Prepared by Committee on Methods of Identification of Bacterial Species.—F.D. Chester, F.P. Gorham, Erwin F. Smith.

Endorsed by the Society for general use at the Annual Meeting, December, 1907.

GLOSSARY OF TERMS.

AGAR HANGING BLOCK,

a small block of nutrient agar cut from a pour plate, and placed on a cover-glass, the surface next the glass having been first touched with a loop from a young fluid culture or with a dilution from the same. It is examined upside down, the same as a hanging drop.

AMEBOID,

assuming various shapes like an ameba.

AMORPHOUS,

without visible differentiation in structure.

ARBORESCENT,

a branched, tree-like growth.

BEADED,

in stab or stroke, disjointed or semiconfluent colonies along the lines of inoculation.

BRIEF,

a few days, a week.

BRITTLE,

growth dry, friable under the platinum needle.

BULLATE,

growth rising in convex prominences, like a blistered surface.

BUTYROUS,

growth of a butter-like consistency.

CHAINS,

Short chains, composed of 2 to 8 elements.

Long chains, composed of more than 8 elements.

CILIATE,

having fine, hair-like extensions, like cilia.

CLOUDY,

said of fluid cultures which do not contain pseudozoogleÆ.

COAGULATION,22

the separation of casein from whey in milk. This may take place quickly or slowly, and as the result either of the formation of an acid or of a lab ferment.

CONTOURED,

an irregular, smoothly undulating surface, like that of a relief map.

CONVEX surface,

the segment of a circle, but flattened.

COPROPHYL,

dung bacteria.

CORIACEOUS,

growth tough, leathery, not yielding to the platinum needle.

CRATERIFORM,

round, depressed, due to the liquefaction of the medium.

CRETACEOUS,

growth opaque and white, chalky.

CURLED,

composed of parallel chains in wavy strands, as in anthrax colonies.

DIASTASIC ACTION,

same as DIASTATIC, conversion of starch into water-soluble substances by diastase.

ECHINULATE,

in agar stroke a growth along line of inoculation, with toothed or pointed margins; in stab cultures growth beset with pointed outgrowths.

EFFUSE,

growth thin, veily, unusually spreading.

ENTIRE,

smooth, having a margin destitute of teeth or notches.

EROSE,

border irregularly toothed.

FILAMENTOUS,

growth composed of long, irregularly placed or interwoven filaments.

FILIFORM,

in stroke or stab cultures a uniform growth along line of inoculation.

FIMBRIATE,

border fringed with slender processes, larger than filaments.

FLOCCOSE,

growth composed of short curved chains, variously oriented.

FLOCCULENT,

said of fluids which contain pseudozoogleÆ, i.e., small adherent masses of bacteria of various shapes and floating in the culture fluid.

FLUORESCENT,

having one color by transmitted light and another by reflected light.

GRAM’S STAIN,

a method of differential bleaching after gentian violet, methyl violet, etc. The + mark is to be given only when the bacteria are deep blue or remain blue after counter-staining with Bismarck brown.

GRUMOSE,

clotted.

INFUNDIBULIFORM,

form of a funnel or inverted cone.

IRIDESCENT,

like mother-of-pearl. The effect of very thin films.

LACERATE,

having the margin cut into irregular segments as if torn.

LOBATE,

border deeply undulate, producing lobes (see Undulate).

LONG,

many weeks, or months.

MAXIMUM TEMPERATURE,

temperature above which growth does not take place.

MEDIUM,

nutrient substance upon which bacteria are grown.

MEMBRANOUS,

growth thin, coherent, like a membrane.

MINIMUM TEMPERATURE,

temperature below which growth does not take place.

MYCELIOID,

colonies having the radiately filamentous appearance of mold colonies.

NAPIFORM,

liquefaction with the form of a turnip.

NITROGEN REQUIREMENTS,

the necessary nitrogenous food. This is determined by adding to nitrogen-free media the nitrogen compound to be tested.

OPALESCENT,

resembling the color of an opal.

OPTIMUM TEMPERATURE,

temperature at which growth is most rapid.

PELLICLE,

in fluid bacterial growth forming either a continuous or an interrupted sheet over the fluid.

PEPTONIZED,

said of curds dissolved by trypsin.

PERSISTENT,

many weeks, or months.

PLUMOSE,

a fleecy or feathery growth.

PSEUDOZOOGLEÆ,

clumps of bacteria, not dissolving readily in water, arising from imperfect separation, or more or less fusion of the components, but not having the degree of compactness and gelatinization seen in zoogleÆ.

PULVINATE,

in the form of a cushion, decidedly convex.

PUNCTIFORM,

very minute colonies, at the limit of natural vision.

RAPID,

developing in twenty-four to forty-eight hours.

RAISED,

growth thick, with abrupt or terraced edges.

RHIZOID,

growth of an irregular branched or root-like character, as in B. mycoides.

RING,

same as RIM, growth at the upper margin of a liquid culture, adhering more or less closely to the glass.

REPAND,

wrinkled.

SACCATE,

liquefaction the shape of an elongated sac, tubular, cylindrical.

SCUM,

floating islands of bacteria, an interrupted pellicle or bacteria membrane.

SLOW,

requiring five or six days or more for development.

SHORT,

applied to time, a few days, a week.

SPORANGIA,

cells containing endospores.

SPREADING,

growth extending much beyond the line of inoculation, i.e., several millimetres or more.

STRATIFORM,

liquefying to the walls of the tube at the top and then proceeding downward horizontally.

THERMAL DEATH-POINT,

the degree of heat required to kill young fluid cultures of an organism exposed for ten minutes (in thin-walled test-tubes of a diameter not exceeding 20mm.) in the thermal water-bath. The water must be kept agitated so that the temperature shall be uniform during the exposure.

TRANSIENT,

a few days.

TURBID,

cloudy with flocculent particles; cloudy plus flocculence.

UMBONATE,

having a button-like, raised centre.

UNDULATE,

border wavy, with shallow sinuses.

VERRUCOSE,

growth wart-like, with wart-like prominences.

VERMIFORM-CONTOURED,

growth like a mass of worms or intestinal coils.

VILLOUS,

growth beset with hair-like extensions.

VISCID,

growth follows the needle when touched and withdrawn, sediment on shaking rises as a coherent swirl.

ZOOGLEÆ,

firm gelatinous masses of bacteria, one of the most typical examples of which is the Streptococcus mesenterioides of sugar vats. (Leuconostoc mesenterioides), the bacterial chains being surrounded by an enormously thickened, firm covering inside of which there may be one or many groups of the bacteria.

NOTES.

(1) For decimal system of group numbers see Table I. This will be found useful as a quick method of showing close relationships inside the genus, but is not a sufficient characterization of any organism.

(2) The morphological characters shall be determined and described from growths obtained upon at least one solid medium (nutrient agar) and in at least one liquid medium (nutrient broth). Growths at 37°C. shall be in general not older than twenty-four to forty-eight hours, and growths at 20°C. not older than forty-eight to seventy-two hours. To secure uniformity in cultures, in all cases preliminary cultivation shall be practised as described in the revised Report of the Committee on Standard Methods of the Laboratory Section of the American Public Health Association, 1905.

(3) The observation of cultural and biochemical features shall cover a period of at least fifteen days and frequently longer, and shall be made according to the revised Standard Methods above referred to. All media shall be made according to the same Standard Methods.

(4) Gelatin stab cultures shall be held for six weeks to determine liquefaction.

(5) Ammonia and indol tests shall be made at end of tenth day, nitrite tests at end of fifth day.

(6) Titrate with N/20 NaOH, using phenolphthalein as an indicator; make titrations at same time from blank. The difference gives the amount of acid produced.

The titration should be done after boiling to drive off any CO₂ present in the culture.

(7) Generic nomenclature shall begin with the year 1872 (Cohn’s first important paper).

Species nomenclature shall begin with the year 1880 (Koch’s discovery of the pour plate method for the separation of organisms).

(8) Chromogenesis shall be recorded in standard color terms.

TABLE I.

The genus according to the system of Migula is given its proper symbol which precedes the number thus:(7)

Source..................................... Date of Isolation.................... Name..................................... Group No.(1)...............

DETAILED FEATURES.

NOTE—Underscore required terms. Observe notes and glossary of terms on opposite side of card.

• I. MORPHOLOGY(2)
  • 1. Vegetative Cells,
    • Medium used............................. temp....................age.................days
    • Form, round, short rods, long rods, short chains, long chains, filaments, commas, short spirals, long spirals, clostridium, cuneate, clavate, curved.
    • Limits of Size.............................
    • Size of Majority.............................
    • Ends, rounded, truncate, concave.

    • Agar Hanging-block	Orientation (grouping)...............
      	Chains (No. of elements)...............
      	Short chains, long chains
      	Orientation of chains, parallel, irregular.

  • 2. Sporangia,
    • medium used............................. temp....................age....................days
    • Form, elliptical, short rods, spindled, clavate, drumsticks.
    • Limits of Size.............................
    • Size of Majority.............................

    • Agar Hanging-block	Orientation (grouping)...............
      	Chains (No. of elements)...............
      	Orientation of chains, parallel, irregular.

    • Location of Endospores, central, polar.
  • 3. Endospores.
    • Form, round, elliptical, elongated.
    • Limits of Size.............................
    • Size of Majority.............................
    • Wall, thick, thin.
    • Sporangium wall, adherent, not adherent.
    • Germination, equatorial, oblique, polar, bipolar, by stretching.
  • 4. Flagella, No...............Attachment polar, bipolar, peritrichiate. How Stained...............
  • 5. Capsules, present on...............
  • 6. Zooglea, Pseudozooglea.
  • 7. Involution Forms, on............in............days at............°C.
  • 8. Staining Reactions.
    • 1:10 watery fuchsin, gentian violet, carbol-fuchsin, Loeffler’s alkaline methylene blue.
    • Special Stains.
      • Gram........................
      • Glycogen........................
      • Fat........................
      • Acid-fast........................
      • Neisser........................
• II. CULTURAL FEATURES(3)
  • 1. Agar Stroke.
    • Growth, invisible, scanty, moderate, abundant.
    • Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid.
    • Elevation of growth, flat, effuse, raised, convex.
    • Lustre, glistening, dull, cretaceous.
    • Topography, smooth, contoured, rugose, verrucose.
    • Optical characters, opaque, translucent, opalescent, iridescent.
    • Chromogenesis(3)........................
    • Odor, absent, decided, resembling........................
    • Consistency, slimy, butyrous, viscid, membranous, coriaceous, brittle.
    • Medium grayed, browned, reddened, blued, greened.
  • 2. Potato.
    • Growth scanty, moderate, abundant, transient, persistent.
    • Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid.
    • Elevation of growth, flat, effuse, raised, convex.
    • Lustre, glistening, dull, cretaceous.
    • Topography, smooth, contoured, rugose, verrucose.
    • Chromogenesis(3)........................Pigment in water insoluble, soluble: other solvents................................................
    • Odor, absent, decided, resembling........................
    • Consistency, slimy, butyrous, viscid, membranous, coriaceous, brittle.
    • Medium, grayed, browned, reddened, blued, greened.
  • 3. Loeffler’s Blood-serum.
    • Stroke invisible, scanty, moderate, abundant.
    • Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid.
    • Elevation of growth, flat, effuse, raised, convex.
    • Lustre, glistening, dull, cretaceous.
    • Topography, smooth, contoured, rugose, verrucose.
    • Chromogenesis(3)..........................
    • Medium grayed, browned, reddened, blued, greened.
    • Liquefaction begins in...............d, complete in...............d,
  • 4. Agar Stab.
    • Growth uniform, best at top, best at bottom: surface growth scanty, abundant: restricted, wide-spread.
    • Line of puncture, filiform, beaded, papillate, villous, plumose, arborescent: liquefaction.
  • 5. Gelatin Stab.
    • Growth uniform, best at top, best at bottom.
    • Line of puncture, filiform, beaded, papillate, villous, plumose, arborescent.
    • Liquefaction crateriform, napiform, infundibuliform, saccate, stratiform: begins in...............d. complete in...............d
    • Medium fluorescent, browned...............
  • 6. Nutrient Broth.
    • Surface growth, ring, pellicle, flocculent, membranous, none.
    • Clouding slight, moderate, strong: transient, persistent: none: fluid turbid.
    • Odor, absent, decided, resembling..................
    • Sediment, compact, flocculent, granular, flaky, viscid on agitation, abundant, scant.
  • 7. Milk.
    • Clearing without coagulation.
    • Coagulation prompt, delayed, absent.
    • Extrusion of whey begins in................days.
    • Coagulum slowly peptonized, rapidly peptonized.
    • Peptonization begins on...............d, complete on...............d.
    • Reaction, 1d......, 2d......, 4d......, 10d......, 20d......
    • Consistency, slimy, viscid, unchanged.
    • Medium browned, reddened, blued, greened.
    • Lab ferment, present, absent.
  • 8. Litmus Milk.
    • Acid, alkaline, acid then alkaline, no change.
    • Prompt reduction, no reduction, partial slow reduction.
  • 9. Gelatin Colonies.
    • Growth slow, rapid.
    • Form, punctiform, round, irregular, ameboid, mycelioid, filamentous, rhizoid.
    • Elevation, flat, effuse, raised, convex, pulvinate, crateriform (liquefying).
    • Edge, entire, undulate, lobate, erose, lacerate, fimbriate, filamentous, floccose, curled.
    • Liquefaction, cup, saucer, spreading.
  • 10. Agar Colonies.
    • Growth slow, rapid (temperature................)
    • Form, punctiform, round, irregular, ameboid, mycelioid, filamentous, rhizoid.
    • Surface smooth, rough, concentrically ringed, radiate, striate.
    • Elevation, flat, effuse, raised, convex, pulvinate, umbonate.
    • Edge, entire, undulate, lobate, erose, lacerate, fimbriate, floccose, curled.
    • Internal structure, amorphous, finely, coarsely granular, grumose, filamentous, floccose, curled.
  • 11. Starch Jelly.
    • Growth, scanty, copious.
    • Diastatic action, absent, feeble, profound.
    • Medium stained.......................
  • 12. Silicate Jelly (Fermi’s Solution).
    • Growth copious, scanty, absent.
    • Medium stained.......................
  • 13. Cohn’s Solution.
    • Growth copious, scanty, absent.
    • Medium fluorescent, non-fluorescent.
  • 14. Uschinsky’s Solution.
    • Growth copious, scanty, absent.
    • Fluid viscid, not viscid.
  • 15. Sodium Chloride in Bouillon.
    • Per cent. inhibiting growth........................
  • 16. Growth in Bouillon over Chloroform, unrestrained, feeble, absent.
  • 17. Nitrogen. Obtained from peptone, asparagin, glycocoll, urea, ammonia salts, nitrogen.
  • 18. Best media for long-continued growth
    ..............................................................................................
  • 19. Quick tests for differential purposes
    ..............................................................................................
    ..........................................................................................
• III. PHYSICAL AND BIOCHEMICAL FEATURES.

  • 1. Fermentation-tubes containing peptone-water or sugar-tree bouillon and	Dextrose	Saccharose	Lactose	Maltose	Glycerin	Mannit
    Gas production, in per cent.
    (H/CO2)
    Growth in closed arm
    Amount of acid produced 1d.
    Amount of acid produced 2d.
    Amount of acid produced 3d.

  • 2. Ammonia production, feeble, moderate, strong, absent, masked by acids.
  • 3. Nitrates in nitrate broth.
    • Reduced, not reduced.
    • Presence of nitrites..................ammonia..................
    • Presence of nitrates..................free nitrogen..................
  • 4. Indol production, feeble, moderate, strong.
  • 5. Toleration of Acids, great, medium, slight.
    • Acids tested..................
  • 6. Toleration of NaOH, great, medium, slight.
  • 7. Optimum reaction for growth in bouillon, stated in terms of Fuller’s scale..........................
  • 8. Vitality on culture media, brief, moderate, long.
  • 9. Temperature relations.
    • Thermal death-point (10 minutes’ exposure in nutrient broth when this is adapted to growth of organism)..........C.
    • Optimum temperature for growth..........°C.; or best growth at 16°C., 20°C., 25°C., 30°C., 37°C., 40°C., 50°C., 60°C.
    • Maximum temperature for growth..........°C.
    • Minimum temperature for growth..........°C.
  • 10. Killed readily by drying: resistant to drying.
  • 11. Per cent. killed by freezing (salt and crushed ice or liquid air)................
  • 12. Sunlight: Exposure on ice in thinly sown agar plates; one-half plate covered (time 15 minutes), sensitive, not sensitive.
    • Per cent. killed................
  • 13. Acids produced................
  • 14. Alkalies produced................
  • 15. Alcohols................
  • 16. Ferments, pepsin, trypsin, diastase, invertase, pectase, cytase, tyrosinase, oxidase, peroxidase, lipase, catalase, glucase, galactase, lab, etc.........................
  • 17. Crystals formed:.........
  • 18. Effect of germicides:

    Substance	Method used	Minutes	Temperature	Killing quantity	Amt. required to restrain growth

• IV. PATHOGENICITY.
  • 1. Pathogenic to Animals.
    • Insects, crustaceans, fishes, reptiles, birds, mice, rats, guinea-pigs, rabbits, dogs, cats, sheep, goats, cattle, horses, monkeys, man..........................
  • 2. Pathogenic to Plants:
    .............................................................
    .........................................................
    .........................................................
  • 3. Toxins, soluble, endotoxins.
  • 4. Non-toxin forming.
  • 5. Immunity bactericidal.
  • 6. Immunity non-bactericidal.
  • 7. Loss of virulence on culture-media: prompt, gradual, not observed in.....................months.