less definite boundaries.
Gyrose: Marked by wavy lines, indefinitely placed (Fig. 146, b).
Marmorated: Showing faint, irregular stripes, or traversed by vein-like
markings, as in marble (Fig. 146, c).
Rivulose: Marked by lines like the rivers of a map.
Rimose: Showing chinks, cracks, or clefts.
[Illustration: FIG. 147.--Types of colonies--curled.]
4. _Filamentous Colonies:_
Filamentous: As already defined.
Floccose: Composed of filaments, densely placed.
Curled: Filaments in parallel strands, like locks or ringlets (Fig.
147).
(E) ~Edges of Colonies.~--
Entire: Without toothing or division (Fig. 148, a).
Undulate: Wavy (Fig. 148, b).
Repand: Like the border of an open umbrella (Fig. 148, c).
Erose: As if gnawed, irregularly toothed (Fig. 148, d).
[Illustration: FIG. 148.--Edges of colonies: a, Entire; b, undulate;
c, repand; d, erose.]
Lobate.
Lobulate: Minutely lobate (Fig. 149, e).
Auriculate: With ear-like lobes (Fig. 149, f).
Lacerate: Irregularly cleft, as if torn (Fig. 149, g).
Fimbriate: Fringed (Fig. 149, h).
Ciliate: Hair-like extensions, radiately placed (Fig. 149, j).
Tufted.
Filamentous: As already defined.
Curled: As already defined.
[Illustration: FIG. 149.--Edges of colonies: e, Lobar-lobulate; f,
auriculate; g, lacerate; h, fimbriate; i, ciliate.]
(F) ~Optical Characters~ (after Shuttleworth).--
1. _General Characters_:
Transparent: Transmitting light.
Vitreous: Transparent and colourless.
Oleaginous: Transparent and yellow; olive to linseed-oil coloured.
Resinous: Transparent and brown, varnish or resin-coloured.
Translucent: Faintly transparent.
Porcelaneous: Translucent and white.
Opalescent: Translucent; greyish-white by reflected light.
Nacreous: Translucent, greyish-white, with pearly lustre.
Sebaceous: Translucent, yellowish or greyish-white.
Butyrous: Translucent and yellow.
Ceraceous: Translucent and wax-coloured.
Opaque.
Cretaceous: Opaque and white, chalky.
Dull: Without lustre.
Glistening: Shining.
Fluorescent.
Iridescent.
2. _Chromogenicity_:
Colour of pigment.
Pigment restricted to colonies.
Pigment restricted to medium surrounding colonies.
Pigment present in colonies and in medium.
~Streak or Smear Cultures.~--
_Gelatine and Agar._--Note general points as indicated under plate
cultivations.
_Inspissated Blood-serum._--Note the presence or absence of liquefaction
of the medium. (The presence of condensation water at the bottom of the
tube must not be confounded with liquefaction of the medium.)
_All Oblique Tube Cultures._--
1. Colonies Discrete: Size, shape, etc., as for plate cultivations
(_vide_ page 261).
2. Colonies Confluent: Surface elevation and character of edge, as for
plate cultivations (_vide_ page 263).
Chromogenicity: As for plate cultures.
~Gelatine Stab Cultures.~--
(A) _Surface Growth._--As for individual colonies in plate cultures
(_vide_ page 261).
[Illustration: FIG. 150.--Stab cultivations--types of growth: a,
Filiform; b, beaded; c, echinate; d, villous; e, arborescent.]
(B) _Line of Puncture._--
Filiform: Uniform growth, without special characters (Fig. 150, a).
Nodose: Consisting of closely aggregated colonies.
Beaded: Consisting of loosely placed or disjointed colonies (Fig. 150,
b).
Papillate: Beset with papillate extensions.
Echinate: Beset with acicular extensions (Fig. 150, c).
Villous: Beset with short, undivided, hair-like extensions (Fig. 150,
d).
Plumose: A delicate feathery growth.
[Illustration: FIG. 151.--Stab cultivations--types of growth: f,
Crateriform; g, saccate; h, infundibuliform; j, napiform; k,
fusiform; l, stratiform.]
Arborescent: Branched or tree-like, beset with branched hair-like
extensions (Fig. 150, e).
(C) _Area of Liquefaction_ (if present).--
Crateriform: A saucer-shaped liquefaction of the gelatine (Fig. 151,
f).
Saccate: Shape of an elongated sack, tubular cylindrical (Fig. 151,
g).
Infundibuliform: Shape of a funnel, conical (Fig. 151, h).
Napiform: Shape of a turnip (Fig. 151, j).
Fusiform: Outline of a parsnip, narrow at either end, broadest below the
surface (Fig. 151, k).
Stratiform: Liquefaction extending to the walls of the tube and downward
horizontally (Fig. 151, l).
(D) _Character of the Liquefied Gelatine._--
1. Pellicle on surface.
2. Uniformly turbid.
3. Granular.
4. Mainly clear, but containing flocculi.
5. Deposit at apex of liquefied portion.
(E) _Production of Gas Bubbles._
~Shake Cultures.~--
1. Presence or absence of liquefaction.
2. Production of gas bubbles.
3. Bulk of growth at the surface--aerobic.
4. Bulk of growth in depths--anaerobic.
~Fluid Media.~
~1. Surface of the Liquid.~--
Presence or absence of froth due to gas bubbles.
Presence or absence of pellicle formation.
Character of pellicle.
~2. Body of the Liquid.~--
Uniformly turbid.
Flocculi in suspension.
Granules in suspension.
Clear, with precipitate at bottom of tube.
Colouration of fluid, presence or absence of.
~3. Precipitate.~--
Character.
Amount.
Colour.
~Carbohydrate Media.~--
Growth.
Reaction.
Gas formation.
Coagulation or not of serum albumen (when serum water media are
employed).
~Litmus Milk Cultivations.~--
{Unaltered.
1. Reaction: {Acid.
{Alkaline.
2. Odour.
3. Formation of gas.
{Unaltered.
4. Consistency: {Peptonised (character of solution).
{Coagulated.
{hard: solid.
5. Clot: Character {soft: floculent.
{ragged and broken up by gas
{bubbles.
(a) Coagulum undissolved.
(b) Coagulum finally peptonised, completely: incompletely.
Resulting solution, clear: turbid.
{Abundant.
{Scanty.
6. Whey: {Clear.
{Turbid.
{Coagulated by boiling, or not.
~BY MICROSCOPICAL METHODS.~
As a council of perfection preparations must be made from pure
cultivations 4, 6, 8, 12, 18, and 24 hours; and subsequently at
intervals of, say, twenty-four hours, during the entire period they are
under observation, and examined--
(A) ~Living.--1.~ In ~hanging drop~, to determine _motility_ or
_non-motility_.
In this connection it must be remembered that under certain conditions
as to environment (e. g., when examined in an unsuitable medium,
atmosphere, temperature, etc.) motile bacilli may fail to exhibit
activity. No organism, therefore, should be recorded as non-motile from
one observation only; a series of observations at different ages and
under varying conditions should form the basis of an opinion as to the
absence of true locomotion.
_Size._--In the case of non-motile or sluggishly motile organisms,
endeavour to measure several individuals in each hanging drop by means
of the eyepiece micrometer or the eikonometer (_vide_ page 63), and
average the results.
If the organism is one which forms spores, observe--
(a) _Spore Formation._--Prepare hanging-drop cultivations (_vide_ page
78) from vegetative forms of the organism, adding a trace of magenta
solution (0.5 per cent.) or other intra vitam stain (see page 77) to the
drop, on the point of the platinum needle, to facilitate the observation
of the phenomenon by rendering the bacilli more distinct.
Place the preparation on the stage of the microscope; if necessary,
using a warm stage.
Arrange illumination, etc., and select a solitary bacillus for
observation, by the help of the 1/6-inch lens.
Substitute the 1/12-inch oil-immersion lens for the sixth, and observe
the formation of the spore; if possible, measure any alteration in size
which may occur by means of the Ramsden micrometer.
(b) _Spore Germination._--Prepare hanging-drop cultivations from old
cultivations in which no living vegetative forms are present, and
observe the process of germination in a similar manner.
The comfort of the microscopist is largely enhanced in those cases where
the period of observation is at all lengthy, by use of some form of eye
screen before the unemployed eye, such as is figured on page 58 (Fig.
49).
If it is impossible to carry out the method suggested above, proceed as
follows:
(a) _Spore Formation._--Plant the organism in broth and incubate under
optimum conditions.
At regular intervals, say every thirty minutes, remove a loopful of the
cultivation and prepare a cover-slip film preparation.
Fix, while still wet, in the corrosive sublimate fixing solution.
Stain with aniline gentian violet, and partially decolourise with 2 per
cent. acetic acid.
Mount and number consecutively; then examine.
(b) _Spore Germination._--Expose a thick emulsion of the spores to a
temperature of 80 deg. C. for ten minutes in the differential steriliser
(_vide_ page 257).
Transfer the emulsion to a tube of sterile nutrient broth and incubate.
Remove specimens from the tube culture at intervals of, say, five
minutes.
Fix, stain, etc., wet, as under (a), and examine.
(B) ~Fixed.--2.~ In ~stained preparations~.
(a) To determine points in _morphology_:
_Shape_ (_vide_ classification, page 131).
_Size_:
(a) Prepare cover-slip film preparations at the various ages, and fix
by exposure to a temperature of 115 deg. C. for twenty minutes in hot-air
oven.
(b) Stain the preparations by Gram's method (if applicable) or with
dilute carbol-fuchsin, and mount in the usual way.
(c) Measure (_vide_ page 66) some twenty-five individuals in each film
by means of the Ramsden's or the stage micrometer and average the
result.
_Pleomorphism_; If noted, record--
The predominant character of the variant forms.
On what medium or media they are observed.
At what period of development.
(b) To demonstrate details of _structure_:
_Flagella_: If noted, record--
Method of staining (_vide_ page 101).
Position and arrangement (_vide_ page 136).
Number.
_Spores_: If noted, record--
Method of staining.
Shape.
Size.
Position within the parent cell.
Condition, as to shape, of the parent cell (_vide_
page 139).
Optimum medium and temperature.
Age of cultivation.
Conditions of environment as to temperature,
atmosphere.
Method of germination (_vide_ page 140).
_Involution Forms_: If noted, record--
Method of staining.
Character (e. g., if living or dead).
Shape.
On what medium they are observed.
Age of medium.
Environment.
_Metachromatic Granules_: If noted, record--
Method of staining.
Character of granules.
Number of granules.
Colour of granules.
~3. Staining Reactions.~--
1. _Gram's Method._--Positive or negative.
2. _Neisser's Method._--If granules are noted, record--
1. Position.
2. Number.
3. _Ziehl-Neelsen's Method._--Acid-fast or decolourised.
4. _Simple Aniline Dyes._--(Noting those giving the best results, with
details of staining processes.)
Methylene-blue }
Fuchsin } and their modifications.
Gentian violet }
Thionine blue }
BY BIOCHEMICAL METHODS.
Test cultivations of the organism for the presence of--
Soluble enzymes--proteolytic, diastatic, invertase.
Organic acids--(a) quantitatively--i. e., estimate the total acid
production; (b) qualitatively for formic, acetic, propionic, butyric,
lactic.
Ammonia.
Neutral volatile substances--ethyl alcohol, aldehyde, acetone.
Aromatic products--indol, phenol.
Soluble pigments.
Test the power of reducing (a) colouring matters, (b) nitrates to
nitrites.
Investigate the gas production--H_{2}S, CO_{2}, H_{2}. Estimate the
ratio between the last two gases.
Prepare all cultivations for these methods of examination under
_optimum_ conditions, previously determined for each of the organisms it
is intended to investigate, as to
(a) Reaction of medium;
(b) Incubation temperature;
(c) Atmospheric environment;
and keep careful records of these points, and also of the age of the
cultivation used in the final examination.
Examine the cultivations for the various products of bacterial
metabolism after forty-eight hours' growth, and ~never omit to examine
"control" (uninoculated) tube or flask of medium from the same batch,
kept for a similar period under identical conditions~.
If the results are negative, test further cultivations at three days,
five days, and ten days.
~1. Enzyme Production.~--
(A) _Proteolytic Enzymes._--(Convert proteins into proteose, peptone
and further products of hydrolysis; e. g., B. pyocyaneus.)
_Media Required_:
Blood-serum and milk-serum which have been carefully
filtered through a porcelain candle.
_Reagents Required_:
Ammonium sulphate.
Thirty per cent. caustic soda solution.
Copper sulphate, 0.5 per cent. aqueous solution.
One per cent. acetic acid solution.
Millon's reagent.
Glyoxylic acid solution.
Concentrated sulphuric acid.
METHOD.--
1. Prepare cultivations in bulk (50 c.c.) in a flask and incubate.
2. Make the liquid faintly acid with acetic acid, then boil. (This
precipitates the unaltered proteins.)
3. Filter.
4. Take 10 c.c. of the filtrate in a test-tube and add 1 c.c. of the
caustic soda, then add the copper sulphate drop by drop.
Pink colour which becomes violet with more copper sulphate =
proteose and peptone.
5. Saturate the rest of the filtrate with ammonium sulphate.
Precipitate = proteose.
6. Filter and divide the filtrate into three parts a, b and c.
a. Repeat the copper sulphate test, using excess of caustic soda to
displace the ammonia from the ammonium sulphate.
Pink colour = peptone.
b. Boil with Millon's reagent.
Red colour = tyrosine.
c. Add glyoxylic acid solution and run in concentrated sulphuric acid.
Violet ring at upper level of acid = tryptophane.
Both the tyrosine and tryptophane may be either in the free state or in
combination as polypeptid or peptone.
(B) _Diastase._--(Converts starch into sugar; e. g., B. subtilis.)
_Medium Required_:
Inosite-free bouillon.
_Reagents Required_:
Starch.
Thymol.
Fehling's solution.
METHOD.--
1. Prepare tube cultivation and incubate.
2. Prepare a thin starch paste and add 2 per cent. thymol to it.
3. Mix equal parts of the cultivation to be tested and the starch paste,
and place in the incubator at 37 deg. C. for six to eight hours.
4. Filter.
Test the filtrate for sugar.
Boil some of the Fehling's solution in a test-tube.
Add the filtrate drop by drop until, if necessary, a quantity has been
added equal in amount to the Fehling's solution employed, keeping the
mixture at the boiling-point during the process.
Yellow or orange precipitate = sugar.
(C) _Invertase._--(Convert saccharose into a mixture of dextrose and
laevulose e. g., B. fluorescens liquefaciens.)
_Medium Required_:
Inosite-free bouillon.
_Reagents Required_:
Cane sugar, 2 per cent. aqueous solution.
Carbolic acid.
METHOD.--
1. Prepare tube cultivations and incubate.
2. Add 2 per cent. of carbolic acid to the sugar solution.
3. Mix equal quantities of the carbolised sugar solution and the
cultivation in a test-tube; allow the mixture to stand for several
hours.
4. Filter.
Test the filtrate for reducing sugar as in the preceding section.
(D) _Rennin and "Lab" Enzymes._--(Coagulate milk independently of the
action of acids; e. g., B. prodigiosus.)
_Media Required_:
Inosite-free bouillon.
Litmus milk.
METHOD.--
1. Prepare tube cultivations and incubate.
2. After incubation heat the cultivation to 55 deg. C. for half an hour,
to sterilise.
3. By means of a sterile pipette run 5 c.c. of the cultivation into each
of three tubes of litmus milk.
4. Place in the cold incubator at 22 deg. C. and examine each day for
ten days.
Absence of coagulation at the end of that period will indicate absence
of rennin ferment formation.
Fermentation Reactions.
As tested upon carbohydrate substances and organic salts.
_Media Required_:
Peptone water containing various percentages (generally 2 per cent.) of
each of the substances referred to under "sugar" media (page 177), also
tubes of peptone water containing 1 per cent. respectively of each of
the following:
Organic salts: Sodium citrate, formate, lactate, malate,
tartrate.
METHOD.--
1. Prepare tube cultivations in each of the above media.
2. Observe from day to day up to the expiration of ten days if
necessary.
3. Note growth, reaction, gas production.
2. Acid Production.
(a) _Quantitative._--
_Medium Required_:
Sugar (glucose) bouillon of known "optimum" reaction.
_Apparatus and Reagents Required_:
As for estimating reaction of media (_vide_ page 150).
METHOD.--
1. Prepare cultivation in bulk (100 c.c.) in a flask; also "control"
flask of medium from same batch.
2. After suitable incubation, heat both flasks in the steamer at 100
deg. C. for thirty minutes to sterilise.
3. Determine the _titre_ of the medium in "inoculated" and "control"
flasks as described in the preparation of nutrient media (_vide_ page
151).
4. The difference between the titre of the medium in the two flasks
gives the total acid production of the bacterium under observation in
terms of normal NaOH.
NOTE.--If the growth is very heavy it may be a difficult
matter to determine the end-point. The cultivation should
then be filtered through a Berkefeld filter candle previous
to step 2, and the filtrate employed in the titration.
(b) _Qualitative_ (of all the organic acids present).--
_Medium Required_:
Sugar (glucose or lactose) bouillon as in quantitative examination.
_Reagents Required_:
Hydrochloric acid, concentrated.
Hydrochloric acid, 25 per cent.
Sulphuric acid, concentrated (pure).
Phosphoric acid, concentrated solution.
Ammonia.
Ammonium sulphate.
Baryta water.
Sodium carbonate, saturated aqueous solution.
Absolute alcohol.
Ether.
Calcium chloride.
Calcium chloride solution.
Zinc carbonate.
Copper sulphate saturated aqueous solution.
Alcoholic thiophene solution (0.15 c.c. in 100 c.c.).
Animal charcoal.
Five per cent. sodium nitroprusside solution.
Potassium bichromate.
Schiff's reagent.
Arsenious oxide.
Ferric chloride, 4 per cent. aqueous solution.
Silver nitrate, 1 per cent. aqueous solution.
Lugol's iodine.
Ten per cent. caustic soda solution.
Hard paraffin wax (melting-point about 52 deg. C.).
METHOD.--
1. Prepare cultivation in bulk (500 c.c.) in a litre flask and add
sterilised precipitated chalk, 10 grammes. Incubate at the optimum
temperature.
2. After incubation throw a piece of paraffin wax (about a centimetre
cube) into the cultivation and connect up the flask with a condenser.
The paraffin, which liquefies and forms a thin layer on the surface of
the fluid, is necessary to prevent the cultivation frothing up and
running unaltered through the condenser during the subsequent process of
distillation.
3. Distill over 200 to 300 c.c.
Use a rose-top burner to minimise the danger of cracking the flask; and
to the same end, well agitate the contents of the flask to prevent the
chalk settling.
The distillate "A" will contain alcohol, etc. (_vide_ page 285); the
residue "a" will contain the volatile and fixed acids.
4. Disconnect the flask and filter. The residue "a" then = filtrate B
and residue b.
[Illustration: FIG. 152.--Arrangement of distillation apparatus for
acids, etc.]
5. Residue b. Wash the residue from the filter paper, dissolve by
heating with dilute hydrochloric acid, and add calcium chloride solution
and ammonia until alkaline.
White precipitate insoluble in acetic acid = oxalic acid.
6. Make up filtrate B to 500 c.c. with distilled water and divide into
two parts.
7. Acidify 250 c.c. with 20 c.c. concentrated phosphoric acid (this
liberates the volatile acids) and distil to small bulk.
The distillate "B" may contain formic, acetic, propionic, butyric and
benzoic acids.
DISTILLATE "B."
(Volatile Acids.)
|
|
1. Add baryta water till alkaline,
and evaporate to dryness.
2. Add 50 c.c. absolute alcohol and allow
to stand, with frequent stirring, for
two to three hours.
3. Filter and wash with alcohol.
|
|
|---------------------------------------|
| |
| |
FILTRATE RESIDUE
| |
| |
may contain barium propionate, may contain barium acetate,
barium butyrate. barium formate, barium benzoate.
| |
| |
1. Evaporate to dryness. 1. Evaporate off alcohol and
dissolve up the residue on
2. Dissolve residue in 150 the filter in hot water and
c.c. water. neutralise.
3. Acidify with phosphoric 2. Divide the solution into
acid and distil. four portions:
4. Saturate distillate with (a) Add ferric chloride solution.
calcium chloride and distill
over a few c.c. ~Brown~ colour = _acetic_ or
_formic_ acids.
5. Test distillate for butyric
acid: ~Buff ppt.~ = _benzoic_ acid
(see ether soluble acids).
Add 3 c.c. alcohol and 4 drops
concentrated sulphuric acid. (b) Add silver nitrate
solution; then add one drop
~Smell of pineapple~ = _butyric_ ammonia water, and boil.
acid.
~Black~ precipitate of metallic
Propionic acid in small silver = _formic_ acid.
quantities cannot be
distinguished from butyric (c) Evaporate to dryness; mix
acid by tests within the with equal quantity of
scope of the bacteriological arsenious oxide and heat
laboratory. on platinum foil.
Unpleasant ~smell of cacodyl~
= _acetic_ acid.
(d) Add a few drops of
mercuric chloride solution
in test-tube, and heat to
70 deg. C.
~Precipitate~ of mercurous
chloride which is slowly
reduced to mercury =
_formic_ acid.
8. If the distillation of "B" is continued as long as acid comes over
(distilled water being occasionally added to the distilling flask) the
distillate can be measured and 50 c.c. used for titration. This will
give the amount of volatile acid formation.
9. The second part of the filtrate "B" (see page 282) should be examined
for lactic, oxalic, succinic, benzoic, salicylic, gallic and tannic
acids, as follows:
~Ether Soluble Acids.~--
1. Evaporate to a thin syrup, acidify strongly with phosphoric acid.
2. Extract with five times its volume of ether by agitation in a
separatory funnel.
3. Evaporate the ethereal extract to a thin syrup.
4. Add 100 c.c. water and mix thoroughly.
5. To a small portion of this solution add slight excess of sodium
carbonate, evaporate to dryness on the water-bath, dissolve in 5-10 c.c.
pure sulphuric acid, add 2 drops saturated copper sulphate solution,
place in a test-tube and heat in a boiling water-bath for 2 minutes,
cool, add 2 or 3 drops of the alcoholic thiophene and warm gently.
Cherry red colour = lactic acid.
If a brown colour is produced on the addition of sulphuric acid, another
sample should be taken and boiled with animal charcoal before
evaporating.
6. If lactic acid is definitely present, prepare zinc lactate by boiling
part of the solution of the ether extract with excess of zinc carbonate,
filtering and evaporating to crystallise. The crystals so obtained have
a characteristic form, and if dried at 110 deg. C, should contain 26.87
per cent. of zinc.
7. Test a portion of the rest of the solution of the ether extract for
oxalic acid (page 282, step 5). Carefully neutralise the remainder and
add ferric chloride solution.
Red brown gelatinous precipitate = succinic acid.
Buff precipitate = benzoic acid, and other acids related to benzoic
acid.
Violet colour = salicylic acid.
Inky black colour or precipitate = gallic acid or tannic acid.
For further identification the melting-points of the crystalline acids,
and the percentage of silver in their silver salts should be determined.
~3. Ammonia Production.~--
_Medium Required_:
Nutrient bouillon.
_Reagent Required_:
Nessler reagent.
METHOD.--
1. Prepare cultivation in bulk (100 c.c.) in a 250 c.c. flask and
incubate together with a control flask.
Test the cultivation and the control for ammonia in the following
manner:
2. To each flask add 2 grammes of calcined magnesia, then connect up
with condensers and distil.
3. Collect 50 c.c. distillate, from each, in a Nessler glass.
4. Add 1 c.c. Nessler reagent to each glass by means of a clean pipette.
Yellow colour = ammonia.
The depth of colour is proportionate to the amount present.