(d) _Serum Agar._--

1. Melt three tubes of nutrient agar, label them 1, 2, and 3, and place them, with three tubes of sterile fluid serum, also labelled 1a, 2a, and 3a, in a water-bath regulated at 45 C.; allow sufficient time to elapse for the temperature of the contents of each tube to reach that of the water-bath.

2. Take serum tube No. 1a and agar tube No. 1. Flame the plugs and remove them from the tubes (retaining the plug of the agar tube in the hand); flame the mouths of the tubes, pour the serum into the tube of liquefied agar and replace the plug of the agar tube.

3. Mix thoroughly and pour plate No. 1 _secundum artem_.

4. Treat the remaining tube of agar and serum in a similar fashion, and pour plates Nos. 2 and 3.

5. Dry the serum agar plates in the incubator running at 60 C. for one hour (see page 232).

6. Inoculate the plates in series as described for gelatine surface plates (page 231).

(e) _Blood Agar, Human._--

1. Melt a tube of sterile agar and pour it into a sterile plate; let it set.

2. Collect a few drops of human blood, under all aseptic conditions, in a sterile capillary teat pipette.

3. Raise the cover of the Petri dish very slightly, insert the extremity of the capillary pipette, and deposit the blood on the centre of the agar surface. Close the dish.

4. Charge a platinum loop with a small quant.i.ty of the inoculum. Raise the cover of the plate, introduce the loop, mix its contents with the drop of blood, remove the loop, close the dish and sterilise the loop.

5. Finally smear the mixture over the surface of the agar with a sterilised L-shaped rod.

6. Label and incubate.

(If considered necessary, two, three, or more similar plates may be inoculated in series.)

(f) _Blood Agar, Animal._--

When preparing citrated blood agar (page 171) it is always advisable to pour several blood agar tubes into plates, which can be stored in the ice chest ready for use at any moment for surface plate cultures.

(g) Hanging-drop or block culture, (_vide_ page 233).

~3. Serial Cultivations.~--These are usually made upon agar or blood-serum, although gelatine may also be used.

The method is as follows:

1. Take at least four "slanted" tubes of media and number them consecutively.

2. Flame all the plugs and see that each can be readily removed.

3. Charge the platinum loop with a small quant.i.ty of the inoculum, observing the usual routine, and plant tube No. 1, smearing thoroughly all over the surface. If any water of condensation has collected at the bottom of the tube, use this as a diluent before smearing the contents of the loop over the surface of the medium.

4. Without sterilising or recharging the loop, inoculate tube No. 2, by making three parallel streaks from end to end of the slanted surface.

5. Plant the remainder of the tubes in the series as "smears" like tube No. 1.

6. Label with distinctive name or number, and date; incubate.

The growth that ensues in the first two or three tubes of the series will probably be so crowded as to be useless. Toward the end of the series, however, discrete colonies will be found, each of which can be transferred to a fresh tube of nutrient medium without risk of contamination from the neighbouring colonies.

~"Working" up Plates.~--

Having succeeded in obtaining a plate (or tube cultivation) in which the colonies are well grown and sufficiently separated from each other, the process of "working up," "p.r.i.c.king out," or "fishing" the colonies in order to obtain subcultures in a state of purity from each of the different bacteria present must now be proceeded with.

Occasionally it happens that this is quite a simple matter. For example, the original mixed cultivation when examined microscopically was found to contain a Gram positive micrococcus, a Gram positive straight bacillus and a Gram negative short bacillus. The third gelatine plate prepared from this mixture, on inspection after four day's incubation, showed twenty-five colonies--seven moist yellow colonies, each sinking into a shallow pit of liquefied gelatine, fourteen flat irridescent filmy colonies, and four raised white slimy colonies. A film preparation (stained Gram) from each variety examined microscopically showed that the yellow liquefying colony was composed of Gram positive micrococci; the flat colony of Gram positive bacilli and the white colony of gram negative bacilli. One of each of these varieties of colonies would be transferred by means of the sterilised loop to a fresh gelatine culture tube, and after incubation the growth in each subculture would correspond culturally and microscopically with that of the plate colony from which it was derived,--the object aimed at would therefore be achieved.

Usually, however, the colonies cannot be thus readily differentiated, and unless they are "worked up" in an orderly and systematic manner much labour will be vainly expended and valuable time wasted. The following method minimises the difficulties involved.

(A) Inspection.

a. Without opening the plate carefully study the various colonies with the naked eye, with the a.s.sistance of a watchmaker's lens or by inverting the plate on the stage of the microscope and viewing with the 1-inch objective through the bottom of the plate and the layer of medium.

b. If gross differences can be detected mark a small circle on the bottom of the plate around the site of each of the selected colonies, with the grease pencil.

c. If no obvious differences can be made out choose nine colonies haphazard and indicate their positions by pencil marks on the bottom of the plate.

(B) Fishing Colonies.--

a. Take a sterile Petri dish and invert it upon the laboratory bench.

Rule two parallel lines on the bottom of the dish with a grease pencil, and two more parallel lines at right angles to the first pair--so dividing the area of the dish into nine portions. Number the top right-hand portion 1, and the central bottom portion 8 (Fig. 139).

Revert the dish. The numbers 1 and 8 can be readily recognised through the gla.s.s and by their positions enable any of the other divisions to be localised by number. This is the stock dish.

b. Slightly raise the cover of the dish, and with a sterile teat-pipette deposit a small drop of sterile water in the centre of each of the nine divisions.

c. With the sterilised platinum spatula raise one of the marked colonies from the "plate 3" and transfer it to the first division in the ruled plate and emulsify it in the drop of water awaiting it. Repeat this process with the remaining colonies, emulsifying a separate colony in each drop of water.

(C) Preliminary Differentiation of Bacteria.--

a. Prepare a cover-slip film preparation from each drop of emulsion in the "stock dish" and number to correspond to the division from which it was taken. Stain by Gram's method.

b. Examine microscopically, using the oil immersion lens and note the numbers of those cover-slips which morphologically and by Gram results appear to be composed of different species of bacteria.

[Ill.u.s.tration: FIG. 139.--Diagram for stock plate.]

(D) Preparing Isolation Subcultures.--

a. Inoculate an agar slope and a broth tube from the emulsion in the stock dish corresponding to each of these specially selected numbers.

b. Ascertain whether the cover-slips from the nine emulsions in the stock dish include all the varieties represented in the cover-slip film preparation made from the original mixture before plating.