Chapter IV: Appendix: 492 (2)

This is a clock of American type in which the face is surrounded by a metal plate having a series of 60 holes at equal distances apart, corresponding to the minutes on the dial. This plate is connected with one of the poles of a dry battery, the other pole of which is connected to the metal case of the clock for the purpose of actuating an ordinary magnet alarm bell. In the centre of each of the holes in the plate a metal rod is fixed, which then passes through an insulating ring and projects inside the clock face, where it makes contact with the hour hand. The clock is mounted on a heavy base, with a key-board containing 20 numbered plugs. If one of the plugs is inserted in a hole in the plate it makes contact with the rod, and when the hour hand of the clock touches the other end the circuit is completed and the bell starts ringing. The period of this friction contact is approximately 20 seconds. The clock can therefore be used for electrically noting the periods of time from one minute by multiples of one minute up to one hour.

~Filtration.~--(a) _Cotton-wool Filter._--Practically the only method in use in the laboratory for the sterilisation of air or of a gas is by filtration through dry cotton-wool or glass-wool, the fibres of which entangle the micro-organisms and prevent their passage.

Perhaps the best example of such a filter is the cotton-wool plug which closes the mouth of a culture tube. Not only does ordinary diffusion take place through it, but if a tube plugged in the usual manner with cotton-wool is removed from the hot incubator, the temperature of the contained air rapidly falls to that of the laboratory, and a partial vacuum is formed; air passes into the tube, through the cotton-wool plug, to restore the equilibrium, and, so long as the plug remains dry, in a germ-free condition. If, however, the plug becomes moist, either by absorption from the atmosphere, or from liquids coming into contact with it, micro-organisms (especially the mould fungi) commence to multiply, and the long thread forms rapidly penetrate the substance of the plug, and gain access to and contaminate the interior of the tube.

METHOD.--

If it is desired to sterilise gases before admission to a vessel containing a pure cultivation of a micro-organism, as, for instance, when forcing a current of oxygen over or through a broth cultivation of the diphtheria bacillus, this can be readily effected as follows:

1. Take a length of glass tubing of, say, 1.5 cm. diameter, in the centre of which a bulb has been blown, fill the bulb with dry cotton-wool (Fig. 32), wrap a layer of cotton-wool around each end of the tube, and secure in position with a turn of thin copper wire or string; then sterilise the piece of apparatus in the hot-air oven.

2. Prepare the cultivation in a Ruffer or Woodhead flask (Fig. 33) the inlet tube of which has its free extremity enveloped in a layer of cotton-wool, secured by thread or wire, whilst the exit tube is plugged in the usual manner.

3. Sterilise a short length of rubber tubing by boiling. Transfer it from the boiling water to a beaker of absolute alcohol.

4. When all is ready remove the rubber tube from the alcohol by means of a pair of forceps, drain it thoroughly, and pass through the flame of a Bunsen burner to burn off the last traces of alcohol.

5. Remove the cotton-wool wraps from the entry tube of the flask and from one end of the filter tube and rapidly couple them up by means of the sterile rubber tubing.

6. Connect the other end of the bulb tube with the delivery tube from the gas reservoir.

The gas in its passage through the dry sterile cotton-wool in the bulb of the filter tube will be freed from any contained micro-organisms and will enter the flask in a sterile condition.

(b) _Porcelain Filter._--The sterilisation of liquids by filtration is effected by passing them through a cylindrical vessel, closed at one end like a test-tube, and made either of porous "biscuit" porcelain, hard-burnt and unglazed (Chamberland system), or of Kieselguhr, a fine diatomaceous earth (Berkefeld system), and termed a "bougie" or "candle" (Fig. 34).

NOTE.--In selecting candles for use in the laboratory avoid
those with metal fittings, since during sterilisation cracks
develop at the junction of the metal and the siliceous
material owing to the unequal expansion.

In this method the bacteria are retained in the pores of the filter while the liquid passes through in a germ-free condition.

It is obvious that to be effective the pores of the filter must be extremely minute, and therefore the rate of filtration will usually be slow. Chamberland filter candles possess finer channels than Berkefeld candles and consequently filter much more slowly. To overcome this disadvantage, either aspiration or pressure, or a combination of these two forces, may be employed to hasten the process.

Doultons white porcelain filters it may be noted are as efficient as the Chamberland candles and filter rather more rapidly.

_Apparatus Required._--

1. Separatory funnel containing the unfiltered fluid.

2. Sterile filter candle (Fig. 34), the open end fitted with a rubber stopper (Fig. 34, a) perforated to receive the delivery tube of the separatory funnel, and its neck passed through a large rubber washer (Fig. 34, b) which fits the mouth of the filter flask.

3. Sterile filter flask of suitable size, for the reception of the filtered fluid, its mouth closed by a cotton-wool plug.

4. Water injector Sprengel (see Fig. 38, c) pump, or Geryk's pump (an air pump on the hydraulic principle, sealed by means of low vapor-tension oil, Fig. 35).

If this latter is employed, a Wulff's bottle, fitted as a wash-bottle and containing sulphuric acid, must be interposed between the filter flask and the pump, in order to prevent moist air reaching the oil in the pump.

5. Air filter (_vide_ page 40) sterilised.

6. Pressure tubing.

7. Screw clamps (Fig. 36).

METHOD.--

1. Couple the exhaust pipe of the suction pump with the lateral tube of the filter flask (first removing the cotton-wool plug from this latter), by means of pressure tubing, interposing, if necessary, the wash-bottle of sulphuric acid.

2. Remove the cotton-wool plug from the neck of the filter flask and adjust the porcelain candle in its place.

3. Attach the nozzle of the separatory funnel to the filter candle by means of the perforated rubber stopper (Fig. 37).

4. Open the tap of the funnel, and exhaust the air from the filter flask and wash-bottle; maintain the vacuum until the filtration is complete.

5. When the filtration is completed close the tap of the funnel; adjust a screw clamp to the pressure tubing attached to the lateral branch of the filter flask; screw it up tightly, and disconnect the acid wash-bottle.

6. Attach the air filter to the open end of the pressure tubing; open the screw clamp gradually, and allow filtered air to enter the flask, to abolish the negative pressure.

7. Detach the rubber tubing from the lateral branch of the flask, flame the end of the branch in the Bunsen, and plug its orifice with sterile cotton-wool.

8. Remove the filter candle from the mouth of the flask, flame the mouth, and plug the neck with sterile cotton-wool.

9. Disinfect the filter candle and separatory funnel by boiling.

If it is found necessary to employ pressure in addition to or in place of suction, insert a perforated rubber stopper into the mouth of the separatory funnel and secure in position with copper wire; next fit a piece of glass tubing through the stopper, and connect the external orifice with an air-pressure pump of some kind (an ordinary foot pump such as is employed for inflating bicycle tyres is one of the most generally useful, for this purpose) or with a cylinder of compressed air or other gas.

In order to filter a large bulk of fluid very rapidly it is necessary to use a higher pressure than glass would stand, and in these cases the metal receptacle designed by Pakes (Fig. 38, a), to hold the filter candle itself as well as the fluid to be filtered, should be employed. (A vacuum must also be maintained in the filter flask, by means of an exhaust pump, during the entire process.)

This piece of apparatus consists of a brass cylinder, capacity 2500 c.c., with two shoulders; and an opening in the neck at each end, provided with screw threads.

A nut carrying a pressure gauge fits into the top screw; and into the bottom is fitted a brass cylinder carrying the filter candle and prolonged downwards into a delivery tube. Leakage is prevented by means of rubber washers.

Into the top shoulder a tube is inserted, bent at right angles and provided with a tap. All the brass-work is tinned inside (Fig. 38, a). In use the reservoir is generally mounted on a tripod stand.

~To Sterilise.~--

1. Insert the filter candle into its cylinder and screw this loosely on.

2. Wrap a layer of cotton-wool around the delivery tube and fasten in position.

3. Remove the nut carrying the pressure gauge and plug the neck with cotton-wool.

4. Heat the whole apparatus in the autoclave at 120 deg. C. for twenty minutes.

METHOD.--

1. Remove the apparatus from the autoclave, and allow it to cool.

2. Screw home the box carrying the bougie.

3. Set the apparatus up in position, with its delivery tube (from which the cotton-wool wrapping has been removed) passing through a perforated rubber stopper in the neck of a filter flask.

4. Fill the fluid to be filtered into the cylinder and screw on the nut carrying the pressure gauge. (This nut should be immersed in boiling water for a few minutes previous to screwing on, in order to sterilise it.)

5. Connect the horizontal arm of the entry tube with a cylinder of compressed oxygen (or carbon dioxide, Fig. 38, b), by means of pressure tubing.

6. Connect the lateral arm of the filter flask with the exhaust pump (Fig. 38, c) and start the latter working.

7. Open the tap of the gas cylinder; then open the tap on the entry tube of the filter cylinder and raise the pressure in its interior until the desired point is recorded on the manometer. Maintain this pressure, usually one or one and a half atmospheres, until filtration is completed, by regulating the tap on the entry tube.

Some forms of filter candle are made with the open end contracted into a delivery nozzle, which is glazed. In this case the apparatus is fitted up in a slightly different manner; the fluid to be filtered is contained in an open cylinder into which the candle is plunged, while its delivery nozzle is connected with the filter flask by means of a piece of flexible pressure tubing (previously sterilised by boiling), as in figure 39.

IV. THE MICROSCOPE.

The essentials of a microscope for bacteriological work may be briefly summed up as follows:

The instrument, of the monocular type, must be of good workmanship and well finished, rigid, firm, and free from vibration, not only when upright, but also when inclined to an angle or in the horizontal position. The various joints and movements must work smoothly and precisely, equally free from the defects of "loss of time" and "slipping." All screws, etc., should conform to the Royal Microscopical Society's standard. It must also be provided with good lenses and a sufficiently large stage. The details of its component parts, to which attention must be specially directed, are as follows:

~1. The Base or Foot~ (Fig. 40, a).--Two elementary forms--the tripod (Fig. 41, a) and the vertical column set into a plate known as the "horse-shoe" (Fig. 41, b)--serve as the patterns for countless modifications in shape and size of this portion of the stand. The chief desiderata--stability and ease of manipulation--are attained in the first by means of the "spread" of the three feet, which are usually shod with cork; in the second, by the dead weight of the foot-plate. The tripod is mechanically the more correct form, and for practical use is much to be preferred. Its chief rival, the Jackson foot (Fig. 41, c), is based upon the same principle, and on the score of appearance has much to recommend it.

~2.~ The ~body tube~ (Fig. 40, b) may be either that known as the "long" or "English" (length 250 mm.), or the "short" or "Continental" (length 160 mm.). Neither length appears to possess any material advantage over the other, but it is absolutely necessary to secure objectives which have been manufactured for the particular tube length chosen. In the high-class microscope of the present day the body tube is usually shorter than the Continental, but is provided with a draw tube which, when fully extended, gives a tube length greater than the English, thus permitting the use of either form of objective.

For practical purposes the tube length = distance from the
end of the nosepiece to the eyeglass of the ocular. This is
the measurement referred to in speaking of "long" or "short"
tube.

~3.~ The ~coarse adjustment~ (Fig. 40, c) should be a rack-and-pinion movement, steadiness and smoothness of action being secured by means of accurately fitting dovetailed bearings and perfect correspondence between the teeth of the rack and the leaves of the pinion (Fig. 42). Also provision should be made for taking up the "slack" (as by the screws _AA_, Fig. 42).

~4.~ The ~fine adjustment~ (Fig. 40, d) should on no account depend upon the direct action of springs, but should be of the lever pattern, preferably the Nelson (Fig. 43). In this form the unequal length of the arms of the lever secures very delicate movement, and, moreover, only a small portion of the weight of the body tube is transmitted to the thread of the vertical screw actuating the movement.

A spindle milled head (Fig. 44) will be found a very useful device to have fitted in place of the ordinary milled head controlling the fine adjustment. In this contrivance the axis of the milled head is prolonged upward in a short column, the diameter of which is one-sixth of that of the head. The spindle can be rapidly rotated between the fingers for medium power adjustments while the larger milled head can be slowly moved when focussing high powers.

~5.~ The ~stage~ (Fig. 40, e) should be square in shape and large in area--at least 12 cm.--flat and rigid, in order to afford a safe support for the Petri dish used for plate cultivations; and should be supplied with spring clips (removable at will) to secure the 3 by 1 glass slides.

A mechanical stage must be classed as a necessity rather than a luxury so far as the bacteriologist is concerned, as when working with high powers, and especially when examining hanging-drop specimens, it is almost impossible to execute sufficiently delicate movements with the fingers. In selecting a mechanical stage, preference should be given to one which forms an integral part of the instrument (Fig. 45) rather than one which needs to be clamped on to an ordinary plain stage every time it is required, and its traversing movements should be controlled by stationary milled heads (Fig. 45, _AA'_). The shape of the aperture is a not unimportant point; it should be square to allow of free movement over the substage condenser. The mechanical stage should be tapped for three (removable) screw studs to be used in place of the sliding bar, so that if desired the Vernier finder (Fig. 45, _BB'_), such as is usually fitted to this class of stage, or a Maltwood finder, may be employed.

~6. Diaphragm.~--Separate single diaphragms must be avoided; a revolving plate pierced with different sized apertures and secured below the stage is preferable, but undoubtedly the best form is the "iris" diaphragm (Fig. 46) which enters into the construction of the substage condenser.

~7.~ The ~substage condenser~ is a necessary part of the optical outfit. Its purpose is to collect the beam of parallel rays of light reflected by the plane mirror, by virtue of a short focus system of lenses, into a cone of large aperture (reducible at will by means of an iris diaphragm mounted as a part of the condenser), which can be accurately focussed on the plane of the object. This focussing must be performed anew for each object, on account of the variation in the thickness of the slides.

The form in most general use is that known as the Abbe (Fig. 47) and consists of a plano-convex lens mounted above a biconvex lens. This combination is carried in a screw-centering holder known as the substage below the stage of the microscope (Fig. 40 f), and must be accurately adjusted so that its optical axis coincides with that of the objective. Vertical movement of the entire substage apparatus effected by means of a rack and pinion is a decided advantage, and some means should be provided for temporarily removing the condenser from the optical axis of the microscope.

With the oil immersion objective, however, an ~achromatic condenser~, giving an illuminating cone of about 0.9, should be used if the full value of the lens is to be obtained. It is generally assumed that a good objective requires an illuminating cone equivalent to two-thirds of its numerical aperture. The best Abbe condenser transmits a cone of about .45 whilst the aperture of the 1/12 inch immersion lenses of different makers varies from 1.0 to 1.4, hence, the efficiency of these lenses is much curtailed if the condenser is merely the Abbe. These improved condensers must be absolutely centered to the objective and capable of very accurate focussing otherwise much of their value is lost.

~8. Mirrors.~--Below the substage condenser is attached a gymbal carrying a reversible circular frame with a plane mirror on one side and a concave mirror on the other (Fig. 40, g). The plane mirror is that usually employed, but occasionally, as for example when using low powers and with the condenser racked down and thrown out of the optical axis, the concave mirror is used.

~9. Oculars, or Eyepieces.~--Those known as the Huyghenian oculars (Fig. 48) will be sufficient for all ordinary work without resorting to the more expensive "compensation" oculars. Two or three, magnifying the "real" image (formed by the objective) four, six, or eight times respectively, form a useful equipment.

As an accessory ~Ehrlich's Eyepiece~ is a very useful piece of apparatus when the enumeration of cells or bacteria has to be carried out. This is an ordinary eyepiece fitted with an adjustable square diaphragm operated by a lever projecting from the side of the mount. Three notches are made in one of the sides of the square and by moving the lever square aperture can be reduced to three-quarters, one-half or one-quarter of the original size.

~10. Objectives.~--Three objectives are necessary: one for low-power work--e. g., 1 inch, 2/3 inch, or 1/2 inch; one for high-power work--e. g., 1/12 inch oil immersion lens; and an intermediate "medium-power" lens--e. g., 1/6 inch or 1/8 inch (dry). These lenses must be carefully selected, especial attention being paid to the following points:

(a) _Correction of Spherical Aberration._--Spherical aberration gives rise to an ill-defined image, due to the central and peripheral rays focussing at different points.

(b) _Correction of Chromatic Aberration._--Chromatic aberration gives rise to a coloured fringe around the edges of objects due to the fact that the different-coloured rays of the spectrum possess varying refrangibilities and that a simple lens acts toward them as a prism.

(c) _Flatness of Field._--The ideal visual field would be large and, above all, _flat_; in other words, objects at the periphery of the field would be as distinctly "in focus" as those in the centre. Unfortunately, however, this is an optical impossibility and the field is always spherical in shape. Some makers succeed in giving a larger central area that is in focus at one time than others, and although this may theoretically cause an infinitesimal sacrifice of other qualities, it should always be sought for. Successive zones and the entire peripheral ring should come into focus with the alteration of the fine adjustment. This simultaneous sharpness of the entire circle is an indication of the perfect centering of the whole of the lenses in the objective.

(d) _Good Definition._--Actual magnification is, within limits, of course, of less value than clear definition and high resolving power, for it is upon these properties we depend for our knowledge of the detailed structure of the objects examined.

(e) _Numerical Aperture_ (_N. A._).--The numerical aperture may be defined, in general terms, as the ratio of the _effective_ diameter of the back lens of the objective to its equivalent focal length. The determination of this point is a process requiring considerable technical skill and mathematical ability, and is completely beyond the powers of the average microscopist.[1]

Although with the increase in power it is correspondingly difficult to combine all these corrections in one objective, they are brought to a high pitch of excellence in the present-day "achromatic" objectives, and so remove the necessity for the use of the higher priced and less durable apochromatic lenses.

In selecting objectives the best "test" objects to employ are:

1. A thin (one cell layer), even } { 1", 2/3", 1/2": "blood film," stained with Jenner's } for { 1/6", 1/8" or Romanowsky's stain. } { 1/12" oil

2. A thin cover-slip preparation } of a young cultivation of } { 1/8" dry _B. diphtheriae_ (showing } for { segmentation) stained with } { 1/12" oil methylene-blue. }

~Accessories.~--_Eye Shade_ (Fig. 49).--This piece of apparatus consists of a pear-shaped piece of blackened metal or ebonite, hinged to a collar which rotates on the upper part of the body tube of the microscope. It can be used to shut out the image of surrounding objects from the unoccupied eye, and when carrying out prolonged observations will be found of real service.

_Nosepiece._--Perhaps the most useful accessory is a nosepiece to carry two of the objectives (Fig. 50), or, better still, all three (Fig. 51). This nosepiece, preferably constructed of aluminium, must be of the covered-in type, consisting of a curved plate attached to the lower end of the body tube--a circular aperture being cut to correspond to the lumen of that tube. To the under surface of this plate is pivoted a similarly curved plate, fitted with three tubulures, each of which carries an objective. By rotating the lower plate each of the objectives can be brought successively in to the optical axis of the microscope.

For critical work and particularly for photo-micrography, however, the interchangeable nosepiece is by no means perfect as it is next to impossible to secure accurate centreing of each lens in the optical axis. For special purposes, therefore, it is necessary to employ a special nosepiece such as that made by Zeiss or Leitz into which each objective slides on its own carrier and upon which it is accurately centred.

_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.

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 glass 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 brass terminals on the wooden frame by platinum wire fused into the glass. 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 deg. 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.

_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-glasses (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.

_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 mu. 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 glass 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-glass is cemented over the scale to protect it from injury.

(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 mu. 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.

(b) By means of the Eyepiece Micrometer.

The ~eyepiece micrometer~ is a circular glass 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 glass disc together with a circular cover-glass 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 diminishing the tube length; and note the number of included divisions.

4. Calculate the value of each division of the eyepiece micrometer in terms of mu, 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 illustration it may be assumed 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.

The eikonometer should then be applied to the eyepiece and the size of the object read off on the eikonometer scale as millimetres, and the actual size calculated by dividing the observed size by the magnification constant for the particular optical combination employed in the observation.

(c) By means of the filar micrometer.

The ~Filar~ or cobweb Micrometer (Ramsden's micrometer) eyepiece (Fig. 58) consists of an ocular having a fine "fixed" wire stretching horizontally across the field (Fig. 59), a vertical reference wire--fixed--adjusted at right angles to the first; and a fine wire, parallel to the reference wire, which can be moved across the field by the action of a micrometer screw; the drum head is divided into one hundred parts, which successively pass a fixed index as the head is turned. In the lower part of the field is a comb with the intervals between its teeth corresponding to one complete revolution of this screw-head.

As in the previous method, the value of each division of the micrometer scale (i. e., the comb) must first be determined for each optical combination. This is effected as follows:

1. Place the filar micrometer and the stage micrometer in their respective positions.

2. Rotate the screw of the filar micrometer until the movable wire coincides with the fixed one, and the index marks zero on the drum head. (If when the drum head is at zero the two wires do not exactly coincide they must be adjusted by loosening the drum screw and resetting the drum.)

3. Focus the scale of each micrometer accurately, and make the lines on them parallel.

4. Rotate the head of the micrometer screw until the movable line has transversed one division of the stage micrometer. Note the number of complete revolutions (by means of the recording comb) and the fractions of a revolution (by means of scale on the head of the micrometer screw), which are required to measure the 0.01 mm.

5. Make several such estimations and average the results.

6. Note the optical combination employed in this experiment and record it carefully, together with the micrometer value in terms of mu.

7. Repeat this process for each of the different optical combinations and record the results.

To measure an object by this method, simply note the number of revolutions and fractions of a revolution of the screw-head required to traverse such object from edge to edge, and express the result as _micra_ by reference to the recorded values for that particular optical combination.

_Microscope Illuminant._--In tropical and subtropical regions diffuse daylight is the best illuminant. In temperate climes however daylight of the desirable quantity is not always available, and recourse must be had to oil lamps, gas lamps--preferably those with incandescent mantles--and electricity; and of these the last is undoubtedly the best. A handy lamp holder which can be manufactured in the laboratory is shown in Fig. 60. It consists of a base board weighted with lead to which is attached the ordinary domestic lamp holder, and behind this is fastened a curved sheet-iron reflector. An obscured metal filament lamp of about 16 candle power gives the most suitable light, and if monochromatic light is needed, the blue grease pencil is streaked over the side of the lamp nearest the microscope; the current is switched on and when the glass bulb is warm, rubbing with a wad of cotton-wool will readily distribute the blue greasy material in an even film over the ground glass.

FOOTNOTES:

[1] Its importance will be realised, however, when it is stated in the words of the late Professor Abbe: "The numerical aperture of a lens determines all its essential qualities; the brightness of the image increases with a given magnification and other things being equal, as the square of the aperture; the resolving and defining powers are directly related to it, the focal depth of differentiation of depths varies inversely as the aperture, and so forth."

[2] Made by Mr. Otto Baumbach, 10, Lime Grove, Manchester.

V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER MICRO-FUNGI.

APPARATUS AND REAGENTS USED IN ORDINARY MICROSCOPICAL EXAMINATION.

The following comprises the essential apparatus and reagents for routine work with which each student should be provided.

1. India-rubber "change-mat" upon which cover-glasses may be rested during the process of staining.

2. Squares of blotting paper about 10 cm., for drying cover-slips and slides.

(The filter paper known as "German lined"--a highly absorbent, closely woven paper, having an even surface and no loose "fluff" to adhere to the specimens--is the most useful for this purpose.)

3. Glass jar filled with 2 per cent. lysol solution for the reception of infected cover-glasses and infected pipettes, etc.

4. A square glazed earthenware box with a loose lining containing 2 per cent. lysol solution for the reception of infected material and used slides. The bottom of the lining is perforated so that when full the lining and its contents can be lifted bodily out of the box, when the disinfectant solution drains away and the slides, etc., can easily be emptied out. The empty lining is then returned to the box with its disinfectant solution (Fig. 61).

5. Bunsen burner provided with "peep-flame" by-pass.

6. Porcelain trough holding five or six hanging-drop slides (Fig. 62).

The best form of hanging-drop slide is a modification of Boettcher's glass ring slide, and is prepared by cementing a circular cell of tin, 13 to 15 mm. diameter, and 1 to 2 mm. in height, to the centre of a 3 by 1 slip by means of Canada balsam. It is often extremely convenient to have two of these cells cemented close together on one slide (Fig. 62, a).

Another form of hanging-drop slide is made in which a
circular or oval concavity or "cell" is ground out of the
centre of a 3 by 1 slip. These are more expensive, less
convenient to work with, and are more easily contaminated by
drops of material under examination, and should be carefully
avoided.

7. Three aluminium rods (Fig. 63), each about 25 cm. long and carrying a piece of 0.015 gauge platino-iridium wire 7.5 cm. in length. The end of one of the wires is bent round to form an oval loop, of about 1 mm. in its short diameter, and is termed a loop or an oese; the terminal 3 or 4 mm. of another wire is flattened out by hammering it on a smooth iron surface to form a "spatula"; the third is left untouched or is pointed by the aid of a file. These instruments are used for inoculating culture tubes and preparing specimens for microscopical examination.

The method of mounting these wires may be described as follows:

Take a piece of aluminium wire 25 cm. long and about 0.25 cm. in diameter, and drill a fine hole completely through the wire about a centimetre from one end. Sink a straight narrow channel along one side of the wire, in its long axis, from the hole to the nearest end, shallow at first, but gradually becoming deeper.

On the opposite side of the wire make a short cut, 2 mm. in length, leading from the hole in the same direction. [The use of a fine dental drill and small circular saw, worked by a dental motor facilitates the manufacture of these aluminium handled instruments.]

Now pass one end of the platinum wire through the hole, turn up about 2 mm. at right angles and press the short piece into the short cut. Turn the long end of the wire sharply, also at right angles, and sink it into the long channel so that it emerges from about the centre of the cut end of the aluminium wire (Fig. 63). A few sharp taps with a watch maker's hammer will now close in the sides of the two channels over the wire and hold it securely.

The platinum wire may be fused into the end of a piece of glass rod, but such a handle is vastly inferior to aluminium and is not to be recommended.]

8. Two pairs of sharp-pointed spring forceps (10 cm. long), one of which must be kept perfectly clean and reserved for handling clean cover-slips, the other being for use during staining operations.

9. A box of clean 3 by 1 glass slips.

10. A glass capsule with tightly fitting (ground on) glass lid, containing clean cover-slips in absolute alcohol.

11. One of Faber's "grease pencils" (yellow, red, or blue) for writing on glass.

12. A wooden rack (Fig. 65) with twelve drop-bottles (Fig. 66) each 60 c.c. capacity, containing

Aniline water.

Gentian violet, saturated alcoholic solution.

Lugol's (Gram's) iodine.

Absolute alcohol.

Methylene-blue, }
Fuchsin, basic, } saturated alcoholic solution.

Neutral red, 1 per cent. aqueous solution.

Leishman's modified Romanowsky stain.

Carbolic acid, 5 per cent. aqueous solution.

Acetic acid, 1 per cent. solution.

Sulphuric acid, 25 per cent. solution.

Xylol.

And two pots with air-tight glass caps (Fig. 67), each provided with a piece of glass rod and filled respectively with Canada balsam dissolved in xylol, and sterile vaseline.

METHODS OF EXAMINATION.

Bacteria, etc., are examined microscopically.

1. In the living state, unstained, or stained.
2. In the "fixed" condition (i. e., fixed, killed,
and stained by suitable methods).

The preparation of a specimen from a tube cultivation for examination by these methods may be described as follows:

~1. Living, Unstained.~--(a) _"Fresh" Preparation._--

1. Clean and dry a 3 by 1 glass slip and place it on one of the squares of filter paper. Deposit a drop of water (preferably distilled) or a drop of 1 per cent. solution of caustic potash, on the centre of the slip, by means of the platinum loop.

TECHNIQUE OF OPENING AND CLOSING A CULTURE TUBE.

2. Remove the tube cultivation from its rack or jar with the
left hand and ignite the cotton-wool plug by holding it to
the flame of the Bunsen burner. Extinguish the flame by
blowing on the plug, whilst rotating the tube on its long
axis, its mouth directed vertically upward, between the
thumb and fingers. (This operation is termed "flaming the
plug," and is intended to destroy any micro-organisms that
may have become entangled in the loose fibres of the
cotton-wool, and which, if not thus destroyed, might fall
into the tube when the plug is removed and so accidentally
contaminate the cultivation.)

3. Hold the tube at or near its centre between the ends of
the thumb and first two fingers of the left hand, and allow
the sealed end to rest upon the back of the hand between the
thumb and forefinger, the plug pointing to the right. Keep
the tube as nearly in the horizontal position as is
consistent with safety, to diminish the risk of the
accidental entry of organisms (Fig. 68).

4. Take the handle of the loop between the thumb and
forefinger of the right hand, holding the instrument in a
position similar to that occupied by a pen or a paint-brush,
and sterilise the platinum portion by holding it in the
flame of a Bunsen burner until it is red hot. Sterilise the
adjacent portion of the aluminium handle by passing it
rapidly twice or thrice through the flame. After sterilising
it, the loop must not be allowed to leave the hand or to
touch against anything but the material it is intended to
examine, until it is finished with and has been again
sterilised.

5. Grasp the cotton-wool plug of the test-tube between the
little finger and the palm of the right hand (whilst still
holding the loop as directed in step 4), and remove it from
the mouth of the tube by a "screwing" motion of the right
hand.

6. Introduce the platinum loop into the tube and hold it in
this position until satisfied that it is quite cool. (The
cooling may be hastened by touching the loop on one of the
drops of moisture which are usually to be found condensed on
the interior of the glass tube, or by dipping it into the
condensation water at the bottom; at the same time care must
be taken in the case of cultures on solid media to avoid
touching either the medium or the growth.)

7. Remove a small portion of the growth by taking up a drop
of liquid, in the case of a fluid culture, in the loop; or
by touching the loop on the surface of the growth when the
culture is on solid medium; and withdraw the loop from the
tube without again touching the medium or the glass sides of
the tube.

8. Replace the cotton-wool plug in the mouth of the tube.

9. Replace the tube cultivation in its rack or jar.

10. Mix the contents of the loop thoroughly with the drop of water on the 3 by 1 slide.

11. Again sterilise the loop as directed in step 4, and replace it in its stand.

12. Remove a cover-slip from the glass capsule by means of the cover-slip forceps, rest it for a moment on its edge, on a piece of filter paper to remove the excess of alcohol, then pass it through the flame of the Bunsen burner. This burns off the remainder of the alcohol, and the cover-slip so "flamed" is now clean, dry, and sterile.

13. Lower the cover-slip, still held in the forceps, on to the surface of the drop of fluid on the 3 by 1 slip, carefully and gently, to avoid the inclusion of air bubbles.

14. Examine microscopically (_vide infra_).

During the microscopical examination, stains and other reagents may be run in under a cover-slip by the simple method of placing a drop of the reagent in contact with one edge of the cover-glass and applying the torn edge of a piece of blotting paper to the opposite side. The reagent may then be observed to flow across the field and come into contact with such of the micro-organisms as lie in its path.

The non-toxic basic dyes most generally employed for the intra-vitam staining of bacteria are

Neutral red, }
Quinoleine blue }
Methylene green } in 0.5 per cent. aqueous solutions.
Vesuvin, }

_Negative Stain_ (Burri).--By this method of demonstration the appearances presented by dark ground illumination (by means of a paraboloid condenser) are closely simulated, since minute particles, bacteria, blood or pus cells etc. stand out as brilliantly white or colourless bodies on a dark grey-brown background.

_Reagent required:_

Any one of the liquid waterproof black drawing inks (Chin-chin, Pelican, etc.). This is prepared for use as follows:

Measure out and mix:

Liquid black ink, 25 c.c.
Tincture of iodine 1 c.c.

Allow the mixture to stand 24 hours, centrifugalise thoroughly, pipette off the supernatant liquid to a clean bottle and then add a crystal of thymol or one drop of formalin as a preservative.

METHOD.--

1. With the sterilised loop deposit one drop of the liquid ink close to one end of a 3 by 1 slide.

2. With the sterilised loop deposit a drop of the fluid culture (or of an emulsion from a solid culture) by the side of the drop of ink (Fig. 69, a); mix the two drops thoroughly by the aid of the loop.

3. Sterilise the loop.

4. Hold the slide firmly on the bench with the thumb and forefinger of the left hand applied to the end nearest the drop of fluid.

5. Take another clean 3 by 1 slide in the right hand and lower its short end obliquely (at an angle of about 60 deg.) transversely on to the mixed ink and culture on the first slide, and allow the fluid to spread across the slide and fill the angle of incidence.

6. Maintaining the original angle, draw the second slide firmly and evenly along the first toward the end farthest from the left hand (Fig. 69, b).

7. Throw the second slide into a pot of disinfectant; allow the first slide to dry in the air.

8. Place a drop of immersion oil on the centre of the film, lower the 1/12-inch objective into the oil and examine microscopically without the intervention of a cover-slip.

(The film of ink may be covered with a long cover-glass and xylol balsam as a permanent preparation.)

(<b) _Hanging-drop Preparation._--

1. Smear a layer of sterile vaseline on the upper surface of the ring cell of a hanging-drop slide by means of the glass rod provided with the vaseline bottle, and place the slide on a piece of filter paper.

2. "Flame" a cover-slip and place it on the filter paper by the side of the hanging-drop slide.

3. Place a drop of water on the centre of the cover-slip by means of the platinum loop.

4. Obtain a small quantity of the material it is desired to examine, in the manner detailed above (pages 74-76, steps 2 to 11 must be followed in their entirety and with the strictest exactitude whenever tube contents are being handled), and mix it with the drop of water on the cover-slip.

5. Raise the cover-slip in the points of the forceps and rapidly invert it on to the ring cell of the hanging-drop slide, so that the drop of fluid occupies the centre of the ring. (Carefully avoid contact between the drop of fluid and either the ring cell or the layer of vaseline. Should this happen, the now _infected_ hanging-drop slide and its cover-slip must be dropped into the pot of lysol and a new preparation made.)

6. Press the cover-slip firmly down into the vaseline on to the top of the ring cell. (This spreads out the vaseline into a thin layer, and besides ensuring the adhesion of the cover-slip, seals the cells and so retards evaporation.)

7. Examine microscopically.

The examination of a "fresh" specimen or a "hanging-drop" preparation is directed to the determination of the following data:

1. The nature of the bacteria present--e. g., cocci, bacilli, etc.

2. The purity of the cultivation; this can only be determined when gross morphological differences exist between the organisms present.

3. The presence or absence of spores; when present, spores show their typical refrangibility exceedingly well by this method.

4. The presence or absence of mobility. In a hanging-drop specimen some form of movement can practically always be observed, and its character must be carefully determined by noting the relative positions of adjacent micro-organisms.

(a) Brownian or molecular movement. Minute particles of solid matter (including bacteria), when suspended in a fluid, will always show a vibratory movement affecting the entire field, but never altering the relative positions of the bacteria. (Cocci exhibit this movement, but with the exception of the Micrococcus agilis, the cocci are non-motile.)

(b) Streaming movement. This is due to currents set up in the hanging drop as a result of jarring of the specimen or of evaporation, or to the fact that the cover-slip is not perfectly level, and although the relative positions of the bacteria may vary, still the flowing movement of large numbers of organisms in some one direction will usually be sufficient to demonstrate the nature of this motion.

(c) Locomotive movement, or ~true motility~, is determined by observing some one particular bacillus changing its position in the field independently of, and in a direction contrary to, other organisms present.

When the examination is completed and the specimen finished with, the "fresh specimen"--i. e., the slide with the cover-slip attached--must be dropped into the lysol pot. In the hanging-drop specimen, however, the cover-slip only is infected, and this may be raised from the ring cell by means of forceps and dropped into the disinfectant.

_Permanent Staining of the Hanging-drop Specimen._--Occasionally it is necessary to fix and stain a hanging-drop preparation. This may be done as follows:

1. Remove the cover-slip from the cell by the aid of the forceps.

2. If the drop is small, fix it by dropping it face downward, whilst still wet, on to the surface of some Gulland's solution or corrosive sublimate solution (_vide_ page 82) in a watch-glass. If the drop is large, place it face upward on the rubber mat, cover it with an inverted watch-glass, and allow it to dry. Then fix it in the alcohol and ether solution (_vide_, page 82).

3. Dip the cover-glass into a beaker containing hot water in order to remove some of the vaseline adhering to it.

4. Wash successively in alcohol, xylol, ether, and alcohol, to remove the last traces of grease.

5. Wash in water.

6. Stain, wash, dry, and mount as for an ordinary cover-slip film preparation (_vide_ pages 83-85).

~2. Killed, Stained.~--In this method three distinct processes are necessary: