List of Laboratory Exercises Given in Connection with the Class Work Included in this Text-book.

DESCRIPTIVE CHART—SOCIETY OF AMERICAN BACTERIOLOGISTS.

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 20 mm.) 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.

A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM. (GROUP NUMBER.)
100.0000000 Endospores produced
200.0000000 Endospores not produced
10.0000000 Aërobic (strict)
20.0000000 Facultative anaërobic
30.0000000 Anaërobic (strict)
1.0000000 Gelatin liquefied
2.0000000 Gelatin not liquefied
0.1000000 Acid and gas from dextrose
0.2000000 Acid without gas from dextrose
0.3000000 No acid from dextrose
0.4000000 No growth with dextrose
0.0100000 Acid and gas from lactose
0.0200000 Acid without gas from lactose
0.0300000 No acid from lactose
0.0400000 No growth with lactose
0.0010000 Acid and gas from saccharose
0.0020000 Acid without gas from saccharose
0.0030000 No acid from saccharose
0.0040000 No growth with saccharose
0.0001000 Nitrates reduced with evolution of gas
0.0002000 Nitrates not reduced
0.0003000 Nitrates reduced without gas formation
0.0000100 Fluorescent
0.0000200 Violet chromogens
0.0000300 Blue chromogens
0.0000400 Green chromogens
0.0000500 Yellow chromogens
0.0000600 Orange chromogens
0.0000700 Red chromogens
0.0000800 Brown chromogens
0.0000900 Pink chromogens
0.0000000 Non-chromogenics
0.0000010 Diastasic action on potato starch, strong
0.0000020 Diastasic action on potato starch, feeble
0.0000030 Diastasic action on potato starch, absent
0.0000001 Acid and gas from glycerin
0.0000002 Acid without gas from glycerin
0.0000003 No acid from glycerin
0.0000004 No growth with glycerin

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

Bacillus coli (Esch.) Mig. becomes B. 222.111102
Bacillus alcaligenes Petr. becomes B. 212.333102
Pseudomonas campestris (Pam.) Sm. becomes Ps. 211.333151
Bacterium suicida Mig. becomes Bact. 222.232103

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.

BRIEF CHARACTERIZATION.
Mark + or 0, and when two terms occur on a line erase the one which does not apply unless both apply.
MORPHOLOGY(2) Diameter over 1µ  
Chains, filaments  
Endospores  
Capsules  
Zooglea, Pseudozooglea  
Motile  
Involution forms  
  Gram’s stain  
CULTURAL FEATURES(3) Broth Cloudy, turbid  
Ring  
Pellicle  
Sediment  
Agar Shining  
Dull  
Wrinkled  
Chromogenic  
Gel. Plate Round  
Proteus-like  
Rhizoid  
Filamentous  
Curled  
Gel. Stab. Surface growth  
Needle growth  
Potato Moderate, absent  
Abundant  
Discolored  
Starch destroyed  
Grows at 37° C.  
Grows in Cohn’s sol.  
Grows in Uschinsky’s sol.  
BIOCHEMICAL FEATURES Liquifaction Gelatin(4)  
Blood-serum  
Casein  
Milk Acid curd  
Rennet curd  
Casein peptonized  
Indol(3)  
Hydrogen sulphide  
Ammonia(3)  
Nitrates reduced(3)  
Fluorescent  
Luminous  
DISTRIBUTION Animal pathogen, epizoon  
Plant pathogen, epiphyte  
Soil  
Milk  
Fresh water  
Salt water  
Sewage  
Iron bacterium  
Sulphur bacterium  

FOOTNOTES.

1 Sir H. A. Blake has called attention to the fact that the “mosquito theory” of malaria is mentioned in a Sanscrit manuscript of about the 6th century A.D.↩

2 Myxomycetes excepted, and they are probably to be regarded as animals—Mycetozoa. ↩

3 Centralblatt f. Bakteriologie, etc. LXIII. 1 Abt. Orig. 1912, 4, idem LXVI. 1 Abt. Orig. 1912, 323. ↩

4 The pronunciation of this word according to English standards is kok-si; the continental pronunciation is kok-kee; the commonest American seems to be kok-ki. We prefer the latter since it is easier and more natural and should like to see it adopted. (Author.) ↩

5 With the possible exception of blue green algæ which have been found with bacteria in the above-mentioned hot springs. Seeds of many plants have been subjected to as low temperatures as those above-mentioned without apparent injury. ↩

6 It is popularly supposed that in canning fruit, vegetables, meats, etc., all the air must be removed, since the organisms which cause “spoiling” cannot grow in a vacuum. The existence of anaërobic and facultative anaërobic bacteria shows the fallacy of such beliefs. ↩

7 “By cellulose is understood a carbohydrate of the general formula C6H10O5 not soluble in water, alcohol, ether, or dilute acids but soluble in an ammoniacal solution of copper oxide. It gives with iodine and sulphuric acid a blue color and with iodine zinc chloride a violet and yields dextrose on hydrolysis.”—H. Fischer. ↩

8 The sulphur bacteria are partially prototrophic for S; probably the iron bacteria also for Fe. Some few soil bacteria have been shown to be capable of utilizing free H, and it seems certain that the bacteria associated with the spontaneous heating of coal may oxidize free C. So far as known no elements other than these six are directly available to bacteria. ↩

9 Only a few kinds of bacteria so far as known are proto-autotrophic. The nitrous and nitric organisms of Winogradsky which are so essential in the soil, and which might have been the first of all organisms so far as their food is concerned, and some of the sulphur bacteria are examples. ↩

10 The term pathogenic is also applied to certain non-parasitic saprophytic bacteria whose products cause disease conditions, as one of the organisms causing a type of food poisoning in man (Clostridium botulinum), which also probably causes “forage poisoning” in domestic animals. ↩

11 The term “fermentation” was originally used to denote the process which goes on in fruit juices or grain extracts when alcohol and gas are formed. Later it was extended to apply to the decomposition of almost any organic substance. In recent years the attempt has been made to give a chemical definition to the word by restricting its use to those changes in which by virtue of a “wandering” or rearrangement of the carbon atoms “new substances are formed which are not constitutents of the original molecule.” It may be doubted whether this restriction is justified or necessary. A definition is at present scarcely possible except when the qualifying adjective is included as “alcoholic fermentation,” “ammoniacal fermentation,” “lactic acid fermentation,” etc. ↩

12 See “Oil and Gas in Ohio,” Bownocker: Geological Survey of Ohio, Fourth Series, Bull. I, pp. 313–314. ↩

13 It is probable that this is the way “Jack o’lanterns” or “Will o’ the wisps” are ignited. Marsh gas is produced as above outlined from the vegetable and animal matter decomposing in swampy places under anaërobic conditions and likewise phosphine. These escape into the air and the “spontaneous combustion” of the phosphine ignites the marsh gas. ↩

14 Dr. H. H. Green, of Pretoria, South Africa, has isolated from “cattle dips” a bacterium that reduces arsenates to arsenites. ↩

15 Dr. Green (l. c.) has also isolated an organism which causes some deterioration of cattle dips by oxidizing arsenites to arsenates. ↩

16 It will be noted that the names of enzymes (except some of those first discovered) terminate in ase which is usually added to the stem of the name of the substance acted on, though sometimes to a word which indicates the substance formed by the action, as lactacidase, alcoholase. ↩

17 Tetanus toxin is about 120 times as poisonous as strychnin, both of which act on the same kind of nerve cells. ↩

18 In the author’s laboratory in the past ten years all sterilization except those few objects in blood and serum work which must be dry, has been done in autoclaves of the type shown in Fig. 81 which are supplied with steam from the University central heating plant. A very great saving of time is thus secured. ↩

19 The author has tested an “electric milk purifier” (Fig. 102) which was as efficient as a first-class pasteurizer and left the milk in excellent condition both chemically and as far as “cream line” was concerned. The cost of operation as compared with steam will depend on the price of electricity. ↩

20 The exact laboratory details for preparing various media are not given in this chapter. It is the object to explain the choice of different materials and the reasons for the various processes to which they are subjected. ↩

21 For a discussion of this method of standardization consult the following:

Additional references will be found in these articles. ↩

22 Term also applied to the solidification of serum in media: e.g., the Hiss inulin medium for the differentiation of pneumococci (see diplococcus of pneumonia). ↩

23 The term “antigen” is also used to designate substances which may take the place of what are supposed to be the true antigens in certain diagnostic reactions (Chapter XXIX, Complement Fixation Test for Syphilis). ↩

24 If the antitoxin is later concentrated (see last paragraph in this chapter) a serum containing as little as 175 units per cc. may be commercially profitable. ↩

25 Tho term “allergie” was introduced by Von Pirquet to designate the state of the animal’s being sensitized and “allergic” as the adjective derived therefrom. It does not seem to the author that there is any advantage gained by the introduction of these terms. ↩