UC-NRLF LABORATORY OUTLINE FOR General Bacteriology MAIN LmRAFJY.AGRICULTU LABORATORY OUTLINE -FOR- GENERAL BACTERIOLOGY LABORATORY OUTLINE GENERAL BACTERIOLOGY BY CONNECTICUT AGRICULTURAL COLLEGE BACTERIOLOGICAL DEPARTMENT SEPTEMBER 1922 BIOLOGY ;...'•• '•''-l G LIBRARY-AGRICULTURE g PREFACE The exercises and descriptions in this laboratory manual are intended primarily for students in General Bacteriology. The work outlined can be covered in an eighteen week period having two two-hour laboratory periods a week, preferably at intervals of two and five days. The course offered at this institution includes two one-hour lectures a week. The lectures and laboratory work are planned as far as possible to parallel and supplement each other. Students in this course do not prepare their culture media or sterilize their glassware for we feel that the beginner, in bacteriology can use the limited time more advantageously in other directions. The description of bacterial species includes some pathogens but only non-pathogenic organisms are used in this course. The glossary of bacteriological terms appended will be found helpful to the student beginning the study of bacteriology. Storrs, Conn., The Authors. September, 1922. 497750 CONTENTS PART I. The microscope Measurement of bacteria Sterilization Lockers and equipment Examination of unstained bacteria Gelatin Agar Melting and solidifying points of agar and gelatin Air analysis Cautions and directions for identification of unknown cultures Descriptive chart Plate 1. Types of growth in stab and streak cultures Age for examination of various cultures Stains Morphology Descriptions of culture media Descriptions of various species of bacteria PART II. Water testing Bacteria in milk. Plating method Direct microscopic examination of milk Effect of temperature on keeping quality of milk Bacteria from various sources Distilled water experiment Bacteria in meats Testing for presence of spores Conversion factors Comparison of metric and English systems Glossary of laboratory bacteriological terms Part I. THE MICROSCOPE The microscope is a delicately adjusted instrument and great care should be exercised in using it to keep it in good condition. The following rules should be observed: 1. When not in use keep in the case. Dust causes unusual wear if allowed to work into the mechanism. 2. Alcohol should never be used on the lacquered parts. If oily material is to be removed use xylol with gentle rubbing. 3. Should the stage or objectives become gummed with im- mersion oil or balsam it can be removed with lens paper moistened with xylol. 4. Use the plane mirror for daylight and the concave for arti- ficial light. 5. Regulate the amount of light by raising or lowering the Abbe condenser. This is done with a screw under left of stage. The higher the condenser the greater the amount of light. Dust in the eyepiece makes the field appear covered with specks. Wipe with lens paper. 6. Do not touch the surface of the lens with the fingers. 7. Objectives of one microscope should not be changed to another even of the same make. 8. To focus with the two highest powered objectives place object to be examined in center of stage. With eyes at the side at the level of the stage lower the objective writh the coarse adjust- ment until it nearly touches the cover glass. Adjust mirror and condenser to give desired amount of light. Focus slowly upward with the fine adjustment until the object becomes clear. With the 1/12 or oil immersion objective a drop of cedar oil between the lens and the cover glass is necessary to prevent dispersion of light. 9. The fine adjustment is used for bringing out details in very small objects consequently has a limited range and is me- chanically delicate. When the fine adjustment screw stops do not force it. 10. Never use oil with any but the oil immersion objectives. With Leitz microscopes the 1/12 objectives (oil immersion) are copper colored with nickel end. With Bausch & Lomb microscopes the oil immersions are nickeled on the lower half. 11. For measuring bacteria a finely ruled scale is used. This is placed ruled side down in top part of eyepiece. The eyepiece may be swung around to make the scale lie in any direction desired. There are ten microns between each of the numbers on the scale. ll Laboratory Outline for General Bacteriology All apparent measurements have to be corrected by multiplying by a microscope constant. This varies for the different microscopes. 12. Clean the oil immersion objective with lens paper imme- diately after using. 13. With the Bausch & Lomb microscope using the 10X eye- piece and the 1/6 objective the magnification is 430 and the working distance is .3 mm. With the 1/12 objective the magnification is 950 and the working distance is .15 mm. MEASUREMENT OF BACTERIA For measuring bacteria use the highest power objective — the 1/12 oil immersion. When an object is magnified 1000 times light is diminished at the same rate and the Abbe condenser is necessary in order to increase the light sufficiently to see the object. Raise the condenser as high as possible. It is regulated by a screw at the left side under the stage. Place a drop of cedar oil on the preparation to be examined. The oil has the same refractive index as the glass and prevents dispersion of the rays of light. Lower the objective into the oil while looking at it from the side. Adjust the mirror to give the desired amount of light. Usually the full amount is best unless the light is exceptionally bright. Use the plane mirror for daylight and the concave for artificial light. With the eye at the microscope focus up slowly with the fine adjustment until the object shows clearly. In the eyepiece is a ruled scale by which bacteria are measured. The eyepiece is ruled to 1/10 millimeter. The space between num- bers on the scale is one millimeter. A micron is 1/1000 of a milli- meter or 1/1,000,000 of a meter, which magnified by the microscope covers about .7 of the space between two lines on the eyepiece micrometer. The magnification of different microscopes varies slightly so all have been tested with a stage micrometer and the factor or microscope constant determined. This microscope con- stant is marked on the stage of the microscope. All apparent measurements have to be multiplied by this factor to get the actual measurements. The following four stained preparations are furnished : No. 19 B. mesentericus 32 Micrococcus agilis 114 Bact. aerogenes 84 Sarcina flava Note the shape and arrangement of the bacteria and measure their size. Bacteria of the same kind vary somewhat in size so 12 Laboratory Outline for General Bacteriology measurements of the smallest and largest are required in addition to the size of the majority. Measure at least ten of the medium sized ones and average them in finding the size of the majority. If bacteria are spherical only one dimension — diameter — is re- quired ; if rod shaped two dimensions — length and width are re- quired. Measure length and width of the same organisms. To be one micron in length the bacterium has to extend from one line to the corresponding point on the next line, that is, across one space and one line. The lines have width and on most of the scales the width of the line is about .3 micron and the width of the space .7 micron. Now if a bacterium is just covered by a line we know the measurement is .3. If it projects beyond the line it is possible to make an accurate estimate of the proportion of the .7 covered which added to the .3 gives the size; or if it just fills the space between two lines it is .7 micron. The scale may be moved in any direction by turning the eyepiece around. Multiply apparent measurements by the microscope constant and record results in the following form : For Rods Smallest Largest Average Length x width Length x width Length x width Arrangement For Cocci Largest Diameter Average Diameter Smallest Diameter Arrangement When through with the microscope carefully wipe cedar oil from lens with lens paper furnished. Also remove oil from slides. Laboratory note books about 6% X S1^ inches are preferred. These are to be left on the table at your place. They are not to be taken from the laboratory. STERILIZATION Sterilization is the destruction of all forms of life. It may be accomplished in various ways. Only the common methods of sterilization by heat will be considered here. Sterilization in a naked flame. The simplest means of sterilizing a metal instrument is to heat it in the flame. This method is usually 13 Laboratory Outline for General Bacteriology recommended for sterilizing platinum needles, spatulas, forceps, etc. When sterilizing a needle heat the entire length of it to redness in the flame. When sterilizing hold needle vertically in flame. Pass the lower part of the handle three or four times through the flame. Before sterilization a platinum needle should be dried by hold- ing it near the flame. This prevents sputtering which scatters organisms, especially when materials such as fat and protein are thrust into the flame. This drying is of special importance when working with pathogenic material. Cool the sterilized needle before using. Do not let the sterilized needle touch anything except the cul- ture from which the inoculation is being made and the medium to be inoculated. Sterilize needle after using before laying it down. By passing instruments like forceps and spatulas several times through a hot flame they may be sterilized. Sterilization by hot air. The usual method of sterilizing all glassware, instruments with metal handles, etc., is by exposure to hot air. A temperature of 160° C. should be maintained for one hour. Heating and cooling is done slowly to avoid breaking the glassware. Sterilization by steam. Under ten pounds pressure for fifteen minutes in an autoclav all life is destroyed unless the material is in large bulk. For flasks and larger volumes of liquid the period of heating is prolonged to twenty minutes. The temperature thus obtained is from 114° to 115° C. Steam without pressure at 100° C. is used. Materials are heated for twenty to thirty minutes on three successive days. On the first day the vegetative cells are killed. The spores remaining develop in time to be killed by the heating on the second day. Heat- ing on the third day is to make certain that all spores have had time to change to the vegetative stage and be killed. LOCKERS AND EQUIPMENT Lockers fitted out with apparatus needed in the work of the course are assigned to students. A deposit of 25c is required for the key. This is refunded at the end of the course when the key is returned. The following materials are in each locker : 1 counting plate 1 hand lens 1 platinum needle 14 Laboratory Outline for General Bacteriology 1 platinum loop 1 test tube rack 2 wire baskets 1 slide box 1 ruler 1 thermometer 2 pipette cases 50 Petri dishes 25 Ice pipettes 6 Ice pipettes graduated to 1/10 cc. 2 2cc pipettes The above list together with the prices is on the inside of the locker door. Check up carefully and if anything is missing report at once. Absolutely no allowance is made for any shortage unless reported at this time. Each student is held responsible for these materials and will be charged at the end of the semester for any- thing broken or missing. Do not leave materials out on the tables or lying around the laboratory. They are returned when the owner- ship is known but in most cases it is not known and the student loses them. The Petri dishes or plates in the lockers are sterile so do not open them. When counting pipettes count from the end without taking them from the case, as they are sterilized ready for use. Bring a towel or cloth for drying glassware. Students are required to wash their Petri dishes and pipettes after using them. Other glassware is cared for by the depart- ment. Care must be used to wash the glassware perfectly clean. If any particles of media remain, sterilizing burns them on and leaves dark brown patches which can only be removed by scraping or by a strong acid cleaning mixture. Petri dishes are to be wiped dry. Mark each one with your locker number. All glassware is marked with red glass marking pencils. These pencils are sold in the laboratory for lOc each. After pipettes are cleaned they are returned to their case with points down. Cases are also marked with the locker number. Place cases of pipettes and Petri dishes which you wish sterilized on the long table in north end of labora- tory. In the book there write your name, locker number, number of dishes, number of cases of pipettes left to be sterilized, and the date. They will be sterilized and returned to your locker. Plates and pipettes must be washed promptly when you are through using them. Forms for recording notes will be given out on the board. Use the forms given. Leave one page in the note book for each experi- ment. 15 Laboratory Outline for General Bacteriology EXAMINATION OF UNSTAINED BACTERIA The purpose is to study bacteria in a living condition ; to dem- onstrate their form, arrangement, and motility. Bacteria have two kinds of motion, the so-called Brownian or molecular movement, and true motility. Brownian movement is shown more or less by all small particles of insoluble matter (in- cluding living non-motile or dead bacteria) in suspension. It is characterized by a vibratory movement; the relative positions of the particles or bacteria are not changed. This type of movement may be distinguished from true motility which is characterized by progressive movement, more or less rapid, of an organism across the field of a microscope, and which changes its position in the field independently of and in a direction contrary to other organisms present. If large numbers of bacteria are moving in one direction it is an indication that they are being carried by currents in the liquid. Method Place a drop of water on a cover glass held in cover-glass forceps. Sterilize platinum loop by heating to redness in gas flame. When cool transfer a small amount of the culture to be examined to the drop of water. Use only enough to cloud the water slightly. Take the culture from the edge of the growth in the lower part of the tube. The bacteria are on the surface of the agar, so do not allow the loop to cut down into the agar. In opening the tube remove the cotton plug and hold it so that the inner part will not touch anything. Never lay it on the table. This is necessary to prevent the culture in the tube from becoming contaminated with other kinds of bacteria or molds. After removing loopful of cul- ture replace cotton plug. Invert the preparation onto a slide and examine with the 1/6 objective. No oil is used with this objective. With the coarse adjustment lower the objective until it nearly touches the cover glass, being careful not to touch it. Then with the eye at the ocular, focus up with the fine adjustment until the bacteria come into view. This focal point will be passed without noticing it if passed too quickly, if the light is too intense, or if the light is too dim. Regulate the amount of light by raising or lowering the condenser and by adjusting the mirror. Unstained bacteria are nearly colorless and appear as faint shadows. Bacteria lose their motility very quickly so if there is a delay of two of three minutes in getting the focus a wrong determination may be made. In such a case it is better to make a new prepara- tion. Do not make but one preparation at a time. 16 Laboratory Outline for General Bacteriology Bacteria move by means of flagella. These are slender, hair- like processes usually several times as long as the bacterial cell. Some species have but one on the end, monotrichiate ; some have a tuft on one end, lophotrichiate ; some have them distributed all over the organism, peritrichiate. The arrangement of the flagella causes a difference in the kind of motion. Special staining methods are necessary in order to see the flagella. Examine No. 19 B. mesentericus 32 M. agilis " 129 M. lactis varians 20 B. proteus 48 B. pullorum 23 B. mycoides Examine preparation from a cake of yeast. Make drawings showing appearance of yeasts, starch grains, and bacteria. GELATIN Gelatin serves two purposes. As a solid culture medium, it is a technical device by which the isolation of a single species of microorganism is made possible. To those organisms which secrete proteolytic enzymes it serves as a nitrogenous food material. Beef extract and peptone are added as food for bacteria. Gelatin was the first substance used for a solid culture medium. This medium was originated in 1882 by Koch and has since revolu- tionized the science of microbiology. Milk sugar and litmus are also added when acid production is to be determined. Gelatin media will melt at a temperature of about 24° C to 26° C solidifying again at 20°C to a clear transparent jelly. Gelatin plates do not have to be inverted during incubation, as do the agar plates, because no water of condensation ordinarily collects on the cover of the Petri dish in case of the gelatin. AGAR Agar is a seaweed product used in preparing various kinds of solid culture media. Ordinarily agar is composed of beef extract, peptone and agar. It becomes fluid at about 85 °C and solidifies below 40 CC, becoming a very solid opaque jelly, which retains its shape well in slants and plates. Agar is commonly used in determining the number of bacteria per cc. in milk, water, and various other substances. A definite 17 Laboratory Outline for General Bacteriology amount of the material to be tested is mixed with the fluid agar and allowed to cool. Each bacterium is held in a certain spot on the agar. Bacterial colonies develop upon several days incubation and each colony represents one bacterium or clump of bacteria originally present. Agar itself is not a food for microorganisms. Its special value is in hardening media used for cultivating organisms which grow at a temperature above melting point of gelatin. This feature has made possible the great strides that have been taken in medical bacteriology. TO DETERMINE THE MELTING AND SOLIDIFYING POINTS OF AGAR AND GELATIN Place tubes of agar and gelatin in wire basket. Set in pan with water above level of media in tubes. Insert thermometer in gelatin. Place pan over gas flame and watch thermometer. When liquefied record temperature. Change thermometer to agar tube and record melting temperature. Flace tubes in cold water and watch for solidifying point first of agar, then of gelatin. Melting point Solidifying point Agar °C. °C. Gelatin °C. °C. AIR ANALYSIS Sterile melted agar tubes will be furnished. Pour seven into Petri dishes using one tube to a dish. In doing this remove cotton stopper without touching end of tube. Raise one side of the cover of the dish just enough to allow the pouring of the agar. This is done to prevent bacteria dropping in from the air. When agar is solid expose plates with cover removed for five minutes in the places designated below. Keep cover right side up while off from plate. The one out of doors should be taken away from roads or buildings. Face the wind in exposing plate so that bacteria will not below from you onto the plate. With red glass marking pencil mark the plates with your name, the date, and place of exposure. After exposing plates place in locker inverted to incubate. 18 Laboratory Outline for General Bacteriology No. Bact. No. Molds Area of Plates Molds per liter Bact. per liter 1 Bacteriology laboratory 2 Out of doors 3 Bottling room of dairy 4 Cow barn 5 Dining room (at meal time) 6 Study 7 Control — not exposed Counting Bacteria and Molds on Air Plates It has been shown that the bacteria from ten liters of air will fall on one hundred square centimeters in five minutes. From this is derived the formula 10 x no. bact. or molds on plate area of plate no. bact. or =molds per liter of air. Measure diameter of plate and from the following table deter mine the area. Diam. plate Area plate 86 mm 58 sq. cm. 87 59 88 61 89 62 90 63.5 91 65 92 66.5 93 68 94 69.5 95 71 96 72.5 97 74 98 75.5 99 77 100 78.5 In counting a plate place it on the glass plate ruled in squares. Remove the cover of the dish. Use the lens holding it near the eye. Bring the plate near enough to focus clearly. . Beginning at the upper part of the plate follow along the lines working back and forth until the whole plate is counted. 19 Laboratory Outline for General Bacteriology From air plates select seven bacterial colonies of different ap- pearances and write descriptions of them in note book. Then make transfers from them to agar slants. Mark tubes so they can be identified with descriptions. To transfer colony sterilize platinum loop by heating entire length of wire to redness in the gas flame. Pass .the lower part of the handle three or four times through the flame. When cool touch loop to the colony to be transferred. Remove cotton plug and hold it so the lower part will not become contaminated by touching anything. Touch the loop to the surface of the agar at the lower part of the tube and make a straight streak up to the top. Care is necessary not to cut down into the agar. Replace cotton stopper. Place tubes in locker to incubate. Glossary on pages 57, 58, 59 is to be learned. These terms will be constantly used so it is necessary to become familiar with them now. There will be a written on it soon, probably at the next laboratory period. 20 CAUTIONS AND DIRECTIONS FOR IDENTIFICATION OF UNKNOWN CULTURES Laboratory Outline for General Bacteriology CAUTIONS AND DIRECTIONS FOR IDENTIFICATION OF UNKNOWN CULTURES J 1. Do keep cultures pure so correct results may be obtained. Agar Slants 2. Do touch only the tops of cotton plugs when removing them from and replacing them in tubes. While out hold between fingers with lower parts away from hand so they will not touch anything. 3. Do burn off cotton adhering to mouth of tube instead of pulling it off with the fingers, rods, etc. 4. Do sterilize the needle or loop by heating entire length of wire to redness and pass lower part of holder through flame three or four times before making a transfer. 5. Do allow needle to cool before using. 6. Do hold needle so nothing touches it while cooling. 7. Do avoid opening tubes in currents of air. Bacteria may be blown in. 8. Do hold tube nearly horizontal while making transfers so bacteria cannot fall in. 9. Do push plugs in until they are firm. 10. Do use care to keep all cultures pure even when you think it is the last time they will be used. 11. Do keep all cultures until advised to discard them. 12. Do make records complete. 13. Do record all negative as well as positive results. 14. Do remember that "chromogenesis" refers to the color of the bacterial growth itself, not to the color of the medium on which the bacteria are growing. 15. Do touch surface of agar slants so lightly that needle or loop never breaks into the medium. Bacteria are usually only on the Surface. (Continued on Page 24) \ 22 Laboratory Outline for General Bacteriology CAUTIONS AND DIRECTIONS FOR IDENTIFICATION OF UNKNOWN CULTURES 1. Don't allow cultures to become contaminated. Later work with contaminated cultures gives incorrect results. The fol- lowing notes give some hints about avoiding contaminations and obtaining satisfactory records. Agar Slants 2. Don't lay cotton plug on the table or let it fall there, or hold the inner part of the plug next the palm of the hand, or allow it to touch anything. If it accidentally touches anything burn off a layer of the cotton before replacing plug in tube. 3. Don't pull off cotton adhering to the mouth of the tube with the fingers, rods, matches, etc. Burn it off. 4. Don't use the needle or loop without first thoroughly steriliz- ing it by heating the entire length to redness and passing the lower part of the holder three or four times through the flame. 5. Don't use the needle or loop when hot enough to kill the bacteria. 6. Don't feel of the needle with the fingers or touch it against something not sterile to see if it is cool enough to use. 7. Don't open tubes in currents of air. Bacteria are blown in. 8. Don't hold tubes vertical when open thus allowing bacteria to fall in. 9. Don't insert plugs so slightly that they fall out when tubes are handled. 10. Don't be careless with cultures when you think it may be the last time you are going to use them. You frequently find they are needed for later work. 11. Don't discard any cultures until you are advised to do so. 12. Don't make partial records of cultures when complete ones are possible. 13. Don't leave record spaces blank when you find that no change has taken place or when you obtain a negative reaction. 14. Don't record the color of the medium under "chromogenesis" or leave the space blank if the bacterial growth is white or colorless. 15. Don't break the surface of the agar slant with the needle or loop. The bacteria are usually entirely on the surface of the agar and mixing them with the agar greatly increases the difficulty of making later inoculations. (Continued on Page 25) 23 Laboratory Outline for General Bacteriology 16. Do take small amounts of bacteria for inoculation. Only a few bacteria are necessary. 17. Do keep bacterial cultures out of the sunlight, j Liquid Media 18. Do keep cultures upright enough so that plugs will not become wet. 19. Same as 1 to 13 inclusive for agar slants. 20. Do keep fermentation tubes so no air enters inner tube. 21. Do sterilize loop after touching it to litmus paper in testing for reaction before putting it back into the tube for another loopful. 22. Do handle incubated cultures gently before taking the ob- servations. This is particularly important with nutrient broth as the appearance may be greatly changed by jarring. 23. Do use uninoculated tubes of media for comparison in making observations and tests. • Plate Cultures 24. Do lift covers only high enough to insert the pipette or needle when adding inoculation material. 25. Do have medium at right temperature when pouring — agar about 40° C., and gelatin between 25° C. and 40° C. 26. Do remove cotton plugs from tubes or flasks without touching lips of tubes or flasks and burn off any adhering cotton that would interfere with pouring. 27. Do lift cover when pouring just high enough so tube or flask will not scrape on edges of dish. 28. Do mix medium well with inoculation so bacteria will be evenly distributed. 29. Do keep medium from getting on edges of plate when pouring or mixing as this glues them together and frequently leads to breakage and contamination. 30. Do pour plates where air is quiet. 31. Do keep plates closed while looking to see if medium has hardened. 32. Do place plates near center of table to harden as edges of table are not level. 33. Do always carry gelatin plates level and right side up. 34. Do keep agar plates inverted after hardening and while in- cubating. (Continued on Page 26) 24 Laboratory Outline for General Bacteriology 16. Don't take all the growth possible for the first inoculation as you will have about nineteen more to make from the same culture. Simply touching the cool sterilized needle to the growth is usually sufficient. 17. Don't allow the sun to shine on cultures. Sunlight destroys certain species of bacteria very quickly. Liquid Media 18. Don't lay tubes of liquid media on table or hold them so that plugs become wet. Contaminations usually follow wet plugs. 19. Same as 1 to 13 inclusive for agar slants. 20. Don't allow air to get into the small inner tube of the fermen- tation tubes. 21. Don't put loop back a second or third time without steriliza- tion after touching it to litmus paper in testing the reaction. 22. Don't shake or handle tubes roughly before taking the obser- vations. 23. Don't make observations or tests of cultures without using uninoculated tubes for controls. Plate Cultures 24. Don't lift covers clear off when adding inoculating material with pipette or loop. 25. Don't pour agar or gelatin when too hot or too cold. 26. Don't touch the lip of the tube or flask when removing cotton stopper. 27. Don't scrape outside of tube or flask on edge of plate when pouring. 28. Don't leave plates without mixing inoculation with medium. 29. Don't slop medium onto edges of plates when pouring or mixing. 30. Don't pour plates in a current of air. 31. Don't remove or lift cover of plate to see if medium has hardened. 32. Don't leave plates anywhere except near the center of table to harden as tables are crowning. This makes the layer of medium uneven. 33. Don't ever carry gelatin plates tipped or invert them to in- cubate. 34. Don't incubate agar plates right side up as condensation water causes trouble by dropping back from the cover, (Continued on Page 27) 25 Laboratory Outline for General Bacteriology 35. Do allow agar to harden in plates without tipping them around and breaking the medium. Gelatin Stab 36. Do have needle straight before puncturing. 37. Do puncture clear to bottom of tube. 38. Do remove needle in line of puncture. 39. Do puncture in center of tube and only once. 40. Do hold tube above medium so warmth of the hand will not melt the gelatin. 41. Do watch cultures carefully to see when liquefaction begins and when it is complete as well as for the form of growth in both liquefying and non-liquefying cultures. Morphology 42. Do come at right times to make agar slants to have them at the required age for studying at laboratory periods. 43. Do prepare films with only enough bacteria to show slight cloudiness. 44. Do spread films over the entire cover glass. 45. Do leave stains on the exact time called for in the directions. 46. Do measure at least ten bacteria to find the size of the majority. 47. Do measure both length and breadth of rod shaped bacteria and put both measurements down. 48. Do use care to fill out all points called for under "Morphology." 49. Do examine preparations for motility as soon as possible after making, as many bacteria lose their motility very quickly ; and do shut down on the light enough so bacteria can be seen. 50. Do handle cover glasses with forceps. 26 Laboratory Outline for General Bacteriology 35. Don't tip agar plates constantly while they are hardening. It breaks the agar so it will not form a solid mass which can be satisfactorily inverted. Gelatin Stab 36. Don't puncture medium with a crooked needle or with the loop. 37. Don't stop puncturing until the needle touches the bottom of the tube. 38. Don't split the medium when removing the needle. 39. Don't puncture near the side of the tube or more than once. 40. Don't hold the tube in the hand until the warmth of the hand melts the gelatin. 41. Don't fail to record the form of growth, when liquefaction begins, and when it is complete. Morphology 42. Don't forget to come extra times when necessary to make cultures so as to have them the right age to use at laboratory periods. 43. Don't use too much or too little material in preparing films. 44. Don't leave all the material of the film in one small thick mass which can never be seen through. Spread. 45. Don't leave stains on for longer or shorter times than called for in the directions. 46. Don't measure less than ten different bacteria before filling in measurements called for. 47. Don't think the size of a rod simply means its length, or that you multiply the length by the width. 48. Don't think that most of the information called for under "Morphology" does not amount to much and needs not to be filled out. 49. Don't make preparations for motility examination until you are ready to examine them, or leave on the full amount of light when trying to find them. 50. Don't ever touch the surface of a cover glass with the fingers. It leaves enough greasiness to prevent the proper spreading of films. 27 Laboratory Outline for General Bacteriology THE CHART The purpose of the descriptive chart is to provide a uniform and concise method of recording nearly all the ordinary and some of the unusual observations concerning the morphology and activi- ties of bacteria. It should familiarize students with bacterial char- acteristics and facilitate in the grouping and identification of species. For preliminary practice in handling bacteria five pure cultures will be given each student. This work includes the Gram's stain, fuchsin stain for measurement, inoculations into and observations on agar slants, gelatin stabs, nutrient broth, milk, litmus milk, potato, and the nitrite test. After this six unknown cultures are given each student. All data called for on the Descriptive Chart is worked out and filled in. When complete the cultures are named by comparing descriptions with those on pages 43, 44, 45, 46. For work on unknown cultures 12-14 laboratory periods are given. No bacteria pathogenic to man are given out to students in this course. 28 Laboratory Outline for General Bacteriology DESCRIPTIVE CHART, ENDORSED BY SOCIETY OF AMERICAN BAG TERIOLOGISTS AT THE ANNUAL MEETING DEC. 30, 1920. Prepared by H. J. Conn, K. N. Atkins, I. J. Kligler, J. F. Norton, G. E. Harmon, Committee on bacteriological technic. MORPHOLOGY Note. Underscore required terms. Vegetative Cells, Medium used.. temp age days. Form, spheres, short rods, long rods, filaments, commas, short spirals, long spirals, curved. Arrangements, single, pairs, chains, fours, clusters, cubical packets. Limits of Size Size of Majority Ends, rounded, truncate, concave. Capsules, present on How stained Sporangia, present, absent. Medium used temp age days. Form, elliptical, short rods, spindled, clavate, drumsticks. Limits of Size Size of Majority Endospores, present, absent. Location of endospores, central, polar. Form, spherical, elliptical, elongated. Limits of Size Size of Majority Wall, thick, thin. Sporangium wall, adherent, not adherent. Motility In broth On agar Flagella. No Attachment, polar, bipolar peritrichiate. How stained Irregular Forms. Present on in days at °C. Form spindled, cuneate, filamentous, branched, or Staining Reactions Gram Acid fast Special Stains CULTURAL CHARACTERISTICS Underscore required terms. Agar Stroke. Incubation temp °C, Age days Growth, scanty, moderate, abundant, none. Form of growth, filiform, echinulate, beaded, spreading, arborescent, rhizoid. Elevation of growth, flat, effuse, raised, convex. Lustre, glistening, dull. Topography, smooth, contoured, rugose. Optical Characters, opaque, translucent, opalescent, iridescent. Chromogensis Photogenic. Fluorescent. 29 Laboratory Outline for General Bacteriology Odor, absent, decided, resembling Consistency, butyrous, viscid, membranous, brittle. Medium, grayed, browned, reddened, blued, greened. Gelatin Stab, Incubation temp °C, Age days Growth, uniform, best at top, best at bottom. Line of puncture, filiform, beaded, papillate, villpus, arborescent. Liquefaction, none, crateriform, napiform, infundibuliform, saccate, strati- form, begins in d complete in d. Depth of liquefaction in tube of 10 mm. diameter evenly inoculated at 20°C for 30 days mm. Medium, fluorescent, browned. Potato, Incubation temp °C, Age days Terms as in Agar stroke. Nutrient Broth, Temp °C, age days Surface growth, ring, pellicle, flocculent membranous, none. Clouding, slight, moderate, strong, transient, persistent, none, fluid turbid. Sediment, compact, flocculent, granular, flaky, viscid on agitation, abun- dant, scant, none. Agar Colonies, temp °C, Age days Growth, slow, rapid. Form, punctiform, circular, irregular, mycelioid, filamentous, rhizoid. Surface, smooth, rough, concentrically ringed, radiate. Elevation, flat, effuse, raised, convex, pulvinate, umbonate. Edge, entire, undulate,lob ate, erose, filamentous, curled. Internal structure, amorphous, finely-, coarsely-, granular, filamentous, curled, concentric. Gelatin Colonies, temp °C, Age days Growth, slow, rapid. Form, punctiform, circular, irregular, mycelioid, filamentous. Elevation, flat, raised, convex, pulvinate, crateriform, (liquefying) Edge, entire, undulate, lobate, erose, filamentous, floccose, curled. Liquefaction, cup, saucer, spreading. Internal structure, amorphous, finely-, coarsely-, granular, filamentous, curled, concentric. PHYSIOLOGY TEMPERATURE RELATIONS Optimum temperature for growth °C. Maximum temperature for growth °C Minimum temperature for growth °C. CHROMOGENESIS Nutrient broth Nutrient gelatin Nutrient agar Potato... PRODUCTION OF INDOL Medium : Indol absent, present in days PRODUCTION OF HYDROGEN SULFIDE Medium : H2S absent, present in days 30 Laboratory Outline for General Bacteriology FERMENTATION Temperature. Medium containing w u CO o H W o M < 2 s g C3 H 1 H £ and: < j H 5 < w CJ Gas First appearance of acid First appearance of alkali . . . . Reaction after days Reaction after days Max. H-ion Cone. . RELATION TO OXYGEN Method used Medium Temperature °C. Aerobic growth ; absent present, better than anaerobic growth. Anaerobic growth: absent, occurs in presence of dextrose, of sucrose, of lactose, of nitrate; better than aerobic growth. DIASTATIC ACTION Breadth of clear zone on starch agar plates, in days : MILK Temperature. . Reaction: 1 day 2 days 4 days 7 days. Acid curd: 1 day 2 days 4 days 7 days. Rennet curd: 1 day 2 days 4 days 7 days. Peptonizat on : 1 day 2 days 4 days 7 days. Reduction of litmus in days; of methylene blue in days. Influence of indicator on growth . . 10 days. ... .10 days. 10 days. 10 days. Nitrite Gas: Gas: Nitrite NITRATE REDUCTION Medium Temperature °C. 1 day 2 days 4 days 7 days 10 days 1 day 2 days 4 days 7 days 10 days Medium Temperature °C. 1 day 2 days 4 days 7 days 10 days 1 day 2 days . .4 days 7 days 10 days Index No.* BRIEF CHARACTERIZATION As each of the following characteristics is determined, indicate in proper marginal square by means of figure, as designated below: PRIMARY CHARACTERISTICS Microscopic Features Form: 1. streptococci; 2, diplococci; 3, micrococci; 4, sarcinae; 5, rods; 6, commas; 7, spirals; 8, branched rods; 9, filamentous Spores: 1. central; 2, polar; 3, absent Flagella: 1, peritrichic; 2, polar; 3, absent Gram stain: 1, positive; 2, negative Miscellaneous Biochemical Reactions Pathogenicity, etc.: 1, for man; 2, for animals; 3, for plants; 4, parasitic but not pathogenic; 5, saprophytic; 6, autotrophic Relation to oxygen: 1, strict aerobe; 2, facultative anaerobe; 3. strict aerobe Gelatin liquefaction: 1, positive; 2, negative In nitrate media: 1, nitrite and gas; 2, nitrite but no gas; 3, neither nitrite nor gas Chromogenesis: 1, flourescent; 2. violet; 3. blue; 4, green; 5, yellow; G, orange; 7, red; 8, brown; 9, pink; 0. none Carbohydrate Reactions Diastatic action: 1. positive; 2, negative From dextrose: 1, acid and gas; 2, acid without gas; 3. no acid From lactose: 1. acid and gas; 2, acid without gas; 3. no acid From sucrose: 1, acid and gas; 2, acid without gas; 3, no acid SECONDARY CHARACTERISTICS Vegetative Cells Diameter: 1, under 0.5M; 2, between 0.5A and 1H\ 3, over 1M Length : 1, less than 2 diameters; 2. more than 2 diameters Chains (4 or more cells): 1. present; 2, absent Capsules: 1. present; 2, absent in o> i Shape: 1, round; 2, oval to cylindrical Diameter: 1, less thin diameter of rod; 2, greater than diameter of rod Cultural Features Agar Stroke Abundance: 1. abundant; 2, moderate; ^3. slight; 4, absent Lustre: 1, glistening; 2, dull Surface: 1, smooth; 2, contoured; 3, rugose Agar colonies: 1, panctiform; 2, round (over 1 mm. diameter); 3, rhizoid; 4, filamentous; 5, curled Gelatin colonies: I. punctiform; 2. round (over 1 mm.); 3, irregular; 4. fila- mentous M 1 Acid: 1, sufficient for curdling; 2. insufficient for curdling; 3, no acid Rennet curd: 1, present; 2. absent Peptonizatioo,: 1, present; 2, absent * Recording the "Index Number" here is optional ; but its use will be found convenient if the charts are to be filed according to the salisnt characteristics of the organisms. The Index Number consists of the firtt thirteen figures from the margin (primary characteristics) copied down in the order of their occurrence in the margin, placing a dash wherever a heavy rule occurs in the margin. Thus, B. coli belongs to the group 5312-41220-1111. PLATE. 1. 0. c. J v^ A. Stab Cultures 1. Filiform 2. Beaded 3. Villous 4. Arborescent 5. Papillate B. Type of Liquefaction C. Stratiform Napiform Infundibuliform Crateriform 5. Saccate Streak Cultures 1. Arborescent 2. Spreading 3. Filiform 4. Beaded 5. Echinulate 33 Laboratory Outline for General Bacteriology AGE OF CULTURES, TO DETERMINE Motility Flagella Spores and sporangia Stains for measurement of vegetative cells Gram's stain 24 hours 24 " 3 days to 1 week. 24 hours 24 " AGE OF VARIOUS CULTURES WHEN EXAMINED Agar slants Potato slants Nutrient broth Gelatin stabs Agar plates Gelatin plates Dunham's peptone for Indol test Lead acetate agar for H0S test. Milk " Litmus milk Nitrate broth Nitrate agar Starch agar for diastatic action Fermentation tubes of dextrose lactose, sacch- arose, glycerine described after 7 days, checked up at 14 days same as agar slants same as agar slants examine every laboratory period until 30 days old, recording any changes examine after 1 week from 2 to 7 days 4 days 7 days 1, 2, 4, 7, and 10 days 1, 2, 4, 7, and 10 days 1, 2, 4, 7, and 10 days 1, 2, 4, 7, arid 10 days 7 days, unless the growth is very rapid, then the test is made sooner. examined 2, 7 and 14 days. TEMPERATURE OF INCUBATION All cultures are to be grown at locker temperature, which is about 20° C. except the one set of agar slants made for testing growth at 37° C. 34 Laboratory Outline for General Bacteriology STAINS Their Composition and Methods of Use Standard Alcoholic Solutions. Standard alcoholic solutions of anilin colors, especially fuchsin, gentian violet, and methylene blue, are made by dissolving 10 grams of dry color in 100 cc of 95% alcohol. These are used as stock solutions from which the various stains are made. Watery Fuchsin. Made by diluting one part of standard alco- holic solution of fuchsin with nine parts of distilled water. Use. Leave on 1 minute, wash with water. Watery Gentian Violet. Made by diluting one part of standard alcoholic solution of gentian violet with nine parts of distilled water. Use. Leave on 1 minute, wash with water. Carbol Fuchsin. Made by diluting one part of standard alco- holic solution of fuchsin with nine parts of a five per cent, solution of carbolic acid. Use. Leave on 1/2 minute, wash with water. Methylene blue, Loeffler's. Made by adding thirty cc of stand- ard alcoholic solution of methylene blue to seventy cc of potassium hydroxide solution (1-10,000). Use. Leave on 10 minutes, wash with water. Gram's stain (Chester). Made by shaking 1 cc of aniline oil with 10-15 cc of distilled water vigorously for several minutes and filtering. To 10 cc of the filtrate add 1 cc of standard alcoholic solution of gentian violet and filter again. This is the stain. The Gram's solution has the following composition: Metallic iodine 1 gram Potassium iodide 2 grams Water, distilled 300 cc Use. Anilin gentian violet Vr> minute, wash with water Gram's mixture (iodine) Alcohol 95 ', about 1 Flagella stain (Duckwall). Mordant is prepared as follows: Desiccated tannic acid 2 grams Water, distilled 15 cc Saturated alcoholic solution of fuchsin 1 cc Normal NaOH solution 2-4 drops 35 Laboratory Outline for General Bacteriology When ready to use mix with Ferrous sulphate, saturated aqueous solution 5 cc Filter. Make stain by adding 2 cc of saturated alcoholic solution of fuchsin to 8 cc of a 5% solution of carbolic acid. Use Mordant V$> — 1 minute, wash with water. Carbol fuchsin 1/2 — 1 minute, wash with water. MORPHOLOGY The size, form, arrangement, etc., of vegetative cells is deter- mined from twenty-four hour old agar slant cultures. Watery fuchsin stain is used as it has been found satisfactory with most bacteria. The Gram stain is made at the same time. This stain is for differential purposes only. Some bacteria keep the stain and some do not. When the bacteria remain dark blue they are recorded as Gram positive, otherwise as Gram negative. Preparation of films. The method of preparing films for these two stains is as follows : Place cover glasses in forceps without touching them with the fingers. With sterile loop place a drop of water on each of two cover glasses. With a cool sterile needle or loop transfer from edge of culture in lower part of tube to the drops of water on the cover glasses. Use only enough to cloud the water slightly. Burn any excess of bacteria from the loop and spread the drops over the surface of the cover glasses. If a drop will not remain spread but rolls together again it shows that the cover glass is greasy and should be discarded. Air dry without heat. Fix film to the cover glass by passing it three times through the gas flame. Keep the film side up. Do not hold in flame longer than you could hold your finger in it without burning. They are now ready to be stained. For staining methods see page 35, After staining drying may be hastened by blotting with filter paper. When dry mount on slide film side down in drop of cedar oil. Label slides carefully. Never pile slides together. Place them in slide boxes. 36 Laboratory Outline for General Bacteriology Motility and flagella staining. Motility is determined from day-old nutrient broth or agar cultures. Motile cultures are stained for flagella. Preparations are made as follows: With a pipette place about 4 drops of water together in a watch glass. Inoculate center of water with culture taken from edge of growth in lower part of agar tube. Be sure the needle is cool before starting and avoid any rapid vigorous movements as the flagella are easily broken off. Use enough bacteria to cloud the center of the water slightly. Let the dilution stand for five minutes, then remove from edge of dilution several loopfuls onto clean cover glasses. Do not spread the loopfuls but let them dry on the several spots where they were placed. The best flagella are usually found near the edges of the drops. Air dry, and pass once through the flame. Cover with freshly mixed mordant \/*> to 1 minute. Wash and stain with carbol f uchsin 1/2 to 1 minute. Wash, dry, and mount. Sporangia and endospores. Determinations on these are usually made from watery fuchsin stains from week-old cultures. Sometimes, however, it is necessary to use younger cultures in order to find sporangia. AGAR STROKE Agar medium is placed in test tubes and cooled in a slanting position. This leaves a large surface. Inoculations are made in one streak the entire length of the slanted surface, care being exercised not to break down into the medium. For comparison of growth at different temperatures two tubes are inoculated from each culture. These are kept at 20 °C. and 37 °C. respectively. De- scriptions are made at 7 and 14 days. GELATIN STAB Gelatin is used in tubes of about 10 mm. diameter. The depth is about 4 cm. Inoculation is made with a straight needle stabbing once in the center to the bottom of the tube. They are watched to determine when liquefaction begins and is complete as well as for the form of both liquefiers and non-liquefiers. Cultures are kept 30 days. POTATO CULTURES Potatoes cut in cylindrical pieces with a slanted surface are sterilized in tubes having moistened cotton on the bottom to prevent drying out of the potato. Inoculation is made in a streak up the slanted surface. After incubation description is made in the same manner and using the same terms as for agar slants. Records 37 Laboratory Outline for General Bacteriology are made on the chart in the blank space with heading "Medium solid." NUTRIENT BROTH Nutrient broth is made from beef extract, peptone, and distilled water. After inoculation the nutrient broth cultures must be handled carefully to avoid shaking down any pellicle or surface growth that may be present. After recording surface growth and clouding, which may be distinguished better by comparison with an uninoculated tube, the character of the sediment may be deter- mined by giving the tube a whirling motion and observing. AGAR PLATES FOR COLONY DESCRIPTIONS AND SUNLIGHT TEST Inoculate a water blank with a small amount of culture taken from the original agar slants. Shake, and with a sterile loop trans- fer once from water blank to a tube of melted agar. Shake agar without having medium touch the cotton plug. Sterilize loop and make two transfers from first agar tube to a second. Pour both agar tubes into plates marking them "a" and "b" in addition to the culture number. When cool paste a thick opaque paper over one-half the bottom of each plate. This is to shut off the light. Expose plates to the sunlight for fifteen minutes, watching to see no shadows creep over them. Place in locker to incubate. After seven days make descriptions of both surface and deep colonies from the plate that shows the most favorable number of bacteria. To determine the percentage of bacteria killed by sunlight count an equal area on both the shaded and sun exposed portion of the Petri dish. Subtract the number of bacteria in sun exposed area from the number in the shaded area and divide by the number of bacteria in the shaded area. Example. Number of bacteria in shaded area 28, number in sun exposed are 12. 28—12=16 16-28=57.1 The result is approximately 57 per cent. GELATIN PLATES These are made in the same manner as the agar plates but are not exposed to sunlight. Incubate in the locker right side up. Liquefying bacteria must be watched carefully to get descriptions before the gelatin is entirely liquefied. NITRATE BROTH Nitrate broth is a medium made by using small amounts of peptone and potassium nitrate in tap water. After cultures have 38 Laboratory Outline for General Bacteriology grown for the required length of time tests are made as follows: With sterile pipette remove 2 cc. of culture to a clean test tube. Add 1 cc. of test solution which is made up of naphthylamin, acetic acid, sulphanilic acid and water. A pink color shows the presence of nitrite. Test an uninoculated tube in the same manner for a con- trol. Observations are made for gas bubbles rising in the nitrate broth cultures. NITRATE AGAR Nitrate agar is the same as standard agar with .1 per cent KNO . added. It is used in slants, which are inoculated by making both a streak on the surface and a stab to the bottom of tube. The test solution is poured over the surface of the medium. Gas pro- duction is shown by cracks in the agar. HYDROGEN SULPHIDE A beef extract agar very rich in peptone and containing lead acetate is used in tubes. This is inoculated by stabbing with a straight needle. A darkening of the medium within a few days shows that hydrogen sulfide has been produced. INDOL PRODUCTION Cultures are grown in Dunham's peptone solution. It is com- posed of peptone, sodium chloride and distilled water. After four days the Ehrlich test is made as follows: Add to the culture 1 cc. of a 2' '< solution of paradimethylaminobenzaldehyde in 95 ' < alcohol. Then add drop by drop y» cc. of concentrated HC1. If indol is pres- ent a pink zone appears which deepens and widens on standing. The test may be confirmed by shaking the culture with chloroform to see if the pigment dissolves. If it proves soluble the test is considered positive. FERMENTATION TUBES The medium is nutrient broth with one per cent of the different sugars added. It is put into tubes in which are small inverted inner tubes which are also filled with the medium. If gas is formed some of it will collect in the inner tube and the percentage can be measured by the gasometer chart shown on page 41. If the bacteria grow only within the inner tube (this is shown by cloudi- ness or sediment) it shows that they can grow only in the absence of oxygen and are anaerobic. When growth occurs only outside the small tube it shows that the bacteria require oxygen and are aerobic. When growth occurs both inside and outside the inner 39 Laboratory Outline for General Bacteriology tube it is called facultative anaerobic. Observations on growth and tests for alkalinity or acidity are made usually at 2, 7, and 14 days. STARCH AGAR Starch agar is like the ordinary agar with .2(/< of soluble starch added. It may be poured hot into plates. When cool it is inocu- lated by making one streak across the center. Do not let the loop cut down into the agar. Invert to incubate. After the culture has grown the required length of time, usually 7 days, it is covered with a solution of iodine. If the starch is unchanged by the bacteria the entire plate will turn blue. When diastase is present a clear zone will appear along the line of inocu- lation. The width of the clear zone shows the intensity of the diastatic action. This is measured in millimeters from the edge of the growth to the edge of the blue. Records should be made at once as the blue reaction disappears within a few minutes. MILK AND LITMUS MILK Fresh skimmed milk is tubed and sterilized. For the litmus milk enough litmus solution is added to give it a distinctly blue color. In testing reaction of milk sterilize loop and take a loopful out onto litmus paper. If it is necessary to take a second loopful sterilize loop before taking it to avoid contaminating the culture. Test an uninoculated tube at the same time for comparison. Fresh milk has what is called an amphoteric reaction, that is, it turns red litmus slightly blue and blue litmus slightly red. If bacteria have produced a change of reaction litmus paper will be turned redder or bluer than the control. Milk and litmus milk tubes from the same culture should show the same reaction. Coagulation is when milk becomes thick like sour milk. Pep- tonizatiori is shown by a clearing or whey like appearance due to destruction of the casein. It generally begins at the surface and works downward. The milk may or may not be curdled. Sometimes when a hard curd is formed whey is pressed out. This is called "extrusion of whey" and can usually be distinguished from pep- tonization by the clearness of the whey. Reduction refers to the destruction of litmus color. When complete the litmus milk looks like the plain milk. Reduction usually begins at the bottom of the tube and works upward. Sometimes the color becomes pale through- out the entire tube. A change of color to red or blue indicates change of reaction not reduction. 40 Laboratory Outline for General Bacteriology THERMAL DEATH POINT The thermal death point of an organism is customarily defined as the least temperature that will destroy it in ten minutes under known conditions. Several factors influence the thermal death point. Old cultures are less resistant than younger ones. An acid medium renders heat much more effective. Moist heat is much more efficient than dry heat. For comparative work it is necessary to use media of uniform reaction and composition. The presence of spores indicates that the organism has two thermal death points ; one for the spores and another lower one for the vegetative cells. The medium recommended by the Committee on Identification of Species of the Society of American Bacteriologists, is nutrient broth. Several tubes are inoculated then heated in water baths at different temperatures ranging usually from 50 °C to 70 °C for 10 minutes. They are then incubated to see if growth takes place. Fermentation Tube Chart PLATE 2. Laboratory Outline for General Bacteriology DESCRIPTIONS OF VARIOUS SPECIES OF BACTERIA The need has long been felt of having for student use a variety of bacterial cultures that have been so carefully worked through that their characteristics are known. With this in view named cultures have been obtained from the American Museum of Natural History in New York and from various other laboratories as well as from our own. ' These have been carefully worked through, in most cases several timesr and by different persons. Work on doubt- ful points has been repeated many times. Anyone familiar with similar work will understand the diffi- culties encountered, such as changes in the cultures due to pro- longed cultivation on artificial media, differences in appearances of cultures and in reactions due to differences in materials, especially peptone, from which the culture media are made, differences due to personal equation, etc. We have retained the names of bacteria as they were sent to us even when in a few cases it is believed they were named incor- rectly. In the condensed form in which the descriptions are tabulated we have not found it practical to always give all the characteristic points and details in regard to a culture. The main points, how- ever, are given in the majority of cases. The rii ire In the ina*. Number, wnioh refers to pathogen! city, has been omitted from these charts. 42 J. .- .J. .^ .i 0! 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