The Elements of Bacteriological Technique Part 9

_Warm Stage_ (Fig. 52).--This is a flat metal case containing a system of tubes through the interior of which water of any required temperature can be circulated. It is made to clamp on to the stage of the microscope by the screws _A A'_, and is perforated with a large hole coinciding with the optical axis of the microscope; a short tube B, projecting from one end of the warm stage permits water of the desired temperature to be conducted from a reservoir through a length of rubber tubing to the interior of the stage and a similar tube at the other end _B'_ of the stage allows exit to the waste water. By raising the temperature of hanging-drop preparations, etc., placed upon it, above that of the surrounding atmosphere, the warm stage renders possible exact observations on spore germination, hanging-drop cultivations, etc.

[Ill.u.s.tration: FIG. 52.--Warm stage.]

A better form is the electrical hot stage designed by Lorrain Smith;[2]

it requires the addition of a lamp resistance and sliding rheostat, also a delicate ammeter reading to .01 of an ampere. It consists of a wooden frame supporting a flat gla.s.s bulb with a long neck bent upward at an obtuse angle (Fig. 53). The bulb is filled with liquid paraffin, which rises in the open neck when expanded by heat. The neck also accommodates the thermometer. Two coils of manganin wire run in the paraffin at opposite sides of the bulb (outside the field of vision), coupled to bra.s.s terminals on the wooden frame by platinum wire fused into the gla.s.s. The resistance of the two coils in series is about 10 ohms. A current of 2-1/2 amperes is needed, and is conducted to the coils in the stage through the rheostat. With the help of the ammeter any desired temperature can be obtained and maintained, up to about 200 C. If immersion oil contact is made between the top lens of the condenser and the lower surface of the bulb, this stage works very well indeed with the 1/12-inch oil immersion lens.

[Ill.u.s.tration: FIG. 53.--Lorrain Smith's warm stage.]

_Dark Ground or Paraboloid Condenser._--This is an immersion substage condenser of high aperture by means of which unstained objects such as bacteria can be shown as bright white particles upon a dense black background. The central rays of light are blocked out by means of an opaque stop while the peripheral rays are reflected from the paraboloidal sides of the condenser and refracted by the object viewed.

To obtain the best results with this type of condenser a powerful illuminant--such as a small arc lamp or an incandescent gas lamp--is needed, together with picked slides of a certain thickness (specified for the particular make of condenser but generally 1 mm.) and specially thin cover-gla.s.ses (not more than 0.17 mm.) The objective must not have a higher NA than 1.0, consequently immersion lenses must be fitted with an internal stop to cut down the aperture.

_Micrometer._--Some form of micrometer for the purpose of measuring bacteria and other objects is also essential. Details of those in general use will be found in the following pages.

[Ill.u.s.tration: FIG. 54--Diamond Object marker.]

_Object Marker_ (Fig. 54).--This is an exceedingly useful piece of apparatus. Made in the form of an objective, the lenses are replaced by a diamond point, set slightly out of the centre, which can be rotated by means of a milled plate. Screwed on to the nosepiece in place of the objective, rotation of the diamond point will rule a small circle on the object slide to permanently record the position of an interesting portion of the specimen. The diamond is mounted on a spring which regulates the pressure, and the size of the circle can be adjusted by means of a lateral screw.

METHODS OF MICROMETRY.

The unit of length as applied to the measurement of microscopical objects is the one-thousandth part of a millimetre (0.001 mm.), denominated a _micron_ (sometimes, and erroneously, referred to as a micro-millimetre), and indicated in writing by the Greek letter . Of the many methods in use for the measurement of bacteria, three only will be here described, viz.:

(a) By means of the Camera Lucida.

(b) By means of the ocular or Eyepiece Micrometer.

(c) By means of the Filar Micrometer (Ramsden's micrometer eyepiece).

For each of these methods a ~stage micrometer~ is necessary. This is a 3 by 1 inch gla.s.s slip having engraved on it a scale divided to hundredths of a millimetre (0.01 mm.), every tenth line being made longer than the intervening ones, to facilitate counting; and from these engraved lines the measurement in every case is evaluated. A cover-gla.s.s is cemented over the scale to protect it from injury.

[Ill.u.s.tration: FIG. 55.--Camera lucida, Abbe pattern.]

(a) By means of the Camera Lucida.

1. Attach a camera lucida (of the Wollaston, Beale, or Abbe pattern) (Fig. 55) to the eyepiece of the microscope.

2. Adjust the micrometer on the stage of the microscope and accurately focus the divisions.

3. Project the scale of the stage micrometer on to a piece of paper and with pen or pencil sketch in the magnified image, each division of which corresponds to 10. Mark on the paper the optical combination (ocular objective and tube length) employed to produce this particular magnification.

4. Repeat this procedure for each of the possible combinations of oculars and objectives fitted to the microscope supplied, and carefully preserve the scales thus obtained.

To measure an object by this method simply project the image on to the scale corresponding to the particular optical combination in use at the moment. Read off the number of divisions it occupies and express them as _micra_.

In place of preserving a scale for each optical combination, the object to be measured and the micrometer scale may be projected and sketched, in turn, on the same piece of paper, taking particular care that the centre of the eyepiece is 25 cm. from the paper on which the divisions are drawn.

[Ill.u.s.tration: FIG. 56.--Eyepiece micrometer, ordinary.]

[Ill.u.s.tration: FIG. 57.--Eyepiece micrometer, net.]

(b) By means of the Eyepiece Micrometer.

The ~eyepiece micrometer~ is a circular gla.s.s disc having engraved on it a scale divided to tenths of a millimetre (0.1 mm.) (Fig. 56), or the entire surface ruled in 0.1 mm. squares (the net micrometer) (Fig. 57).

It can be fitted inside the mount of any ocular just above the aperture of the diaphragm and must be adjusted exactly in the focus of the eye lens.

Some makers mount the gla.s.s disc together with a circular cover-gla.s.s in such a way that when placed in position in any Huyghenian eyepiece of their own manufacture, the scale is exactly in focus for normal vision.

Special eyepieces are also obtainable having a sledging adjustment to the eye lens for focussing the micrometer.

The value of one division of the micrometer scale must first be ascertained for each optical combination by the aid of the stage micrometer, thus:

1. Insert the eyepiece micrometer inside the ocular and adjust the stage micrometer on the stage of the microscope.

2. Focus the scale of the stage micrometer accurately; the lines will appear to be immediately below those of the eyepiece micrometer. Make the lines on the two micrometers parallel by rotating the ocular.

3. Make two of the lines on the ocular micrometer coincide with those bounding one division of the stage micrometer; this is effected by increasing or diminis.h.i.+ng the tube length; and note the number of included divisions.

4. Calculate the value of each division of the eyepiece micrometer in terms of , by means of the following formula:

x = 10 y.

Where x = the number of included divisions of the eyepiece micrometer.

y = the number of included divisions of the stage micrometer.

5. Note the optical combination employed in this experiment and record it with the calculated micrometer value.

Repeat this process for each of the other combinations. Carefully record the results.

To measure an object by this method read off the number of divisions of the eyepiece micrometer it occupies and express the result in _micra_ by a reference to the standard value for the particular optical combination employed.

Zeiss prepares a compensating eyepiece micrometer for use with his apochromatic objectives, the divisions of which are so computed that (with a tube length of 160 mm.) the value of each is equivalent to as many _micra_ as there are millimetres in the focal length of the objective employed.

_Wright's Eikonometer_ is really a modification of the eyepiece micrometer for rapidly measuring microscopical objects by direct inspection, having previously determined the magnifying power of the particular optical combination employed. It is a small piece of apparatus resembling an eyepiece, with a sliding eye lens, which can be accurately focussed on a micrometer scale fixed within the instrument.

When placed over the microscope ocular the divisions of this scale measure the actual size of the virtual image in millimetres.

In order to use this instrument for direct measurement, it is first necessary to determine the magnifying power of each combination of ocular, tube length and objective.

Place a stage micrometer divided into hundredths of a millimetre on the microscope stage and focus accurately.

Rest the eikonometer on the eyepiece. Observation through the eikonometer shows its micrometer scale superposed on the image of the stage micrometer.

Rotate the eikonometer until the lines on the two scales are parallel, and make the various adjustments to ensure that two lines on the eikonometer scale coincide with two lines on the stage micrometer.

For the sake of ill.u.s.tration it may be a.s.sumed that five of the divisions on the stage micrometer accurately fill one of the divisions of the eikonometer scale; this indicates a magnifying power of 500 as the constant for that particular optical combination, and a record should be made of the fact.

The magnification constants of the various other optical combinations should be similarly made and recorded.

To measure any object subsequently it should be first focussed carefully in the ordinary way.