The Elements of Bacteriological Technique Part 62

This method of pouring what may be termed "whole" plates (since colonies may appear both on the surface and in the depths of the medium) is essential to the accurate study of the formation of colonies under various conditions, but when the main object of the separation of the bacteria is to obtain subcultivations from a number of individual bacteria, "surface" plates must be prepared, since here colony formation is restricted to the surface of the medium. The method adopted varies slightly according to whether the medium employed is gelatine or agar, or one of the derivatives or variants of the latter.

(a) ~Gelatine Surface Plates.~--

1. Liquefy three tubes of nutrient gelatine.

2. Pour each tube into a separate Petri dish and allow it to solidify.

Then turn each plate and its cover upside down.

[Ill.u.s.tration: FIG. 126.--Surface plate spreader.]

3. When quite cold raise the bottom of plate 1, revert it and deposit a drop of the inoculum (whether a fluid culture or an emulsion from solid culture) upon the surface of the gelatine with a platinum loop--close to one side of the plate; replace the bottom half of the Petri dish in its cover.

4. Take a piece of thin gla.s.s rod, stout platinum wire or best of all a piece of aluminium wire (say 2 mm. diameter) about 28 cm. long. Bend the terminal 4 cm. at right angles to the remainder, making an L-shaped rod (Fig. 126). Sterilise the short arm and adjacent portion of the long arm, in the Bunsen flame, and allow it to cool.

5. Now raise the bottom of the Petri dish in the left hand, leaving the cover on the laboratory bench, and holding it vertically, smear the drop of inoculum all over the surface of the gelatine with the short arm of the spreader by a rotatory motion, (Fig. 127). Replace the dish in its cover.

6. Raise the bottom of plate 2 and rub the infected spreader all over the surface of the gelatine--then go on in like manner to the third plate in the series.

7. Sterilise the spreader.

8. Label and incubate the plates.

[Ill.u.s.tration: FIG. 127.--Spreading surface plate.]

After incubation, plate No. 1 will probably yield an enormous number of colonies; plate 2 will show fewer colonies, since only those bacteria adhering to the rod after rubbing over plate 1 would be deposited on its surface, and by the time the rod reached plate 3 but very few organisms should remain upon it. So that the third plate as a rule will only show a very few scattered colonies, eminently suitable for detailed study.

(b) ~Agar Surface Plates.~--

1. Liquefy three tubes of nutrient agar--nutrose agar or the like.

2. Pour each tube into a separate Petri dish and allow it to solidify.

3. When quite solid invert each dish, raise the bottom half and rest it obliquely on its inverted cover (Fig. 128) and place it in this position in an incubator at 60 C. for forty-five minutes (or in an incubator at 42 C. for two hours). This evaporates the water of condensation and gives the medium a firm, dry surface.

4. On removing the plates from the incubator close each dish and place it--still upside down--on the laboratory bench.

[Ill.u.s.tration: FIG. 128.--Drying surface plate of agar.]

5. Inoculate the plates in series of three, as described for gelatine surface plates 3-8.

Hanging-drop Cultivation.

~Apparatus Required.~--

Hanging-drop slides.

Cover-slips.

Section rack (Fig. 75).

Blotting paper.

Bell gla.s.s to cover slides.

Original culture.

Tubes of broth, or liquefied gelatine or agar.

Forceps.

Platinum loop.

Bunsen burner.

Grease pencil.

Sterile vaseline.

Lysol.

(a) ~Fluid Media.~--

1. Prepare first and second dilutions of the inoculum as directed for plate cultivations (_vide_ pages 228-229, sections 4 to 6), subst.i.tuting tubes of nutrient broth for the liquefied gelatine.

2. Sterilise a hanging-drop slide by washing thoroughly in water and drying, then plunging it into a beaker of absolute alcohol, draining off the greater part of the spirit, grasping the slide in a pair of forceps, and burning off the remainder of the alcohol in the flame.

3. Place the hanging-drop slide on a piece of blotting paper moistened with 2 per cent. lysol solution and cover it with a small bell gla.s.s that has been rinsed out with the same solution and _not dried_.

4. Raise the bell gla.s.s slightly and smear sterile vaseline around the rim of the metal cell by means of a sterile spatula of stout platinum wire.

5. Remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the alcohol; again raise the bell gla.s.s and place the sterile cover-slip on the blotting paper by the side of the hanging-drop slide.

6. Remove a drop of the broth from the second dilution tube with a large platinum loop; raise the bell gla.s.s and deposit the drop on the centre of the cover-slip. Sterilise the loop.

7. Raise the bell gla.s.s sufficiently to allow of the cover-slip being grasped with forceps, inverted, and adjusted over the cell of the hanging-drop slide. Remove the bell gla.s.s altogether and press the cover-slip firmly on to the cell.

8. Either incubate and examine at definite intervals, or observe continuously with the microscope, using a warm stage if necessary (Fig.

53).

(b) ~Solid Media.~--Observing precisely similar technique, a few drops of liquefied gelatine or agar from the second dilution tube may be run over the surface of the sterile cover-slip and a hanging-drop plate cultivation thereby prepared.

This method is extremely useful in connection with the study of yeasts, when the circular cell on the hanging-drop slide should be replaced by a rectangular cell some 38 by 19 mm., and the gelatine spread over a cover-slip of similar size. After sealing down the preparation, the upper surface of the cover-slip may be ruled into squares by the aid of the grease pencil or a writing diamond and numbered to facilitate the subsequent identification of the colonies which are observed to develop from solitary germs.

~Hanging-block Culture~ (Hill).--

_Apparatus required_: As for hanging-drop cultivation with the addition of a scalpel.

Carry out the method as far as possible under cover of a bell gla.s.s, to avoid aerial contamination.

1. Liquefy a tube of nutrient agar (or gelatine) and pour into a Petri dish to the depth of about 4 mm. and allow to set.

2. With a sharp scalpel cut out a block some 8 mm. square, from the entire thickness of the agar layer.

3. Raise the agar block on the blade of the scalpel and transfer it, under side down, to the centre of a sterile slide.