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.

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° 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.

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.

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.

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° 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.—

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.

4. Alcohol, etc., Production.—Divide the distillate "A" obtained in the course of a previous experiment (vide page 282, step 3) into four portions and test for the production of alcohol, acetaldehyde, acetone.

1. Add Lugol's iodine, then a little NaOH solution, and stir with a glass rod till the colour of the iodine disappears.

Pale-yellow crystalline precipitate of iodoform, with its characteristic smell, appearing in the cold, indicates acetaldehyde, or acetone; appearing only on warming indicates alcohol.

The precipitate may be absent even when the odour is pronounced.

2. Add Schiff's reagent.

Violet or red colour = aldehyde.

3. To 10 c.c. of solution add 2.5 c.c., 25 per cent. sulphuric acid, and a crystal or two of potassium bichromate and distil. Reduction of the bichromate to a green colour and a distillate, which smells of acetaldehyde and reacts with Schiff's reagent, shows the presence of alcohol in the original liquid.

4. Add a few drops of sodium nitroprusside solution, make alkaline with ammonia, then saturate with ammonium sulphate crystals. Acetone gives little colour on the addition of ammonia, but after the addition of ammonium sulphate a deep permanganate colour, which takes ten minutes to reach its full intensity. Aldehyde gives a carmine red unaltered by ammonium sulphate.

5. Indol Production.—

Method.—

Prepare several test-tube cultivations of the organism to be tested, and incubate.

Test for indol by means of the Rosindol reaction in the following manner. (If the culture has been incubated at 37°C., it must be allowed to cool to the room temperature before applying the test.)

1. Remove 2 c.c. of the cultivation by means of a sterile pipette and transfer to a clean tube, then,

2. Add 2 c.c. paradimethylamino-benzaldehyde solution.

3. Add 2 c.c. potassium persulphate solution.

The presence of indol is indicated by the appearance of a delicate rose-pink colour throughout the mixture which deepens slightly on standing.

5a. Phenol Production.—

Method.—

1. Prepare cultivation in a Bohemian flask containing at least 50 c.c. of medium, and incubate.

Test for phenol in the following manner:

2. Add 5 c.c., 25 per cent. sulphuric acid to the cultivation and connect up the flask with a condenser.

3. Distil over 15 to 20 c.c. Divide the distillate into three portions a, b and c.

4. Add to (a) 0.5 c.c. Millon's reagent and boil.

Red colour = phenol.

5. Add to (b) about 0.5 c.c. ferric chloride solution. Violet colour = phenol.

(If the distillate be acid the reaction will be negative.)

6. Add to (c) bromine water. Crystalline white ppt. of tribromo-phenol = phenol.

1. Prepare inosite-free bouillon cultivation, say 200 or 300 c.c., in a flask as before.

2. Render definitely acid by the addition of acetic acid and connect up the flask with a condenser.

3. Distil over 50 to 70 c.c.

Distillate will contain both indol and phenol.

4. Render the distillate strongly alkaline with caustic potash and redistil.

Distillate will contain indol; residue will contain phenol.

5. Test the distillate for indol (vide ante).

6. Saturate the residue, when cold, with carbon dioxide and redistil.

7. Test this distillate for phenol (vide ante).

6. Pigment Production.—

1. Prepare tube cultivations upon the various media and incubate under varying conditions as to temperature (at 37° C. and at 20°C.), atmosphere (aerobic and anaerobic), and light (exposure to and protection from).

Note the conditions most favorable to pigment formation.

2. Note the solubility of the pigment in various solvents, such as water (hot and cold), alcohol, ether, chloroform, benzol, carbon bisulphide.

3. Note the effect of acids and alkalies respectively upon the pigmented cultivation, or upon solutions of the pigment.

4. Note spectroscopic reactions.

7. Reducing Agent Formation.—

(a) Colour Destruction.—

1. Prepare tube cultivations in nutrient bouillon tinted with litmus, rosolic acid, neutral red, and incubate.

2. Examine the cultures each day and note whether any colour change occurs.

(b) Nitrates to Nitrites.—

Method.—

1. Prepare tube cultivations and incubate together with control tubes (i. e., uninoculated tubes of the same medium, placed under identical conditions as to environment).

This precaution is necessary as the medium is liable to take up nitrites from the atmosphere, and an opinion as to the absence of nitrites in the cultivation is often based upon an equal colouration of the medium in the control tube.

Test both the culture tube and the control tube for the presence of nitrites.

2. Add a few drops of sulphuric acid to the medium in each of the tubes.

3. Then run in 2 or 3 c.c. metaphenylene diamine into each tube. Brownish-red colour = nitrites.

The depth of colour is proportionate to the amount present.

8. Gas Production.—

(A) Carbon Dioxide and Hydrogen.—

Method.—

1. Inoculate the surface of the medium in the bulb of a fermentation tube and incubate.

2. Mark the level of the fluid in the closed branch of the fermentation tube, at intervals of twenty-four hours, and when the evolution of gas has ceased, measure the length of the column of gas with the millimetre scale.

Express this column of gas as a percentage of the entire length of the closed branch.

3. To analyse the gas and to determine roughly the relative proportions of CO₂ and H₂, proceed as follows:

Fill the bulb of the fermentation tube with caustic soda solution.

Close the mouth of the bulb with a rubber stopper.

Alternately invert and revert the tube six or eight times, to bring the soda solution into intimate contact with the gas.

Return the residual gas to the end of the closed branch, and measure.

The loss in volume of gas = carbon dioxide.

The residual gas = hydrogen.

Transfer gas to the bulb of the tube, and explode it by applying a lighted taper.

(B) Sulphuretted Hydrogen.—

1. Inoculate tubes of media, and incubate together with control tubes.

2. Examine from day to day, at intervals of twenty-four hours.

The liberation of the H₂S will cause the yellowish-white precipitate to darken to a brownish-black, or jet black, the depth of the colour being proportionate to the amount of sulphuretted hydrogen present.

Quantitative: For exact quantitative analyses of the gases produced by bacteria from certain media of definite composition, the methods devised by Pakes must be employed, as follows:

An Orsat-Lunge working with mercury instead of water, provided with two gas tubes of extra length (capacity 120 and 60 c.c. respectively and graduated throughout, both being water-jacketed) or other gas analysis apparatus, capable of dealing with CO₂, O₂, H₂, and N₂.

Method.—

1. Inoculate the medium in the flask in the usual manner, by means of a platinum needle, taking care that the neck of the flask and the rubber stopper are thoroughly flamed before and after the operation.

2. Fill the iron cylinder with mercury.

3. Place the bell jar mouth downward in the mercury—first seeing that there is free communication between the interior of the jar and the external air—and suck up the mercury into the tap; then shut off the tap.

4. Plug the open end of the three-way tap with melted wax.

5. Connect up the horizontal arm of the culture flask with that of the gas receiver by means of the pressure tubing (after removing the cotton-wool plug from the rubber tube), as shown in Fig. 153.

6. Give the three-way tap half turn to open communication between flask and receiver, and seal all joints by coating with a film of melted wax. When the tap is turned, the mercury in the receiver will naturally fall.

7. Place the entire apparatus in the incubator. (Two hours later, by which time the temperature of the apparatus is that of the incubator, mark the height of the mercury on the receiver.)

8. Examine the apparatus from day to day and mark the level of the mercury in the receiver at intervals of twenty-four hours.

9. When the evolution of gas has ceased, remove the apparatus from the incubator; clear out the wax from the nozzle of the three-way tap (first adjusting the tap so that no escape of gas shall take place) and connect it with the Orsat.

10. Remove, say, 100 c.c. of gas from the receiver, reverse the tap and force it into the culture flask. Remove 100 c.c. of mixed gases from the culture flask and replace in the receiver.

Repeat these processes three or four times to ensure thorough admixture of the contents of flask and receiver.

11. Now withdraw a sample of the mixed gases into the Orsat and analyse.

In calculating the results be careful to allow for the volume of air contained in the flask at the commencement of the experiment.

For the collection of gases formed under anaerobic conditions a slightly different procedure is adopted:

1. Fix a culture flask (500 c.c. capacity) with a perforated rubber stopper carrying an L-shaped piece of manometer tubing, each arm 5 cm. in length.

2. Prepare a second L-shaped piece of tubing, the short arm 5 cm. and the long arm 20 cm., and connect its short arm to the horizontal arm of the tube in the culture flask by means of a length of pressure tubing, provided with a screw clamp.

3. Fill the culture flask completely with boiling medium and pass the long piece of tubing through the plug of an Erlenmeyer flask (150 c.c. capacity) which contains 100 c.c. of the same medium.

4. Sterilise these coupled flasks by the discontinuous method, in the usual manner.

Immediately the last sterilisation is completed, screw up the clamp on the pressure tubing which connects them, and allow them to cool.

As the fluid cools and contracts it leaves a vacuum in the neck of the flask below the rubber stopper.

5. To inoculate the culture flask, withdraw the long arm of the bent tube from the Erlenmeyer flask and pass it to the bottom of a test-tube containing a young cultivation (in a fluid medium similar to that contained in the culture flask) of the organism it is desired to investigate.

6. Slightly release the clamp on the pressure tubing to allow 4 or 5 c.c. of the culture to enter the flask.

7. Clamp the rubber tube tightly; remove the bent glass tube from the culture tube and plunge it into a flask containing recently boiled and quickly cooled distilled water.

8. Release the clamp again and wash in the remains of the cultivation until the culture flask and tubing are completely filled with water.

9. Clamp the rubber tubing tightly and take away the long-armed glass tubing.

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:

Reaction of medium.

Resistance to lethal agents:

(a) Desiccation.

(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:

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.—

Apparatus Required:

Method.—

1. Prepare at least seven tube cultivations upon solid media and deposit them in Bulloch's apparatus.

2. Connect up the inlet tube of the Bulloch's jar with the sterile gas filter, and this again with the delivery tube of the gasometer or gas generator.

3. Open both stop-cocks of the Bulloch's apparatus and pass the gas through until it has completely replaced the air in the bell jar as shown by the result of analyses of samples collected from the exit tube.

4. Incubate under optimum conditions as to temperature.

5. Examine the cultivations at intervals of twenty-four hours, until the completion of seven days.

6. Remove one tube from the interior of the apparatus each day. If no growth is visible, incubate the tube under optimum conditions as to temperature and atmosphere, and in this way determine the length of exposure to the action of the gas necessary to kill the organisms under observation.

7. Control these results.

II. Temperature.—

(A) Range.—

1. Prepare a series of ten tube cultivations, in fluid media, of optimum reaction.

2. Arrange a series of incubators at fixed temperatures, varying 5° C. and including temperatures between 5° C. and 50° C.

(In the absence of a sufficient number of incubators utilise the water-bath employed in testing the thermal death-point of vegetative forms.)

3. Incubate one tube cultivation of the organism aerobically or anaerobically, as may be necessary, in each incubator, and examine at half-hour intervals for from five to eighteen hours.

4. Note that temperature at which growth is first observed macroscopically (Optimum temperature).

5. Continue the incubation until the completion of seven days. Note the extremes of temperature at which growth takes place (Range of temperature).

6. Control these results—if considered necessary arranging the series of incubators to include each degree centigrade for five degrees beyond each of the extremes previously noted.

(B) Optimum.—

1. Prepare a second series of ten tube cultivations under similar conditions as to reaction of medium.

2. Incubate in a series of incubators in which the temperature is regulated at intervals of 1° C. for five degrees on either side of optimum temperature observed in the previous experiment (A, step 4).

3. Observe again at half-hour intervals and note that temperature at which growth is first visible to the naked eye = Optimum temperature.

(C) Thermal Death-point (t. d. p.)—

Moist—Vegetative Forms:

The t. d. p. here is that temperature which with certainty kills a watery suspension of the organisms in question after an exposure of 10 minutes.

Method.—

1. Prepare tube cultivations on solid media of optimum reaction; incubate forty-eight hours under optimum conditions as to temperature and atmosphere.

2. Examine preparations from the cultivation microscopically to determine the absence of spores.

3. Pipette 5 c.c. salt solution into each of twelve capsules.

4. Suspend three loopfuls of the surface growth (using a special platinum loop, vide page 316) in the normal saline solution by emulcifying evenly against the moist walls of each capsule.

5. Transfer emulsion from each capsule to sterile 250 c.c. flask, and mix.

6. Pipette 5 c.c. emulsion into each of twelve sterile test-tubes numbered consecutively.

7. Adjust the first tube in the water-bath, regulated at 40° C, by means of two rubber rings around the tube, one above and the other below the perforated top of the bath, so that the upper level of the fluid in the tube is about 4 cm. below the surface of the water in the bath, and the bottom of the tube is a similar distance above the bottom of the bath.

8. Arrange a control test-tube containing 5 c.c. sterile saline solution under similar conditions. Plug the tube with cotton-wool and pass a thermometer through the plug so that its bulb is immersed in the water.

9. Close the unoccupied perforations in the lid of the water-bath by means of glass balls.

10. Watch the thermometer in the test-tube until it records a temperature of 40° C. Note the time. Ten minutes later remove the tube containing the suspension, and cool rapidly by immersing its lower end in a stream of running water.

11. Pour three gelatine (or agar) plates containing respectively 0.2, 0.3, and 0.5 c.c. of the suspension, and incubate.

12. Pipette the remaining 4 c.c. of the suspension into a culture flask containing 250 c.c. of nutrient bouillon, and incubate.

13. Observe these cultivations from day to day. "No growth" must not be recorded as final until after the completion of seven days' incubation.

14. Extend these observations to the remaining tubes of the series, but varying the conditions so that each tube is exposed to a temperature 2° C. higher than the immediately preceding one—i. e., 42° C., 44° C., 46° C., and so on.

15. Note that temperature, after exposure to which no growth takes place up to the end of seven days' incubation, = the thermal death-point.

16. If greater accuracy is desired, a second series of tubes may be prepared and exposed for ten minutes to fixed temperatures varying only 0.5° C., through a range of 5° C. on either side of the previously observed death-point.

Moist—Spores: The thermal death-point in the case of spores is that time exposure to a fixed temperature of 100° C. necessary to effect the death of all the spores present in a suspension.