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 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 ident.i.ty 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 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 quant.i.ty (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 a.n.a.lytical schema--whilst the full differential details are set out in tabular form.

a.n.a.lYTICAL 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 s.p.a.ces.

+-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ | | M | D | L | G | M | L | S | R | D | |A = acid reaction | o | e | ae | a | a | a | a | a | e | |G = gas formation | t | x | v | l | l | c | c | f | t | | | i | t | u | a | t | t | c | f | r | | | l | r | l | c | o | o | h | i | i | | | i | o | o | t | s | s | a | n | n | | | t | s | s | o | e | e | r | o | | | | y | e | e | s | | | o | s | | | | | | | 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.