c. The presence or absence of gas formation, as indicated by a froth
on the surface of the medium, and the collection of gas in the inner "gas" tube.
9. Replace those tubes which show no signs of growth in the incubator. Examine after another period of twenty-four hours (total forty-eight hours incubation) with reference to the same points.
10. Remove culture tubes which show visible growth from the Buchner's tubes, whether acid production and gas formation are present or not.
11. Examine all tubes which show growth by hanging-drop preparations. Note such as show the presence of chains of cocci.
12. Prepare surface plate cultivations upon nutrose agar from each tube that shows growth either macroscopically or microscopically, and incubate for twenty-four hours aerobically at 37 deg. C.
13. Examine the growth on the plates either with the naked eye or with the help of a small hand lens. Practice will facilitate the recognition of colonies of the coli group, the typhoid group and the paratyphoid group; also those due to the growth of streptococci. The investigation from this stage proceeds along two divergent lines of enquiry--the first being concerned with the identity of the bacilli--typhoid bacilli, the second with that of the cocci.
A. _B. Coli and its allies._
14. Pick off coliform or typhiform colonies; make streak or smear subcultivations upon nutrient agar; incubate aerobically for twenty-four hours at 37 deg. C.
15. Examine the growth in each tube carefully both macroscopically and microscopically. If the growth is impure, replate on nutrose agar, pick off colonies and subcultivate again. When the growth in a tube is pure, add 5 c.c. sterile normal saline solution or sterile broth, and emulsify the entire surface growth with it.
16. Utilise the emulsion for the preparation of a series of subcultivations upon the media enumerated below, using the ordinary loop to make the subcultures upon solid media, but adding one-tenth of a cubic centimetre of the emulsion to each of the fluid media by means of a sterile pipette.
Gelatine streak. Agar streak. Potato. Nutrient broth. Litmus milk. Dextrose peptone solution. Laevulose peptone solution. Galactose peptone solution. Maltose peptone solution. Lactose peptone solution. Saccharose peptone solution. Raffinose peptone solution. Dulcite peptone solution. Mannite peptone solution. Glycerin peptone solution. Inulin peptone solution. Dextrin peptone solution.
17. Differentiate the bacilli after isolation by means of their cultural reactions and biological characters into members of:
I. The Escherich Group.
B. coli communis. B. coli communior. B. lactis aerogenes. B. cloacae.
II. The Gaertner Group.
Bacillus enteritidis (of Gaertner). B. paratyphosus A. B. paratyphosus B. Bacillus cholerae suum.
III. The Eberth Group.
B. typhosus. B. dysenteriae (Shiga). B. dysenteriae (Flexner). B. faecalis alcaligines.
18. Confirm these results by testing the organisms isolated against specific agglutinating sera obtained from experimentally inoculated animals.
If a positive result is obtained when using this method, it only needs a simple calculation to determine the smallest quantity (down to 0.1 c.c.) of the sample that contains at least one of the microbes of indication. For instance, if growth occurs in all the tubes from 4 to 10, and that growth is subsequently proved to be due to the multiplication of B. coli, then it follows that at least one colon bacillus is present in every 10 c.c. of the water sample, but not in every 5 c.c. If, on the other hand, the presence of the B. coli can only be proved in flask No. 7, then the average number of colon bacilli present in the sample is at least one in every 50 c.c. (i. e., twenty per litre), but not one in every 25 c.c. and so on.
The general outline of the method of identifying the members of the coli-typhoid group is given in the form of an analytical schema--whilst the full differential details are set out in tabular form.
ANALYTICAL SCHEME FOR ISOLATION OF MEMBERS OF THE COLI AND TYPHOID GROUPS.
Nutrose agar. | ----------------------------------- | | Red colonies. Blue colonies. Escherich group. Gaertner and Eberth groups. || | ====================--------------- || Lactose peptone solution. || ====================--------------- || | Gas. No gas. || | B. coli communis and its allies. | || Gaertner and Eberth groups. Acid and gas in glucose peptone solution. | Acid and coagulation in milk. | General turbidity and indol in bouillon. Glucose peptone solution. | ==================================| || | || | Gas. No gas. || | Gaertner group. Eberth group. || | =================== ---------------- || || | | || || | | Litmus milk. Peptone solution. Litmus milk. Peptone solution. || || | | Acid at first. General turbidity. Acid. General turbidity. Alkaline later. No indol. No coagulation. No indol. No coagulation. Serum reaction. Serum reaction.
_B. Streptococci._
19. Pick off streptococcus colonies and subcultivate upon nutrient agar exactly as directed in steps 14, 15 and 16.
20. Differentiate the streptococci isolated into members of the saprophytic group of short-chained cocci, or members of the parasitic (pathogenic) group of long-chained cocci, by means of their cultural characters, and record their numerical frequency in the manner indicated for the members of the coli-typhoid group.
DIFFERENTIAL TABLE OF COLI-TYPHOID GROUP
Transcriber's note: Table split to fit 80 spaces.
+-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ | | M | D | L | G | M | L | S | R | D | |A = acid reaction | o | e | a | a | a | a | a | a | e | |G = gas formation | t | x | e | l | l | c | c | f | t | | | i | t | v | a | t | t | c | f | r | | | l | r | u | c | o | o | h | i | i | | | i | o | l | t | s | s | a | n | n | | | t | s | o | o | e | e | r | o | | | | y | e | s | s | | | o | s | | | | | | e | e | | | s | e | | | | | | | | | | e | | | | | +-----+-----+-----+-----+-----+-----+-----+-----+ | | | A G | A G | A G | A G | A G | A G | A G | A G | +-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ |_The Escherich Group._ | | | | | | | | | | | B. coli communis | + | + + | + + | + + | + + | + + | O | + + | + + | | B. coli communior | + | + + | + + | + + | + + | + + | + + | + + | + + | | B. lactis aerogenes | - | + + | + + | + + | + + | + + | O | O | + + | | B. acidi lactici | - | + + | + + | + + | + + | + + | O | O | O | | B. pneumoniae | - | + + | + + | + + | + + | + + | + + | + + | + + | | B cloaceae(A) | + | + + | + + | + + | + + | + + | + + | + + | + + | | | | | | | | | | | | |_The Gaertner Group._ | | | | | | | | | | | B. enteritidis | + | + + | + + | + + | + + | O | O | O | O | | B. paratyphosus A | + | + + | + + | + + | + + | O | O | O | O | | B. paratyphosus B | + | + + | + + | + + | + + | O | O | O | O | | B. cholerae suum | + | + + | + + | + + | + + | O | O | | O | | B. suipestifer | + | + + | + + | + + | + + | O | O | | O | | | | | | | | | | | | |_The Eberth Group._ | | | | | | | | | | | B. typhosus | + | + | + | + | + | O | O | O | + | | B. dysenteriae (Shiga) | - | + | + | + | O | O | O | O | O | | B. dysenteriae (Flexner)| - | + | + | + | + | O | O | +/- | O | | B. faecalis alkaligines | + | O | O | O | O | O | O | O | O | | | | | | | | | | | | +-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ | Table Notes: |(B)| (C) | +-------------------------+---+-----------------------------------------------+
+-------------------------+-----+-----+-----+-----+-----+-----+---+-----------+ | | I | S | G | D | M | S | I |Litmus Milk| |A=acid reaction | n | a | l | u | a | o | n | | |G=gas formation | u | l | y | l | n | r | d | | | | l | i | c | c | n | b | o +-----+-----+ | | i | c | e | i | i | i | l |Early|Late | | | n | i | r | t | t | t | | | | | | | n | i | e | e | e | | | | | | | | n | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |-----+-----+-----+-----+-----+-----+ | | | | | A G | A G | A G | A G | A G | A G | | | | +-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+ |_The Escherich Group_ | | | | | | | | | | | B. coli communis | O | O | + + | + + | + + | + + | + | + | + C | | B. coli communior | O | O | + + | + + | + + | + + | + | + | + C | | B. lactis aerogenes | O | O | O | O | + + | + + | - | + | + C | | B. acidi lactici | O | O | O | + + | + + | + + | + | + | + C | | B. pneumoniae | O | O | + + | + + | + + | + + | - | + | + C | | B cloaceae[A] | O | O | + + | O | + + | - + | + | + | + C | | | | | | | | | | | | |_The Gaertner Group._ | | | | | | | | | | | B. enteritidis | O | O | O | + + | + + | + + | - | +/- | - | | B. paratyphosus A | O | +/- | O | + + | + + | + + | - | + | O | | B. paratyphosus B | O | O | O | + + | + + | + + | - | + | - | | B. cholerae suum | O | O | O | O | O | + + |+/-| + | - | | B. suipestifer | O | O | O | + + | + + | + + | - | + | - | | | | | | | | | | | | |_The Eberth Group._ | | | | | | | | | | | B. typhosus | O | O | O | O | + | + | - | + | + | | B. dysenteriae (Shiga) | O | O | O | O | O | O | - | + | - | | B. dysenteriae (Flexner)| O | O | O | O | + | O |+/-| + | - | | B. faecalis alkaligines | O | O | O | O | O | O | - | - | - | | | | | | | | | | | | +-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+ | Table Notes: | |(D)| (E) | +-------------------------+-----------------------------------+---+-----------+
Table Notes:
(A) * Liquefies gelatine.
(B) + = motile. - = non-motile.
(C) + = acid or gas production. +/- = slight acid production. O = no change.
(D) + = indol production. +/- = slight indol production. - = no indol formed.
(E) + = acid production. - = alkali production. O = no change in reaction. C = clot.
21. Determine the pathogenicity for mice (subcutaneous inoculation) and rabbits (intravenous inoculation) of the streptococci isolated.
On the facing insert page is reproduced a blank from the author's Laboratory Water Analysis Book, by means of which an exact record can be kept, with a minimum of labour, of every sample examined.
B. ~Concentration Method.~
The remaining organisms referred to on page 426 are more conveniently sought for by the concentration method.
_Collection of the Sample._--The quantity of water required for this method of examination is about 2000 c.c., and the vessel usually chosen for its reception is an ordinary blue glass Winchester quart bottle, sterilised in the hot-air oven, and over this a paper or parchment cap fastened with string. The bottle may be packed in a wooden box or in an ordinary wicker case. The method of collecting the sample is identical with that described under the heading of Quantitative Examination; there is, however, not the same imperative necessity to pack the sample in ice for transmission to the laboratory.
_Apparatus required_:
Sterile Chamberland or Doulton "white" porcelain open mouth filter candle, fitted with rubber washer.
Rubber cork to fit mouth of the filter candle, perforated with one hole.
Kitasato serum flask, 2500 c.c. capacity.
Geryk air pump or water force pump.
Wulff's bottle, fitted as wash-bottle, and containing sulphuric acid (to act as a safety valve between filter and pump).
Pressure tubing, clamps, pinch-cock.
Retort stand, with ring and clamp.
Rubber cork for the neck of Winchester quart, perforated with two holes and fitted with one 6 cm. length of straight glass tubing, and one V-shaped piece of glass tubing, one arm 32 cm. in length, the other 52 cm., the shorter arm being plugged with cotton-wool. The rubber stopper must be sterilised by boiling and the glass tubing by hot air, before use.
Flask containing 250 c.c. sterile broth.
Test-tube brush to fit the lumen of the candle, enclosed in a sterile test-tube (and previously sterilised by dry heat or by boiling).
Case of sterile pipettes, 10 c.c. in tenths.
Case of sterile pipettes, 1 c.c. in tenths.
Case of sterile pipettes, 1 c.c. in hundredths.
Tubes of various nutrient media (according to requirements).
Twelve Buchner's tubes with rubber stoppers.
Pyrogallic acid tablets.
Caustic soda tablets.
METHOD.--
1. Fit up the filtering apparatus as in the accompanying diagram (Fig. 209), interposing the wash-bottle with sulphuric acid between the filter flask and the force-pump (in the position occupied in the diagram by the central vertical line), and placing another screw clamp on the rubber tubing connecting the lateral arm of the filter flask with the wash-bottle.
2. Filter the entire 2000 c.c. of water through the filter candle.
3. When the nitration is completed, screw up the clamps and so occlude the two pieces of pressure tubing.
4. Reverse the position of the glass tubes in the Wulff's bottle so that the one nearest the air pump now dips into the sulphuric acid.
5. Slowly open the metal clamps and allow air to gradually pass through the acid, and enter filter flask, and so restore the pressure.
6. Unship the apparatus, remove the cork from the mouth of the candle.
7. Pipette 10 c.c. of sterile broth into the interior of the candle, and by means of the sterile test-tube brush (Fig. 210) emulsify the slimy residue which lines the candle, with the broth.
Practically all the bacteria contained in the original 2000 c.c. of water are now suspended in 10 c.c. of broth, so that 1 c.c. of the suspension is equivalent, so far as the contained organisms are concerned, to 200 c.c. of the original water. (Some bacteria will of course be left behind on the walls of the filter and in its pores.)
Up to this point the method is identical, irrespective of the particular organism whose presence it is desired to demonstrate; but from this point onward the methods must be specially adapted to the isolation of definite groups of organisms or of individual bacteria.
The Coli-Typhoid Group.--
1. Number nine tubes of bile salt broth (_vide_ page 180), consecutively from 1 to 9.
2. To No 1 add 1 c.c. } of the original water sample 2 add 2 c.c. } before the nitration is commenced. 3 add 5 c.c. }
3. To the remaining tubes of bile salt broth add varying quantities of the suspension by means of suitably graduated sterile pipettes, as follows:
No. 4 0.05 c.c. (equivalent to 10 c.c. of the original water sample). No. 5 0.125 c.c. (equivalent to 25 c.c. of the original water sample). No. 6 0.25 c.c. (equivalent to 50 c.c. of the original water sample). No. 7 0.5 c.c. (equivalent to 100 c.c. of the original water sample). No. 8 1.0 c.c. (equivalent to 200 c.c. of the original water sample). No. 9 2.5 c.c. (equivalent to 500 c.c. of the original water sample).
4. Put up each tube anaerobically in a Buchner's tube and incubate at 42 deg. C.
5. The subsequent steps are identical with those described under the Enrichment method (see page 428 to 431; Steps 8 to 18).
_Alternative Methods._--
A few of the older methods for the isolation of the members of the coli-typhoid groups are referred to but they are distinctly inferior to those already described.
(A) The Carbolic Method:
1. Take ten tubes of carbolised bouillon (_vide_ page 202) and number them consecutively from 1 to 10.
2. Inoculate each tube with a different amount of the water sample or suspension, as in the previous method.
3. Incubate aerobically at 37 deg. C.
4. Examine the culture tubes after twenty-four hours' incubation.
5. From those tubes which shows signs of growth, pour plates in the usual manner, using carbolised gelatine (_vide_ page 202) in place of the ordinary gelatine, and incubate at 20 deg. C. for three, four, or five days as may be necessary.
6. Subcultivate from any colonies that make their appearance, and determine their identity on the lines laid down in the previous method.
(B) Parietti's Method:
1. Take nine tubes of Parietti's bouillon (_vide_ page 202)--i. e., three each of those containing 0.1 c.c., 0.2 c.c., and 0.5 c.c. of Parietti's solution respectively. Mark plainly on the outside of each tube the quantity of Parietti's solution it contains.
2. To each tube add a different amount of the original water, or of the suspension, and incubate at 37 deg. C.
3. Examine the culture tubes after twenty-four and forty-eight hours' incubation, and plate in nutrient carbolised or potato gelatine from such as have grown.
4. Pick off suspicious colonies, if any such appear on the plates, subcultivate them upon the various media, and identify them.
(C) Elsner's Method: This method simply consists in substituting Elsner's potato gelatine (_vide_ page 204) for ordinary nutrient gelatine in any of the previously mentioned methods.
(D) Cambier's Candle Method:
Treat a large volume of the water sample by the concentration method (_vide_ page 434).
1. Remove the rubber stopper from the mouth of the filter candle, introduce 10 c.c. sterile bouillon into its interior, and emulsify the bacterial sediment; replug the mouth of the candle with a wad of sterile cotton-wool.
2. Remove the filter candle from the filter flask and insert it into the mouth of a flask or a glass cylinder containing sterile bouillon sufficient to reach nearly up to the rubber washer on the candle.
3. Incubate for twenty-four to thirty-six hours at 37 deg. C.
4. From the now turbid bouillon in the glass cylinder pour gelatine plates and incubate at 20 deg. C.
5. Subcultivate and identify any suspicious colonies that appear.
(The method depends upon the assumption that members of the typhoid and coli groups find their way through the porcelain filter from the interior to the surrounding bouillon at a quicker rate than the associated bacteria.)
B. ~Enteritidis Sporogenes.~--
1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.
2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (_vide_ page 257), and expose to a temperature of 80 deg. C. for twenty minutes.
3. Number ten tubes of litmus milk consecutively from 1 to 10.
4. Remove the test-tube from the benzole bath and shake well to distribute the spores evenly through the fluid.
5. To each tube of litmus milk add a measured quantity of the suspension corresponding to the amounts employed in isolating the coli group (_vide_ page 437).
6. Incubate each tube anaerobically at 37 deg. C. Anaerobic conditions can be obtained by putting the cultures up in Buchner's tubes or in Bulloch's apparatus. If, however, whole milk has been used in making the litmus milk the layer of cream that rises to the surface will be sufficient to ensure anaerobiosis; whilst if separated milk has been employed it will be sufficient to pour a layer of sterile vaseline or liquid paraffin on the surface of the fluid.
7. Examine after twenty-four hours' incubation. Note (if B. enteritidis sporogenes is present)--
(a) Acid reaction of the medium as indicated by the colour of the litmus or its complete decolourisation.
(b) Presence of clotting, and the separation of clear whey.
(c) Presence of gas, as indicated by fissures and bubbles in the coagulum, and possibly masses of coagulum driven up the tube almost to the plug.
8. Replace the tubes which show no signs of growth in the incubator for a further period of twenty-four hours and again examine with reference to the same points.
9. Remove those tubes which give evidence of growth from the Buchner's tubes and carefully pipette off the whey; examine the whey microscopically.
10. Inoculate two guinea-pigs each subcutaneously with 0.5 c.c. of the whey and observe the result.
~Vibrio Cholerae.~--
1. Number ten tubes of peptone water consecutively from 1 to 10.
2. To each of the tubes of peptone water add a measured quantity of the suspension, corresponding to those amounts employed in isolating the members of the coli group (_vide_ page 437).
3. Incubate aerobically at 37 deg. C. for twenty-four hours. Examine the tubes carefully for visible growth, especially delicate pellicle formation, which if present should be examined microscopically for vibrios, both by stained preparations or by fresh specimens with dark ground illumination.
4. Inoculate fresh tubes of peptone water from such of the tubes as exhibit pellicle formation--from the pellicle itself--and incubate at 37 deg. C. for twenty-four hours.
5. Test the peptone water itself for the presence of indol and nitrite by the addition of pure concentrated H_{2}SO_{4}.
5. Prepare gelatine and agar plates in the usual way from such of these tubes as show pellicle formation.
6. Pick off from the plates any colonies resembling those of the Vibrio cholerae and subcultivate upon all the ordinary laboratory media.
7. Test the vibrio isolated against the serum of an animal immunised to the Vibrio cholerae for agglutination.
~B. Anthracis.~--
1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.
2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (_vide_ page 257), and expose to a temperature of 80 deg. C. for twenty minutes.
3. Inoculate a _young_ white rat subcutaneously (on the inner aspect of one of the hind legs) with 1 c.c. of the emulsion. Observe during life, and, if the animal succumbs, make a complete post-mortem examination.
4. Melt three tubes of nutrient agar in boiling water and cool to 42 deg. C.
5. Number the tubes 1, 2, and 3. To No. 1 add 0.2 c.c., to No. 2 add 0.3 c.c., and to No. 3 add 0.5 c.c. of the suspension, and pour plates therefrom.
6. Incubate at 37 deg. C. for twenty-four or forty-eight hours.
7. Pick off any colonies resembling those of anthrax and subcultivate on all the ordinary laboratory media.
8. Inoculate another young white rat as in 3, using two loopfuls of the agar subcultivation emulsified with 1 c.c. sterile bouillon. Observe during life, and if the animal succumbs, make a complete post-mortem examination.
~B. Tetani.~--
1. Proceed as detailed above in steps 1 and 2 for the isolation of the B. anthracis.
2. Add 1 c.c. of the suspension to each of three tubes of glucose formate broth, and incubate anaerobically in Buchner's tubes at 37 deg. C.
3. From such of the tubes as show visible growth (with or without the production of gas) after twenty-four hours' incubation inoculate guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1 c.c. of the bouillon cultivation as a dose. Observe carefully during life, and, if death occurs, make a complete post-mortem examination.
4. From the same tubes pour agar plates and incubate anaerobically in Bulloch's apparatus, at 37 deg. C.
5. Subcultivate suspicious colonies on the various media, incubate anaerobically, making control cultivations on glucose formate agar, stab and streak, to incubate aerobically and carry out further inoculation experiments with the resulting growths.
EXAMINATION OF MILK.
"One-cow" or "whole" milk, if taken from the apparently healthy animal (that is, an animal without any obvious lesion of the udder or teats) with ordinary precautions as to cleanliness, avoidance of dust, etc., contains but few organisms. In dealing with one-cow milk, from a suspected, or an obviously diseased animal, a complete analysis should include the examination (both qualitative and quantitative) of samples of (a) fore-milk, (b) mid-milk, (c) strippings, and, if possible, from each quarter of the udder. "Mixed" milk, on the other hand, by the time it leaves the retailer's hands, usually contains as many micro-organisms as an equal volume of sewage and indeed during the examination it is treated as such.
It is possible however to collect and store mixed milk in so cleanly a manner that its germ content does not exceed 5000 micro-organisms per cubic centimetre. Such comparative freedom from extraneous bacteria is usually secured by the purveyor only when he resorts to the process of pasteurisation (heating the milk to 65 deg. C. for twenty minutes or to 77 deg. C. for one minute) or the simpler plan of adding preservatives to the milk. Information regarding the employment of these methods for the destruction of bacteria should always be sought in the case of mixed milk samples, and in this connection the following tests will be found useful:
1. _Raw Milk_ (Saul).
To 10 c.c. milk in a test tube, add 1 c.c. of a 1 per cent. aqueous solution of ortol (ortho-methyl-amino-phenol sulphate), recently prepared and mix. Next add 0.2 c.c. of a 3 per cent. peroxide of hydrogen solution. The appearance of a brick red color within 30 seconds indicates raw milk. Milk heated to 74 deg. C. for thirty minutes undergoes no alteration in color; if heated to 75 deg. C. for ten minutes only, the brick red color appears after standing for about two minutes.
2. _Boric Acid._
Evaporate to dryness, 50 c.c. of the milk which has been rendered slightly alkaline to litmus, then incinerate.
Dissolve in distilled water, add slight excess of dilute hydrochloric acid and again evaporate to dryness.
Dissolve the residue in a small quantity of hot water and moisten a piece of turmeric paper with the solution. Dry the turmeric paper. _Rose_ or _cherry-red_ color = borax or boric acid.
3. _Formaldehyde_ (Hehner).
To 10 c.c. milk in a test tube add 5 c.c. concentrated _commercial_ sulphuric acid slowly, so that the two fluids do not mix. Hold the tube vertically and agitate very gently. _Violet zone_ at the junction of the two liquids = formaldehyde.
4. _Hydrogen Peroxide._
To 10 c.c. milk (diluted with equal quantities of water) in a test tube add 0.4 c.c. of a 4 per cent. alcoholic solution of benzidine and 0.2 c.c. acetic acid. _Blue coloration_ of the mixture = hydrogen peroxide.
5. _Salicylic Acid._
Precipitate the caseinogen by the addition of acetic acid and filter. To the filtrate add a few drops of 1 per cent. aqueous solution of ferric chloride. _Purple coloration_ = salicylic acid.
6. _Sodium Carbonate or Bicarbonate._
To 10 c.c. of the milk in a test tube add 10 c.c. of alcohol and 0.3 c.c. of a 1 per cent. alcoholic solution of rosolic acid. _Brownish_ color = pure milk; _rose_ color = preserved milk.
Quantitative.--
_Collection of Sample._--
The apparatus used for the collection of a retail mixed milk sample consists of a cylindrical copper case, 16 cm. high and 9 cm. in diameter, provided with a "pull-off" lid, containing a milk dipper, also made of copper; and inside this, again, a wide-mouthed, stoppered glass bottle of about 250 c.c. capacity (about 14 cm. high by 7 cm. diameter), having a tablet for notes, sand-blasted on the side. The copper cylinder and its contents, secured from shaking by packing with cotton-wool, are sterilised in the hot-air oven (Fig. 26).
When collecting a sample,
1. Remove the cap from the cylinder.
2. Draw out the cotton-wool.
3. Lift out the bottle and dipper together.
4. Receive the milk in the sterile dipper, and pour it directly into the sterile bottle.
5. Enter the particulars necessary for the identification of the specimen, on the tablet, with a lead pencil, or pen and ink.
6. Pack the apparatus in the ice-box for transmission to the laboratory in precisely the same manner as an ordinary water sample.
"Whole" milk may with advantage be collected in the sterile bottle directly since the mouth is sufficiently wide for the milker to direct the stream of milk into it.
~Condensed milk~ must be diluted with sterile distilled water in accordance with the directions printed upon the label, then treated as ordinary milk.
_Apparatus Required_:
Case of sterile capsules (25 c.c. capacity). Case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre). Case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre). Flask containing 250 c.c. sterile bouillon. Tall cylinder containing 2 per cent. lysol solution. Plate-levelling stand. Case of sterile plates. Tubes nutrient gelatine or gelatine agar. Tubes of wort gelatine. Tubes of nutrient agar. Water-bath regulated at 42 deg. C. Bunsen burner. Grease pencil.
METHOD.--
1. Arrange four sterile capsules in a row; number them I, II, III, and IV.
2. Fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon into each of the three remaining capsules.
3. Remove 1 c.c. milk from one of the bottles by means of a sterile pipette and add it to the bouillon in capsule I; mix thoroughly by repeatedly filling and emptying the pipette.
4. Remove 0.1 c.c. of the milky bouillon from capsule I, add it to the contents of capsule II, and mix as before.
5. In like manner add 0.1 c.c. of the contents of capsule II to capsule III; and then 0.1 c.c. of the contents of capsule III to capsule IV.
Then 1 c.c. of dilution I contains 0.1 c.c. milk sample. 1 c.c. of dilution II contains 0.001 c.c. milk sample. 1 c.c. of dilution III contains 0.00001 c.c. milk sample. 1 c.c. of dilution IV contains 0.0000001 c.c. milk sample.
6. Melt the gelatine and the agar tubes in boiling water; then transfer to the water-bath and cool them down to 42 deg. C.
7. Number the gelatine tubes consecutively 1 to 12.
8. Inoculate the tubes with varying quantities of the material as follows:
To tube No. 1 add 1.0 c.c. of the milk sample. 2 add 0.1 c.c. of the milk sample. { 3 add 1.0 c.c. from capsule I. { 4 add 0.1 c.c. from capsule I. { 5 add 1.0 c.c. from capsule II. { 6 add 0.1 c.c. from capsule II. { 7 add 0.5 c.c. from capsule III. { 8 add 0.3 c.c. from capsule III. { 9 add 0.2 c.c. from capsule III. { 10 add 0.5 c.c. from capsule IV. { 11 add 0.3 c.c. from capsule IV. { 12 add 0.2 c.c. from capsule IV.
9. Pour plates from the gelatine tubes; label, and incubate at 20 deg. C.
10. Liquefy five wort gelatine tubes and to them add 1.0 c.c. of the milk sample and a similar quantity of the diluted milk from capsules I, II, and III and IV respectively.
11. Pour plates from the wort gelatine; label, and incubate at 20 deg. C.
12. Inoculate the liquefied agar tubes as follows:
To tube No. 1 add 0.1 c.c. of the milk sample. 2 add 0.1 c.c. from capsule I. 3 add 0.1 c.c. from capsule II. 4 add 0.1 c.c. from capsule III. 5 add 1.0 c.c. from capsule IV. } 6 add 0.1 c.c. from capsule IV. }
13. Pour plates from the agar tubes; label, and incubate at 37 deg. C.
14. After twenty-four hours' incubation "inspect," and after forty-eight hours' incubation, "count" the agar plates and estimate the number of "organisms growing at 37 deg. C." present per cubic centimetre of the sample of milk.
15. After three, four, or five days' incubation, "count" the gelatine plates and estimate therefrom the number of "organisms growing at 20 deg. C." present per cubic centimetre of the sample of milk.
16. After a similar interval "count" the wort gelatine plates and estimate the number of moulds and yeasts present per cubic centimetre of the sample of milk.
NOTE.--Many observers prefer to employ gelatine agar (see page 193) for the quantitative examination. In this case gelatine-agar plates should be poured from tubes containing the quantities of material indicated in step 8, incubated at 28 deg. C. to 30 deg. C. and after five days the "total number of organisms developing at 28 deg. C." recorded.
~Qualitative.~--The qualitative bacteriological examination of milk is chiefly directed to the detection of the presence of one or more of the following pathogenic bacteria and when present to the estimation of their numerical frequency.
Members of the Coli-typhoid group. Vibrio cholerae. Streptococcus pyogenes longus. Micrococcus melitensis. Staphylococcus pyogenes aureus. Bacillus enteritidis sporogenes. Bacillus diphtheriae. Bacillus tuberculosis.
Some of these occur as accidental contaminations, either from the water supply to the cow farm, or from the farm employees, whilst others are derived directly from the cow.
In milk, as in water examinations, two methods are available, viz.: Enrichment and Concentration--the former is used for the demonstration of bacteria of intestinal origin, the latter for the isolation of the micro-organisms of diphtheria and tubercle. The first essential in the latter process is the concentration of the bacterial contents of a large volume of the sample into a small compass; but in the case of milk, thorough centrifugalisation is substituted for filtration.
_Apparatus Required_:
A large centrifugal machine. This machine, to be of real service in the bacteriological examination of milk, must conform to the following requirements:
1. The centrifugal machine must be of such size, and should carry tubes or bottles of such capacity, as to enable from 200 to 500 c.c. of milk to be manipulated at one time.
2. The rate of centrifugalisation should be from 2500 to 3000 revolutions per minute.
3. The portion of the machine destined to carry the tubes should be a metal disc, of sufficient weight to ensure good "flank" movement, continuing over a considerable period of time. In other words, the machine should run down very gradually and slowly after the motive power is removed, thus obviating any disturbance of the relative positions of particulate matter in the solution that is being centrifugalised.
4. The machine should preferably be driven by electricity, or by power, but in the case of hand-driven machines--
(a) The gearing should be so arranged that the requisite speed is obtained by not more than forty or fifty revolutions of the crank handle per minute, so that it may be maintained for periods of twenty or thirty minutes without undue exertion.
(b) The handle employed should be provided with a special fastening (e. g., a clutch similar to that employed for the free wheel of a bicycle), or should be readily detachable so that, on ceasing to turn, the handle should not, by its weight and air resistance, act as a brake and stop the machine too suddenly.
One of the few satisfactory machines of this class is shown in figure 212.
Sterile centrifugal tubes, of some 60-70 c.c. capacity, tapering to a point at the closed end, plugged with cotton-wool.
Small centrifugal machine to run two tubes of 10 c.c. capacity at 2500 to 3000 revolutions per minute preferably driven by electricity, of the type figured on page 327 (Fig. 162).
Sterile centrifugal tubes of 10 c.c. capacity with the distal extremity contracted to a narrow tube and graduated in hundredths of a cubic centimetre (Fig. 213).
Sterilised cork borer.
Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).
Case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre).
Sterile teat pipettes.
Flask of sterile normal saline solution.
METHOD.--
1. Fill 50 c.c. of the milk sample into each of four tubes, and replace the cotton-wool plugs by solid rubber stoppers (sterilised by boiling), and fit the tubes in the centrifugal machine.
NOTE.--One or two cubic centimetres of paraffinum liquidum introduced into the buckets of the centrifuge before the glass tubes are inserted will obviate any risk of breakage to the latter.
2. Centrifugalise the milk sample for thirty minutes at a speed of 2500 revolutions per minute.
3. Remove the motive power and allow the machine to slow down gradually.
4. Remove the tubes of milk from the centrifuge. Each tube will now show (Fig. 214):
(a) A superficial layer of cream (varying in thickness with different samples) condensed into a semi-solid mass, which can be shown to contain some organisms and a few leucocytes.
(b) A central layer of separated milk, thin, watery, and opalescent, and containing extremely few bacteria.
(c) A sediment or deposit consisting of the great majority of the contained bacteria and leucocytes, together with adventitious matter, such as dirt, hair, epithelial cells, faecal debris, etc.
5. Withdraw the rubber stopper and remove a central plug of cream from each tube by means of a sterile cork borer; place these masses of cream in two sterile capsules. Label C^{1} and C^{2}.
6. Remove all but the last one or two c.c. of separated milk from each tube, by means of sterile pipettes.
7. Mix the deposits thoroughly with the residual milk, pipette the mixture from each pair of tubes into one sterile 10 c.c. tube (graduated) by means of sterile teat pipettes, then fill to the 10 c.c. mark with sterile normal saline solution and mix together. Label D^{1} and D^{2}.
8. Place the two tubes of mixed deposit in the centrifuge, adjust by the addition or subtraction of saline solution so that they counterpoise exactly, and centrifugalise for ten minutes.
NOTE.--Each tube now contains the deposit from 100 c.c. of the milk sample and the amount can be read off in hundredths of a centimetre. The multiplication of this figure by 100 will give the amount of "Apparent Filth," in "parts per million"--the usual method of recording this quality of milk.
9. Pipette off all the supernatant fluid and invert the tube to drain on to a pad of sterilised cotton-wool, contained in a beaker. (This wool is subsequently cremated.)
10. Examine both cream (C^{1}) and deposit (D^{1}) microscopically--
(a) In hanging-drop preparations.
(b) In film preparations stained carbolic methylene-blue, by Gram's method, by Neisser's method, and by Ziehl-Neelsen's method.
Note the presence or absence of altered and unaltered vegetable fibres; pus cells, blood discs; cocci in groups or chains, diphtheroid bacilli, Gram negative bacilli or cocci, spores and acid fast bacteria.
11. Adapt the final stages of the investigation to the special requirements of each individual sample, thus:
~1. Members of the Coli-typhoid Group.~--
1. Emulsify the deposit from the second centrifugal tube (D^{2}) with 10 c.c. sterile bouillon and inoculate three tubes of bile salt broth as follows:
To Tube No. 1 add 2.5 c.c. milk deposit emulsion (=25 c.c. original milk.) To Tube No. 2 add 1.0 c.c. milk deposit emulsion (=10 c.c. original milk.) To Tube No. 3 add 0.5 c.c. milk deposit emulsion (= 5 c.c. original milk.)
2. Inoculate tube of bile salt broth No. 4 with 1 c.c. of the original milk.
3. Inoculate further tubes of bile salt broth with previously prepared dilutions (see page 445) as follows:
To tube No. 5 add 1.0 c.c. from capsule I. To tube No. 6 add 0.1 c.c. from capsule I. To tube No. 7 add 1.0 c.c. from capsule II. To tube No. 8 add 0.1 c.c. from capsule II. To tube No. 9 add 1.0 c.c. from capsule III. To tube No. 10 add 0.1 c.c. from capsule III. To tube No. 11 add 1.0 c.c. from capsule IV. To tube No. 12 add 0.1 c.c. from capsule IV.
and incubate anaerobically (in Buchner's tubes) at 42 deg. C. for a maximum period of forty-eight hours.
4. If growth occurs complete the investigation as detailed under the corresponding section of water examination (see pages 428 to 431).
NOTE.--The B. coli communis, derived from the alvine discharges of the cow, is almost universally present in large or small numbers, in retail milk. Its detection, therefore, unless in enormous numbers, (when it indicates want of cleanliness), is of little value.
~2. Vibrio Cholerae.~--Inoculate tubes of peptone water by using the same amounts as in the search for members of the Coli-typhoid groups (_vide ante_ 1-3); incubate aerobically at 37 deg. C. and complete the examination as detailed under the corresponding section of water examination (see page 439).
~3. B. Enteritidis Sporogenes.~--Inoculate tubes of litmus milk with similar amounts to those used in the previous searches, omitting tube No. 1 (_vide ante_ 1-3) place in the differential steriliser at 80 deg. C. for ten minutes and then incubate anaerobically at 37 deg. C. for a maximum period of forty-eight hours. Complete the investigation as detailed under the corresponding section of water examination (see page 438).
~4. B. Diphtheriae.~--
(A) 1. Plant three sets of serial cultivations, twelve tubes in each set, from (a) cream C^{2}, (b) deposit D^{1} upon oblique inspissated blood-serum, and incubate at 37 deg. C.
2. Pick off any suspicious colonies which may have made their appearance twelve hours after incubation, examine microscopically and subcultivate upon blood-serum and place in the incubator; return the original tubes to the incubator.
3. Repeat this after eighteen hours' incubation.
4. From the resulting growths make cover-slip preparations and stain carbolic methylene-blue, Neisser's method, Gram's method. Subcultivate such as appear to be composed of diphtheria bacilli in glucose peptone solution. Note those in which acid production takes place.
5. Inoculate guinea-pigs subcutaneously with one or two cubic centimetres forty-eight-hour-old glucose bouillon cultivation derived from the first subcultivation of each glucose fermenter, and observe the result.
6. If death, apparently from diphtheritic toxaemia, ensues, inoculate two more guinea pigs with a similar quantity of the lethal culture. Reserve one animal as a control and into the other inject 1000 units of antidiphtheritic serum. If the control dies and the treated animal survives, the proof of the identity of the organism isolated with the Klebs-Loeffler bacillus becomes absolute.
7. Inoculate guinea-pigs subcutaneously with filtered glucose bouillon cultivations (toxins?) and observe the result.
(B) 1. Emulsify the remainder of the deposit with 5 c.c. sterile bouillon and inoculate two guinea-pigs, thus: guinea-pig a, subcutaneously with 1 c.c. emulsion; guinea-pig b, subcutaneously with 2 c.c. emulsion; and observe the result.
2. If either or both of the inoculated animals succumb, make complete post-mortem examination and endeavour to isolate the pathogenic organisms from the local lesion. Confirm their identity as in A5 and 6 (_vide supra_).
~5. Bacillus Tuberculosis.~--
(A) 1. Inoculate each of three guinea-pigs (previously tested with tuberculin, to prove their freedom from spontaneous tuberculosis) subcutaneously at the inner aspect of the bend of the left knee, with 1 c.c. of the deposit emulsion remaining in one or other tube (D^{1} or D^{2}).
2. Introduce a small quantity of the cream into a subcutaneous pocket prepared at the inner aspect of the bend of the right knee of each of these three animals. Place a sealed dressing on the wound.
3. Observe carefully, and weigh accurately each day.
4. Kill one guinea-pig at the end of the second week and make a complete post-mortem examination.
5. If the result of the examination is negative or inconclusive, kill a second guinea-pig at the end of the third week and examine carefully.
6. If still negative or inconclusive, kill the third guinea-pig at the end of the _sixth_ week. Make a careful post-mortem examination. Examine material from any caseous glands microscopically and inoculate freely on to Dorset's egg medium.
NOTE.--Every post-mortem examination of animals infected with tuberculous material should include the naked eye and microscopical examination of the popliteal, superficial and deep inguinal, iliac, lumbar and axillary glands on each side of the body, also the retrohepatic, bronchial and sternal glands, the spleen, liver and lungs (Fig. 215).
(B) 1. Intimately mix all the available cream and deposit from the milk sample, and transfer to a sterile Erlenmeyer flask.
2. Treat the mixture by the antiformin method (_vide_ Appendix, page 502).
3. Inoculate each of two guinea-pigs, intraperitoneally, with half of the emulsion thus obtained.
4. Kill one of the guinea-pigs at the end of the first week and examine carefully.
5. Kill the second guinea-pig at the end of the second week and examine carefully.
6. Utilise the remainder of the deposit for microscopical examination and cultivations upon Dorset's egg medium.
NOTE.--No value whatever attaches to the result of a microscopical examination for the presence of the B. tuberculosis unless confirmed by the result of inoculation experiments.
~6. Streptococcus Pyogenes Longus.~--
(A) 1. Spread serial surface plates upon nutrose agar. Also plant serial cultivations upon sloped nutrient agar (six tubes in series).
2. If the resulting growth shows colonies which resemble those of the streptococcus, make subcultivations upon agar and in bouillon, in the first instance, and study carefully.
(B) 1. Plant a large loopful of the deposit D^{2} into each of three tubes of glucose formate bouillon, and incubate anaerobically (in Buchner's tubes) for twenty-four hours at 37 deg. C.
2. If the resulting growth resembles that of the streptococcus, make subcultivations upon nutrient agar.
3. Prepare subcultivations of any suspicious colonies that appear, upon all the ordinary media, and study carefully.
If the streptococcus is successfully isolated, inoculate serum bouillon cultivations into the mouse, guinea-pig, and rabbit, to determine its pathogenicity and virulence.
~7. Staphylococcus Pyogenes Aureus.~--
1. Examine carefully the growth upon the serial blood serum cultivations prepared to isolate B. diphtheriae and the serial agar cultivations to isolate streptococci after forty-eight hours' incubation.
2. Pick off any suspicious orange coloured colonies, plant on sloped agar, and incubate at 20 deg. C. Observe pigment formation.
3. Prepare subcultivations from any suspicious growths upon all the ordinary media, study carefully and investigate their pathogenicity.
~8. Micrococcus Melitensis.~--The milk from an animal infected with M. melitensis usually contains the organisms in large numbers and but few other bacteria.
1. Spread several sets of surface plates upon nutrose agar, each from one loopful of the deposit in tube D^{1} or D^{2}.
2. Spread several sets of surface plates upon nutrose agar, each from one drop of the original milk sample.
3. Incubate aerobically at 37 deg. C. and examine daily up to the end of ten days.
4. Pick off suspicious colonies, examine them microscopically and subcultivate upon nutrose agar in tubes; upon glucose agar and in litmus milk.
5. Test the subsequent growth against the serum of an experimental animal inoculated against M. melitensis to determine its agglutinability.
6. If apparently M. melitensis, inoculate growth from a nutrose agar culture after three days incubation intracranially into the guinea-pig.
ICE CREAM.
~Collection of the Sample.~--
1. Remove the sample from the drum in the ladle or spoon with which the vendor retails the ice cream, and place it at once in a sterile copper capsule, similar to that employed for earth samples (_vide_ page 471).
2. Pack for transmission in the ice-box.
3. On arrival at the laboratory place the copper capsules containing the ice cream in the incubator at 20 deg. C. for fifteen minutes--that is, until at least some of the ice cream has become liquid.
~Qualitative and Quantitative Examination.~--Treat the fluid ice cream as milk and conduct the examination in precisely the same manner as described for milk (_vide_ page 443).
EXAMINATION OF CREAM AND BUTTER.
~Collection of the Sample.~--Collect, store, and transmit samples to the laboratory, precisely as is done in the case of ice cream.
~Quantitative.~--
_Apparatus Required_:
Sterile test-tube. Sterilised spatula. Water-bath regulated at 42 deg. C. Case of sterile plates. Case of sterile graduated pipettes, 1 c.c. (in hundredths). Tubes of gelatine-agar (+10 reaction). Plate-levelling stand, with its water chamber filled with water at 42 deg. C.
METHOD.--
1. Transfer a few grammes of the sample to a sterile test-tube by means of the sterilised spatula.
2. Place the tube in the water-bath at 42 deg. C. until the contents are liquid.
3. Liquefy eight tubes of gelatine-agar and place them in the water-bath at 42 deg. C, and cool down to that temperature.
4. Inoculate the gelatine-agar tubes with the following quantities of the sample by the help of a sterile pipette graduated to hundredths of a cubic centimetre--viz.,
To tube No. 1 add 1 c.c. liquefied butter. 2 add 0.5 c.c. liquefied butter. 3 add 0.3 c.c. liquefied butter. 4 add 0.2 c.c. liquefied butter. 5 add 0.1 c.c. liquefied butter. 6 add 0.05 c.c. liquefied butter. 7 add 0.03 c.c. liquefied butter. 8 add 0.02 c.c. liquefied butter. 9 add 0.01 c.c. liquefied butter.
5. Pour a plate cultivation from each of the gelatine-agar tubes and incubate at 28 deg. C.
6. "Count" the plates after three days' incubation, and from the figures thus obtained estimate the number of organisms present per cubic centimetre of the sample.
~Qualitative.~--
_Apparatus Required_:
Sterile beaker, its mouth plugged with sterile cotton-wool.
Counterpoise for beaker.
Scales and weights.
Sterilised spatula.
Water-bath regulated at 42 deg. C.
Separatory funnel, 250 c.c. capacity, its delivery tube protected against contamination by passing it through a cotton-wool plug into the interior of a small Erlenmeyer flask which serves to support the funnel. This piece of apparatus is sterilised _en masse_ in the hot-air oven.
Large centrifugal machine.
Sterile tubes (for the centrifuge) closed with solid rubber stoppers.
Case of sterile pipettes, 10 c.c.
Case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre).
METHOD.--
1. Weigh out 100 grammes of the sample in a sterile beaker.
2. Plug the mouth of the beaker with sterile cotton-wool and immerse the beaker in a water-bath at 42 deg. C. until the contents are completely liquefied.
3. Fill the liquefied butter into the sterile separatory funnel.
4. Transfer the funnel to the incubator at 37 deg. C. and allow it to remain there for four days.
At the end of this time the contents of the funnel will have separated into two distinct strata.
(a) A superficial oily layer, practically free from bacteria.
(b) A deep watery layer, turbid and cloudy from the growth of bacteria.
5. Draw off the subnatant turbid layer into sterile centrifugal tubes, previously warned to about 42 deg. C., and centrifugalise at once.
6. Pipette off the supernatant fluid and fill the tubes with sterile 1 per cent. sodium carbonate solution previously warmed slightly; stopper the tubes and shake vigourously for a few minutes.
7. Centrifugalise again.
8. Pipette off the supernatant fluid; filling the tubes with warm sterile bouillon, shake well, and again centrifugalise, to wash the deposit.
9. Pipette off the supernatant fluid.
10. Prepare cover-slip preparations, fix and clear as for milk preparations, stain carbolic methylene-blue, Gram's method, Ziehl-Neelsen's method, and examine microscopically with a 1/12 inch oil-immersion lens.
11. Proceed with the examination of the deposit as in the case of milk deposit (see pages 450 _et seq._).
EXAMINATION OF UNSOUND MEATS.
(INCLUDING TINNED OR POTTED MEATS, FISH, ETC.)
The bacterioscopic examination of unsound food is chiefly directed to the detection of those members of the Coli-typhoid group--B. enteritidis of Gaertner and its allies--which are usually associated with epidemic outbreaks of food poisoning, and such anaerobic bacteria as initiate putrefactive changes in the food which result in the formation of poisonous ptomaines, consequently the quantitative examination pure and simple is frequently omitted.
A. Cultural Examination.
Quantitative.--
_Apparatus Required_:
Sterilised tin opener, (if necessary.)
Erlenmeyer flask (500 c.c. capacity) containing 200 c.c. sterile bouillon and fitted with solid rubber stopper.
Counterpoise.
Scissors and forceps.
Scales and weights.
Water steriliser.
Hypodermic syringe.
Syringe with intragastric tube.
Rat forceps.
Case of sterile capsules.
Filtering apparatus as for water analysis.
Case of sterile plates.
Case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre).
Case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre).
Plate-levelling stand.
Tubes of nutrient gelatine.
Tubes of nutrient agar.
Water-bath regulated at 42 deg. C.
Bulloch's apparatus.
METHOD.--
1. Place the flask containing 200 c.c. sterile broth on one pan of the scales and counterpoise accurately.
2. Mince a portion of the sample by the aid of sterile scissors and forceps, and add the minced sample to the bouillon in the flask to the extent of 20 grammes.
3. Make an extract by standing the flask in the incubator running at 42 deg. C. (or in a water-bath regulated to that temperature) for half an hour, shaking its contents from time to time. Better results are obtained if an electrical shaker is fitted inside the incubator and the flask kept in motion throughout the entire thirty minutes.
Now every centimetre contains the bacteria washed out from 0.1 gramme of the original food.
4. Inoculate tubes of liquefied gelatine as follows:
To tube No. 1 add 1.0 c.c. of the extract. 2 add 0.5 c.c. of the extract. 3 add 0.3 c.c. of the extract. 4 add 0.2 c.c. of the extract. 5 add 0.1 c.c. of the extract.
Pour plates from these tubes and incubate at 20 deg. C.
5. Prepare a precisely similar set of agar plates and incubate at 37 deg. C.
6. Pipette 5 c.c. of the extract into a sterile tube, heat in the differential steriliser at 80 deg. C. for ten minutes.
7. From the heated extract prepare duplicate sets of agar and gelatine plates and incubate anaerobically in Bulloch's apparatus at 37 deg. C. and 20 deg. C. respectively.
8. After three days' incubation examine the agar plates both aerobic and anaerobic and enumerate the colonies developed from spores (7), and from vegetative forms and spores (5), and calculate and record the numbers of each group per gramme of the original food.
9. After seven days' incubation (or earlier if compelled by the growth of liquefying colonies) enumerate the gelatine plates in the same way.
10. Subcultivate from the colonies that make their appearance and identify the various organisms.
11. Continue the investigations with reference to the detection of pathogenic organisms as described under water (page 429 _et seq._).
Qualitative.--
I. _Cultural._
The micro-organisms sought for during the examination of unsound foods comprise the following:
Members of the Coli-typhoid groups (chiefly those of the Gaertner class).
B. anthracis.
Streptococci
Anaerobic Bacteria:
B. enteritidis sporogenes. B. botulinus. B. cadaveris.
The methods by which these organisms if present may be identified and isolated have already been described under the corresponding section of water examination with the exception of those applicable to B. botulinus, and B. cadaveris. These can only be isolated satisfactorily from the bodies of experimentally inoculated animals.
II _Experimental._
_Tissue._--
1. Feed rats and mice on portions of the sample and observe the result.
2. If any of the animals die, make complete post-mortem examinations and endeavour to isolate the pathogenic organisms.
_Extract._--
1. Introduce various quantities of the bouillon extract into the stomachs of several rats, mice and guinea-pigs repeatedly over a period of two or three days by the intragastric method of inoculation (see page 367) and observe the result. Guinea-pigs and mice are very susceptible to infection by B. botulinus by this method; rabbits less so.
2. Inoculate rats, mice, and guinea-pigs subcutaneously into deep pockets, and intraperitoneally with various quantities of the bouillon extract, and observe the result.
3. Filter some of the extract through a Chamberland candle and incubate the filtrate to determine the presence of soluble toxins.
4. If any of the animals succumb to either of these methods of inoculation, make careful post-mortem examinations and endeavour to isolate the pathogenic organisms.
THE EXAMINATION OF OYSTERS AND OTHER SHELLFISH.
On opening the shell of an oyster a certain amount of fluid termed "liquor" is found to be present. This varies in amount from a drop to many cubic centimetres (0.1 c.c. to 10 c.c.)--in the latter case the bulk of the fluid is probably the last quantum of water ingested by the bivalve before closing its shell. In order to obtain a working average of the bacteriological flora of a sample, ten oysters should be taken and the body, gastric juice and liquor should be thoroughly mixed before examination. The examination, as in dealing with other food stuffs, is directed to the search for members of the Coli-typhoid group, sewage streptococci and perhaps also B. enteritidis sporogenes.
_Apparatus Required_:
Two hard nail brushes.
Liquid soap.
Sterile water in aspirator jar with delivery nozzle controlled by a spring clip.
Sterile oyster knives.
Sterile glass dish, with cover, sufficiently large to accommodate ten oysters.
Sterile forceps.
Sterile scissors.
Sterile towels or large gauze pads.
Sterile graduated cylinders 1000 c.c. capacity, with either the lid or the bottom of a sterile Petri dish inverted over the open mouth as a cover.
Glass rods.
Corrosive sublimate solution, 1 per mille.
Bile salt broth tubes.
Litmus milk tubes.
Surface plates of nutrose agar.
Case of sterile pipettes, 1 c.c. (in tenths of a c.c.)
Case of sterile pipettes, 10 c.c. (in tenths of a c.c.)
Case of sterile glass capsules.
Erlenmeyer flasks, 250 c.c. capacity.
Double strength bile salt broth.
METHOD.--
1. Thoroughly clean the outside of the oyster shells by scrubbing each in turn with liquid soap and nail brush under a tap of running water. Then, holding an oyster shell in a pair of sterile forceps wash every part of the outside of the shell with a stream of sterile water running from an aspirator jar; deposit the oyster inside the sterile glass dish. Repeat the process with each of the remaining oysters.
2. Before proceeding further, cleanse the hands thoroughly with clean nail brush, soap and water, then plunge them in lysol 2 per cent. solution, and finally in sterile water.
3. Spread a sterile towel on the bench.
4. Remove one of the oysters from the sterile glass dish and place it, resting on its convex shell, on the towel. Turn a corner of the sterile towel over the upper flat shell to give a firmer grip to the left hand, which holds the shell in position.
5. With the sterile oyster knife (in the right hand) open the shell and separate the body of the oyster from the inner surface of the upper flat shell. Bend back and separate the flat shell, leaving the body of the oyster in and attached to the concave shell. Avoid spilling any of the liquor.
(Some dexterity in opening oysters should be acquired before undertaking these experiments).
6. Cut up the body of the oyster with sterile scissors into small pieces and allow the liquor freed from the body during the process to mix with the liquor previously in the shell.
7. Transfer the comminuted oyster and the liquor to the cylinder.
8. Treat each of the remaining oysters in similar fashion.
9. Mix the contents of the cylinder thoroughly by stirring with a sterile glass rod. The total volume will amount to about 100 c.c.
10. Use 0.1 c.c. of the mixed liquor to inseminate each of a series of three nutrose surface plates.
11. Inoculate 0.1 c.c. of the mixed liquor into each of three tubes of litmus milk.
12. Add sterile distilled water to the contents of the cylinder up to 1000 c.c. and stir thoroughly with a sterile glass rod and allow to settle. The bacterial content of each oyster may be regarded, for all practical purposes, as comprised in 100 c.c. of fluid.
13. Arrange four glass capsules in a row and number I, II, III, IV. Pipette 9 c.c. sterile distilled water into each.
14. To capsule No. I add 1 c.c. of the diluted liquor, etc. from the cylinder, and mix thoroughly. To capsule II add 1 c.c. of dilution in capsule I and mix thoroughly. Carry over 1 c.c. of fluid from capsule II to capsule III, afterwards adding 1 c.c. of fluid from capsule III to capsule IV.
15. Label tubes of bile salt broth and inoculate with the following amounts of diluted oysters:
No. 6 with 10 c.c. cylinder fluid = 0.1 oyster. No. 5 with 1 c.c. cylinder fluid = 0.01 oyster. No. 4 with 1 c.c. capsule I fluid = 0.001 oyster. No. 3 with 1 c.c. capsule II fluid = 0.0001 oyster. No. 2 with 1 c.c. capsule III fluid = 0.00001 oyster. No. 1 with 1 c.c. capsule IV fluid = 0.000001 oyster.
16. Transfer 100 c.c. cylinder fluid (= 1 oyster) to an Erlenmeyer flask and add 50 c.c. double strength bile salt broth, and label 7.
17. Duplicate all the above indicated cultures.
18. Put up the tube cultures in Buchner's tubes and incubate anaerobically at 42 deg. C.
If growth occurs in tube 1 the organism finally isolated, e. g., B. coli, must have been present to the extent of one million per oyster.
19. Complete the examination for members of the Coli-typhoid group and sewage streptococci, as directed under Water Examination, page 429 (steps 11-21).
20. Inoculate a series of 6 tubes of litmus milk with quantities of the material similar to those indicated in step 15; heat to 80 deg. C. for ten minutes, and incubate under anaerobic conditions at 37 deg. C. Examine for the presence of B. enteritidis sporogenes as directed under Water Examination, page 438 (steps 7-10).
EXAMINATION OF SEWAGE AND SEWAGE EFFLUENTS.
Quantitative.--
_Collection of the Sample._--As only small quantities of material are needed, the samples should be collected in a manner similar to that described under water for quantitative examination and transmitted in the ice apparatus used in packing those samples.
_Apparatus Required._--As for water (_vide_ page 420).
METHOD.--
1. Arrange four sterile capsules in a row and number them I, II, III, IV.
2. Pipette 9 c.c. sterile bouillon into capsule No. I.
3. Pipette 9.9 c.c. sterile bouillon into capsules II, III, and IV.
4. Add 1 c.c. of the sewage to capsule No. I by means of a sterile pipette, and mix thoroughly.
5. Take a fresh sterile pipette and transfer 0.1 c.c. of the mixture from No. I to No. II and mix thoroughly.
6. In like manner transfer 0.1 c.c. from No. II to No. III, and then 0.1 c.c. from No. III to No. IV.
Now 1 c.c. of dilution No. I contains 0.1 c.c. of the original sewage. 1 c.c. of dilution No. II contains 0.001 c.c. of the original sewage. 1 c.c. of dilution No. III contains 0.00001 c.c. of the original sewage. 1 c.c. of dilution No. IV contains 0.0000001 c.c. of the original sewage.
7. Pour a set of gelatine plates from the contents of each capsule, three plates in a set, and containing respectively 0.2, 0.3, and 0.5 c.c. of the dilution. Label carefully; incubate at 20 deg. C. for three, four, or five days.
8. Enumerate the organisms present in those sets of plates which have not liquefied, probably those from dilution III or IV, and calculate therefrom the number present per cubic centimetre of the original sample of sewage.
Qualitative.--The qualitative examination of sewage is concerned with the identification and enumeration of the same bacteria dealt with under the corresponding section of water examination; it is consequently conducted on precisely similar lines to those already indicated (_vide_ pages 426 to 441).
EXAMINATION OF AIR.
Quantitative.--
_Apparatus Required_:
Aspirator bottle, 10 litres capacity, fitted with a delivery tube, and having its mouth closed by a perforated rubber stopper, through which passes a short length of glass tubing.
Erlenmeyer flask, 250 c.c. capacity (having a wide mouth properly plugged with wool), containing 50 c.c. sterile water.
Rubber stopper to fit the mouth of the flask, perforated with two holes, and fitted as follows:
Take a 9 cm. length of glass tubing and bend up 3 cm. at one end at right angles to the main length of tubing. Pass the long arm of the angle through one of the perforations in the stopper; plug the open end of the short arm with cotton-wool.
Take a glass funnel 5 or 6 cm. in diameter with a stem 12 cm. in length and bend the stem close up to the apex of the funnel, in a gentle curve through a quarter of a circle; pass the long stem through the other perforation in the rubber stopper.
A battery jar or a small water-bath to hold the Erlenmeyer flask when packed round with ice.
Supply of broken ice.
Rubber tubing.
Screw clamps and spring clips, for tubing.
Water steriliser.
Retort stand and clamps.
Apparatus for plating (as for enumeration of water organisms, _vide_ page 420).
METHOD.--
1. Fill 10 litres of water into the aspirating bottle and attach a piece of rubber tubing with a screw clamp to the delivery tube. Open the taps fully and regulate the screw clamp, by actual experiment, so that the tube delivers 1 c.c. of water every second. The screw clamp is not touched again during the experiment.
At this rate the aspirator bottle will empty itself in just under three hours. Shut off the tap and make up the contents of the aspirator bottle to 10 litres again.
2. Sterilise the fitted rubber cork, with its funnel and tubing, by boiling in the water steriliser for ten minutes.
3. Remove the cotton-wool plug from the flask, and replace it by the rubber stopper with its fittings. Make sure that the end of the stem of the funnel is immersed in the bouillon.
4. Place the flask in a glass or metal vessel and pack it round with pounded ice. Arrange the flask with its ice casing just above the neck of the aspirator bottle.
5. Connect up the free end of the glass tube from the flask--after removing the cotton-wool plug--with the air-entry tube in the mouth of the aspirating bottle (Fig. 216).
6. Open the tap fully, and allow the water to run.
Replenish the ice from time to time if necessary.
(In emptying itself the aspirator bottle will aspirate 10 litres of air slowly through the water in the Erlenmeyer flask.)
7. When the aspiration is completed, disconnect the flask and remove it from its ice packing.
8. Liquefy three tubes of nutrient gelatine and add to them 0.5 c.c., 0.3 c.c., and 0.2 c.c., respectively, of the water from the flask, by means of a sterile graduated pipette, as in the quantitative examination of water. Pour plates.
9. Pour a second similar set of gelatine plates.
10. Incubate both sets of plates at 20 deg. C.
11. Enumerate the colonies present in the two sets of gelatine plates after three, four, or five days and average the results from the numbers so obtained; estimate the number of micro-organisms present in 1 c.c., and then in the 50 c.c. of broth in the flask.
12. The result of air examination is usually expressed as the number of bacteria present per cubic metre (i. e., kilolitre) of air; and as the number of organisms present in the 50 c.c. water only represent those contained in 10 litres of air, the resulting figure must be multiplied by 100.
Qualitative.--
1. Proceed exactly as in the quantitative examination of air (_vide supra_), steps 1 to 10.
2. Pour plates of wort agar with similar quantities of the air-infected water, and incubate at 37 deg. C.
3. Pour plates of nutrient agar with similar quantities of the water and incubate at 37 deg. C.
4. Pour similar plates of wort gelatine and incubate at 20 deg. C.
5. Pick off the individual colonies that appear in the several plates, subcultivate them on the various media, and identify them.
EXAMINATION OF SOIL.
The bacteriological examination of soil yields information of value to the sanitarian during the progress of the process of homogenisation of "made soil" (e. g., a dumping area for the refuse of town) and determines the period at which such an area may with propriety and safety be utilised for building purposes; or to the agriculturalist in informing him of the suitability of any given area for the growth of crops.
The surface of the ground, exposed as it is to the bactericidal influence of sunlight and to rapid alternations of heat and cold, rain and wind, contains but few micro-organisms. Again, owing to the density of the molecules of deep soil and lack of aeration on the one hand, and the filtering action of the upper layers of soil and bacterial antagonism on the other, bacterial life practically ceases at a depth of about 2 metres. The intermediate stratum of soil, situated from 25 to 50 cm. below the surface, invariably yields the most numerous and the most varied bacterial flora.
~Collection of Sample.~--A small copper capsule 6 cm. high by 6 cm. diameter, with "pull-off" cap secured by a bayonet catch, previously sterilised in the hot-air oven, is the most convenient receptacle for samples of soil.
The instrument used for the actual removal of the soil from its natural position will vary according to whether we require surface samples or soil from varying depths.
(a) For ~surface~ samples, use an iron scoop, shaped like a shoe horn, but provided with a sharp spine (Fig. 217). This is wrapped in asbestos cloth and sterilised in the hot-air oven. When removed from the oven, wrap a piece of oiled paper, silk, or gutta-percha tissue over the asbestos cloth, and secure it with string, as a further protection against contamination.
On reaching the spot whence the samples are to be taken, the coverings of the scoop are removed, and the asbestos cloth employed to brush away loose stones and debris from the selected area. The surface soil is then broken up with the point of the scoop, scraped up and collected in the body of the scoop, and transferred to the sterile capsule for transmission.
(b) For ~deep~ samples collected at various distances from the surface, an experimental trench may be cut to the required depth and samples collected at the required points on the face of the section. It is, however, preferable to utilise some form of borer, such as that designed by Fraenkel (Fig. 218).
_Fraenkel's Earth Borer._--This instrument consists of a stout hard-steel rod, 150 cm. long, marked in centimetres from the drill-pointed extremity. It is provided with a cross handle (adjustable at any point along the length of the rod by means of a screw nut). The terminal centimeters are thicker than the remainder of the rod, and on one side a vertical cavity about 0.5 cm. deep is cut. This is covered by a flanged sleeve so long as the borer is driven into the soil clockwise, and is opened for the reception of the sample of soil, when the required depth is reached, by reversing the screwing motion, and again closed before withdrawal of the borer from the earth by resuming the original direction of twist. It can be sterilised in a manner similar to that adopted for the scoop, or by repeatedly filling the cavity with ether and burning it off.
~Quantitative.~--Four distinct investigations are included in the complete quantitative bacteriological examination of the soil:
1. The enumeration of the aerobic organisms.
2. The enumeration of the spores of aerobes.
3. The enumeration of the anaerobic organisms (including the facultative anaerobes).
4. The enumeration of the spores of anaerobes.
Further, by a combination of the results of the first and second, and of the third and fourth of these, the ratio of spores to vegetative forms is obtained.
_Apparatus Required_:
Case of sterile capsules (25 c.c. capacity).
Case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre).
Case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre).
Flask containing 250 c.c. sterile bouillon.
Tall cylinder containing 2 per cent. lysol solution.
Plate-levelling stand.
12 sterile plates.
Tubes of nutrient gelatine.
Tubes of wort gelatine.
Tubes of nutrient agar.
Tubes of glucose formate gelatine.
Tubes of glucose formate agar.
Water-bath regulated at 42 deg. C.
Bunsen burner.
Grease pencil.
Sterile mortar and pestle (agate).
Sterile wide-mouthed Erlenmeyer flask (500 c.c. capacity).
Sterile metal funnel with short wide bore delivery tube to just fit mouth of flask.
Solid rubber stopper to fit the flask (sterilised by boiling).
Pair of scales.
Counterpoise (Fig. 107).
Sterile metal (nickel) spoon or spatula.
Fractional steriliser (Fig. 140).
METHOD.--
1. Arrange four sterile capsules numbered I, II, III, and IV; pipette 9 c.c. sterile bouillon into the first capsule, and 9.9 c.c. into each of the remaining three.
2. Pipette 100 c.c. sterile bouillon into the Erlenmeyer flask.
3. Remove the cotton-wool plug from the flask and replace it by the sterile funnel.
4. Place flask and funnel on one pan of the scales, and counterpoise accurately.
5. Empty the sample of soil into the mortar and triturate thoroughly.
6. By means of the sterile spatula add 10 grammes of the earth sample to the bouillon in the flask.
The final results will be more reliable if steps 2, 3, 4, and 5 are performed under a hood--to protect from falling dust, etc.
7. Remove the funnel from the mouth of the flask; replace it by the rubber stopper and shake vigourously; then allow the solid particles to settle for about thirty minutes. One cubic centimetre of the turbid broth contains the washings from 0.1 gramme of soil.
8. Pipette off 1 c.c. of the supernatant bouillon, termed the "soil water," and add it to the contents of capsule I; mix thoroughly.
9. Remove 0.1 c.c. of the infected bouillon from capsule I and add it to capsule II, and mix.
10. In like manner add 0.1 c.c. of the contents of capsule II to capsule III, and then 0.1 c.c. of the contents of capsule III to capsule IV.
Then 1 c.c. fluid from capsule I contains soil water from .01 gm. earth. Then 1 c.c. fluid from capsule II contains soil water from .0001 gm. earth. Then 1 c.c. fluid from capsule III contains soil water from .000001 gm. earth. Then 1 c.c. fluid from capsule IV contains soil water from .00000001 gm. earth.
(A) _Aerobes (Vegetative Forms and Spores)._--
11. Pour a set of gelatine plates from the contents of each capsule--two plates in a set, and containing respectively 0.1 c.c. and 0.4 c.c. of the diluted soil water. Label and incubate.
12. Pour similar sets of wort gelatine plates from the contents of capsules II and III, label, and incubate at 20 deg. C.
13. Pour similar sets of agar plates from the contents of capsules II and III; label and incubate at 37 deg. C.
14. Weigh out a second sample of soil--10 grammes--dry over a water-bath until of constant weight and calculate the ratio
wet soil weight --------------- dry soil weight
15. "Count" the plates after incubation for three, four, or five days, and correcting the figures thus obtained by means of the "wet" to "dry" soil ratio estimate--
(a) The number of aerobic micro-organisms present per gramme of the soil.
(b) The number of yeasts and moulds present per gramme of the soil.
(c) The number of aerobic organisms "growing at 37 deg. C." present per gramme of the soil.
(B) _Anaerobes (Vegetative Forms and Spores)._--
16. Pour similar sets of plates in glucose formate gelatine and agar and incubate in Bulloch's anaerobic apparatus.
(C) _Aerobes and Anaerobes (Spores Only)._--
17. Pipette 5 c.c. soil water into a sterile tube.
18. Place in the differential steriliser at 80 deg. C. for ten minutes.
19. Pour two sets of four gelatine plates containing 0.1, 0.2, 0.5, and 1 c.c. respectively of the soil water; label and incubate at 20 deg. C., one set aerobically, the other anaerobically in Bulloch's apparatus.
20. "Count" the plates (delay the enumeration as long as possible) and estimate the number of spores of aerobes and anaerobes respectively present per gramme of the soil.
21. Calculate the ratio existing between spores and spores + vegetative forms under each of the two groups, aerobic and anaerobic micro-organisms.
~Qualitative Examination.~--The qualitative examination of soil is usually directed to the detection of one or more of the following:
Members of the Coli-typhoid group.
Streptococci.
Bacillus anthracis.
Bacillus tetani.
Bacillus oedematis maligni.
The nitrous organisms.
The nitric organisms.
1. Transfer the remainder of the soil water (88 c.c.) to a sterile Erlenmeyer flask by means of a sterile syphon.
2. Fix up the filtering apparatus as for the qualitative examination of water, and filter the soil water.
3. Suspend the bacterial residue in 5 c.c. sterile bouillon (technique similar to that described for the water sample, _vide_ pages 434-436).
Every cubic centimetre of suspension now contains the soil water from nearly 1 gramme of earth.
The methods up to this point are identical no matter which organism or group of organisms it is desired to isolate; but from this stage onward the process is varied slightly for each particular bacterium.
~I. The Coli-typhoid Group.~--
~II. Streptococci.~--
~III. Bacillus Anthracis.~--
~IV. Bacillus Tetani.~--
The methods adopted for the isolation of these organisms are identical with those already described under water (page 437 _et seq._).
~V. Bacillus Oedematis Maligni.~--Method precisely similar to that employed for the B. tetani.
~VI. The Nitrous Organisms.~--
1. Take ten tubes of Winogradsky's solution No I (_vide_ page 198) and number them consecutively from 1 to 10.
2. Inoculate each tube with varying quantities of the material as follows:
To tube No. 1 add 1.0 c.c. of the soil water. To tube No. 2 add 0.1 c.c. of the soil water. To tube No. 3 add 1.0 c.c. from Capsule I. To tube No. 4 add 0.1 c.c. from Capsule I. To tube No. 5 add 1.0 c.c. from Capsule II. To tube No. 6 add 0.1 c.c. from Capsule II. To tube No. 7 add 1.0 c.c. from Capsule III. To tube No. 8 add 0.1 c.c. from Capsule III. To tube No. 9 add 1.0 c.c. from Capsule IV. To tube No. 10 add 0.1 c.c. from Capsule IV.
Label and incubate at 30 deg. C.
~VII. The Nitric Organisms.~--
3. Take ten tubes of Winogradsky's solution No II, number them consecutively from 1 to 10 and inoculate with quantities of soil water similar to those enumerated in section VI step 2. Label and incubate at 30 deg. C.
4. Examine after twenty-four and forty-eight hours' incubation. From those tubes that show signs of growth make subcultivations in fresh tubes of the same medium and incubate at 30 deg. C.
5. Make further subcultivations from such of those tubes as show growth, and again incubate.
6. If growth occurs in these subcultures, make surface smears on plates of Winogradsky's silicate jelly (_vide_ page 198).
7. Pick off such colonies as make their appearance and subcultivate in each of these two media.
TESTING FILTERS.
Porcelain filter candles are examined with reference to their power of holding back _all_ the micro-organisms suspended in the fluids which are filtered through them, and permitting only the passage of germ-free filtrates. In order to determine the freedom of the filter from flaws and cracks which would permit the passage of bacteria no matter how perfect the general structure of the candle might be, the candle must first be attached by means of a long piece of pressure tubing, to a powerful pump, such as a foot bicycle pump, fitted with a manometer. The candle is then immersed in a jar of water and held completely submerged whilst the internal pressure is gradually raised to two atmospheres by the action of the pump. Any crack or flaw will at once become obvious by reason of the stream of air bubbles issuing from it.
The examination for permeability is conducted as follows:
_Apparatus Required_:
Filtering apparatus: The actual filter candle that is used must be the one it is intended to test and must be previously carefully sterilised; the arrangement of the apparatus will naturally vary with each different form of filter, one or other of those already described (_vide_ pages 42-48).
Plate-levelling stand.
Case of sterile plates.
Case of sterile pipettes, 10 c.c. (in tenths).
Case of sterile pipettes, 1 c.c. (in tenths).
Tubes of nutrient gelatine.
Flask containing sterile normal saline solution.
Sterile measuring flask, 1000 c.c. capacity.
METHOD.--
1. Prepare surface cultivations, on nutrient agar in a culture bottle, of the Bacillus mycoides, and incubate at 20 deg. C., for forty-eight hours.
2. Pipette 5 c.c. sterile normal saline into the culture bottle and emulsify the entire surface growth in it.
3. Pipette the emulsion into the sterile measuring flask and dilute up to 1000 c.c. by the addition of sterile water.
4. Pour the emulsion into the filter reservoir and start the filtration.
5. When the filtration is completed, pour six agar plates each containing 1 c.c. of the filtrate.
6. Incubate at 37 deg. C. until, if necessary, the completion of seven days.
7. If the filtrate is not sterile, subcultivate the organism passed and determine its identity with the test bacterium before rejecting the filter--since the filtrate may have been accidentally contaminated.
8. If the filtrate is sterile, resterilise the candle and repeat the test now substituting a cultivation of B. prodigiosus--a bacillus of smaller size.
9. If the second test is satisfactory, test the candle against a cultivation of a very small coccus, e. g., Micrococcus melitensis, in a similar manner; in this instance continuing the incubation of cultivations from the filtrate for fourteen days.
TESTING OF DISINFECTANTS.
Methods have already been detailed (page 310) for the purpose of studying the vital resistance offered by micro-organisms to the lethal effect of germicides. But it frequently happens that the bacteriologist has to determine the relative efficiency of "disinfectants" from the standpoints of the sanitarian and commercial man rather than from the research worker's point of view. In pursuing this line of investigation, it is convenient to compare the efficiency, under laboratory conditions, of the proposed disinfectant with that of some standard germicide, such as pure phenol. In so doing, and in order that the work of different observers may be compared, conditions as nearly uniform as possible should be aimed at. The method described is one that has been in use by the writer for many years past, modified recently by the adoption of some of the recommendations of the Lancet Commission on the Standardisation of Disinfectants--particularly of the calculation for determining the phenol coefficient.
This method has many points in common with that modification of the "drop" method known as the Rideal-Walker test.
~General Considerations.~--
These may be grouped under three headings: Test Germ, Germicide, and Environment.
1. _Test Germ._--~B. coli.~
As disinfectants are tested for sanitary purposes, it is obvious that a member of the coli-typhoid group should be selected as the test germ. B. coli is selected on account of its relative nonpathogenicity, the ease with which it can be isolated and identified by different observers in various parts of the world, the stability of its fundamental characters, and evenness of its resistance when utilised for these tests; finally since the colon bacillus is an organism which is slightly more resistant to the lethal action of germicides than the more pathogenic members of this group, a margin of safety is introduced into the test which certainly enhances its value.
B. coli should be recently isolated from a normal stool, and plated at least twice to ensure the purity of the strain; and a stock agar culture prepared which should be used throughout any particular test. For any particular experiment prepare a smear culture on agar and incubate at 37 deg. C. for 24 hours anaerobically. Then emulsify the whole of the surface growth in 10 c.c. of sterile water. Transfer the emulsion to a sterile test-tube with some sterile glass beads and shake thoroughly to ensure homogenous emulsion. Transfer to a centrifuge tube and centrifugalise the emulsion to throw down any masses of bacteria which may have escaped the disintegrating action of the beads. Pipette off the supernatant emulsion for use in the test.
_2. Germicide._--
_a. Disinfectant to be tested._--
The first essential point is to test the unknown disinfectant, which may be referred to as germicide-x, on the lines set out on page 311 to determine its inhibition coefficient.
This constant having been fixed, prepare various solutions of germicide-x with sterilised distilled water by accurate volumetric methods, commencing with a solution somewhat stronger than that representing the inhibition coefficient. The solutions must be prepared in fairly large bulk, not less than 5 c.c. of the disinfectant being utilised for the preparation of any given percentage solution.
_b. Standard Control._--~Phenol.~
The standard germicide used for comparison should be one which is not subject to variation in its chemical composition, and the one which has obtained almost universal use is Phenol.
The following table shows the effect of different percentages of carbolic acid upon B. coli for varying contact times, compiled from an experiment conducted under the standard conditions referred to under Environment. The results closely correspond to those recorded by the Lancet Commission on Disinfectants, 1909.
+----------------------------------- | Contact time in minutes. Percentage of phenol +------+---+---+---+---+---+---+---- | 2-1/2| 5 |10 | 15| 20| 25| 30| 35 ---------------------+------+---+---+---+---+---+---+---- 1.20 | - | - | - | - | - | - | - | - 1.10 | - | - | - | - | - | - | - | - 1.0 | + | - | - | - | - | - | - | - 0.9 | + | - | - | - | - | - | - | - 0.85 | + | + | - | - | - | - | - | - 0.80 | + | + | + | - | - | - | - | - 0.75 | + | + | + | + | + | - | - | - 0.7 | + | + | + | + | + | + | - | - 0.65 | + | + | + | + | + | + | + | - ---------------------+------+---+---+---+---+---+---+----
- = No growth, i. e., bacteria killed. + = Growth, i. e., bacteria still living.
From this it will be seen that the following percentage solutions will need to be prepared, namely: 1.1 per cent., 1.0 per cent., 0.9 per cent., 0.75 per cent., 0.7 per cent., as controls for each experiment.
Prepare solutions of varying percentages by weighing out the quantity of carbolic acid required for each and dissolving in 100 c.c. of pure distilled water in an accurately standardised measuring flask. The solutions must be prepared freshly as required each day.
~Environment.~--
_a. General._--
Close the windows and doors of the laboratory in which the investigation is carried out, to avoid draughts. Flush over the work bench and adjacent floor with 1:1000 solution of corrosive sublimate. Caution the assistant, if one is employed, to avoid unnecessary movement or speech.
_b. Contact Temperature_, ~15-18 deg. C.~--
This is the temperature at which contact between the germicide and the test germ takes place, and is of importance, since some germicides (_e. g._, Phenol) appear to be more powerful at high temperatures. 18 deg. C.--practically the ordinary room temperature--is a temperature at which the multiplication of B. coli is a comparatively slow process, but variation of a degree above this temperature or of two or three degrees below is of no moment. If the room temperature is below 15 deg. C. when the experiments are in progress, arrange a water-bath regulated at 18 deg. C. for the reception of the tubes containing the mixture of germ and germicide; if above 19 deg. C. immerse the tubes in cold water, to which small pieces of ice are added from time to time to prevent the temperature rising above 18 deg. C.
_c. Relative Proportional Bulk of Test Germ and Germicide_, ~50:1.~--
Five cubic centimetres is a convenient amount of germicidal solution to employ, and to this 0.1 c.c. of the emulsion of test germ should be added.
_d. Bulk of Sample Removed from Germ + Germicide Mixture at Each of the Time Periods_, ~0.1 c.c.~--
This is sufficient to afford a fair sample of the germ content of the mixture, and at the same time is insufficient to exert any inhibitory action when transferred to the subculture medium.
_e. Subculture Medium._ ~Bile Salt Broth.~--
A _fluid_ medium is essential in order to obtain immediate dilution of the germicide carried over; at the same time it is advantageous to employ a selective medium which favours the growth of the test germ to the exclusion of organisms likely to contaminate the preparation, and if possible one which affords characteristic cultural appearances.
Bile Salt Broth (page 180) combines these desiderata; it permits only the growth of intestinal bacteria, whilst the formation of an acid reaction and the production of gas in subcultures prepared from the germ-germicide mixture is fairly complete evidence of the presence of living B. coli.
The amount of medium present in each test-tube is a matter of importance, since the medium not only provides pabulum for the test germ, but also acts as a diluent to the germicide, to reduce its strength below its inhibition coefficient. For routine work each subculture tube contains 10 c.c. of medium, but it is obvious that if germicide-x possesses an inhibition coefficient of 0.1 per cent. the addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium would effectually prevent the subsequent growth of the test germ after a contact period insufficient to destroy its vitality. Hence the preliminary tests may in some instances indicate the necessity for the presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture tubes.
_f. Incubation Temperature_, ~37 deg. C.~--
_g. Observation Period of the Subcultivations_, ~Seven Days.~--
In order to determine whether or no the test germs have been destroyed, observations must always be continued--when growth appears to be absent--up to the end of seven days before recording "no growth."
_h. Identification of the Organisms Developing in the Subcultivations after Contact in the Germ + Germicide Solution._--
This is based on the naked eye characters of the growth in the bile salt broth, supplemented where necessary by plating methods, further subcultivations upon carbohydrate media and agglutination experiments. The sign (+) is used to indicate that growth of the test organism occurred in the subcultivations, and the sign (-) to indicate that the test germs have been destroyed and no subsequent growth has taken place.
METHOD.--
_Apparatus Required_:
Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter).
Test-tube rack (Fig. 219).
Sterile graduated pipettes in case, 1 c.c. (in tenths).
Sterile graduated pipettes in case, 5 c.c. (in c.c.).
Circular rubber washers, 2.5 cm. diameter with central hole, sterilised by boiling immediately before use, then transferred to sterilised glass double dish.
Electric signal clock or stop watch.
Sterile forceps.
Sterilised glass beads.
Shaking machine.
Grease pencil.
_Material Required_:
Percentage solutions of germicide-x (_vide_ page 481).
Percentage solutions of pure phenol (_vide_ page 482).
Aqueous emulsion of B. coli (_vide_ page 481).
Tubes of bile salt broth.
~Preliminary Tests.~--
_a. Inhibition Coefficient._--
Determine the lowest percentage of germicide-x which inhibits growth of B. coli in the bile salt broth, and the highest percentage which fails to inhibit (page 311). On the result of this experiment determine the bulk of medium required in the subculture tubes and the percentage solutions to be employed in the trial trip. Assuming the inhibition coefficient to be 1:1000, it will be quite safe to employ the ordinary culture tubes containing 10 c.c. medium in the subsequent experiments.
_b. Trial Trip._--
Determine the lethal effect of a series of five solutions of germicide-x (say 1:100, 1:250, 1:300, 1:500, 1:600) at contact times of 2-1/2, 5, 25 and 30 minutes in the following manner:
1. Arrange five test-tubes marked A to E in the lower tier of the test-tube rack.
2. Into tube A pipette 5 c.c. germicide-x 1:100 solution.
Into tube B pipette 5 c.c. germicide-x 1:200 solution.
Into tube C pipette 5 c.c. germicide-x 1:300 solution.
Into tube D pipette 5 c.c. germicide-x 1:500 solution.
Into tube E pipette 5 c.c. germicide-x 1:600 solution.
3. Arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20.
4. Place a square wire basket of about 50 tubes capacity close to the left of the test-tube rack, for the reception of the inoculated tubes.
5. Take a sterile 1 c.c. pipette from the case, pick up a sterile rubber washer with forceps and push the point of the pipette into the central hole.
6. Put down the forceps on the bench with the sterile points projecting over the edge. Without taking the tube from the rack remove the cotton-wool plug from tube A, and lower the pipette, with the rubber washer affixed, on to the open mouth of the tube; with the help of the forceps to steady the washer, push the pipette on through the hole until the point of the pipette has reached to within a few millimetres of the bottom of the tube (see fig. 219).
7. Adjust in the same way a pipette and a washer in the mouth of each of the other tubes, B, C, D and E.
8. Set the electric signal clock to ring for the commencement of the experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes.
9. Take up 0.5 c.c. of B. coli emulsion in sterile pipette graduated in tenths of a cubic centimetre and stand by.
10. As soon as the bell rings lift the pipette from tube A with the left hand and from the charged pipette held in the right hand deliver 0.1 c.c. of B. coli emulsion into the 1:100 solution. Then replace the pipette and washer.
11. Raise the tube with the left hand and shake it to mix germ and germicide, whilst returning the delivery pipette in the right hand.
12. Repeat the process with tubes B, C, D and E; then drop the infected delivery pipette in the lysol jar. The inoculation of the five tubes can be carried out very expeditiously, but a period of 10 seconds must be allowed for each tube.
13. When the bell rings at 2-1/2 minutes blow through the pipette in tube A (this agitates the germ + germicide mixture and ensures the collection of a fair sample); allow the mixture to enter the pipette, and as the column of fluid extends well above the terminal graduation, the right forefinger adjusted over the butt-end of the pipette before it is lifted will retain more than 0.1 c.c. of the mixture within the bore when the point of the pipette is clear of the fluid in the tube. Touch the point of the pipette on the inner wall of the tube, and allow any excess of fluid to escape, only retaining 0.1 c.c. in the pipette.
14. At the same time, with the left hand remove Bile Salt Tube No. 1 from the upper tier of the rack, take out the cotton-wool plug with the hand already holding the pipette (the relative positions of pipette, plug and culture tubes being practically the same as those of platinum loop, plug and culture tube shown in Fig. 68, page 74).
15. Insert the point of the pipette into the subculture tube, and blow out the mixture into the medium--replug the tube and drop it into the wire basket. Replace the washer-pipette in tube A.
As soon as the point of the pipette has entered the mouth of tube A it may be released, since it has already been so adjusted that it just clears the bottom of the test-tube, and the elastic washer will prevent any damage to the tube.
Steps 13, 14 and 15 occupy on an average 10 seconds.
16. Repeat steps 13, 14 and 15 with each of the other tubes B, C, D and E.
17. Repeat these various steps 13-16 when the bell rings at 5, 25 and 30 minutes.
18. Place all the inoculated tubes in the incubator at 37 deg. C.
19. Examine the tubes at intervals of 24 hours, and record the results in tabular form as shown in Table page 491 (the figures in the squares indicate the number of hours at which the changes in the medium due to the growth of B. coli first appeared).
20. If a consideration of the tabulated results indicates strengths of Germicide-x lethal at 2-1/2 and 30 minutes the final test can be arranged, but if this result has not been attained, sufficient evidence will probably be available to enable a second trial test to be planned which will give the required information.
~Final Test.~--
c. _Determination of Phenol Coefficient._--
_X-Disinfectant._--This comprises two distinct tests, one of the Germicide-x, the other of the standard phenol.
1. Arrange five test-tubes clearly marked in the lower tier of the rack.
2. Pipette into each 5 c.c. respectively of the five percentage solutions of x-disinfectant which the trial run has already shown will include those affording lethal values at 2-1/2 and 30 minutes.
3. Arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20.
4. Arrange further 20 tubes of bile salt broth numbered 21-40 in two rows in a second smaller rack which can be stood on the upper tier of the rack as soon as the first 20 tubes have been inoculated.
5. Place a square wire basket of about 50 tube capacity close to the left of the test-tube rack, for the reception of the inoculated tubes.
6. Adjust a sterile 1 c.c. pipette in the mouth of each of the tubes, A, B, C, D and E, by means of a washer, as previously described.
7. Set the electric signal clock to ring for the commencement of the experiment and subsequently at 2-1/2, 5, 10, 15, 20, 25, 30 and 35 minutes.
8. Complete precisely as indicated in Trial Runs, steps 9-19.
_Control Phenol._--
Immediately the subculture tube from the 30-minute contact period have been inoculated, carry out a precisely similar experiment, in which five percentage strengths of Phenol, (e. g., 1.1, 1.0, 0.9, 0.75, 0.7) are arranged in the lower tier of the test-tube rack in place of the five strengths of Germicide-x.
Calculate the phenol coefficient by the following method:
(a) Divide the figure representing the percentage strength of the weakest lethal dilution of the carbolic acid control at the 2-1/2-minute contact period by the figure representing the percentage strength of the weakest lethal dilution of the x-disinfectant at the same period. The quotient = phenol coefficient at 2-1/2 minutes.
(b) Similarly obtain the phenol coefficient at 30 minutes contact period.
(c) Record the mean of the two coefficients obtained in (a) and (b) as the _mean phenol coefficient_, or simply as the ~Phenol Coefficient~.
The details of the Final Test of an actual determination are set out in the accompanying table.
TABLE 27
Organism employed, B. Coli Communis.
Culture Medium, Nutrient Agar (+10). Age, 24 hrs. Temp. of Incubation, 37 deg. C.
Quantities used { Culture } Emulsion 0.1 c.c. + 5 c.c. Germicide. { Emulsion }
Room Temperature during Experiments, 17 deg. C.
Germicide Strength Time of exposure Incubation 2-1/2 5 10 15 20 25 30 35 Time Temp. 1 Germicide-x 4% -- -- -- -- -- -- -- -- 7 days. 37 deg. C. 2 Germicide-x 3% 48 -- -- -- -- -- -- -- 7 days. 37 deg. C. 3 Germicide-x 2% 24 24 24 24 48 72 7 days. 37 deg. C. 4 Germicide-x 1% 24 24 24 24 72 24 72 7 days. 37 deg. C. 5 Germicide-x 0.5% 24 24 24 24 24 24 24 24 24 hours. 37 deg. C.
1 Phenol 1.10% -- -- -- -- -- -- -- -- 7 days. 37 deg. C. 2 Phenol 1.00% 24 7 days. 37 deg. C. 3 Phenol 0.75% 24 24 24 24 48 7 days. 37 deg. C. 4 Phenol 0.70% 24 24 24 24 24 72 7 days. 37 deg. C. 5 Phenol 0.65% 24 24 24 24 24 48 24 24 2 days. 37 deg. C.
((1.10/4.00) + (0.7/2.0)) 0.27 + 0.35 .62 Phenol Coefficient = ------------------------ = ----------- = --- = 0.31 2 2 2
APPENDIX.
METRIC AND IMPERIAL SYSTEMS OF WEIGHTS AND MEASURES.
The initial unit of the metric system is the Metre (_m._) or unit of length, representing one-fourth-millionth part of the circumference of the earth round the poles.
The unit of mass is the Gramme (_g._), and represents the weight of one cubic centimetre of water at its maximum density (viz. 4 deg. C. and 760 mm. mercury pressure).
The unit of the measure of capacity is the Litre (_l._), and represents the volume of a kilogramme of distilled water at its maximum density.
The decimal subdivisions of each of the units are designated by the Latin prefixes _milli_ = 1/1000; _centi_ = 1/100; _deci_ = 1/10; the multiples of each unit by the Greek prefixes _deka_ = 10; _hecto_ = 100; _kilo_ = 1000; _myria_ = 10,000.
For a comparison of the values of some of the more frequently employed expressions of the Metric System and the Imperial System, the following may be found convenient for reference:
~Length:~
1 millimetre (= 1 mm.) = 1/25 of an inch.
1 centimetre (= 1 cm.) = 2/5 of an inch.
1 inch (1") = 25 millimetres or 2-1/2 centimetres.
~Mass:~
1 milligramme (= 1 mg.) = 0.01543 grain (or approximately 1/64 grain).
1 gramme (= 1 g.) = 15.4323 grains.
1 "kilo" or kilogramme (= 1 kgm.) = 2 pounds, 3-1/4 ounces avoirdupois.
1 pound avoirdupois (= 1 lb.) = 453.592 grammes.
1 ounce avoirdupois (= 1 oz.) = 28.35 grammes.
1 grain = 0.0648 gramme or 64.8 milligrammes.
~Capacity:~
1 cubic centimetre (= 1 c.c.) = 16.9 minims imperial measure.
1 litre (= 1 _l._) = 35.196 fluid ounces imperial measure.
1 fluid ounce imperial measure (= 1 [Symbol: ounce]) = 28.42 cubic centimetres.
1 pint imperial measure (= 1 O.) = 568.34 cubic centimetres.
1 gallon imperial measure (= 1 C.) = 4.546 litres, or 10 pounds avoirdupois, of pure water at 62 deg. F. and under an atmospheric pressure of 30 inches of mercury.
FACTORS FOR CONVERTING FROM ONE SYSTEM TO THE OTHER.
To convert grammes into grains x 15.432. To convert grammes into ounces avoirdupois x 0.03527. To convert kilogrammes into pounds x 2.2046. To convert cubic centimetres into fluid ounces imperial x 0.0352. To convert litres into fluid ounces imperial x 35.2. To convert metres into inches x 39.37. To convert grains into grammes x 0.0648. To convert avoirdupois ounces into grammes x 28.35. To convert troy ounces into grammes x 31.104. To convert fluid ounces into cubic centimetres x 28.42. To convert pints into litres x 0.568. To convert inches into metres x 0.0254.
TABLE FOR THE CONVERSION OF DEGREES CENTIGRADE INTO DEGREES FAHRENHEIT.
_X. deg. C. = ((9x/5) + 32) deg. F._
| Cent. | Faht. || Cent. | Faht. || Cent. | Faht. | | 0 | 32.0 || 34 | 93.2 || 68 | 154.4 | | 1 | 33.8 || 35 | 95.0 || 69 | 156.2 | | 2 | 35.6 || 36 | 96.8 || 70 | 158.0 | | 3 | 37.4 || 37 | 98.6 || 71 | 159.8 | | 4 | 39.2 || 38 | 100.4 || 72 | 161.6 | | 5 | 41.0 || 39 | 102.2 || 73 | 163.4 | | 6 | 42.8 || 40 | 104.0 || 74 | 165.2 | | 7 | 44.6 || 41 | 105.8 || 75 | 167.0 | | 8 | 46.4 || 42 | 107.6 || 76 | 168.8 | | 9 | 48.2 || 43 | 109.4 || 77 | 170.6 | | 10 | 50.0 || 44 | 111.2 || 78 | 172.4 | | 11 | 51.8 || 45 | 113.0 || 79 | 174.2 | | 12 | 53.6 || 46 | 114.8 || 80 | 176.0 | | 13 | 55.4 || 47 | 116.6 || 81 | 177.8 | | 14 | 57.2 || 48 | 118.4 || 82 | 179.6 | | 15 | 59.0 || 49 | 120.2 || 83 | 181.4 | | 16 | 60.8 || 50 | 122.0 || 84 | 183.2 | | 17 | 62.6 || 51 | 123.8 || 85 | 185.0 | | 18 | 64.4 || 52 | 125.6 || 86 | 186.8 | | 19 | 66.2 || 53 | 127.4 || 87 | 188.6 | | 20 | 68.0 || 54 | 129.2 || 88 | 190.4 | | 21 | 69.8 || 55 | 131.0 || 89 | 192.2 | | 22 | 71.6 || 56 | 132.8 || 90 | 194.0 | | 23 | 73.4 || 57 | 134.6 || 91 | 195.8 | | 24 | 75.2 || 58 | 136.4 || 92 | 197.6 | | 25 | 77.0 || 59 | 138.2 || 93 | 199.4 | | 26 | 78.8 || 60 | 140.0 || 94 | 201.2 | | 27 | 80.6 || 61 | 141.8 || 95 | 203.0 | | 28 | 82.4 || 62 | 143.6 || 96 | 204.8 | | 29 | 84.2 || 63 | 145.4 || 97 | 206.6 | | 30 | 86.0 || 64 | 147.2 || 98 | 208.4 | | 31 | 87.8 || 65 | 149.0 || 99 | 210.2 | | 32 | 89.6 || 66 | 150.8 || 100 | 212.0 | | 33 | 91.4 || 67 | 152.6 || | |
TABLE FOR THE CONVERSION OF DEGREES FAHRENHEIT INTO DEGREES CENTIGRADE.
_X deg. F. = (5(x - 32))/9 deg. C._
Faht.| Cent.|| Faht.| Cent.|| Faht.|Cent. || Faht.| Cent.|| Faht.| Cent. 32 | 0.|| 68 | 20.0 || 104 | 40.0 || 140 | 60.0 || 176 | 80.0 33 | 0.6 || 69 | 20.6 || 105 | 40.6 || 141 | 60.6 || 177 | 80.6 34 | 1.1 || 70 | 21.1 || 106 | 41.1 || 142 | 61.1 || 178 | 81.1 35 | 1.7 || 71 | 21.7 || 107 | 41.7 || 143 | 61.7 || 179 | 81.7 36 | 2.2 || 72 | 22.2 || 108 | 42.2 || 144 | 62.2 || 180 | 82.2 37 | 2.8 || 73 | 22.8 || 109 | 42.8 || 145 | 62.8 || 181 | 82.8 38 | 3.3 || 74 | 23.3 || 110 | 43.3 || 146 | 63.3 || 182 | 83.3 39 | 3.9 || 75 | 23.9 || 111 | 43.9 || 147 | 63.9 || 183 | 83.9 40 | 4.4 || 76 | 24.4 || 112 | 44.4 || 148 | 64.4 || 184 | 84.4 41 | 5.0 || 77 | 25.0 || 113 | 45.0 || 149 | 65.0 || 185 | 85.0 42 | 5.6 || 78 | 25.6 || 114 | 45.6 || 150 | 65.6 || 186 | 85.6 43 | 6.1 || 79 | 26.1 || 115 | 46.1 || 151 | 66.1 || 187 | 86.1 44 | 6.7 || 80 | 26.7 || 116 | 46.7 || 152 | 66.7 || 188 | 86.7 45 | 7.2 || 81 | 27.2 || 117 | 47.2 || 153 | 67.2 || 189 | 87.2 46 | 7.8 || 82 | 27.8 || 118 | 47.8 || 154 | 67.8 || 190 | 87.8 47 | 8.3 || 83 | 28.3 || 119 | 48.3 || 155 | 68.3 || 191 | 88.3 48 | 8.9 || 84 | 28.9 || 120 | 48.9 || 156 | 68.9 || 192 | 88.9 49 | 9.4 || 85 | 29.4 || 121 | 49.4 || 157 | 69.4 || 193 | 89.4 50 | 10.0 || 86 | 30.0 || 122 | 50.0 || 158 | 70.0 || 194 | 90.0 51 | 10.6 || 87 | 30.6 || 123 | 50.6 || 159 | 70.6 || 195 | 90.6 52 | 11.1 || 88 | 31.1 || 124 | 51.1 || 160 | 71.1 || 196 | 91.1 53 | 11.7 || 89 | 31.7 || 125 | 51.7 || 161 | 71.7 || 197 | 91.7 54 | 12.2 || 90 | 32.2 || 126 | 52.2 || 162 | 72.2 || 198 | 92.2 55 | 12.8 || 91 | 32.8 || 127 | 52.8 || 163 | 72.8 || 199 | 92.8 56 | 13.3 || 92 | 33.3 || 128 | 53.3 || 164 | 73.3 || 200 | 93.3 57 | 13.9 || 93 | 33.9 || 129 | 53.9 || 165 | 73.9 || 201 | 93.9 58 | 14.4 || 94 | 34.4 || 130 | 54.4 || 166 | 74.4 || 202 | 94.4 59 | 15.0 || 95 | 35.0 || 131 | 55.0 || 167 | 75.0 || 203 | 95.0 60 | 15.6 || 96 | 35.6 || 132 | 55.6 || 168 | 75.6 || 204 | 95.6 61 | 16.1 || 97 | 36.1 || 133 | 56.1 || 169 | 76.1 || 205 | 96.1 62 | 16.7 || 98 | 36.7 || 134 | 56.7 || 170 | 76.7 || 206 | 96.7 63 | 17.2 || 99 | 37.2 || 135 | 57.2 || 171 | 77.2 || 207 | 97.2 64 | 17.8 || 100 | 37.8 || 136 | 57.8 || 172 | 77.8 || 208 | 97.8 65 | 18.3 || 101 | 38.3 || 137 | 58.3 || 173 | 78.3 || 209 | 98.3 66 | 18.9 || 102 | 38.9 || 138 | 58.9 || 174 | 78.9 || 210 | 98.9 67 | 19.4 || 103 | 39.4 || 139 | 59.4 || 175 | 79.4 || 211 | 99.4 | || | || | || | || 212 |100.0
~Percentage Formula~ for addition of salts, etc., to completed media.
~Formula for preparing any desired percentage~ of a given salt, etc., in tubed media; e. g., to make 4 per cent. solution of KNO_{3} in a series of tubes of broth each containing 10 c.c. of medium, when there is already available a 25 per cent. stock aqueous solution of potassium nitrate.
(_N_ + ~X~) _Y_ _A_ (~X~) --------------- = ---------- 100 100
_N_ = number of cubic centimetres contained in each tube.
~X~ = amount of stock solution to be added to each tube.
_Y_ = percentage required in the medium.
_A_ = percentage of stock solution.
Then
(10 + ~X~) 4 25 ~X~ ------------ = ------ 100 100
Therefore, 40 + 4~X~ = 25~X~.
Therefore, 21~X~ = 40.
~X~ = 1.9 c.c.
This allows for solution added to the original bulk of medium.
Therefore, 10 c.c. broth + 1.9 c.c. of a 25 per cent. aqueous solution KNO_{3} makes 11.9 c.c. medium containing 4 per cent. KNO_{3}.
~TABLES FOR PREPARING DILUTIONS~
(of Serum, Disinfectants or other substances.)
In estimating the agglutinin content or _titre_ of a serum, testing disinfectants and for many other purposes, it becomes necessary to prepare a series of dilutions of the material under examination, and in order to avoid unnecessary expenditure of labour it is convenient to adhere to some definite scale of increment, such for example as the following:
From dilutions of 1:10 to 1:80 rise by increments of 5.
From dilutions of 1:80 to 1:200 rise by increments of 10.
From dilutions of 1:200 to 1:400 rise by increments of 25.
From dilutions of 1:400 to 1:500 rise by increments of 50.
From dilutions of 1:500 to 1:1000 rise by increments of 100.
From dilutions of 1: 1000 to 1:5000 rise by increments of 250.
From dilutions of 1: 5000 to 1:10,000 rise by increments of 1000.
From dilutions of 1:10,000 to 1:100,000 rise by increments of 5000.
From dilutions of 1:100,000 to 1:1,000,000 rise by increments of 100,000.
When dealing with a substance of unknown powers--and this is especially true with regard to agglutinating sera--it is customary to run a preliminary test, using a few widely separated dilutions such as may be obtained in the following manner:
FIRST DILUTION--I.
1 c.c. serum + 9 c.c. normal saline solution = 10 per cent. solution or 1: 10 dilution (of which 1 c.c. contains 0.1 c.c. of the original serum).
When dealing with fluids other than serum the diluent is usually distilled water; whilst if the original substance is a solid the instructions would read:
1 gram o.s. + 10 c.c. distilled water = 10 per cent. solution, etc.
SECOND DILUTION--II.
1 c.c. first dilution + 9 c.c. normal saline solution = 1 per cent. solution or 1: 100 dilution.
THIRD DILUTION--III.
1 c.c. second dilution + 9 c.c. normal saline solution = 1 per mille solution or 1: 1000 dilution.
FOURTH DILUTION--IV.
1 c.c. second dilution + 9 c.c. normal saline solution = 0.1 per mille solution or 1: 10,000 dilution.
The following tables showing the secondary dilutions that can readily be prepared from each of these four primary dilutions for use in the subsequent determination of the exact _titre_ will probably be found of service by those who are not ready mathematicians.
TABLES FOR PREPARING DILUTIONS.
+---------------------------------- | TABLE I | TABLE II Using 10 % stock solution | Using 1% stock solution First } | Second } dilution } + Diluent | dilution } + Diluent | -----------------------------------+---------------------------------- | 1: 10 = 1 c.c. + 0 c.c. | 1: 100 = 1 c.c. + 0 c.c. 1: 15 = 1 c.c. + 0.5 c.c. | 1: 110 = 1 c.c. + 0.1 c.c. 1: 20 = 1 c.c. + 1.0 c.c. | 1: 120 = 1 c.c. + 0.2 c.c. 1: 25 = 1 c.c. + 1.5 c.c. | [1: 125 = 1 c.c. + 0.25 c.c.] 1: 30 = 1 c.c. + 2.0 c.c. | 1: 130 = 1 c.c. + 0.3 c.c. 1: 35 = 1 c.c. + 2.5 c.c. | 1: 140 = 1 c.c. + 0.4 c.c. 1: 40 = 1 c.c. + 3.0 c.c. | 1: 150 = 1 c.c. + 0.5 c.c. 1: 45 = 1 c.c. + 3.5 c.c. | 1: 160 = 1 c.c. + 0.6 c.c. 1: 50 = 1 c.c. + 4.0 c.c. | 1: 170 = 1 c.c. + 0.7 c.c. 1: 55 = 1 c.c. + 4.5 c.c. | [1: 175 = 1 c.c. + 0.75 c.c.] 1: 60 = 1 c.c. + 5.0 c.c. | 1: 180 = 1 c.c. + 0.8 c.c. 1: 65 = 1 c.c. + 5.5 c.c. | 1: 190 = 1 c.c. + 0.9 c.c. 1: 70 = 1 c.c. + 6.0 c.c. | 1: 200 = 1 c.c. + 1.0 c.c. 1: 75 = 1 c.c. + 6.5 c.c. +--------------------------------- 1: 80 = 1 c.c. + 7.0 c.c. | 1: 200 = 1 c.c. + 1.0 c.c. ------------------------------+ 1: 225 = 1 c.c. + 1.25 c.c. 1: 80 = 1 c.c. + 7.0 c.c. | 1: 250 = 1 c.c. + 1.5 c.c. 1: 90 = 1 c.c. + 8.0 c.c. | 1: 275 = 1 c.c. + 1.75 c.c. 1: 100 = 1 c.c. + 9.00 c.c. | 1: 300 = 1 c.c. + 2.0 c.c. 1: 110 = 1 c.c. + 10.0 c.c. | 1: 325 = 1 c.c. + 2.25 c.c. 1: 120 = 1 c.c. + 11.0 c.c. | 1: 350 = 1 c.c. + 2.5 c.c. [1: 125 = 1 c.c. + 11.5 c.c.] | 1: 375 = 1 c.c. + 2.75 c.c. 1: 130 = 1 c.c. + 12.0 c.c. | 1: 400 = 1 c.c. + 3.0 c.c. 1: 140 = 1 c.c. + 13.0 c.c. +--------------------------------- 1: 150 = 1 c.c. + 14.0 c.c. | 1: 400 = 1 c.c. + 3.0 c.c. 1: 160 = 1 c.c. + 15.0 c.c. | 1: 450 = 1 c.c. + 3.5 c.c. 1: 170 = 1 c.c. + 16.0 c.c. | 1: 500 = 1 c.c. + 4.0 c.c. [1: 175 = 1 c.c. +-16.5 c.c.] +--------------------------------- 1: 180 = 1 c.c. + 17.0 c.c. | 1: 500 = 1 c.c. + 4.0 c.c. 1: 190 = 1 c.c. + 18.0 c.c. | 1: 600 = 1 c.c. + 5.0 c.c. 1: 200 = 1 c.c. + 19.0 c.c. | 1: 700 = 1 c.c. + 6.0 c.c. ----------------- ------------+ [1: 750 = 1 c.c. + 6.5 c.c.] 1: 200 = 1 c.c. + 19.0 c.c. | 1: 800 = 1 c.c. + 7.0 c.c. 1: 225 = 1 c.c. + 21.5 c.c. | 1: 900 = 1 c.c. + 8.0 c.c. 1: 250 = 1 c.c. + 24.0 c.c. | 1: 1000 = 1 c.c. + 9.0 c.c. 1: 275 = 1 c.c. + 26.5 c.c. +-------------------------------- 1: 300 = 1 c.c. + 29.0 c.c. | 1: 1000 = 1 c.c. + 9.0 c.c. 1: 325 = 1 c.c. +-31.5 c.c. | 1: 2000 = 1 c.c. + 19.0 c.c. 1: 350 = 1 c.c. + 34.0 c.c. | 1: 3000 = 1 c.c. + 29.0 c.c. 1: 375 = 1 c.c. + 36.5 c.c. | 1: 4000 = 1 c.c. + 39.0 c.c. 1: 400 = 1 c.c. + 39.0 c.c. | 1: 5000 = 1 c.c. + 49.0 c.c. ------------------------------+-------------------------------- 1: 400 = 1 c.c. + 39.0 c.c. | 1: 450 = 1 c.c. + 44.5 c.c. | 1: 500 = 1 c.c. + 49.0 c.c. |
---------------------------------+------------------------------- | TABLE III | TABLE IV Using 0.1% stock solution | Using 0.01% stock solution Third } | Fourth } dilution } + Diluent | Dilution } + Diluent | ---------------------------------+------------------------------- | 1: 1000 = 1 c.c. + 0 c.c. | 1: 10,000 = 1 c.c. + 0 c.c. 1: 1250 = 1 c.c. + 0.25 c.c. | 1: 15,000 = 1 c.c. + 0.5 c.c. 1: 1500 = 1 c.c. + 0.5 c.c. | 1: 20,000 = 1 c.c. + 1.0 c.c. 1: 1750 = 1 c.c. + 0.75 c.c. | 1: 25,000 = 1 c.c. + 1.5 c.c. 1: 2000 = 1 c.c. + 1.0 c.c. | 1: 30,000 = 1 c.c. + 2.0 c.c. 1: 2250 = 1 c.c. + 1.25 c.c. | 1: 35,000 = 1 c.c. + 2.5 c.c. 1: 2500 = 1 c.c. + 1.5 c.c. | 1: 40,000 = 1 c.c. + 3.0 c.c. 1: 2750 = 1 c.c. + 1.75 c.c. | 1: 45,000 = 1 c.c. + 3.5 c.c. 1: 3000 = 1 c.c. + 2.0 c.c. | 1: 50,000 = 1 c.c. + 4.0 c.c. 1: 3250 = 1 c.c. + 2.25 c.c. | 1: 55,000 = 1 c.c. + 4.5 c.c. 1: 3500 = 1 c.c. + 2.5 c.c. | 1: 60,000 = 1 c.c. + 5.0 c.c. 1: 3750 = 1 c.c. + 2.75 c.c. | 1: 65,000 = 1 c.c. + 5.5 c.c. 1: 4000 = 1 c.c. + 3.0 c.c. | 1: 70,000 = 1 c.c. + 6.0 c.c. 1: 4250 = 1 c.c. + 3.25 c.c. | 1: 75,000 = 1 c.c. + 6.5 c.c. 1: 4500 = 1 c.c. + 3.5 c.c. | 1: 80,000 = 1 c.c. + 7.0 c.c. 1: 4750 = 1 c.c. + 3.75 c.c. | 1: 85,000 = 1 c.c. + 7.5 c.c. 1: 5000 = 1 c.c. + 4.0 c.c. | 1: 90,000 = 1 c.c. + 8.0 c.c. --------------------------------+ 1: 95,000 = 1 c.c. + 8.5 c.c. 1: 5000 = 1 c.c. + 4.0 c.c. | 1: 100,000 = 1 c.c. + 9.0 c.c. 1: 6000 = 1 c.c. + 5.0 c.c. +----------------------------------- 1: 7000 = 1 c.c. + 6.0 c.c. | 1: 100,000 = 0.1 c.c. + 0.9 c.c. [1: 7500 = 1 c.c. + 6.5 c.c.] | 1: 200,000 = 0.1 c.c. + 1.9 c.c. 1: 8000 = 1 c.c. + 7.0 c.c. | [1: 250,000 = 0.1 c.c. + 2.4 c.c.] 1: 9000 = 1 c.c. + 8.0 c.c. | 1: 300,000 = 0.1 c.c. + 2.9 c.c. 1: 10,000 = 1 c.c. + 9.0 c.c. | 1: 400,000 = 0.1 c.c. + 3.9 c.c. ------------------------------- + 1: 500,000 = 0.1 c.c. + 4.9 c.c. 1: 10,000 = 1 c.c. + 9.0 c.c. +----------------------------------- 1: 15,000 = 1 c.c. + 14.0 c.c. | 1: 500,000 = 0.1 c.c. + 4.9 c.c. 1: 20,000 = 1 c.c. + 19.0 c.c. | 1: 600,000 = 0.1 c.c. + 5.9 c.c. 1: 25,000 = 1 c.c. + 24.0 c.c. | 1: 700,000 = 0.1 c.c. + 6.9 c.c. 1: 30,000 = 1 c.c. + 29.0 c.c. | [1: 750,000 = 0.1 c.c. + 7.4 c.c.] --------------------------------+ 1: 800,000 = 0.1 c.c. + 7.9 c.c. | 1: 900,000 = 0.1 c.c. + 8.9 c.c. | 1:1,000,000 = 0.1 c.c. + 9.9 c.c. -+-------------------------------------
TEMPERATURE PRESSURE TABLE.
---------------+--------------+---------------------+------------- Temperature | | Pounds per sq. in. | Centigrade | Mm. of Hg. | absolute pressure | Atmospheres | | | ---------------+--------------+---------------------+------------- | | | 98 deg. | 707.1 | 13.7 | 0.93 99 deg. | 733.1 | 14.2 | 0.96 100 deg. | 760.0 | 14.7 | 1.00 | | | 101 deg. | 787.8 | 15.2 | 1.03 102 deg. | 816.0 | 15.8 | 1.07 103 deg. | 845.2 | 16.3 | 1.11 104 deg. | 875.4 | 16.9 | 1.15 105 deg. | 906.4 | 17.5 | 1.19 | | | 106 deg. | 938.3 | 18.1 | 1.23 107 deg. | 971.1 | 18.8 | 1.27 108 deg. | 1004.9 | 19.4 | 1.32 109 deg. | 1039.6 | 20.1 | 1.36 110 deg. | 1075.3 | 20.8 | 1.41 | | | 111 deg. | 1112.0 | 21.5 | 1.46 112 deg. | 1149.8 | 22.2 | 1.51 113 deg. | 1188.6 | 22.9 | 1.56 114 deg. | 1228.4 | 23.7 | 1.61 115 deg. | 1269.4 | 24.5 | 1.67 | | | 116 deg. | 1311.4 | 25.3 | 1.72 117 deg. | 1354.6 | 26.2 | 1.78 118 deg. | 1399.0 | 27.0 | 1.84 119 deg. | 1444.5 | 27.9 | 1.90 120 deg. | 1491.2 | 28.8 | 1.96 | | | 121 deg. | 1539.2 | 29.7 | 2.02 122 deg. | 1588.4 | 30.7 | 2.09 123 deg. | 1638.9 | 31.7 | 2.15 124 deg. | 1690.7 | 32.7 | 2.22 125 deg. | 1743.8 | 33.7 | 2.29 ---------------+--------------+---------------------+-------------
TABLE FOR DESICCATION AT LOW TEMPERATURES IN VACUO.
+--------------------------+ | Temperature | | | Centigrade | Mm. of Hg. | +-------------+------------+ | 21 deg. | 18.4 | | 22 deg. | 19.6 | | 23 deg. | 20.8 | | 24 deg. | 22.1 | | 25 deg. | 23.5 | | | | | 26 deg. | 24.9 | | 27 deg. | 26.4 | | 28 deg. | 28.0 | | 29 deg. | 29.7 | | 30 deg. | 31.5 | | | | | 31 deg. | 33.3 | | 32 deg. | 35.3 | | 33 deg. | 37.3 | | 34 deg. | 39.5 | | 35 deg. | 41.7 | | | | | 36 deg. | 44.1 | | 37 deg. | 46.6 | | 38 deg. | 49.2 | | 39 deg. | 51.9 | | 40 deg. | 54.8 | | | | | 41 deg. | 57.8 | | 42 deg. | 61.0 | | 43 deg. | 64.3 | | 44 deg. | 67.7 | | 45 deg. | 71.3 | | | | | 46 deg. | 75.1 | | 47 deg. | 79.0 | | 48 deg. | 83.1 | | 49 deg. | 87.4 | | 50 deg. | 91.9 | +-------------+------------+
ANTIFORMIN METHOD
For the detection of B. Tuberculosis.
_Antiformin_ was introduced into bacteriological technique by Uhlenhuth in 1908 for the purpose of demonstrating tubercle bacilli when present in small numbers, in sputum or other material. It is a powerful oxidising agent and rapidly destroys most bacteria, but tubercle and other acid-fast organisms resist its lethal action for considerable periods, and upon this fact the method is based.
_To prepare Antiformin_ measure out and mix:--
Eau de Javelle (Liquor sodae chlorinatae--B.P.) 50 c.c. Sodic hydrate 15 per cent. aqueous solution 50 c.c.
METHOD.
1. Introduce the sputum or other material (e. g. milk deposit and cream; pus; minced gland or other organ; caseous material; broken down foci, etc.) into a sterile tube and then add an equal volume of antiformin.
2. Close the tube with a rubber cork and shake vigorously (a sample of antiformin that does not "foam" at this stage is of little use). Disintegration of the material at once starts, associated bacteria are destroyed and the mixture rapidly becomes a homogenous but turbid fluid--a process which may be hastened by:--
3. Placing the tube in the incubator at 37 deg. C. for 30 minutes--shaking from time to time.
4. Centrifugalise the fluid thoroughly, at high speed.
5. Pipette off the supernatant fluid, fill up with sterile distilled water, cork the tube and shake to distribute the deposit throughout the water. Again centrifugalise.
6. Repeat steps 4 and 5 twice more.
7. Employ one portion of the final deposit to inoculate guinea pigs.
8. Plant the remainder of the deposit freely on Dorset's Egg medium; cap and incubate at 37 deg. C.
NOTE.--If only microscopical films are needed, fill up the centrifuge tube with Ligroin (a petroleum ether) in place of sterile distilled water in step 5 and prepare the films from the _surface_ of the fluid, to stain by the Ziehl-Neelsen process.
INDEX
Abbe's condenser, 7
Abbott's stain for spores, 107
Aberration, chromatic, 56 spherical, 55
Absolute alcohol as a fixative, 82 as an antiseptic, 27
Absorbent paper for drying cover-slips, 69
A. C. E. mixture, 345
Acetic acid for clearing films, 82
Achromatic condenser, 54
Acid haematin, 96 production, analysis table, 283 by bacteria, 145 investigation of, 280 qualitative examination, 283, 284 quantitative examination, 280
Acid-fast bacilli in tissues, to stain, 124
Action of various gases on bacteria, 295
Active immunisation, illustrative example, 322
Adjustable water bath, 299
Aerobic cultures, 221
Aerogenic bacteria, 131
Aesculin agar, 204
Agar gelatine (guarniari), 194 methods of preparation, 167 surface plates, 232
Agar-agar, preparation of, 167
Agglutination reaction, macroscopical, 386 microscopical, 385
Agglutinin, 381
Air, analysis of, 468 filter, 40 pump, Geryk, 43
Albumin solution, Mayer's, 120
Alcohol production, test for, 285
Alkaline pyro, 239
Alum carmine, 96
Ammonia production test for, 285
Amphitrichous bacteria, 136
Anaerobic cultures, 236 Botkin's method, 243 Buchner's method, 238 Bulloch's method, 245 Hesse's method, 237 McLeod's method, 240 media, 180 Novy's method, 244
Anaerobic cultures, Roux's biological method, 237 physical method, 237 vacuum method, 238 Wright's method, 239
Anaesthetics, 345
Analysis of air, apparatus for, 469 method of, 468 qualitative bacteriological, 470 quantitative bacteriological, 468 of butter, qualitative bacteriological, 458 quantitative bacteriological, 457 of cream, qualitative bacteriological, 458 quantitative bacteriological, 457 of fish, 460 of ice cream, qualitative bacteriological, 457 of meat, apparatus for, 460 method of, 460 qualitative bacteriological, 462 of milk, apparatus for, 444 collection of samples, 441 method of, 441 qualitative bacteriological, 446. quantitative bacteriological, 444 of oysters, 463 of sewage, qualitative bacteriological, 467 quantitative bacteriological, 466 of shellfish, 463 of soil, apparatus for, 473 collection of samples, 471 method of, 470 qualitative bacteriological, 476 quantitative bacteriological, 473 of water, apparatus for, 420, 427 collection of samples, 416 method of, 416 qualitative bacteriological, 426
Analysis of water, quantitative bacteriological, 420
Aniline dyes, 83 Gentian violet, 95 water, to prepare, 108
Animal tissue media (Frugoni), 210
Animals, natural infections of, 337
Antiformin method for B. tuberculosis, 502
Antigen, definition of, 324
Antiseptics, 27 action of, 310
Apparent filth in milk, 450
Arnold's steam steriliser, 34
Arthrogenous spores, 138
Ascitic bouillon, 210 fluid agar (Wassermann), 213
Ascomycetae, 128
Ascopores, 129
Asparagin Media (Frankel and Voges), 183 (Uschinsky), 183
Aspergillus, 127
Atmospheric conditions, 295
Attenuating the virulence of organisms, 321
Autoclave, 37 to use, 37
Automatic pipettes, 13
Autopsies, 396
Autopsy, card index for, 402
Bacilli, morphology of, 132
Bacillus anthracis in soil, 477 in water, 440 coli in water, detection of, 429 diphtheriae in milk, 452 enteritidis in water, 437 sporogenes in milk, 452 in water, 438 oedematis maligni in soil, 477 tetani in soil, 477 in water, 441 tuberculosis in milk, 453 antiformin method, 502 typhosus in water, 441
Bacteria, anatomy of, 134 classification of, 131 grouping of, for study, 410 in tissues, demonstration of, 114 influence of environment on, 142 metabolic products of, 143 methods of identification, 259 microscopical examination of, stained, 81 unstained, 74 physiology of, 136
Bacteria, simple stains for, 90
Bacterial emulsion, preparation of, 389 enzymes, 144, 277 ferments, 144 food stuffs, 142 toxins, 144
Bacteriological analyses, general considerations, 415 examination of blood, 377
Base of microscope, 50
Basidium, 128
Beer wort, preparation of, 175
Beetroot media, 200
Beggiotoa, morphology of, 133
Benzole bath, 256
Berkefeld filter, 42
Beyrinck's solution I, 197 II, 198
Bile salt agar (MacConkey), 205 broth, double strength, 199 (MacConkey), 180
Biochemical examination of cultures, 276
Biochemistry of bacteria, 276
Biological differentiation of bacteria, 249
Bipolar germination, 140
Bismarck brown, 94
Blastomycetes, morphology of, 129
Blood agar, 171, 214 plates, animal, 251 human, 250 (Washbourn), 214 bacteriological examination of, 377 cells, washing of, 388 collection of, for serological examination, 379 films, preparations of, 376 staining of, 97 histological examination of, 373 pipettes, 11 serological examination of, 378 stains, 97
Blood-serum (Councilman and Mallory), 208 inspissated, 168 (Loeffler), 208 (Lorrain Smith), 208
Blowpipe table, 9
Body tube of microscope, 50
Bohemian flask, 4
Boiling water, 33
Bone marrow, films, preparation of, 400
Bordet-Gengou reaction, 393
Boric acid in milk, test for, 442
Botkin's anaerobic method, 243
Bouillon, preparation of, 163
Brain extract, 149
Bread paste, 193
Brilliant green agar (Conradi), 206 bile salt agar (Fawcus), 206
Brownian movement, 79
Buchner's anaerobic method, 238
Bulloch's anaerobic method, 245 tubes for permanent preparations, 407
Bunge's mordant, 104
Burri's Chinese ink stain, 77
Butter, analysis of, 457 qualitative analysis of, 458 quantitative analysis of, 457
Cadaver, preparation of, for autopsy, 397
Cages for guinea-pigs, 343 for laboratory animals, 341 for mice, 342 for rabbits, 343 for rats, 342
Calculated figure for weight of medium mass, 166, 167
Cambier's candle method of isolating coli-typhoid groups, 438
Camera lucida, 62
Capaldi-Proskauer medium, No I, 186 No II, 187
Capillary pipettes, 10 graduated, 13
Capitate bacilli, 139
Capsule formation, 134 of bacteria, 134 thermo-regulator, 218
Capsules, collodion, inoculation of, 357 preparation of, 357 glass, 6 to clean infected, 20 new, 18 to stain, 99 to sterilise, 31
Carbohydrate media, preparation of, 177
Carbolic acid as a germicide, 27, 481 method of isolating coli-typhoid group, 437
Carbolised agar, 202 bouillon, 202 gelatine, 202
Carbon dioxide in cultures, test for, 289
Card index, 336, 402
Carrot media, 200
Cedarwood oil for immersion lens, 88
Cell wall of bacteria, 134
Celloidin sacs, manufacture of, 358
Cellular incubator, 216
Centrifugal machine for blood and serum work, 327 for milk work, 447
Centrifugalised milk, 449
Centrigade degrees, conversion of, 494
Chemical products of bacteria, 145
China green agar (Werbitski), 207
Chloroform as an antiseptic, 27
Chromatic aberration, 56
Chromogenic bacteria, 131
Chromoparous bacteria, 144
Chromophorous bacteria, 144
Citrated blood agar, 191
Cladothrix, morphology, 193
Classification of bacteria, 131 of fungi, 126
Clavate bacilli, 139
Clearing films with acetic acid, 82
Clostridium, 139
Coarse adjustment, 51
Cobweb micrometer, 66
Cocaine, 345
Cocci, morphology of, 131
Coccidium infection, 339
Coefficient, inferior lethal, 312 of inhibition, 311 phenol, 489 superior lethal, 313
Cohn's solution, 191
Cold incubator, 217
Coli-typhoid group, differential table, 433 in milk, 451 in soil, 477 isolation of, 432 members of, 430
Collection of blood for bacteriological examination, 378 for media making, 168 of milk samples, 443 of pathological material during life, 373 of pus, 373 of soil sample, 471 of water samples, 416
Collodion capsules, 357 sacs, manufacture of, 357
Colonies of bacteria, edges, 267
Coloured light, action of, 309
Columella, 127
Comparative haemocytology, 374
Complement, definition of, 325 fixation test, 393
Concentration method in water, analysis, 434
Condenser achromatic, 54 dark ground, 60 paraboloid, 60 substage, 54
Condidium, 128
Continuous sterilisation, 36
Contrast stains, 93
Corrosive sublimate (Lang), 82
Cotton-wool filter, 40
Counterstaining films, 84
Counting plate colonies, 423
Cover-slip films, 81 to clean new, 22 used, 24
Crates for test-tubes, 31
Cream, analysis of, 457 qualitative analysis of, 458 quantitative analysis of, 457
Crenothrix morphology, 133
Criteria of infection, 370
Criterion of immunity, 324
Cultural characters, macroscopical examination, 261
Culture flask, Guy's, 5 Kolle, 4 Roux, 5
Cuneate bacilli, 139
Cutaneous inoculation, 352
Dark ground condenser, 60 illumination, 87
Daughter cells, 129
Daylight, diffuse, action of, 308
Decimal scales, 340
Decolourising agents, 84
Definition of objective, 56
Depilatory powder, 346
Description of plate culture, 261
Descriptive terms, 261
Desiccation, effects of, 306 table, 501
Desiccator, Mueller's, 307
Dextrose solution, preparation of, 178
Diaphragm, iris, 53
Diastatic enzymes, tests for, 278
Differential atmosphere cultivation, 257 incubation, 255 media, 255 staining, 108 sterilisation, 256
Diluting chamber, 248
Dilution by teat pipette, 383 of serum, 382 tables, 498
Dilutions, preparations of, 496
Diphtheria, bacillus of, in milk, 452
Diplobacilli, morphology of, 133
Diplococci, morphology of, 133
Diplococcus pneumoniae, immunisation against, 322
Discontinuous sterilisation, 36
Discs of plaster-of-Paris, 192
Disinfectants, action of, 310 chemical, 27 testing of, 480
Dissociating fluid, Price Jones; 400
Dosage of inoculum, 316
Double nosepiece, 58 stains for spores, 106 sugar agar (Russell), 207
Drop-bottle, 73
Dry heat, 28
Dunham's solution, 177
Dyes, aniline, 83
Earthenware box for dirty slides, 70
Earthy salts agar (Lipman and Brown), 197
Edge of individual colonies, characters of, 267
Egg albumin agar, 213 broth, (Lipschuetz), 213 media (Dorset), preparation of, 174 inspissated, 212 (Lubenau), 209 (Tarchanoff and Kolesnikoff), 212 to clear nutrient media with, 166
Ehrlich's eyepiece, 55
Eikonometer, 65
Eisenberg's milk-rice medium, 189
Electric dental engine, 360 signal clock, 38 warm stage, 59
Elevation of colonies, 263
Eisner's gelatine, 204 method of isolating coli: typhoid group, 438
Endogenous spores, 138 varieties of, 139
Endo-germination, 139
English proof agar, Blaxall, 193
Enumerating colonies on plates, 423 discs, Jeffer's, 424 Pakes', 424
Enrichment method in water analysis, 427
Enumeration of micro-organisms, 423
Environmental conditions, 142
Enzyme production, investigation of, 277
Eosin, 93
Equatorial germination, 140
Erlenmeyer flask, 4
Ernstschen Koerner, 136
Esmarch's roll culture, 226 water collecting bottle, 417
Estimation of reaction of media, 280
Ether flame, 28 soluble acids, 284
Eucaine, 345
Exalting virulence of organisms, 320
Examination of milk, 441
Experimental infections, study of, during life, 370 inoculation of animals, 332
Extracellular toxins, 144
Eyepiece, Ehrlich, 55 _micrometer_, 63
Eyepieces, 55
Eye-shade, 57
Fahrenheit degrees, conversion of, 495
Feeding experiments, 369
Fermentation reactions, 279 tubes, 17
Field of objective, 56
Filar micrometer, 66
Filling tubes, etc., with medium, 160
Film preparations, 81 fixing, 81 making, 81 mounting, 85 staining, 83
Filter candle, closed, 47 open, 43 testing efficiency of, 478 to disinfect, 28 to sterilise, 29 flask, 6 papers, to fold, 156
Filters, cotton-wool, 40 porcelain, 42 testing of, 478
Filtration, 40 by aspiration, 42 of media, 156 under pressure, 45
Fine adjustment, 51 spindle head, 52
Fish, analysis of, 460 bouillon, 190
Fish gelatine, 190 gelatine-agar, 190
Fishing colonies, 253
Fission, reproduction by, 136
Fixation, 81 by heat, 81 of tissues, 114
Fixing fluids, for films, 82
Flagella, classification of bacilli by, 136 to stain, 101
Flask Bohemian, 4 Erlenmeyer, 4 filter, 6 Kitasato'a serum, 6 Kolle's culture, 4
Flasks and test tubes, to plug, 24 to clean dirty, 20 new, 18 to sterilise, 31
Fleischwasser, 148
Fluid cultures, description of, 271 media, 146
Foot of microscope, 50
Formaldehyde in milk, Hehner's test for, 442
Formalin method of preserving cultures, 407 tissues, 404
Fractional sterilisation, 33
Fraenkel and Voge's solution, 183
Fraenkel's earth borer, 472
Freezing method for sections, 115
French Mannite Agar (Sabouraud), 193 proof agar (Sabouraud), 193
Fresh preparations of bacteria, 74
Friedlaender's capsule stain for sections, 123
Frost's mounting fluid, 406
Frozen sections, rapid method, 116
Fuchsin, 92 agar (Braun), 205 sulphite agar (Endo), 206
Gas analysis, qualitative, 290 quantitative, 290 collecting apparatus, 291 generators, 242 production by bacteria, 289 tubes for media, 161
Gasperini's solution, 193
Gelatin agar, 193 preparation of, 164 surface plates, 231
General anaesthetics, 345
Gentian violet, 91
German lined paper, 69
Germicides, 27 testing power of, 480
Germination, 140
Geryk air-pump, 43
Glass apparatus in common use, 3 to clean, 18
Glass-cutting knife, 8
Glucose formate agar (Kitasato), 180 bouillon (Kitasato), 180 gelatine (Kitasato), 180
Glycerinated potato, 209
Glycerine agar, 209 blood-serum, 208 bouillon, 209 potato bouillon, 203 broth, 203
Goadby's gelatine, 214
Gonidium, 128
Goniodophore, 128
Graduated capillary pipettes, 13 pipettes, 6
Gram-Claudius' differential stain, 109
Gram's differential stain, 108
Gram-Weigert for sections, 121, 122
Gram-Weigert's differential stain, 109 modified, 110
Grease pencils, 72
Grouping of bacteria for study, 410
Guarded trepine, 360
Guarniari's agar gelatine, 194
Guinea-pig cages, 343 holder, 350
Gulland's solution, 82
Gum solution, preparation of, 116
Guy's culture bottle, 5
Gypsum blocks (Engel and Hansen), 192
Haematin, 95
Haematocytometer, 248
Haematoxilin, 95
Haemolysin, definition of, 326 preparation of, 327 storage of, 331
Haemolytic serum, titration of, 328
Hanging-block culture (Hill), 235
Hanging-drop cultures, 233 examination of, 86, 79 preparation of, 78 permanent staining of, 80 slides, 70
Hardening tissues, 114
Haricot agar, 200 bouillon, 200
Hay infusion, 200
Hearson's water bath, 299
Heat effect of, 299
Hehner's test, 442
Heiman's serum agar, 210
Hesse's anaerobic culture method, 237
Histological examination of blood, 373
Holder for guinea-pigs, 350
Hot air, 29 steriliser, 30 to use, 31 incubator, 217
Hot-water funnel, 158
Human blood agar plates, 250
Huyghenian eyepiece, 55
Hydrogen, generating apparatus, 242 in culture, test for, 289 peroxide in milk, test for, 442
Hyphomycetes, morphology of, 126 reproduction of, 126
Ice-box, for water samples, 419
Ice cream, analysis of, 457
Illuminant for microscope, 67
Immune body, 393
Immunisation, methods of, 321
Imperial system, 492 factors for converting, 493
Impression films, 85
Incubators, 216
Index cards, 336, 403
Indol, test for, 286
Infection, definition of, 370 general observations during life, 371 results of, 404
Influence of environment on bacterial growth, 142
Inhalation, fluid inoculum, 365 powdered inoculum, 366
Inhibition coefficient, 310, 311
Inoculation card index, 336 cutaneous, 352 intracranial, 360 intramuscular, 355 intraocular, 362 intraperitoneal, 355 intrapulmonary, 363 intravenous, 363 of collodion capsules, 357 subcutaneous, 353 syringe, 344
Inoculum, character of, 346 preparation of, 346
Inosite-free media--bouillon (Durham), 183
Inseparate toxins, 144
Intermittent sterilisation, 36
Intracellular toxins, 144
Intracerebral inoculation, 362
Intracranial inoculation, 360
Intragastric inoculation, large animals, 367 Marks method, 367
Intramuscular inoculation, 355
Intraocular inoculation, 362
Intraperitoneal inoculation, 355
Intrapulmonary inoculation, 363
Intravenous inoculation, 363
In vacuo anaerobia cultures, 289
Invertin enzymes, tests for, 279
Involution forms, 137
Iodine solution, 108
Iron bouillon, 185 peptone solution (Pakes), 185
Isolation by animal experiments, 258 by differential atmosphere, 257 incubation, 255 media, 255 sterilisation, 256 by dilution, 248 by plate cultures, 250 subcultures, preparation of, 254
Jeffer's counting disc, 424
Jenner's stain, 97
Jores' mounting fluid, 405
Kaiserling fixing solution, 405
Kanthack's serum agar, 211
Killed cultivations, 318
Kipp's hydrogen apparatus, 242
Kitasato's serum flask, 6
Klebs-Loeffler bacillus in milk, 452
Koch's steam steriliser, 34
Kohle's culture flask, 4
Lab enzymes, test for, 279
Laboratory animals, 335 comparative haematocytology of, 374 normal temperature, 372 regulations, 1
Lactose litmus agar (Wurtz), 203 bouillon, 203 gelatine (Wurtz), 203
Lakmus Molke, 203
Lang's solution, 82
Lead bouillon, 185 peptone solution, 186
Leishman's stain, 98 for sections, 125
Lemco broth, 163
Leptothrix, morphology, 133
Lethal dose, minimal, 316
Leviditi's staining method, 124
Light, action of, 308
Liquefiable media, 147
Liquid soap, 346
Lithium carmine, 96
Litmus bouillon, 186 gelatine, 202 milk cultures, description of, 272 preparation of, 172 nutrose agar (Drigalski-Conradi), 205 whey, 195 agar, 196 gelatine, 196 (Petruschky), 195
Local anaesthetics, 345 reaction to infection, 372
Locomotive movement, 80
Loeffler's capsule stain, 103 serum, 208
Lophotrichous bacilli, 136
Lorrain Smith electric warm stage, 59 serum, 208
Lugol's solution, to prepare, 108
Lysol, 27
MacConkey's capsule stain, 99 media, 180, 199, 205
MacCrorrie's capsule stain, 103
Macroscopical examination of cultures, 261
Malachite green agar (Loeffler), 207
Malt extract solution (Herschell), 196
Margin of individual colonies, 267
Martin's filtering apparatus, 320
Material for inoculation, 346
Mayer's albumin, 120
Mean phenol coefficient, 490
Measuring bacteria, 61
Meat, bacteriological analysis of, 460 extract preparation of, 148 reaction of, 149
Mechanical separation of bacteria, 249 stage, 52
Media, filtration of, 156 preparation of, 163 aerobic culture, 222 aesculin agar, 204 agar-agar, 167 agar gelatine (Guarniari), 194
Media, preparation of anaerobic culture, 180 animal tissue (Frugoni), 210 ascitic bouillon, 210 fluid agar (Wassermann), 213 asparagin (Fraenkel and Voge's), 183 (Uschinsky), 183 beer wort, 175 beetroot, 200 Beyrinck's solution I, 197 II, 198 bile salt agar (MacConkey), 205 broth (MacConkey), 180 double strength, 199 blood agar (Washbourn), 214 blood-serum, 168 (Councilman and Mallory), 208 (Loeffler), 208 (Lorrain Smith), 208 bouillon, 163 bread paste, 193 brilliant green agar (Conradi), 206 bile salt agar (Fawcus), 206 Capaldi-Proskauer, No. I, 186 No. II, 187 carbohydrate, 177 carbolised agar, 202 bouillon, 202 gelatine, 202 carrot, 200 China green agar (Werbitski), 207 citrated blood agar, 171 Cohn's solution, 191 dextrose solution, 178 double sugar agar (Russell), 207 earthy salt agar (Lipman and Brown), 197 egg Dorset, 174 Lubenau, 209 egg-albumen, inspissated, 212 (Tarchanoff and Kolesnikoff), 212 egg-albumin agar, 213 broth (Lipschuetz), 213 English proof agar (Blaxall), 193 fish bouillon, 190 gelatine, 190 agar, 190 fluid, 146 French mannite agar (Sabouraud), 193
Media, preparation of French proof agar (Sabouraud), 193 Fuchsin agar (Braun), 205 sulphite agar (Endo), 206 gelatine, 193 agar, 193 glucose formate agar (Kitasato), 180 bouillon (Kitasato), 180 gelatine (Kitasato), 180 glycerinated broth, 209 potato, 209 glycerine agar, 209 blood-serum, 208, 209 bouillon, 209 potato bouillon, 203 gypsum blocks (Engel and Hansen), 192 haricot agar, 200 bouillon, 200 hay infusion, 200 inosite free-bouillon (Durham), 183 iron bouillon, 185 peptone solution (Pakes), 185 lactose litmus agar (Wurtz), 203 bouillon, 203 gelatine (Wurtz), 203 lakmus molke, 203 lead bouillon, 185 peptone solution, 186 lemco broth, 163 liquefiable, 147 litmus bouillon, 186 gelatine, 202 milk, 172 nutrose agar (Drigalski-Conradi), 205 whey, 195 agar, 196 gelatine, 196 (Petruschky), 195 malachite green agar (Loeffler), 207 malt extract solution (Herschell), 196 milk, 172 rice (Eisenberg), 189 (Soyka), 189 Naegeli's solution, 191 Naehrstoff agar (Hesse and Niedner), 199 neutral litmus solution, 179 nitrate bouillon, 185 peptone solution (Pakes), 186 nutrient, 146 agar-agar, 167
Media, preparation of nutrient bouillon, 163 gelatine, 164 nutrose agar (Eyre), 172 oleic acid agar (Fleming), 201 Omeliansky's nutrient fluid, 189 Parietti's bouillon, 202 parsnip, 200 Pasteur's solution, 191 peptone rosolic acid water, 186 water (Dunham), 177 plaster-of-Paris discs, 192 potato, 174 gelatine (Elsner), 204 (Goadby), 214 proteid free broth (Uschinsky), 183 rosolic acid peptone solutions, 186 serum, bouillon, 210 dextrose water, (Hiss), 188 sugar, (Hiss), 188 water, 170 serum-agar (Heiman), 210 (Kanthack and Stevens), 211 (Libman), 212 (Wertheimer), 211 silicate jelly (Winogradsky), 198 solid, 147 special, 182 stock nutrient, 163 sugar, 177 agar, 185 (dextrose) bouillon, 184 gelatine, 184 sulphindigotate agar, 181 bouillon (Weyl), 181 gelatine (Weyl), 181 tissue (Noguchi), 214 turnip, 200 urine agar, 188 bouillon, 187 gelatine, 187 (Heller), 188 wheat bouillon (Gasperini), 193 whey agar, 195 gelatine, 195 wine must, 192 Winogradsky's solution (for nitric organisms), 198 (for nitrous organisms), 198 wood ash agar, 201 wort agar, 176 gelatine, 176
Media, preparation of yeast water (Pasteur), 191 standardisation of, 154 storage of, in bulk, 159 storing tubes of, 161 sore boxes, 162 titration of, 150 tubing of nutrient, 160
Merismopedia, morphology of, 132
Mesophilic bacteria, 143 pathogenic effects, 315
Metabolic end-products, 145
Metachromatic granules, 136
Metal instruments, to sterilise, 28
Metatrophic bacteria, 131
Methods of cultivation, 221 of identification of bacteria, 259 of inoculation, 352 of isolation, 248 of sterilisation, 26
Methylene-blue, 90
Metric system, 492 factors for converting, 493
Meyer's carmine, 96
Microbes of indication, 426
Micrococci, morphology, 132
Micrococcus, melitensis in milk, 456
Micrometer, filar, 66 net, 63 ocular, 63 stage, 62
Micrometry, methods of, 61
Micron, 61
Microscope, 49
Microscopical examination of bacteria, 86 stained, 88 unstained, 86 observations of cultures, 272
Milk, analysis of, qualitative, 446 quantitative, 444 condensed, analysis of, 444 media, 193 preparation of, 172 rice (Eisenberg), 193 (Soyka), 189 samples, collection of, 443 sedimenting tubes, 449
Minimal lethal dose, 316
Mirror for microscope, 55
Moeller's stain for spores, 107
Moist heat, 32
Molecular movement, 79
Monotrichous bacilli, 136
Motility, examination for, 79 true, 80
Moulds, examination of, 126 for paraffin imbedding, 117, 119
Mounting film preparations, 85 paraffin sections, 119
Mouse cages, 342 holder, 351 scales, 341
Mucor mucedo, 126
Mucorinae, 126
Mueller's desiccator, 307
Muffle furnace, 28
Muirs's capsule stain, 100 flagella stain, 101
Museum preparations of bacteria, 407 of tissues, 404 sealing of, 406
Mycelium, 126
Mycoprotein, 135
Naegeli's solution, 191
Naehrstoff agar (Hesse and Niedner), 199
Naked flame, 28
Neisser's stain modified, 111
Net micrometer, 63
Neutral litmus solution, preparation of, 179 red, 94
Nitrate bouillon, 185 peptone solution (Pakes), 186
Nitric organisms in soil, 478
Nitrosoindol reaction, 287
Nitrous organisms in soil, 477
Normal averages (_t.p.r._), 372 serum, 375
Nosepiece, 57 double, 58 triple, 58
Navy's anaerobic method, 244 jars, 245
Nuclei, to stain, 105
Nucleus of bacteria, 135
Numerical aperture, 56
Nutrient media, 146
Nutrose agar (Eyre), preparation of, 172
Object marker, 61
Objectives, 55
Oblique tube cultures, 223
Ocular micrometer, 63
Oculars, 55
Oese, platinum, 71
Oidium, 128
Oil of garlic, 27 of mustard, 27
Oleic acid agar (Fleming), 201
Omeliansky's nutrient fluid, 189
Operation tables (Eyre's), 352 (Tatin's), 351
Opsonic index, 393
Opsonic index, determination of, 390
Opsonin, 387
Optical characters of colonies, 267
Optimum reaction of medium, determination of, 305 temperature, determination of, 298
Organisms of suppuration, 409
Orsat-Lunge gas apparatus, 292
Orth's carmine, 96
Oxford stain for Actinomyces, 112
Oysters, analysis of, 463
Pakes' counting disc, 424 filter reservoir, 45
Papier chardin, 158
Pappenheim's stain, 111
Paraboloid condenser, 60
Parachromophorous bacteria, 144
Paraffin method for sections, 117 sections, mounting of, 119 to stain, 121
Paratrophic bacteria, 131
Parietti's bouillon, 202 method of isolating coli-typhoid group, 437
Parsnip medium, 200
Passages of virus, 320
Pasteur-Chamberland filter, 42
Pasteur's pipettes, 10 solution, 191
Pathogenesis, investigation of, 315
Pathogenic bacteria, 131 study of, 408
Pediococci, morphology of, 132
Penicillium, 128
Peptone rosolic acid water, 186 water (Dunham), preparation of, 177
Percentage formula, 496
Perchloride of mercury, 27
Perisporaceae, 127
Peritrichous bacilli, 136
Permanent preparations of bacteria, 407 of tissues, 404
Petri's dishes, 6
Phagocytic index, 392
Phenol coefficient, 489 production, test for, 287
Photogenic bacteria, 131, 144
Physiological filter, 156
Picric acid solution, 121 (Spengler's), 112
Picrocarmine, 97
Pigment production, observations on, 288
Pipettes, automatic, 13 blood, 11 capillary, 10 cases for, 7 graduated, 6 capillary, 13 Pasteur's, 10 sedimentation, 16 standard graduated, 7 teat, 10 throttle, 13 to clean infected, 20 new, 18 to sterilise, 31
Piridin method of staining spirochaetes, 124
Pitfield's flagella stain, 103
Plasmolysis, 135
Plaster-of-Paris discs, 192
Plate box, 7 cultures, description of, 261 preparation of, 226 levelling stand, 228
Plates, Petri's, 6 to clean infected, 20 new, 18 to sterilise, 31
Platinum needles, 71 method of mounting, 71
Pleomorphism, 133
Polar germination, 140 granules, 136
Polkoerner, 136
Polychrome blood stains, 97
Pooled serum, 379
Porcelain filter, 42 Berkefeld, 42 Chamberland, 42 Doulton, 42
Post-mortem examination of experimental animals, 396
Potato gelatine (Eisner), 204 (Goadby), 214 medium, preparation of, 174
Potted meat, analysis of, 460
Pouring plates, 227
Preparation of experimental animals, 335
Preservatives in milk, 442
Pressure temperature table, 500
Primary colours, action of, 309
Proteid free broth (Uschinsky), 183
Proteolytic enzymes, tests for, 277
Prototrophic bacteria, 131
Psychrophilic bacteria, 143 pathogenic effects, 315
Pus, collection of, 373
Pyrogallic acid solution, 293
Qualitative analysis of air, 470 of milk, 446 of sewage, 467 of soil, 476 of unsound meat, 462 of water, 426
Quantitative analysis of air, 468 of milk, 444 of sewage, 466 of soil, 473 of unsound meat, 460
Rabbit cages, 343 scabies, treatment of, 338 scales, 340
Raising virulence of organisms, 320
Ramsden's micrometer, 66
Range of medium reaction, measurement of, 305 of temperature, measurement of, 298
Rat cages, 342
Raw milk, Saul's test for, 442
Reaction of medium, 305 optimum, 305 range of, 305 scale, 153
Reduced pressure and temperature table, 501
Reducing agents, production, 389 tests for, 289
Reduction of nitrates, 389
Reichert's thermo-regulator, 218
Relation of bacteria to environment, 142
Removal of material from culture tubes, 74
Rennin enzymes, tests for, 279
Reproduction of bacteria, 136
Resistance glass, 6 to lethal agents, 306
Resting stage of bacteria, 137
Restrictions upon experimental inoculations, 334
Ribbert's capsule stain, 101
Roll cultures, 226
Rosolic acid peptone solution, 186
Rosindol reaction, 286
Roux's anaerobic culture method, 237 culture bottle, 5
Sabouraud's medium, 193
Saccharomyces, morphology of, 129
Safranine, 94
Salicylic acid in milk, test for, 443
Saprogenic bacteria, 131
Sarcinae, morphology of, 132
Saul's test, 442
Scales, decimal, 340 trip, 164
Scalpels, to sterilise, 32, 33
Schallibaum's solution, 121
Scheme for study of bacteria, 259
Schizomycetes, classification of, 131 morphology of, 131
Scissors, to sterilise, 32
Sealing museum jars, 406
Searing iron, 397
Sections, special staining methods for, 121
Sedimentation pipettes, 16 tubes, 9
Selecting objectives, 57
Sensitising red blood cells, 395
Serial cultivations, 251
Serological examination of blood, 378
Serum agar (Heiman), 210 (Kanthack and Stevens), 211 (Libman), 212 plates, 250 (Wertheimer), 211 bouillon, 210 collection of, 379 dextrose water (Hiss), 188 inspissator, 169 sugar media (Hiss), 188 water, preparation of, 170
Sewage, analysis of, qualitative, 467 quantitative, 466
Shake cultivations, 225 description of, 271
Shape of colonies, 262
Shaving experimental animals, 349
Shellfish, analysis of, 463
Silicate jelly (Winogradsky), 198
Single stain for spores, 106
Size of colonies, 262
Slanted tube cultures, 223
Slides, to clean new, 22 used, 23
Smear culture, 224 description of, 268
Soap liquid, 346
Soda solution, storage of stock, 154
Sodium bicarbonate in milk, test for, 443
Soil, analysis of, qualitative, 476 quantitative, 473 collection of samples, 471
Solid media, 147
Soluble toxins, 144
Soyka's milk rice, 189
Spear-headed spatula, 402
Special media, 182
Specific serum, 379 dilution of, 382
Spherical aberration, 55
Spirillum, morphology of, 133
Spirochaeta, morphology of, 133
Spirochaetes in tissues, to stain, 124
Spleen extract, 149
Sporangium, 127
Spore formation, arthrogenous, 138 endogenous, 138 method of, 138, 273 germination, method of, 140, 274 observation of, 140, 273
Spores, characters of, 139 classification of, 139 double stain for, 106 to stain, 106
Stab culture, 224 description of, 265
Stage micrometer, 62 of microscope, 52
Staining methods, 90 paraffin sections, 121 reactions of bacteria, 274
Stains intra-vitam, 77 negative (Burri), 77 rack for, 72
Standard graduated pipettes, 7 soda solution, 154
Standardisation of media, 154
Standardising bouillon, 155
Staphylococci, morphology, 132
Staphylococcus in milk, 456
Steam steriliser, Arnold, 35 Koch, 35 to use, 35 streaming, 35
Sterigma, 127
Sterilisation by chemicals, 27 by dry heat, 28 by filters, 40 by moist heat, 32 by streaming steam, 35 by superheated steam, 36 of albuminous liquids, 32 of gases, 40
Sterilising agents, 26
Stichcultur, 224
Stock dilutions, 497 nutrient media, 163 plate for isolation work, 253
Storage of media in bulk, 159 of tubed media, 161
Store boxes for media, 161
Streak culture, 224 description of, 268
Streaming movement, 80 steam, 35
Streptobacilli, morphology, 133
Streptococci in soil, 477 in water, detection of, 432 morphology of, 132
Streptococcus pyogenes longus in milk, 455
Streptothrix, morphology of, 133
Strichcultur, 223
Structure, internal, of colonies, 265
Study of pathogenic bacteria, 408
Subcutaneous inoculation, 353
Subdural inoculation, 361
Substage condenser, 54
Sugar agar, 185 dextrose bouillon, 184 gelatine, 184 media, preparation of, 177
Sulphindigotate agar, 181 bouillon (Weyl), 181 gelatine (Weyl), 181
Sulphuretted hydrogen in cultures, test for, 290
Sunlight, action of, 309
Superheated steam, 36
Superior lethal coefficient, 310, 313
Suppuration, organisms of, 409
Surface characters of colonies, 264 plates, 230
Surgical motor, electric, 360
Swarm spores, 127
Syringe for subcutaneous inoculation of solid material, 354 hypodermic, 344
Tatin's operating table, 351
Taxonomy, 262
Teat-pipettes, 10
Temperature, action of, 299 optimum, 298 pressure table, 500 range, 298 taking, 340
Test objects for objectives, 57
Testing filters, 478
Test-tubes, 3 to clean infected, 19 new, 18 to plug, 24 to sterilise, 31
Tetracocci, morphology of, 132
Thermal death-point, 143 determination of, 298 of spores, 301, 304 of vegetative forms, 298, 303
Thermophilic bacteria, 143
Thermo-regulators, Hearson's capsule, 218 Reichert's, 218
Thionine blue, 92
Thiothrix, morphology of, 133
Thresh's water collecting bottle, 418
Throttle pipettes, 13
Tinned meat, analysis of, 460
Tissue medium (Noguchi), 214 stains, 95
Tissues for sectioning, fixing, 114 freezing, 116 hardening, 114 imbedding, 118 preparation of, 114 washing, 115
Titration of media, 150
Torulae, differentiation from saccharomyces, 130
Total acidity, 280
Toxins, testing of, 318
Trephines, 360
Triple nosepiece, 58
True motility, 80
Tube cultures, preparation of, 222 length, 50
Tubercle bacillus in milk, 453 to stain, 110, 124
Tuberculous guinea-pig, cadaver of, 454
Tubing nutrient media, 160
Turnip media, 200
Unna-Pappenheim's stain for sections, 123
Unsound meat, analysis of, 460
Urine agar, 188 gelatine, 187 (Heller), 188 media bouillon, 187
Uschinsky's solution, 183
Valency of specific sera, 386
Van Ermengem's flagella stain, 104
Vegetative stage of bacteria, 136
Vesuvin, 94
Vibrio cholerae in milk, 452 in water, 439 morphology of, 133
Virulence, attenuating, 321 of organisms, 320 raising, 320
Vivisection license, 334
Voges holder, 350
Volatile oils as disinfectants, 27
Warm stage, 58
Washing red blood cells, 388 tissues, 115
Water, analysis of, qualitative, 426 quantitative, 416 steriliser, 33
Weighing animals, 340
Welch's capsule stain, 101
Wertheimer's serum agar, 211
Wheat bouillon (Gasperini), 193
Whey agar, 195 gelatine, 195
Wine must, 192
Winogradsky's solution I, 198 II, 198
Wire crates for test-tubes, 31
Wood ash agar, 201
Working up plates, 252
Wort agar, 176 gelatine, 176
Wright's anaerobic method, 239
Yeast water (Pasteur), 191
Ziehl-Neelsen's stain, 110
Zoogloea, 134
Zymogenic bacteria, 131
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Hill's Histology and Organography
~A Manual of Histology and Organography.~ By CHARLES HILL, M. D., formerly Assistant Professor of Histology and Embryology, Northwestern University, Chicago. 12mo of 468 pages, 337 illustrations. Flexible leather, $2.00 net.
~THE NEW (2d) EDITION~
Dr. Hill's work is characterized by a completeness of discussion rarely met in a book of this size. Particular consideration is given the mouth and teeth.
~Pennsylvania Medical Journal~
"It is arranged in such a manner as to be easy of access and comprehension. To any contemplating the study of histology and organography we would commend this work."
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GET THE NEW THE BEST STANDARD American Illustrated Dictionary
~New (7th) Edition--5000 Sold in Two Months~
~The American Illustrated Medical Dictionary.~ A new and complete dictionary of the terms used in Medicine, Surgery, Dentistry, Pharmacy, Chemistry, Veterinary Science, Nursing, and kindred branches; with over 100 new and elaborate tables and many handsome illustrations. By W. A. NEWMAN DORLAND, M.D., Editor of "The American Pocket Medical Dictionary." Large octavo, 1107 pages, bound in full flexible leather. Price, $4.50 net; with thumb index, $5.00 net.
~IT DEFINES ALL THE NEW WORDS--IT IS UP TO DATE~
The American Illustrated Medical Dictionary defines hundreds of the newest terms not defined in any other dictionary--bar none. These new terms are live, active words, taken right from modern medical literature.
It gives the capitalization and pronunciation of all words. It makes a feature of the derivation or etymology of the words. In some dictionaries the etymology occupies only a secondary place, in many cases no derivation being given at all. In the American Illustrated practically every word is given its derivation.
Every word has a separate paragraph, thus making it easy to find a word quickly.
The tables of arteries, muscles, nerves, veins, etc., are of the greatest help in assembling anatomic facts. In them are classified for quick study all the necessary information about the various structures.
Every word is given its definition--a definition that _defines_ in the fewest possible words. In some dictionaries hundreds of words are not defined at all, referring the reader to some other source for the information he wants at once.
~Howard A, Kelly, M. D.~, _Johns Hopkins University, Baltimore._
"The American Illustrated Dictionary is admirable. It is so well gotten up and of such convenient size. No errors have been found in my use of it."
~J. Collins Warren, M. D., LL.D., F.R.C.S. (Hon.)~, _Harvard Medical School_
"I regard it as a valuable aid to my medical literary work. It is very complete and of convenient size to handle comfortably. I use it in preference to any other."
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Stengel's Text-Book of Pathology
~Fifth Edition~
~A Text-Book of Pathology.~ By ALFRED STENGEL, M. D., Professor of Medicine in the University of Pennsylvania. Octavo volume of 979 pages, with 400 text-illustrations, many in colors, and 7 full-page colored plates. Cloth, $5.00 net; Sheep or Half Morocco, $6.50 net.
~WITH 400 TEXT-CUTS, MANY IN COLORS, AND 7 COLORED PLATES~
In this work the practical application of pathologic facts to clinical medicine is considered more fully than is customary in works on pathology. While the subject of pathology is treated in the broadest way consistent with the size of the book, an effort has been made to present the subject from the point of view of the clinician. In the second part of the work the pathology of individual organs and tissues is treated systematically and quite fully under subheadings that clearly indicate the subject-matter to be found on each page. In this edition the section dealing with General Pathology has been most extensively revised, several of the important chapters having been practically rewritten.
~The Lancet, London~
"This volume is intended to present the subject of pathology in as practical a form as possible, and more especially from the point of view of the 'clinical pathologist.' These objects have been faithfully carried out, and a valuable text-book is the result. We can most favorably recommend it to our readers as a thoroughly practical work on clinical pathology."
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Stiles' Nutritional Physiology
~Nutritional Physiology.~ By PERCY GOLDTHWAIT STILES, Assistant Professor of Physiology at Simmons College, Boston. 12mo of 295 pages, illustrated. Cloth, $1.25 net.
~ILLUSTRATED~
This new work expresses the most advanced views on this important subject. It discusses in a concise way the processes of digestion and metabolism. The key-word of the book throughout is "energy"--its source and its conservation.
"It is remarkable for the fineness of its diction and for its clear presentation of the subject, relieved here and there by a quaintly humorous turn of phrase that is altogether delightful."--_Colin C. Stewart, Ph. D., Dartmouth College._
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Jordan's General Bacteriology
~A Text-Book of General Bacteriology.~ By EDWIN O. JORDAN, PH.D., Professor of Bacteriology in the University of Chicago and in Rush Medical College. Octavo of 623 pages, illustrated. Cloth, $3.00 net.
~NEW (3d) EDITION~
Professor Jordan's work embraces the entire field of bacteriology, the non-pathogenic as well as the pathogenic bacteria being considered, giving greater emphasis, of course, to the latter. There are extensive chapters on methods of studying bacteria, including staining, biochemical tests, cultures, etc.; on the development and composition of bacteria; on enzymes and fermentation-products; on the bacterial production of pigment, acid and alkali; and on ptomaines and toxins. Especially complete is the presentation of the serum treatment of gonorrhea, diphtheria, dysentery, and tetanus. The relation of bovine to human tuberculosis and the ocular tuberculin reaction receive extensive consideration.
This work will also appeal to academic and scientific students. It contains chapters on the bacteriology of plants, milk and milk-products, air, agriculture, water, food preservatives, the processes of leather tanning, tobacco curing, and vinegar making; the relation of bacteriology to household administration and to sanitary engineering, etc.
~Prof. Severance Burrage~, _Associate Professor of Sanitary Science, Purdue University._
"I am much impressed with the completeness and accuracy of the book. It certainly covers the ground more completely than any other American book that I have seen."
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Buchanan's Veterinary Bacteriology
~Veterinary Bacteriology.~ By ROBERT E. BUCHANAN, PH.D., Professor of Bacteriology in the Iowa State College of Agriculture and Mechanic Arts. Octavo, 516 pages, 214 illustrations. Cloth, $3.00 net.
~THE BEST PUBLISHED~
Professor Buchanan discusses thoroughly all bacteria causing diseases of the domestic animals. He goes minutely into the consideration of immunity, opsonic index, reproduction, sterilization, antiseptics, biochemic tests, culture-media, isolation of cultures, the manufacture of the various toxins, antitoxins, tuberculins, and vaccines that have proved of diagnostic or therapeutic value. Then, in addition to bacteria and protozoa proper, he considers molds, mildews, smuts, rusts, toadstools, puff-balls, and the other fungi pathogenic for animals.
~B. F. Kaupp, D. V. S.~, _State Agricultural College, Fort Collins._
"It is the best in print on the subject. What pleases me most is that it contains all the late results of research. It fills a long felt want."
Heisler's Embryology
~A Text-Book of Embryology.~ By JOHN C. HEISLER, M.D., Professor of Anatomy in the Medico-Chirurgical College, Philadelphia. Octavo volume of 435 pages, with 212 illustrations, 32 of them in colors. Cloth, $3.00 net.
~THIRD EDITION--WITH 212 ILLUSTRATIONS, 32 IN COLORS~
This edition represents all the advances recently made in the science of embryology. Many portions have been entirely rewritten, and a great deal of new and important matter added. A number of new illustrations have also been introduced and these will prove very valuable. Heisler's Embryology has become a standard work.
~G. Carl Huber, M.D.~, _Professor of Embryology at the Wistar Institute, University of Pennsylania._
"I find this edition of 'A Text-Book of Embryology,' by Dr. Heisler, an improvement on the former one. The figures added increase greatly the value of the work. I am again recommending it to our students."
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Boehm, Davidoff, _and_ Huber's Histology
~A Text-Book of Human Histology.~ Including Microscopic Technic. By DR. A. A. BOEHM and DR. M. VON DAVIDOFF, of Munich, and G. CARL HUBER, M.D., Professor of Embryology at the Wistar Institute, University of Pennsylvania. Handsome octavo of 528 pages, with 361 beautiful original illustrations. Flexible cloth, $3.50 net.
~SECOND EDITION, ENLARGED~
The work of Drs. Boehm and Davidoff is well known in the German edition, and has been considered one of the most practically useful books on the subject of Human Histology. This second edition has been in great part rewritten and very much enlarged by Dr. Huber, who has also added over one hundred original illustrations. Dr. Huber's extensive additions have rendered the work the most complete students' text-book on Histology in existence.
~Boston Medical and Surgical Journal~
"Is unquestionably a text-book of the first rank, having been carefully written by thorough masters of the subject, and in certain directions it is much superior to any other histological manual."
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Wells' Chemical Pathology
~Chemical Pathology.~--Being a Discussion of General Pathology from the Standpoint of the Chemical Processes Involved. By H. GIDEON WELLS, PH.D., M.D., Assistant Professor of Pathology in the University of Chicago. Octavo of 616 pages. Cloth, $3.25 net.
~JUST READY--NEW (2d) EDITION~
Dr. Wells' work is written for the physician, for those engaged in research in pathology and physiologic chemistry, and for the medical student. In the introductory chapter are discussed the chemistry and physics of the animal cell, giving the essential facts of ionization, diffusion, osmotic pressure, etc., and the relation of these facts to cellular activities. Special chapters are devoted to _Diabetes_ and to _Uric-acid Metabolism and Gout_.
~Wm. H. Welch, M.D.~ _Professor of Pathology, Johns Hopkins University._
"The work fills a real need in the English literature of a very important subject, and I shall be glad to recommend it to my students."
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Lusk's Elements of Nutrition
~Elements of the Science of Nutrition.~ By GRAHAM LUSK, PH.D., Professor of Physiology at Cornell Medical School. Octavo volume of 302 pages. Cloth, $3.00 net.
~THE NEW (2d) EDITION--TRANSLATED INTO GERMAN~
Prof. Lusk presents the scientific foundations upon which rests our knowledge of nutrition and metabolism, both in health and in disease. There are special chapters on the metabolism of diabetes and fever, and on purin metabolism. The work will also prove valuable to students of _animal dietetics_ at agricultural stations.
~Lewellys F. Barker, M. D.~ _Professor of the Principles and Practice of Medicine, Johns Hopkins University._
"I shall recommend it highly to my students. It is a comfort to have such a discussion of the subject in English."
Daugherty's Economic Zoology
~Economic Zoology.~ By L. S. DAUGHERTY, M. S., PH. D., Professor of Zoology, State Normal School, Kirksville, Mo., and M. C. DAUGHERTY, author with Jackson of "Agriculture Through the Laboratory and School Garden." Part I: _Field and Laboratory Guide_. 12mo of 237 pages, interleaved. Cloth, $1.25 net. Part II: _Principles._ 12mo of 406 pages, illustrated. Cloth, $2.00 net.
~ILLUSTRATED~
There is no other book just like this. Not only does it give the salient facts of structural zoology and the development of the various branches of animals, but also the natural history--the _life and habits_--thus showing the interrelations of structure, habit, and environment. In a word, it gives the principles of zoology and _their actual application_. The economic phase is emphasized.