Before the introduction of solid media the only method of ob_The elements of bacteriological technique

cultivations was by "dilution"--by no means a reliable method.

"Dilution" consisted in estimating approximately the number of bacteria

present in a given volume of fluid (by means of a graduated-celled slide

resembling a haematocytometer, Fig. 138), and diluting the fluid by the

addition of sterile water or bouillon until a given volume (usually 1

c.c.) of the dilution contained but one organism. By planting this

volume of the fluid into several tubes or flasks of nutrient media, it

occasionally happened that the resulting growth was the product of one

individual microbe. A method so uncertain is now fortunately replaced by

many others, more reliable and convenient, and in those methods selected

for description here, the segregation and isolation of the required

bacteria may be effected--

A. ~By Mechanical Separation.~

1. By surface plate cultivation:

(a) Gelatine.

(b) Agar.

(d) Blood agar.

(e) Hanging-drop or block.

[2. By Esmarch's roll cultivation:

This archaic method (see page 226) is no longer employed for the

isolation of bacteria.]

3. By serial cultivation.

B. ~By Biological Differentiation.~

4. By differential media.

(a) Selective.

(b) Deterrent.

5. By differential incubation.

6. By differential sterilisation.

7. By differential atmosphere cultivation.

8. By animal inoculation.

The selection of the method to be employed in any specific instance will

depend upon a variety of circumstances, and often a combination of two

or more will ensure a quicker and more reliable result than a rigid

adherence to any one method. Experience is the only reliable guide, but

as a general rule the use of either the first or the third method will

be found most convenient, affording as each of them does an opportunity

for the simultaneous isolation of several or all of the varieties of

bacteria present in a mixture.

~1. Surface Plate Cultivations.~--

(a) _Gelatine_ (_vide_ page 164).

(b) _Agar_ (_vide_ page 167).

These plates are prepared in a manner precisely similar to that adopted

for nutrient gelatine and agar surface plates (_vide_ pages 231-233).

(d) _Serum Agar._--

1. Melt three tubes of nutrient agar, label them 1, 2, and 3, and place

them, with three tubes of sterile fluid serum, also labelled 1a, 2a,

and 3a, in a water-bath regulated at 45 deg. C.; allow sufficient time to

elapse for the temperature of the contents of each tube to reach that of

the water-bath.

2. Take serum tube No. 1a and agar tube No. 1. Flame the plugs and

remove them from the tubes (retaining the plug of the agar tube in the

hand); flame the mouths of the tubes, pour the serum into the tube of

liquefied agar and replace the plug of the agar tube.

3. Mix thoroughly and pour plate No. 1 _secundum artem_.

4. Treat the remaining tube of agar and serum in a similar fashion, and

pour plates Nos. 2 and 3.

5. Dry the serum agar plates in the incubator running at 60 deg. C. for

one hour (see page 232).

6. Inoculate the plates in series as described for gelatine surface

plates (page 231).

(e) _Blood Agar, Human._--

1. Melt a tube of sterile agar and pour it into a sterile plate; let it

set.

2. Collect a few drops of human blood, under all aseptic conditions, in

a sterile capillary teat pipette.

3. Raise the cover of the Petri dish very slightly, insert the extremity

of the capillary pipette, and deposit the blood on the centre of the

agar surface. Close the dish.

4. Charge a platinum loop with a small quantity of the inoculum. Raise

the cover of the plate, introduce the loop, mix its contents with the

drop of blood, remove the loop, close the dish and sterilise the loop.

5. Finally smear the mixture over the surface of the agar with a

sterilised L-shaped rod.

6. Label and incubate.

(If considered necessary, two, three, or more similar plates may be

inoculated in series.)

(f) _Blood Agar, Animal._--

When preparing citrated blood agar (page 171) it is always advisable to

pour several blood agar tubes into plates, which can be stored in the

ice chest ready for use at any moment for surface plate cultures.

(g) Hanging-drop or block culture, (_vide_ page 233).

~3. Serial Cultivations.~--These are usually made upon agar or

blood-serum, although gelatine may also be used.

The method is as follows:

1. Take at least four "slanted" tubes of media and number them

consecutively.

2. Flame all the plugs and see that each can be readily removed.

3. Charge the platinum loop with a small quantity of the inoculum,

observing the usual routine, and plant tube No. 1, smearing thoroughly

all over the surface. If any water of condensation has collected at the

bottom of the tube, use this as a diluent before smearing the contents

of the loop over the surface of the medium.

4. Without sterilising or recharging the loop, inoculate tube No. 2, by

making three parallel streaks from end to end of the slanted surface.

5. Plant the remainder of the tubes in the series as "smears" like tube

No. 1.

6. Label with distinctive name or number, and date; incubate.

The growth that ensues in the first two or three tubes of the series

will probably be so crowded as to be useless. Toward the end of the

series, however, discrete colonies will be found, each of which can be

transferred to a fresh tube of nutrient medium without risk of

contamination from the neighbouring colonies.

~"Working" up Plates.~--

Having succeeded in obtaining a plate (or tube cultivation) in which the

colonies are well grown and sufficiently separated from each other, the

process of "working up," "pricking out," or "fishing" the colonies in

order to obtain subcultures in a state of purity from each of the

different bacteria present must now be proceeded with.

Occasionally it happens that this is quite a simple matter. For example,

the original mixed cultivation when examined microscopically was found

to contain a Gram positive micrococcus, a Gram positive straight

bacillus and a Gram negative short bacillus. The third gelatine plate

prepared from this mixture, on inspection after four day's incubation,

showed twenty-five colonies--seven moist yellow colonies, each sinking

into a shallow pit of liquefied gelatine, fourteen flat irridescent

filmy colonies, and four raised white slimy colonies. A film preparation

(stained Gram) from each variety examined microscopically showed that

the yellow liquefying colony was composed of Gram positive micrococci;

the flat colony of Gram positive bacilli and the white colony of gram

negative bacilli. One of each of these varieties of colonies would be

transferred by means of the sterilised loop to a fresh gelatine culture

tube, and after incubation the growth in each subculture would

correspond culturally and microscopically with that of the plate colony

from which it was derived,--the object aimed at would therefore be

achieved.

Usually, however, the colonies cannot be thus readily differentiated,

and unless they are "worked up" in an orderly and systematic manner much

labour will be vainly expended and valuable time wasted. The following

method minimises the difficulties involved.

(A) Inspection.

a. Without opening the plate carefully study the various colonies with

the naked eye, with the assistance of a watchmaker's lens or by

inverting the plate on the stage of the microscope and viewing with the

1-inch objective through the bottom of the plate and the layer of

medium.

b. If gross differences can be detected mark a small circle on the

bottom of the plate around the site of each of the selected colonies,

with the grease pencil.

c. If no obvious differences can be made out choose nine colonies

haphazard and indicate their positions by pencil marks on the bottom of

the plate.

(B) Fishing Colonies.--

a. Take a sterile Petri dish and invert it upon the laboratory bench.

Rule two parallel lines on the bottom of the dish with a grease pencil,

and two more parallel lines at right angles to the first pair--so

dividing the area of the dish into nine portions. Number the top

right-hand portion 1, and the central bottom portion 8 (Fig. 139).

Revert the dish. The numbers 1 and 8 can be readily recognised through

the glass and by their positions enable any of the other divisions to be

localised by number. This is the stock dish.

b. Slightly raise the cover of the dish, and with a sterile

teat-pipette deposit a small drop of sterile water in the centre of each

of the nine divisions.

c. With the sterilised platinum spatula raise one of the marked

colonies from the "plate 3" and transfer it to the first division in the

ruled plate and emulsify it in the drop of water awaiting it. Repeat

this process with the remaining colonies, emulsifying a separate colony

in each drop of water.

a. Prepare a cover-slip film preparation from each drop of emulsion in

the "stock dish" and number to correspond to the division from which it

was taken. Stain by Gram's method.

b. Examine microscopically, using the oil immersion lens and note the

numbers of those cover-slips which morphologically and by Gram results

appear to be composed of different species of bacteria.

[Illustration: FIG. 139.--Diagram for stock plate.]

(D) Preparing Isolation Subcultures.--

a. Inoculate an agar slope and a broth tube from the emulsion in the

stock dish corresponding to each of these specially selected numbers.

b. Ascertain whether the cover-slips from the nine emulsions in the

stock dish include all the varieties represented in the cover-slip film

preparation made from the original mixture before plating.

c. If some varieties are missing prepare a second stock dish from

other colonies on plate 3, and repeat the process until each

morphological form or tinctorial variety has been secured in subculture.

_d._ Place the stock dishes in the ice chest to await the results of

incubation. (If any of the subcultures fail, further material can be

obtained from the corresponding emulsion; or if it has dried, by

moistening it with a further drop of sterile distilled water.)

_e._ Incubate all the subcultures and identify the organisms picked out.

4. Differential Media.--

(a) _Selective._--Some varieties of media are specially suitable for

certain species of bacteria and enable them to overgrow and finally

choke out other varieties; e. g., wort is the most suitable

medium-base for the growth of torulae and yeasts and should be employed

when pouring plates for the isolation of these organisms. To obtain a

pure cultivation of yeast from a mixture containing bacteria as well, it

is often sufficient to inoculate wort from the mixture and incubate at

37 deg. C. for twenty-four hours. Plant a fresh tube of wort from the

resulting growth and incubate. Repeat the process once more, and from

the growth in this third tube plant a streak on wort gelatine, and

incubate at 20 deg. C. The resulting growth will almost certainly be

a pure culture of the yeast.

(b) _Deterrent._--The converse of the above also obtains. Certain

media possess the power of inhibiting the growth of a greater or less

number of species. For instance, media containing carbolic acid to the

amount of 1 per cent. will inhibit the growth of practically everything

but the Bacillus coli communis.

~5. Differential Incubation.~--

In isolating certain bacteria, advantage is taken of the fact that

different species vary in their optimum temperature. A mixture

containing the Bacillus typhosus and the Bacillus aquatilis sulcatus,

for example, may be planted on two slanted agar tubes, the one incubated

at 40 deg. C., and the other at 12 deg. C. After twenty-four hours'

incubation the first will show a pure cultivation of the Bacillus

typhosus, whilst the second will be an almost pure culture of the

Bacillus aquatilis.

6. Differential Sterilisation.--

(a) _Non-sporing Bacteria._--Similarly, advantage may be taken of the

varying thermal death-points of bacteria. From a mixture of two

organisms whose thermal death-points differ by, say, 4 deg. C.--e. g.,

Bacillus pyocyaneus, thermal death-point 55 deg. C., and Bacillus

mesentericus vulgatus, thermal death-point 60 deg. C.--a pure cultivation

of the latter may be obtained by heating the mixture in a water-bath to

58 deg. C. and keeping it at that point for ten minutes. The mixture is

then planted on to fresh media and incubated, when the resulting growth

will be found to consist entirely of the B. mesentericus.

(b) _Sporing Bacteria._--This method finds its chief practical

application in the differentiation of a spore-bearing organism from one

which does not form spores. In this case the mixture is heated in a

water-bath at 80 deg. C. for fifteen to twenty minutes. At the end of this

time the non-sporing bacteria are dead, and cultivations made from the

mixture will yield a growth resulting from the germination of the spores

only.

Differential sterilisation at 80 deg. C. is most conveniently carried out

in a water-bath of special construction, designed by Balfour Stewart (Fig.

140). It consists of a double-walled copper vessel mounted on legs, and

provided with a tubulure communicating with the space between the walls.

This space is nearly filled with benzole (boiling-point 80 deg. C.; pure

benzole, free from thiophene must be employed for the purpose, otherwise

the boiling-point gradually and perceptibly rises in the course of

time), and to the tubulure is fitted a long glass tube, some 2 metres

long and about 0.75 cm. diameter, serving as a condensing tube (a tube

half this length if provided with a condensing bulb at the centre will

be equally efficient). The interior of the vessel is partly filled with

water and covered with a lid which is perforated for a thermometer. This

latter dips into the water and records its temperature. A very small

Bunsen flame under the apparatus suffices to keep the benzole boiling

and the water within at a constant temperature of 80 deg. C. The bath is

thus always ready for use.

METHOD.--To use the apparatus.

1. Place some of the mixture itself, if fluid, containing the spores, or

an emulsion of the same if derived from solid material, in a test-tube.

2. Immerse the test-tube in the water contained in the benzole bath,

taking care that the upper level of the liquid in the tube is at least 2

cm. beneath the surface of the water in the copper vessel.

3. The temperature of the water, of course, falls a few degrees after

opening the bath and introducing a tube of colder liquid, but after a

few minutes the temperature will have again reached 80 deg. C.

4. When the thermometer again records 80 deg. C., note the time, and

fifteen minutes later remove the tube containing the mixture from the

bath.

5. Make cultures upon suitable media; incubate.

[Illustration: FIG. 140.--Benzole bath.]

7. Differential Atmosphere Cultivation.--

(a) By adapting the atmospheric conditions to the particular organism

it is desired to isolate, it is comparatively easy to separate a strict

aerobe from a strict anaerobe, and _vice versa_. In the first case,

however, it is important that the cultivations should be made upon

solid media, for if carried out in fluid media the aerobes multiplying

in the upper layers of fluid render the depths completely anaerobic, and

under these conditions the growth of the anaerobes will continue

unchecked.

(b) When it is desired to separate a facultative anaerobe from a

strict anaerobe, it is generally sufficient to plant the mixture upon

the sloped surface agar, incubate aerobically at 37 deg. C., and examine

carefully at frequent intervals. At the first sign of growth,

subcultivations must be prepared and treated in a similar manner. As a

result of these rapid subcultures, the facultative anaerobe will be

secured in pure culture at about the third or fourth generation.

required from a mixture of facultative and strict anaerobes, pour plates

of glucose formate agar (or gelatine) in the usual manner, place them in

a Bulloch's or Novy's jar, and incubate at a suitable temperature. Pick

off the colonies of the required organism when the growth appears, and

transfer to tubes of the various media.

Incubate under suitable conditions as to temperature and atmosphere.

~8. Animal Inoculation.~--

Finally, when dealing with pathogenic organisms, it is often advisable

to inoculate some of the impure culture (or even some of the original

_materies morbi_) into an animal specially chosen on account of its

susceptibility to the particular pathogenic organism it is desired to

inoculate. Indeed, with some of the more sensitive and strictly

parasitic bacteria this method of animal inoculation is practically the

only method that will yield a satisfactory result.

XVI. METHODS OF IDENTIFICATION AND STUDY.

In order to identify an organism after isolation, tube, plate, and other

cultivations must be prepared, incubated under suitable conditions as to

temperature and environment, and examined from time to time (a)

~macroscopically~, (b) by ~microscopical methods~, (c) by ~chemical

methods~, (d) by ~physical methods~, (e) by ~inoculation methods~, and

the results of these examinations duly recorded.

It must be stated definitely that no micro-organism can be identified by

any _one_ character or property, whether microscopical, biological or

chemical, but that on the contrary its entire life history must be

carefully studied and then its identity established from a consideration

of the sum total of these observations.

In order to give to the recorded results their maximum value it is

essential that they should be exact and systematic, therefore some such

scheme as the following should be adhered to; and especially is this

necessary in describing an organism not previously isolated and studied.

SCHEME OF STUDY.

Designation:

Originally isolated by (_observer's name_) in (_date_), from (_source of

organism_).

~1. Cultural Characters.~--(_Vide_ Macroscopical Examination

of Cultivation, page 261.)

Gelatine plates, }

Gelatine streak, } at 20 deg. C.

Gelatine stab, }

Gelatine shake, }

Agar plates, }

Agar streak or smear, }

Agar stab, }

Inspissated blood-serum, } at 20 deg. C. and 37 deg. C.

Bouillon, }

Litmus milk, }

Potato, }

Special media for the purpose of demonstrating

characteristic appearances.

~2. Morphology~.--(_Vide_ Microscopical Examination of

Cultivations, page 272.)

Vegetative forms:

Shape.

Size.

Motility.

Flagella (if present).

Capsule (if present).

Involution forms.

Pleomorphism (if observed).

Sporing forms (if observed). Of which class?

Staining reactions.

~3. Chemical Products of Growth.~--(_Vide_ Chemical

Examination of Cultivations, page 276.)

Chromogenesis.

Photogenesis.

Enzyme formation.

Fermentation of carbohydrates:

Acid formation.

Alkali formation.

Indol formation.

Phenol formation.

Reducing and oxidising substances.

Gas formation.

~4. Biology.~--(_Vide_ Physical Examination of Cultures, page

295.)

Atmosphere.

Temperature.

Reaction of nutrient media.

Resistance to lethal agents:

Physical:

Desiccation.

Light.

Colours.

Chemical germicides.

Vitality.

~5. Pathogenicity:~

Susceptible animals, subsequently arranged in order of susceptibility.

Immune animals.

Experimental inoculation, symptoms of disease.

Post-mortem appearances.

Virulence:

Length of time maintained.

Optimum medium?

Minimal lethal dose.

Exaltation and attenuation of virulence?

Toxin formation.

MACROSCOPICAL EXAMINATION OF CULTIVATIONS.

In describing the naked-eye and low-power appearances of the bacterial

growth the descriptive terms introduced by Chester (and included in the

following scheme) should be employed.

SOLID MEDIA.

~Plate Cultures.~--

_Gelatine._--Note the presence or absence of liquefaction of the

surrounding medium. If liquefaction is present, note shape and character

(_vide_ page 269, "stab" cultures).

_Agar._--No liquefaction takes place in this medium. The liquid found on

the surface of the agar (or at the bottom of the tube in agar tube

cultures) is merely water which has been expressed during the rapid

solidification of the medium and has subsequently condensed.

_Gelatine and Agar._--Examine the colonies at intervals of twenty-four

hours.

(a) With the naked eye.

(b) With a hand lens or watchmaker's glass.

dissecting microscope.

Distinguish superficial from deep colonies and note the characters of

the individual colonies.

(A) ~Size.~--The diameter in millimetres, at the various ages.

(B) ~Shape.~--

Punctiform: Dimensions too slight for defining form by naked eye;

minute, raised, hemispherical.

Round: Of a more or less circular outline.

Elliptical: Of a more or less oval outline.

Irregular: Outlines not conforming to any recognised shape.

Fusiform: Spindle-shaped, tapering at each end.

Cochleate: Spiral or twisted like a snail shell (Fig. 141, a).

[Illustration: FIG. 141.--Types of colonies: a, Cochleate; b,

amoeboid; c, mycelioid.]

Amoeboid: Very irregular, streaming (Fig. 141, b).

Mycelioid: A filamentous colony, with the radiate character of a mould

(Fig. 141, c).

Filamentous: An irregular mass of loosely woven filaments (Fig. 142,

a).

Floccose: Of a dense woolly structure.

Rhizoid: Of an irregular, branched, root-like character (Fig. 142, b).

Conglomerate: An aggregate of colonies of similar size and form (Fig.

142, c).

Toruloid: An aggregate of colonies, like the budding of the yeast plant

(Fig. 142, d).

Rosulate: Shaped like a rosette.

[Illustration: FIG. 142.--Types of colonies: a, Filamentous; b,

rhizoid; c, conglomerate; d, toruloid.]

1. _General Character of Surface as a Whole_:

Flat: Thin, leafy, spreading over the surface (Fig. 143, a).

Effused: Spread over the surface as a thin, veily layer, more delicate

than the preceding.

Raised: Growth thick, with abrupt terraced edges (Fig. 143, b).

Convex: Surface the segment of a circle, but very flatly convex (Fig.

143, c).

Pulvinate: Surface the segment of a circle, but decidedly convex (Fig.

143, d).

Capitate: Surface hemispherical (Fig. 143, e).

Umbilicate: Having a central pit or depression (Fig. 143, f).

Conical: Cone with rounded apex (Fig. 143, g).

Umbonate: Having a central convex nipple-like elevation (Fig. 143, h).

2. _Detailed Characters of Surface_:

Smooth: Surface even, without any of the following distinctive

characters.

Alveolate: Marked by depressions separated by thin walls so as to

resemble a honeycomb (Fig. 144).

Punctate: Dotted with punctures like pin-pricks.

Bullate: Like a blistered surface, rising in convex prominences, rather

coarse.

Vesicular: More or less covered with minute vesicles due to gas

formation; more minute than bullate.

[Illustration: FIG. 143.--Surface elevation of colonies: a, Flat; b,

raised; c, convex; d, pulvinate; e, capitate; f, umbilicate;

g, conical; h, umbonate.]

[Illustration: FIG. 144.--Types of colonies--alveolate.]

Verrucose: Wart-like, bearing wart-like prominences.

Squamose: Scaly, covered with scales.

Echinate: Beset with pointed prominences.

Papillate: Beset with nipple or mamma-like processes.

Rugose: Short irregular folds, due to shrinkage of surface growth.

Corrugated: In long folds, due to shrinkage.

Contoured: An irregular but smoothly undulating surface, resembling the

surface of a relief map.

Rimose: Abounding in chinks, clefts, or cracks.

(D) ~Internal Structure of Colony~ (_Microscopical_).--

Refraction Weak: Outline and surface of relief not strongly defined.

Refraction Strong: Outline and surface of relief strongly defined;

dense, not filamentous colonies.

[Illustration: FIG. 145.--Types of colonies: a, Grumose; b,

moruloid; c, clouded.]

1. _General_:

Amorphous: Without any definite structure, such as is specified below.

Hyaline: Clear and colourless.

Homogeneous: Structure uniform throughout all parts of the colony.

Homochromous: Colour uniform throughout.

2. _Granulations or Blotchings_:

Finely granular.

Coarsely granular.

Grumose: Coarser than the preceding, with a clotted appearance, and

particles in clustered grains (Fig. 145, a).

Moruloid: Having the character of a mulberry, segmented, by which the

colony is divided in more or less regular segments (Fig. 145, b).

Clouded: Having a pale ground, with ill-defined patches of a deeper tint

(Fig. 145, c).

[Illustration: FIG. 146.--Types of colonies: a, Reticulate; b,

gyrose; c, marmorated.]

3. _Colony Marking or Striping_:

Reticulate: In the form of a network, like the veins of a leaf (Fig.

146, a).

Before the introduction of solid media the only method of obtaining pure