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_Size_:
(a) Prepare cover-slip film preparations at the various ages, and fix by exposure to a temperature of 115 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) quant.i.tatively--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.~--
