10. Prepare the gas receiver as in the previous method (in this case, however, the mercury should be warmed slightly) and fill the horizontal arm of the receiver with hot water.

11. Connect up the culture flask with the horizontal arm of the gas receiver.

12. Remove the screw clamp from the rubber tubing, adjust the three-way tap, seal all joints with melted wax, and incubate.

13. Complete the investigation as described for the previous method.

BY PHYSICAL METHODS.

Examine cultivations of the organism with reference to its growth and development under the following headings:

Atmosphere:

(a) In the presence of oxygen.

(b) In the absence of oxygen.

(c) In the presence of gases other than oxygen.

Temperature:

(a) Range.

(b) Optimum.

(c) Thermal death-point:

Moist: Vegetative forms.

Spores.

Dry: Vegetative forms.

Spores.

Reaction of medium.

Resistance to lethal agents:

(a) Desiccation.

(b) Light: Diffuse.

Direct.

Primary colours.

(c) Heat.

(d) Chemical antiseptics and disinfectants.

Vitality in artificial cultures.

~I. Atmosphere.~--The question as to whether the organism under observation is (a) an obligate aerobe, (b) a facultative anaerobe, or (c) an obligate anaerobe is roughly decided by the appearance of cultivations in the fermentation tubes. Obvious growth in the closed branch as well as in the bulb or in the inverted gas tube as well as in the bulk of the medium will indicate that it is a facultative anaerobe; whilst growth only occurring in the bulb or in the closed branch shows that it is an obligate aerobe or anaerobe respectively. This method, however, is not sufficiently accurate for the present purpose, and the examination of an organism with respect to its behaviour in the absence of oxygen is carried out as follows:

_Apparatus Required:_

Buchner's tubes.

Bulloch's apparatus.

Exhaust pump.

Pyrogallic acid.

Dekanormal caustic soda.

_Media Required:_

Glucose formate agar.

Glucose formate gelatine.

Glucose formate bouillon.

METHOD.--

1. Prepare four sets of cultivations:

(A) Sloped glucose formate agar, and incubate aerobically at 37 C.

Sloped glucose formate gelatine, and incubate aerobically at 20 C.

(B) Sloped glucose agar to incubate anaerobically at 37 C.

Sloped glucose formate gelatine to incubate anaerobically at 20 C.

(C) Sloped glucose formate agar to incubate anaerobically at 37 C.

Glucose formate bouillon to incubate anaerobically at 37 C.

(D) Sloped glucose formate gelatine to incubate anaerobically at 20 C.

Glucose formate bouillon to incubate anaerobically at 20 C.

2. Seal the cultures forming set B in Buchner's tubes (_vide_ page 239).

3. Seal the cultures forming set C in Bulloch's apparatus; exhaust the air by means of a vacuum pump, and provide for the absorption of any residual oxygen by the introduction of pyrogallic acid and caustic soda in solution (_vide_ page 245). Treat set D in the same way.

4. Observe the cultivations macroscopically and microscopically at intervals of twenty-four hours until the completion, if necessary, of seven days' incubation.

5. Control these results.

_Gases Other than Oxygen._--