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—H2S, CO2, H2. 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°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 lævulose 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° 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° 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° 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° 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.

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° 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° 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.

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.—

Media Required:

Inosite-free bouillon (vide page 183).
Or peptone water (vide page 177).

Reagents Required:

Potassium persulphate, saturated aqueous solution.
Paradimethylamino-benzaldehyde solution. This is prepared by mixing:

Paradimethylamino-benzaldehyde 4 grammes
Absolute alcohol 380 c.c.
Hydrochloric acid, concentrated 80 c.c.

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.

Indol is tested for in many laboratories by the ordinary nitrosoindol reaction which, however, is not so delicate a method as that above described. The test is carried out as follows:

1. Remove the cotton-wool plug from the tube, and run in 1 c.c. pure concentrated sulphuric acid down the side of the tube by means of a sterile pipette. Place the tube upright in a rack, and allow it to stand, if necessary, for ten minutes.

A rose-pink or red colour at the junction of the two liquids = indol (plus a nitrite).

2. If the colour of the medium remains unaltered, add 2 c.c. of a 0.01 per cent. aqueous solution sodium nitrite, and again allow the culture to stand for ten minutes.

Red colouration = indol.

Note.—In place of performing the test in two stages as given above, 2 c.c. concentrated commercial sulphuric, hydrochloric, or nitric acid (all of which hold a trace of nitrite in solution), may be run into the cultivation. The development of a red colour within twenty minutes will indicate the presence of indol.

5a. Phenol Production.—

Medium Required:

Nutrient bouillon.

Reagents Required:

Hydrochloric acid, concentrated.
Millon's reagent.
Ferric chloride, 1 per cent. aqueous solution.

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.

Note.—If both indol and phenol appear to be present in cultivations of the same organism, it is well to separate them before testing. This may be done in the following manner:

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.—

Medium Required:

Nitrate bouillon (vide page 185).
Or nitrate peptone solution (vide page 186).

Reagents Required:

Sulphuric acid (25 per cent.).
Metaphenylene diamine, 5 per cent. aqueous solution.

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.—

Apparatus Required:

Fermentation tubes (vide page 161) containing sugar bouillon (glucose, lactose, etc.). The medium should be prepared from inosite-free bouillon (vide page 183).

Reagent Required:

n/2 caustic soda.

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.—

Media Required:

Iron peptone solution (vide page 185).
Lead peptone solution.

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:

Apparatus Required:

Bohemian flask (300 to 1500 c.c. capacity) containing from 100 to 400 c.c. of the medium. The mouth of the flask is fitted with a perforated rubber stopper, carrying an L-shaped piece of glass tubing (the short arm passing just through the stopper). To the long arm of the tube is attached a piece of pressure tubing some 8 cm. in length, plugged at its free end with a piece of cotton-wool. Measure accurately the total capacity of the flask and exit tube, also the amount of medium contained. Note the difference.

Gas receiver. This is a bell jar of stout glass, 14 cm. high and 9 cm. in diameter. At its apex a glass tube is fused in. This rises vertically 5 cm., and is then bent at right angles, the horizontal arm being 10 cm. in length. A three-way tap is let horizontally into the vertical tube just above its junction with the bell jar.

An iron cylinder just large enough to contain the bell jar.

About 15 kilos of metallic mercury.

Melted paraffin.

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.