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.
[Illustration: FIG. 58.--Ramsden's Filar micrometer.]
[Illustration: FIG. 59.--Ramsden's micrometer field, a, fixed wire;
b, reference wire (fixed); c, travelling wire.]
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.
[Illustration: FIG. 60.--Electric microscope lamp.]
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.)
[Illustration: FIG. 61.--Disinfectant Jar.]
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).
[Illustration: FIG. 62.--Hanging-drop slides: a, Double cell seen from
above; b, single cell seen from the side.]
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.
[Illustration: FIG. 63.--Ends of platinum rods. a, loop; b, spatula;
c, needle.]
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.
[Illustration: FIG. 64.--Platinum rod in aluminium handle--method of
mounting.
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.
[Illustration: FIG. 65.--Staining rack, rubber change mat and lysol
pot.]
[Illustration: FIG. 66.--Drop bottle.]
[Illustration: FIG. 67.--Canada balsam pot.]
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.
[Illustration: FIG. 68.--Holding tubes for removing bacterial growth, as
seen from the front.]
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.
[Illustration: FIG. 69.--Spreading negative film.]
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:
"Preparing" and "fixing" the film.
Staining.
Mounting.
_Preparing the Film._--
1. Flame a cover-slip and place it on a piece of filter paper.
2. Place a drop of water on the centre of the cover-slip by means of
platinum loop.
3. Obtain a small quantity of the material to be examined upon a
sterilised platinum loop (see pages 74-76, steps 2 to 11) and mix it
with the drops of water on the cover-slip.
4. Spread the drop of emulsion evenly over the cover-slip in the form of
a square film to within 1 mm. of each edge of the cover-slip.
5. Allow it to dry completely in the air.
_Fixing._--Fix by passing the cover-slip, held in the fingers, three or
four times through the flame of a Bunsen burner.
In some instances (e. g., when the films after staining are intended
for micrometric observations) it is almost essential to fix by exposure
to a uniform temperature of 115 deg. C., for twenty minutes. This is best
done in a carefully regulated hot-air oven.
Fixation may also be effected by immersing in some fixative fluid, such
as one of the following:
1. Absolute alcohol, for five to fifteen minutes.
{ equal parts, for five to thirty
2. Absolute alcohol, { minutes (e. g., for blood or
Ether, { milk).
3. Osmic acid, 1 per cent. aqueous solution, for thirty seconds.
4. Corrosive sublimate, saturated aqueous solution, for five minutes.
5. Corrosive sublimate (Lang), for five minutes. This solution is
prepared by dissolving:
Sodium chloride 0.75 gramme
Hydrarg. perchloride 12.00 grammes
Acetic acid 5.00 grammes
In distilled water 100.00 c.c.
Filter.
6. Gulland's solution, for five minutes. This solution is prepared by
mixing:
Absolute alcohol 25.0 c.c.
Ether 25.0 c.c.
Corrosive sublimate, 20 per cent. alcoholic solution 0.4 c.c.
7. Formalin 10 per cent. aqueous solution (= 4 per cent. aqueous
solution of formaldehyde since formalin is a 40 per cent. solution of
the gas in water).
Either of these methods of fixation coagulates the albuminous material
and ensures perfect adhesion of the film to the cover-slip.
_Clearing._--Wash the cover-slip thoroughly in running water and proceed
with the staining.
If the film has been prepared from broth, liquefied gelatine, or pus or
other morbid exudations, saturate the film after fixation with acetic
acid 2 per cent. and allow it to act for two minutes.
Wash with alcohol, then let the alcohol remain on the cover-slip for two
minutes. (This will "clear" the groundwork and give a much sharper and
cleaner film than would otherwise be obtained.)
If the film has been prepared from blood or bloodstained fluid, treat
with acetic acid 2 per cent. for two minutes after fixation. Wash with
water, dry, and proceed with the staining. (This will remove the
haemoglobin and facilitate examination.)