Neelsen method, 152 Tuberculosis, 151 inoculation, 155 Tuberculous sputum, examination of, 152 Tubes, etched, 55 Typhoid bacilli, 115, 144 fever, 115, 144 ULVINA aceti, 176 Unna's polychrome methylene-blue, 172 Urine, tubercle bacteria in, 156 VAN ERMENGEM'S method for fla- gella, 30 modification of, 30 Vaselin, 15 Vibrio cholerz Asiaticze, 180 comma, 180 dunbari, 180 finkleri, 180 flavescens, 181 flavus, 181 metschnikovi, 180 proteus, 180 saprophiles 6, 180 y, 180 tonsillaris, 180 Virulence, test for, Bact. diphtherize, 139 WATER analysis, qualitative, 115 quantitative, 114 bacteria, classification of, 112 192 Water bath, 68 collection of, for analysis, 115 contamination of, by sewage, I15 Welch's method for capsules, 31 White acid, 55 Whitney's method for blood, 161 Widal reaction, 145, 149 slide, 148, 149 Wilson and Randolph's method for measuring bacteria, 163 Wire, platinum, 14 Wolfhiigel’s counting plate, 116, 117 INDEX. Wright's method for growing anaé- robes, 79 YEASTS, 165, 166 cultures of, 166 identification of, 168 staining, 166 ZENKER’S fluid, 20, 173 Ziehl-Neelsen method for sections, 156 for sputum, 152 Ziehl’s carbol-fuchsin, 172 Zylonite, 15 SAUNDERS’ BOOKS on GYNECOLOGY and OBSTETRICS W. B. SAUNDERS COMPANY West Washington Square Philadelphia 9, Henrietta Street Covent Garden, London SAUNDERS’ ANNOUNCEMENTS HAVE AN ANNUAL CIRCULATION OF OVER 5,000,000 T= recent growth of our foreign business necessitated some further provision for bringing our new books before the English-speaking pro- _ fession abroad. In addition to our front cover and inside space in the British Medical Journal and the London Lancet, we have, therefore, recently arranged for front cover and inside space in the Indian Medical Gazette, the China Medical Journal, and the Bulletin of the Manila Medical Soctety— each a leading journal in its field.: The extension of our advertising has always gone hand in hand with the expansion of our business both at home and abroad. In 1905 we advertised in 10 journals; in 1906 and in 1907, in 11 journals; in 1908, in 13 journals; in 1909, in 16 journals ; in 1910, in 17 journals; in 1911, in 18 journals; and in 1912, in 20 journals, so that the Saunders Announcements now have an annual circulation of over 5,000,000, or nearly 100,000 every week in the year. A Complete Catalogue of our Publications will be Sent upon Request 2 DISEASES OF WOMEN DeLee’s New Obstetrics Text-Book of Obstetrics. By JosrpH B. DrLezz, M.D., Professor of Obstetrics at Northwestern University Medical School, Chicago. Large octavo of 1060 pages, with 913 illus- trations, 150 in colors. Cloth, $8.00 net; Half Morocco, $9.50 net. JUST READY—SUPERB You will pronounce this new book the most claborate, the most superbly illustrated work on Obstetrics you have ever seen. Especially will you value the 913 illustrations, all, with but few exceptions, original, and the best work of leeding medical artists. Some 150 of these illustrations.are in color. Such a magnificent collection of obstetric pictures—and with readly practical value—has never before appeared in one book. You will find the text extremely practical throughout. Déagnoszs is fea- tured, and the relations of obstetric conditions and accidents to general medi- cine, surgery, and the specialties are brought into prominence. Regarding 7reatment : You get here the very latest advances in this’ field, and you can rest assured every method of treatment, every step in operative technic, is just right. Dr. DeLee’s twenty-one years’ experience as a teacher and obstetrician guarantees this. Worthy of your particular attention are the descriptive legends under the illustrations. These are unusually full, and by studying the pictures serially with their detailed legends you are better able to follow the operations than by referring to the pictures from a distant text—the usual method, These illustrations, with their explanatory legends, you will find particularly valu- able for the quick reference work your daily practice demands. The subject-matter of the book is divided into four parts: The Physiology of Pregnancy, Labor and the Puerperium ; the Conduct of Pregnancy, Labor and the Puerperium; the Pathology of Pregnancy, Labor and the Puer- perium ; and Operative Obstetrics. This arrangement, you see, is extremely practical. GYNECOLOGY. Cullen’s Uterine Adenomyoma Uterine Adenomyoma. By Tuomas S. Cutten, M. D., Associate Professor of Gynecology, Johns Hopkins University. Octavo of 275 pages, with original illustrations by Hermann Becker and August Horn. Cloth, $5.00 net. ILLUSTRATED BY BECKER AND HORN Dr. Cullen’s large clinical experience and his extensive original work along the lines of gynecologic pathology have enabled him to present his subject with originality and precision. The work gives the early literature on adenomyoma, traces the disease through its various stages, and then gives the detailed findings in a large number of cases personally examined by the author. Formerly the physician and surgeon were unable to determine the cause of uterine bleeding, but after following closely the clinical course of the disease, Dr. Cullen has found that the majority of these cases can be diagnosed clinically, The results of these observations he presents in this work, - The Lancet, London ; “A good example of how such a monograph should be written. It is an excellent work, worthy of the high reputation of the author and of the school from which it emanates.” Cullen’s Cancer of the Uterus Cancer of the Uterus. By Tuomas S. CuLLen, M. B., Associate Professor of Gynecology, Johns Hopkins University. Large octavo of 693 pages, with over 300 colored and half-tone -text-cuts and eleven lithographs. Cloth, $7.50 net; Half Morocco, $8.50 net. Howard A. Kelly, M. D. Professor of Gynecologic Surgery, Johns Hopkins University, “Dr. Cullen’s book is the standard work on the greatest problem which faces the sur- gical world to-day. Any one who desires to attack this great problem must have this book.’ 4 SAUNDERS’ BOOKS ON Kelly and Cullen’s Myomata of the Uterus Myomata of the Uterus. By Howarp A. KE ty, M. D., Professor of Gynecologic Surgery at Johns Hopkins University; and Tuomas S. CuLLen, M. B., Associate in Gynecology at Johns Hopkins University. Large octavo of about 700 pages, with 388 original illustrations by August Horn and Hermann Becker. Cloth, $7.50 net; Half Morocco, $9.00 net. A MASTER WORK ILLUSTRATED BY AUGUST HORN AND HERMANN BECKER This monumental work, the fruit of over ten years of untiring labors, will remain for many years the last word upon the subject. Written by those men who have brought, step by step, the operative treatment of uterine myoma to such perfection that the mortality is now less than one per cent., it stands out as the record of greatest achievement of recent times. The illustrations have been made with wonderful accuracy in detail by Mr. August Horn and Mr. Hermann Becker, whose superb work is so well known that comment is unnecessary. For painstaking accuracy, for attention to every detaii, and as an example of the practical results accruing from the associa- tion of the operating amphitheater with the pathologic laboratory, this work will stand as an enduring testimonial. Surgery, Gynecology, and Obstetrics “Tt must be considered as the most comprehensive work of the kind yet published. It will always be a mine of wealth to future students.” New York Medical Journal “Within the covers of this monograph every form, size, variety, and complication of uterine fibroids is discussed. It isa splendid example of the rapid progress of American professional thought.” Bulletin Medical and Chirurgical Faculty of Maryland “Few medical works in recent years have come toour notice so complete in detail, so well illustrated, so practical, and so far reaching in their teaching to general practitioner, specialist, and student alike.”’ GYNECOLOGY. 5 Bandler’s Medical Gynecology b Medical Gynecology. By S. Wvyius Banpizr, M. D., Adjunct Professor of Diseases of Women, New York Post- Graduate Medical School and Hospital. Octavo of 702 pages, with 150 original illustrations. Cloth, $5.00 net; Half Morocco, $6.50 net. THE NEW (2d) EDITION This new work by Dr. Bandler is just the book that the physician en- gaged in general practice has long needed. It is truly the practitioner's gyne- colagy—planned for him, written for him, and illustrated for him. There are many gynecologic conditions that do not call for operative treatment ; yet, because of lack of that special knowledge required for their diagnosis and treatment, the general practitioner has been unable to treat them intelligently. This work gives just the information the practitioner needs. American Journal of Obstetrics ‘“. “He has shown good judgment in the selection of his data. He has placed most ‘emphasis on diagnostic and therapeutic aspects. He has presented his facts in a manner ” to be readily grasped by the general practitioner.” Bandler’s Vaginal Celiotomy Vaginal Celiotomy. By S.Wytuis BaNDLER, M.D. Octavo of 450 pages, with 148 illustrations. Cloth, $5.00 net. SUPERB ILLUSTRATIONS The vaginal route, because of its simplicity, ease of execution, absence of shock, more certain results, and the opportunity for conservative measures, constitutes a field which should appeal to all surgeons, gynecologists, and obstetricians. Posterior vaginal celiotomy is of great importance in the re- moval of small tubal and ovarian tumors and cysts, and is an important step in the performance of vaginal myomectomy, hysterectomy, and hystero- myomectomy, Anterior vaginal celiotomy with thorough separation of: the bladder is the only certain method of correcting cystocele. The Lancet, London ee eee “ has done good service in writing this book, which gives a very clear : ican Oe ee aoecation: which, may be undertaken through the vagina, He makes out a strong case for these operations. 6 SAUNDERS’ BOOKS ON Ashton’s Practice of Gynecology The Practice of Gynecology. By W. EasTERLY AsHTON, M.D., LL.D., Professor of Gynecology in the Medico-Chirurgi- cal College, Philadelphia. Handsome octavo volume of 1100 pages, containing 1058 original line drawings. Cloth, $6.50 net; Half Morocco, $8.00 net. JUST READY—THE NEW (5th) EDITION Among the important new matter may be mentioned the De Keating-Hart fulguration treatment, Coley’s mixed toxins for sarcoma of the genito-urinary organs, the cutireaction of von Pirquet in the diagnosis of tuberculosis, “606”? for syphilis, the hormone theory, the Fowler-Murphy treatment of suppurative peritonitis, tincture of iodin in sterilization, and Baldy’s new round ligament operation for retrodisplacement. Nothing is left to be taken for granted, the author not only telling his readers in every instance what should be done, but also precisely Zow to do it. A distinctly original feature of the book is the illustrations, numbering 1058 line drawings made especially under the author’s personal supervision. From its first appearance Dr. Ashton’s book set a standard in practical medical books ; that he as produced a work of unusual value to the medical practitioner is shown by the demand for new editions. Howard A. Kelly, M. D., Professor of Gynecologic Surgery, Fohns Hopkins University “It is different from anything that has as yet appeared. The illustrations are particu- larly clear and satisfactory. One specially good feature is the pains with which you describe so many details so often left to the imagination.” Charles B. Penrose, M. D., Formerly Professor of Gynecology, University of Pennsylvania. “I know of no book that goes so thoroughly and satisfactorily into all the details ot everything cc d with the subj In this respect your book differs from the others.” George M. Edebohls, M.D. Professor of Diseases of Women, New York Post-Graduate Medical School. “T have looked it through and must congratulate you upon having produced a text book most admirably adapted to teach gynecology to those who must get their knowledge, even to the minutest and most elementary details, from books.” DISEASES OF WOMEN. 7 Webster’s Diseases of Women Diseases of Women. By J. Crarence Wesster, M. D. (Epin.), F. R. C. P. E., Professor of Gynecology and Obstetrics in Rush Medical College. Octavo of 712 pages, with 372 illus- trations. Cloth, $7.00 net; Half Morocco, $8.50 net. FOR THE PRACTITIONER Dr. Webster has written this work especially for the general practitioner, discussing the clinical features of the subject in their widest relations to general practice rather than from the standpoint of specialism. The magni- ficent illustrations, three hundred and seventy-two in number, are nearly all original. Drawn by expert anatomic artists under Dr. Webster’s direct super- vision, they portray the anatomy of the parts and the steps in the operations with rare clearness and exactness. Howard A. Kelly, M.D., Professor of Gynecologic Surgery, Johns Hopkins University. “It is undoubtedly one of the best works which has been put on the market within recent years, showing from start to finish Dr. Webster’s well-known thoroughness. The illustrations are also of the highest order.” Webster’s Obstetrics A Text-Book of Obstetrics. By J. CLarence WEBSTER, M. D. (Ep1n.), Professor of Obstetrics and Gynecology in Rush Medical College. Octavo of 767 pages, illustrated. Cloth, $5.00 net; Half Morocco, $6.50 net. Medical Record, New York “The author’s remarks on asepsis and antisepsis are admirable, the chapter on eclamp- sia is full of good material, and. . . the book can be cordially recommended as a safe guide.”’ 8 SAUNDERS’ BOOKS ON Hirst’s Obstetrics Just Ready—The New (7th) Edition A Text-Book of Obstetrics. By Barron Cooxe Hirst, M. D., Professor of Obstetrics in the University of Pennsylvania. Handsome octavo, 1025 pages, with goo illustrations, 46 in colors. Cloth, $5.00 net; Half Morocco, $6.50 net. INCLUDING RELATED GYNECOLOGIC OPERATIONS Immediately on its publication this work took its place as the leading text- book on the subject. Both in this country and abroad it is recognized as the most satisfactorily written and clearly illustrated work on obstetrics in the language. The illustrations form one of the features of the book. They are numerous and the most of them are original. In this edition the book has been thoroughly revised. Recognizing the inseparable relation between ob- stetrics and certain gynecologic conditions, the author has included all the gynecologic operations for complications and consequences of childbirth, together with a brief account of the diagnosis and treatment of all the path- ologic phenomena peculiar to women. British Medical Journal “ The illustrations in Dr. Hirst’s volume are far more numerous and far better exe- cuted, and therefore more instructive, than those commonly found in the works of writers on obstetrics in our own country.” Hirst’s Diseases of Women A Text-Book of Diseases of Women. By Barton Cooke Hirst, M. D. Octavo of 745 pages, 701 illustrations, many in colors. Cloth, $5.00 net; Half Morocco, $6.50 net. SECOND EDITION As diagnosis and treatment are of the greatest importance in considering diseases of women, particular attention has been devoted to these divisions. The palliative treatment, as well as the radical operation, is fully described, enabling the general practitioner to treat many of his own patients without referring them to a specialist. Medical Record, New York “Its merits can be appreciated only by a careful perusal. . . . Nearly one hundrea pages are devoted to technic, this chapter being in some respects superior to the descrip- tions in other text-books.” GYNECOLOGY. 9 Kelly and Noble’s Gynecology and Abdominal Surgery Gynecology and Abdominal Surgery. Edited by Howarp A. Ke.ty, M. D., Professor of Gynecology in Johns Hopkins University ; and Cuartes P. Nosiz, M.D., formerly Clinical Professor of Gynecology in the Woman’s Medical College, Phila- delphia. Two imperial octavo volumes of g50 pages each, con- taining 880 illustrations, mostly original. Per volume: Cloth, $8.00 net ; Half Morocco, $9.50 net. BOTH VOLUMES NOW READY WITH 880 ORIGINAL ILLUSTRATIONS BY HERMANN BECKER AND MAX BRODEL In view of the intimate association of gynecology with abdominal surgery the editors have combined these two important subjects in one work. For this reason the work will be doubly valuable, for not only the gynecologist and general practitioner will find it an exhaustive treatise, but the surgeon also will find here the latest technic of the various abdominal operations. It possesses a number of valuable features not to be found in any other publication cover- ing the same fields. It contains a chapter upon the bacteriology and one upon the pathology of gynecology, dealing fully with the scientific basis of gyne- cology. In no other work can this information, prepared by specialists, be found as separate chapters. There is « large chapter devoted entirely to medical gynecology, written especially for the physician engaged in general practice. Heretofore the general practitioner was compelled to search through an entire work in order to obtain the information desired. Aédominal sur- gery proper, as distinct from gynecology, is fully treated, embracing operations upon the stomach, upon the intestines, upon the liver and bile-ducts, upon the pancreas and spleen, upon the kidney, ureter, bladder, and the peritoneum. Special attention has been given to modern technic. The illustrations are the work of Mr. Hermann Becher and Mr. Max Brodel. American Journal of the Medical Sciences “TET a to say that the work has been thoroughly done + the names of the authors and a edt iaiarantee this; but much may be said in praise of the method of presen- tation, and attention mav be called to the inclusion of matter not to be found elsewhere.” 4 ‘ j « 10 SAUNDERS’ BOOKS ON GET THE NEW THE BEST American STANDARD Illustrated Dictionary The New (6th) Edition, Reset The American Illustrated Medical Dictionary. A new and complete dictionary of the terms used in Medicine, Surgery, Dentistry, Pharmacy, Chemistry, Veterinary Science, Nursing, and all kindred branches; with over 100 new and elaborate tables and many handsome illustrations. By W. A. NEwMAN Dorianp, M.D., Editor of ‘‘ The American Pocket Medical Dictionary.’’ Large octavo, 986 pages, bound in full flexible leather. Price, $4.50 net; with thumb index, $5.00 net. A KEY TO MEDICAL LITERATURE Gives a Maximum Amount of Matter in a Minimum Space ENTIRELY RESET—A NEW WORK, WITH ADDED FEATURES We really believe that Dorland’s Dictionary is the most useful single book that the medical practitioner can own. We are confident you will get more real use out of it than out of any one book you ever bought. Nearly every medical paper to-day contains special words which are new to most readers. If you want to get the best out of your journals and text-books, Dorland’s Dictionary should be on your desk for ready reference. This new edition defines all the new words, and is a safe key to capitalization, pronunciation, and etymology. PERSONAL OPINIONS Howard A. Kelly, M. D., Professor of Gynecologic Surgery, Johns Hopkins University, Baltimore, ‘©Dr. Dorland’s dictionary is admirable. It is so well gotten up and of such conve- nient size. No errors have been found in my use of it.” J. Collins Warren, M.D., LL.D., F.R.C.S. (Hon.) Professor of Surgery, Harvard Medical School. “T regard it as a valuable aid to my medical literary work. It is very complete and of convenient size to handle comfortably. I use it in preference to any other,” GYNECOLOGY AND OBSTETRICS. II Penrose’s Diseases of Women Sixth Revised Edition A Text-Book of Diseases of Women. By Cuartes B. Penrose, M. D., Pu. D., formerly Professor of Gynecology in the University of Pennsylvania; Surgeon to the Gynecean Hos- pital, Philadelphia. Octavo volume of 550 pages, with 225 fine original illustrations. Cloth $3.75 net. UP TO DATE—ACCURATE Regularly every year a new edition of this excellent text-book is called for, and it appears to be in as great favor with physicians as with students. Indeed, this book has taken its place as the ideal work for the general prac- titioner. The author presents the best teaching of modern gynecology, un- trammeled by antiquated ideas and methods. In every case the most modern and progressive technique is adopted, and the main points are made clear by excellent illustrations, Howard A. Kelly, M.D., Professor of Gynecologic Surgery, Johns Hopkins University, Baltimore. “T shall value very highly the copy of Penrose’s ‘ Diseases of Women’ received. I have already recommended it to my class as THE BEST book,” : Davis’ Operative Obstetrics Operative Obstetrics. By Epwaxp P. Davis, M.D., Pro- fessor of Obstetrics at Jefferson Medical College, Philadelphia. Octavo of 483 pages, with 264 illustrations. Cloth, $5.50 net. INCLUDING SURGERY OF NEWBORN Dr. Davis’ new work on Operative Obstetrics is a most practical one and no , niee hag BeSn spared to make it the handsomest work on the subject, as expe Every step in every operation is described minutely, and the technic wells me hee new illustrations. Dr. Davis’ name is sufficient guarantee b ? shows By g above the ordinary. for somethin 12 SAUNDERS’ BOOKS ON Dorland’s Modern Obstetrics Modern Obstetrics: General and Operative. By W. A. Newman Dortanp, A. M., M. D., Professor of Obstetrics at Loyola University, Chicago. Handsome octavo volume of 797 pages, with zor illustrations. Cloth, $4.00 net. Second Edition, Revised and Greatly Enlarged In this edition the book has been entirely rewritten and very greatly enlarged. Among the new subjects introduced are the surgical treatment of puerperal sepsis, infant mortality, placental transmission of diseases, serum- therapy of puerperal sepsis, etc. Journal of the American Medical Association ‘This work deserves commendation, and that it has received what it deserves at the hands of the profession is attested by the fact that a second edition is called for within such a short time. Especially deserving of praise is the chapter on puerperal sepsis,” Davis’ Obstetric and Gynecologic Nursing Obstetric and Gynecologic Nursing. By Epwarp P. Davis, A. M., M. D., Professor of Obstetrics in the Jefferson Medical College and Philadelphia Polyclinic; Obstetrician and Gynecologist, Philadelphia Hospital. 12mo of 436 pages, illus- trated. Buckram, $1.75 net. THIRD REVISED EDITION This volume gives a very clear and accurate idea of the manner to meet the conditions arising during obstetric and gynecologic nursing, The third edition has been thoroughly revised. The Lancet, London ‘Not only nurses, but even newly qualified medical men, would learn a great deal by a perusal of this book. It is written in a clear and pleasant style, and is a work we can recommend,” GYNECOLOGY AND OBSTETRICS 13 Garrigues’ Diseases of Women Third Edition A TExT-Book oF DisEAsEs or Women. By HENRY J. GARRIGUES, A. M., M. D., Gynecologist to St, Mark’s Hospital and to the German Dispensary, New York City. Handsome octavo, 756 pages, with 367 engravings and colored plates. Cloth, $4.50 net; Half Morocco, $6.00 net. Thad. A. Reamy, M. D., Professor of Gynecology, Medical College of Ohio. “ One of the best text-books for students and practitioners which has been published in the English language; it is condensed, clear, and comprehensive. The profound learning and great clinical experience of the distinguished author find expression in this book.” Macfarlane’s Gynecology for Nurses A REFERENCE HAND-Book oF GYNECOLOGY FoR Nurses. By CATH- ARINE MACFARLANE, M. D., Gynecologist to the Woman’s Hospital of Philadelphia. 32mo of 150 pages, with 7o illustrations. Flexible leather, $1.25 net. A. M. Seabrook, M. D., Woman's Medical College of Philadelphia, “Tt isa most admirable little book, covering ina concise but attractive way the sub- ject from the nurse’s standpoint,” Second American Text-Book of Gynecology Edition AMERICAN TEXT-BOOK OF GYNECOLOGY. Edited by J. M. Baupy, M. D. Iwperial octavo of 718 pages, with 341 text-illustrations and 38 plates. Cloth, $6.00 net. American Text-Book of Obstetrics Second Edition THe AMERICAN TEXT-BookK oF OBSTETRICS. In two volumes. Edited by RicHarp C. Norris, M. D.; Art Editor, Robert L. Dick- inson, M. D. Two octavos of about 600 pages each; nearly goo illus- trations, including 49 colored and half-tone plates. Per volume: Cloth, $3.50 net. Matthew D. Mann, M. D., Professor of Obstetrics and Gynecology, University of Buffalo. “J like it exceedingly and have recommended the first volume as a text-book, It is certainly a most excellent work, I know of none better.” 14 SAUNDERS’ BOOKS ON Schaffer and Webster’s Operative Gynecology Atlas and Epitome of Operative Gynecology. By Dr. Q. ScuArrer, of Heidelberg. Edited, with additions, by J. CLARENCE WEBSTER, M. D. (Ep1n.), F. R.C. P. E., Professor of Obstetrics and Gynecology in Rush Medical College, in affili- ation with the University of Chicago. 42 colored lithographic plates, many text-cuts, a number in colors, and 138 pages of text. In Saunders’ Hand-Atlas Series. Cloth, $3.00 net. Much patient endeavor has been expended by the author, the artist, and the lithographer in the preparation of the plates for this Atlas. They are based on hundreds of photographs taken from nature, and illustrate most faithfully the various surgical situations. Dr. Schaffer has made a specialty of demon- strating by illustrations. Medical Record, New York “‘The volume should prove most helpful to students and others in grasping details usually to be acquired only in the amphitheater itself.” : : De Lee’s Obstetrics for Nurses Obstetrics for Nurses. By JosepH B. DeLeg, M.D., Professor of Obstetrics in the Northwestern University Medical School, Chicago; Lecturer in the Nurses’ Training Schools of Mercy, Wesley, Provident, Cook County, and Chicago Lying-In Hospitals. 12mo of 512 pages, fully illustrated. Cloth, $2.50 net. THE NEW (3d) EDITION While Dr. DeLee has written his work especially for nurses, the practi- tioner will also find it useful and instructive, since the duties of a nurse often devolve upon him in the early years of his practice. The illustrations are nearly all original and represent photographs taken from actual scenes. The text is the result of the author’s many years’ experience in lecturing to the nurses of five different training schools, J. Clifton Edgar, M. D., Professor of Obstetrics and Clinical Midwifery, Cornell University, New York. . _‘‘Itis far and away the best that has come to my notice, and I shall take great pleasure in recommending it to my nurses, and students as well,”” GYNECOLOGY AND OBSTETRICS. 1§ Schaffer and Edgar’ Labor and Operative Obstetrics Atlas and Epitome of Labor and Operative Obstetrics. By Dr. O. ScHArFer, of Heidelberg. From the Fifth Revised and Enlarged German Edition. "Edited, with additions, by J. Cuirron Epcar, M. D., Professor of Obstetrics and Clinical Mid- wifery, Cornell University Medical School, New York. With 14 lithographic plates in colors, 139 other illustrations, and 111 pages of text. Cloth, $2.00 net. Jn Saunders’ Hand-Atlas Series. This book presents the act of parturition and the various obstetric opera- tions in.a series of easily understood illustrations, accompanied by a text treating the subject from a practical standpoint. American Medicine “The method of presenting obstetric operations is admirable. The drawings, repre- senting original work, have the commendable merit of illustrating instead of confusing.” Schaffer and Edgar’ Obstetric Diagnosis and Treatment Atlas and Epitome of Obstetric Diagnosis and Treat- ment. By Dr. O. ScHArrer, of Heidelberg. From the Second Revised German Edition. Edited, with additions, by J. Cuir- Ton Epcar, M.D., Professor of Obstetrics and Clinical Mid- wifery, Cornell University Medical School, N.Y. With 122 colored figures on 56 plates, 38 text-cuts, and 315 pages of text. Cloth, $3.00 net. J Saunders’ Hand-Atlas Series. This book treats particularly of obstetric operations, and, besides the wealth of beautiful lithographic illustrations, contains an extensive text of great value, This text deals with the practical, clinical side of the subject. New York Medical Journal . ‘© The illustrations are admirably executed, as they are in all of these atlases, and the text can safely be commended, not only as elucidatory of the plates, but as expounding the scientific midwifery of to-day.” 16 SAUNDERS’ BOOKS ON GYNECOLOGY AND OBSTETRICS. American Pocket Dictionary New (7th) Edition THE AMERICAN PocKET MEDICAL DICTIONARY. Edited by W. A. Newman Dortanp, A. M.,M.D. With 610 pages. Full leather, limp, with gold edges, $1.00 net; with patent thumb index, $1.25 net. James W. Holland, M. D., Professor of Chemistry and Toxicology, at the Jefferson Medical College, Philadelphia. “Tam struck at once with admiration at the compact size and attractive exterior I can recommend it to our students without reserve.” Cragin’s Gynecology New (7th) Ed. ESSENTIALS OF GYNECOLOGY. By Epwin B. CraGin, M. D., Pro- fessor of Obstetrics, College of Physicians and Surgeons, New York. Crown octavo, 240 pages, 62 illustrations. Cloth, $1.co net. x Saunders Question-Compend Series. Galbraith’s Four Epochs of Woman’s Life S2cond THE Four Epocus or ‘WomAn’s LirE: A StTupy IN HYGIENE, Maidenhood, Marriage, Maternity, Menopause. By ANNA M. GAL- BRAITH, M. D, With an Introductory Note by Joun H. Musser, M. D., University of Pennsylvania. I2mo of 247 pages. Cloth, $1.50 net. Schaffer and Norris’ Gynecology Saunders’ Atlases ATLAS AND EPITOME OF GyNECOLOGY. By Dr. O. SCHAFFER, of Heidelberg. Edited, with additions, by RICHARD C. Norris, A. M., M. D., Assistant Professor of Obstetrics, University of Pennsylvania. 207 colored illustrations on 90 plates, 65 text-cuts, and 272 pages of text. Cloth, $3.50 net. Ashton’s Obstetrics New (7th) Ed. ESSENTIALS OF OBSTETRICS. By W. EASTERLY AsHTON, M. D., Pro- fessor of Gynecology in the Medico-Chirurgical College, Philadelphia. Crown octavo, 252 pages, 109 illustrations. Cloth, $1.00 net. lx Saunders’ Question-Compend Series. Southern Practitioner ‘An excellent little volume, containing correct and practical knowledge. An admir- able compend, and the best condensation we have seen.” Barton and Wells’ Medical Thesaurus A THESAURUS OF MEDICAL WORDS AND PHRASES. By WILFRED M. Barton, M. D., Assistant to Professor of Materia Medica and Thera- peutics, Georgetown University, Washington, D. C.; and WALTER A. WELLS, M. D., Demonstrator of Laryngology, Georgetown University, Washington, D.C. 12mo of 534 pages. Flexible leather, $2.50 net; with thumb index, $3.00 net. : eo taeale Et hash maka aia ore ate ered AAR Fae = ; FY Ryten nme Eek SR ATMS SLES GS To 2S ar Nene pet er Ge pe | De ete Fieve em ciety 4 mt 2 mente as aetaee pape — aa ieee ne eter =o peda : Ae. Des OE aarial toa tenho ataeoraiaer ae ; iad preren es re ent ees ponte se eget oe aie Puy aoe Toe ata A — Shiai a Wie ai yacey ag foe ee ae ee ort een argo, ; oe Te Betas 2k inn Fo aay *, EF the J 53 Opes Ape kon er See ane es PEARS: ; 2 ire magnets Sy Nite 98 RA Seren per ate mn ; " A AR: Seep s eur Poe i ech ne eRe LRM SRA AR Hr ST ee Ah SAP Ae SAN KAN wee Sears SIS e SYRRRAR oc tes STIS rat ate hee e teed ety ete neneey wee Tete ponaamerns ‘ - Sa re aa Sa BER eee Oe See errr Faeries uachars 2 5 eres : eo RANKER Le Petry br a Ts *- (ERT ae ES ee ee WES GRR wee = =a at Sls cles bite. tpciian Uelibes Eatarask Sb oe Cae See + PPP Se a Ae piel SE ey ee rene eras