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.
[Illustration: FIG. 39.--Closed candle arranged for filtering.]
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:
[Illustration: FIG. 40.--Microscope stand.]
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:
[Illustration: FIG. 41.--Foot, three types.]
~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.
[Illustration: FIG. 42.--Coarse adjustment.]
[Illustration: FIG. 43.--Fine adjustment.]
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.
[Illustration: FIG. 44.--Spindle head to fine adjustment.]
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.
[Illustration: FIG. 45.--Mechanical stage.]
[Illustration: FIG. 46.--Iris diaphragm.]
~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.
[Illustration: FIG. 47--Optical part of Abbe illuminator.]
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.
[Illustration: FIG. 48.--Huyghenian eyepiece.]
(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.
[Illustration: FIG. 49.--Eye shade.]
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.
[Illustration: FIG. 50.--Double nosepiece.]
[Illustration: FIG. 51.--Triple nosepiece.]
_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.
[Illustration: 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 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.
[Illustration: 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-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.
[Illustration: 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 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.
[Illustration: 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 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.
[Illustration: FIG. 56.--Eyepiece micrometer, ordinary.]
[Illustration: FIG. 57.--Eyepiece micrometer, net.]
(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.