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Chapter XII The Isolation and Cultivation of Single Organisms
CHAPTER XI1
The Isolation and Cultivation of Single Organisms K. I.
JOHNSTONE
Department of Bacteriology, The School of Medicine, Leeds, England I. Introduction . . 11. A Review of the Chief Methods Devised for Single-organism Culture A. Selection of an isolated organism on a randomly inoculated nutrient agar gel B. Formation of droplets with a micropipette . . C. Isolation by micromanipulation on an agar gel surface . D. Destruction of all organisms except the one selected
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111. The Agar Block Dissection Techniques . A. The agar gel . . B. Casting of blocks of gel * C. Optical equipment . . D. Use of a second microscope as micromanipulator . . E. Isolation with a simple angulated microneedle and location of . the isolates . F. Isolation with a microloop on pre-marked sites . . G. Evidence for the reliability of the methods References
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I. INTRODUCTION The cultivation of bacteria from single organisms is especially valuable in the purification of strains, since the only absolute criterion of the purity of a culture is the certainty that it has been derived from the progeny of a single organism. Failure to apply this criterion may lead to much waste of effort in research, especially in regard to the clostridia, apparently pure strains of which may carry latent contaminants for long periods. All strains upon which research is to be based should therefore be rigorously purified at the outset. The study of the properties of individual organisms, as distinct from those of bacterial populations, is a little-explored field, requiring a rapid and accurate method of isolation to enable batches of isolates to be tested, e.g., the heat resistance of bacterial spores.
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Because of the current belief that single-organism culture is necessarily tedious and involves the use of costly and complex apparatus, the technique is often shunned. However, the methods available range from those requiring only the simplest apparatus readily improvised in the laboratory, to those requiring elaborate micromanipulators and ancillary apparatus. The former are only suitable when a few isolations are to be made at rare intervals. When many isolations are required, more complex equipment is necessary to minimize fatigue of the operator. In all cases, it is essential that the method be absolutely reliable and that adequate controls are used to establish the validity of the isolations. For multiple and rapid bacterial isolations under sterile conditions, it is essential that the microinstrument should be readily removable from the sterile chamber to facilitate interchange of culture materials and should be as readily replaceable. Many of the types of micromanipulator now available are unnecessarily complex for this work and the agar gel dissection methods here described in detail do not require a conventional instrument. A simple type of microforge is, however, a necessity for the satisfactory production of microinstruments.
11. A REVIEW OF THE CHIEF METHODS DEVISED FOR SINGLE-0 RGANI SM CULTURE
A. Selection of an isolated organism on a randomly inoculated nutrient agar gel This method, employing a block cut from a lightly-inoculated nutrient agar plate (Orskov, 1922) and improved by the use of a cast agar block (Gardner, 1925), requires only the simplest apparatus, which can be assembled at very slight cost in the laboratory. The disadvantages are that (a) the method of location of the isolate by drawing a pattern of scratches on the glass slide is tedious, (b) observations on the growing microcolony may extend over many hours, (c) the subculture of the microcolony is a blind operation and (d) aerial contamination of the exposed gel may occur.
B. Formation of droplets with a micropipette 1. At an &-glass interfuce (Mulone, 1918) Using a second microscope as manipulator, microdroplets are deposited on the lower surface of a sterile coverglass and are searched for those containing only single organisms. Such droplets are each drawn into separate sterile micropipettes, the tips of which are broken off in nutrient broth tubes. The advantage lies in the use of the x 100 oil-immersion objective, which is not possible in methods operating above the air-gel interface. The
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disadvantages are (a) the difficulty in excluding the presence of a second organism at the margin of each droplet, (b) the rapid drying of the minute droplets and (c) the sacrifice of a micropipette for each organism isolated.
2. In an oil chamber (a2 Fonbrune, 1949) The coverglass forms the roof of a chamber filled with sterile liquid paraffin and droplets are formed between the glass and the paraffin by means of a micropipette, being examined at intervals until multiplication of a single organism is observed. Such a droplet is removed using a fresh sterile pipette and is cultured. The advantages are (a) the use of the x 100 oil-immersion objective, (b) the complete elimination of drying of the droplets and (c) the ease with which the successive cell divisions may be observed. The disadvantages are that (a) the charged pipette must traverse the paraffin seal during formation of the droplets and the possibility exists of contamination of the oil and subsequent contamination of the subculturing pipette, (b) a clear fluid medium alone can be used and (c) the conditions are anaerobic, and strictly aerobic organisms may not grow. The method is more suitable for observations on the process of multiplication in silu, without recovery of the resulting microcultures.
3. On a mechanically propelled Cellophane strip (Reyniers and Trexler, 1943) Microdroplets are formed mechanically on the lower surface of a strip of Cellophane, which then passes through the microscope and is taken up on a spool. When a droplet is recognized as containing a single organism, it is cut out on a disc of Cellophane by a micro-fly cutter and falls direct into a culture tube. The method attains the highest degree of mechanization, but relies on the recognition of droplets containing single organisms and requires specialized and complex apparatus. C. Isolation by micromanipulation on an agar gel sudace 1. Below the air-gel interfme (Dickinson, 1926) Using the lower surface of a coverglass coated with a thin layer of nutrient agar, enclosed to reduce drying of the gel, selected organisms are carried in turn across the agar surface in the water column formed by contact of a vertical needle tip with the gel. The advantages are that (a) the isolates are taken to sterile portions of the gel remote from the inoculum, (b) the organisms can be cultured in situ, or portions of the agar may be cut out carrying isolates and cultured separately, (c) the x 100 oil-immersion objective can be used and (d) aerial contamination is excluded. The disadvantages are that (a) there is no rapid and positive method of location of the isolates, their positions being marked with ink on the upper surface of
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the coverglass, (b) manipulations below the air-gel interface are difficult to carry out and (c) the necessarily thin layer of agar renders dissection of the gel tedious, especially if many isolations are required.
2. Above the air-gel interface (a) Technique of Koblmuller and Vierthaler (2933).The technique involves the use of a metal needle on a nutrient gel, and cultivation of the isolates in situ. Selected organisms on the upper surface of a nutrient agar gel are drawn in turn to isolated sites on the sterile surface by means of a metallic needle inclined at an angle to the surface. The selected organism floats in the water exuded from the gel around the needle tip. The isolates are cultivated on the agar surface. The advantage lies in the greater ease of manipulation on the upper surface of the gel. The disadvantages are that (a) the method of location of the isolates by ink marks is tedious, (b) a metal microneedle is less easy to prepare than one of glass and (c) a dry objective must be used.
(b) Technique ofJohnstone (2943,2953). The technique involves the use of a glass needle and positive location of the isolates on a non-nutrient gel, which is subsequently dissected. Selected organisms are carried across the upper surface of a cast block of clarified non-nutrient agar gel, with an angulated glass microneedle. The location of each isolate is defined by melting two pits in the gel surface, the organism lying between them. The isolates are cultivated by dissection of the gel, as shown by the pairs of pits, and transfer of portions, each carrying a pair of pits and therefore an organism, to separate tubes of medium. The advantages are that (a) a glass microneedle is used which is readily prepared, (b) manipulations are carried out on the plane upper surface of the gel and are readily observed, (c) the method of location is positive and can be made rapid by mechanical means and (d) any type of culture medium may be used for cultivation of the isolates. The disadvantage, in common with other methods operating above the air-gel interface, is that immersion objectives cannot be used. (c) Technique of Holdom and Johnstone (2967).The technique involves the use of a glass microloop on a non-nutrient gel with pre-marked isolation sites and subsequent dissection of the gel. The cast block of agar gel is pre-marked with 24 isolation sites in a sterile punch. With a glass microloop, prepared in a high-power microforge, selected organisms are carried rapidly above the gel to the isolation sites and are there ejected from the loop on to the agar surface. Dissection of the gel and cultivation of the isolates follow. The advantages are that (a) the isolations are carried out rapidly and (b) when many isolations of the same
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strain are required, the microloop can be charged with 6-12 selected organisms which are deposited singly on the isolation sites, with great saving of time. The disadvantage is that the microloop must be prepared in a high-power microforge, but such a loop will serve for several thousand isolations.
D. Destruction of all organisms except the one selected This method (Topley et al., 1921)is unique, in that the selected organism, located in a gelatin film under a quartz coverglass, is protected by a globule of mercury on the coverglass from ultraviolet radiation, which kills all other organisms in the film. After incubation, the resulting microcolony is subcultured. The advantage lies in the simple apparatus required. The disadvantages are that (a) careful controls are necessary to establish the validity of each experiment, especially in the presence of spores, (b) incubation is at 25°C and (c) the method is not suitable if many isolations are required. 111. THE AGAR BLOCK DISSECTION TECHNIQUES A. The agar gel The gel must be free from both living and dead bacteria, the latter being abundant in most samples of agar and causing confusion during micromanipulation. They are removed by clarification of a fluid 2.0% (w/v) aqueous solution of New Zealand agar with 2.0% (w/v) of Hyflo Super Cel diatomaceous earth (Koch-Light Laboratories Ltd, Colnbrook, Buckinghamshire) at 60°C for 4 days, with gentle inversion of the bottle twice daily (Feinberg, 1956). This is followed by filtration through Hyflo Super Cel sandwiched between layers of paper pulp and yields a gel free from bacteria, when autoclaved. The reaction should be pH 7.0-7-4. No nutrients are added, since multiplication of the organisms is not desired during manipulations, and a batch of agar can be stored in sealed ampoules for several years, being used for manipulation of a great variety of bacteria. After trial with the microinstrument, the concentration of agar is lowered, by adding water, to the optimum for the method of isolation used. B. Casting of blocks of gel A clean 7.6 x 2.5 cm glass slide is separated from a clean 7.6 x 3.8 cm slide by two glass strips 2.0 mm thick to form a casting cell (Fig. l), the slides being held in apposition to the strips by wire paper clips and the whole sterilized by dry heat in a Petri dish. Diamond-ruled lines on the outer surfaces of the cell serve as guides in sectioning the agar slab. Clarified agar at 100°C is pipetted into the casting cell at 60°C and, when set, the excess of gel beyond the margin of the smaller slide is removed with
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a sterile scalpel. The slides are drawn apart with a sliding motion and the gel, which remains adherent to one slide, is cut into blocks 2.5 x 1.3 cm and 2.0 mm thick. Each block is lifted with the back of the scalpel blade and is
F I ~1.. A glass casting cell for the agar gel. The portion occupied by the gel is
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FIG.2. The Perspex cell and coverglass for protection of the agar block during manipulations. A, the access slot for the microinstrument, or marking needle; B, the coverglass.
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deposited on a separate sterile 7.6 x 3.8 cm slide, enclosed in a Petri dish with a Duralumin humidifying trough containing glass wool moistened with distilled water. A t all stages it is essential that thegel beprotectedfrom desiccation, as otherwise the plane upper surface will be &formed and the concentration of agar will rise, producing a surface unsuitablefor micromanipulation. Protection of the agar block from airborne contamination and from desiccation during manipulation is provided by a sterile Perspex cell (Fig. 2), which rests on the supporting slide around the block and carries a 5.5 x 2.5 cm No. 0 coverglass. For optical system 1, the lower surface of the coverglass is 0.9-1.0 mm above the gel surface, but for system 2, this height can be greatly increased, thus allowing more working space for the needle tip. A slot, A, (Fig. 2) gives access to the shaft of the microneedle, a second slot at the opposite end of the cell giving access to the platinum marking needle.
C. Optical equipment Phase-contrast microscopy renders earlier methods, including darkground illumination, obsolete. Positive phase contrast shows bacteria clearly on the gel surface as dark objects against a green background, except for the mature spore, which appears bright before germination. The gel surface appears faintly mottled. Two systems may be used.
1. A refroacting phase-contrast objective x 40 The numerical aperture (N.A.) must be at least 0.65, and the working distance 1.0 mm, excluding the coverglass thickness. This has the mechani-
cal disadvantage of a very confined space for operation of the microneedle and also of condensation on the lower surface of the coverglass, requiring a warm-air jet playing on the upper surface of the glass to disperse the water droplets. With a high-absorption phase-plate, the Parachromatic objective x 40 (Watson) gives good resolution.
2. A Dyson long-working-distance phase-contrast objective x 40 (VicRers Instruments) Incorporating both reflecting and refracting units, this overcomes both mechanical and thermal disadvantages with a working distance of 12.8 mm, but the N.A. is reduced to 0.57 and the light intensity must be greatly increased to compensate for losses by reflection, with possible lethal action on sensitive organisms. The phase-contrast condenser must also have a long working distance, to operate through the slide and a 2 mm thickness of agar gel. The illuminant should be of high intensity with a cooling trough, followed by a Chance’s ON 20 heat-absorbing filter, and a yellow-green filter to improve optical performance.
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D. Use of a second microscope as micromanipulator A second microscope on a smooth and rigid bench surface contributes the sensitive vertical movement to the microinstrument through its fine adjustment. A nosepiece attachment (Fig. 3) is screwed firmly into the objective thread and the bar A is clamped by the screw B so as to project towards the axis of the observing microscope. This bar is bored to receive the microneedle holder, or the platinum marking needle, as required. Centration of the microneedle tip in the field of the observing microscope is effected by
FIG.3. The nosepiece attachment (left), the microneedle holder (centre) and the microneedle handle (right). C, the r.m.8. objective thread; D, the clamping screw for the microneedle holder; E, the clamping screw for the microneedle.
sliding the manipulating microscope on the bench by finger pressure from both hands applied to the foot of the instrument, while movement of the tip is observed in the field. Movement in the horizontal plane is obtained by means of the mechanical stage of the observing microscope, which carries the agar block on the sterile slide and therefore gives motion in two dimensions to the gel surface relative to the needle tip, which remains centred in the field.
E. Isolation with a simple angulated microneedle and location of the isolates The selected organism is carried across the sterile gel surface in the minute pool of water that exudes from the gel around the glass microneedle where it is in contact with the gel. The isolate is under constant observation during its passage and the presence of a second organism of comparable size cannot be
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overlooked. This method is especially suitable for pure strain isolations, when only a limited number is required.
1. The angulated microneedle Soft-glass rod, 6 mm diameter, is drawn out by hand to 0.9-1.0 mm diameter thus forming the handle of the microneedle. The thin rod is again drawn out after being heated in a minute coal-gas flame burning at the tip of a glass capillary tube, to form the needle shaft 3.8 cm long and 0.15 mm diameter at the free end (Fig. 4a). The tip (Fig. 4b) is formed in a low-power
FIG.4. (a). An angulated glass microneedle. A, the handle; B, the shaft; C, the tip. (b). The tip of the microneedle enlarged, showing its spatial relation to the coverglass, D, and the agar gel surface, E. The distance F is the clearance of the shaft above the gel.
microforge (Johnstone, 1953) and consists of a portion tapering rapidly from 150 pm to 20 pm at an angle of 60" to the axis of the shaft, and a point inclined at 30" to the same axis, tapering rapidly to 1.5 pm at its tip. The clearance of approximately 400 p m is essential to avoid contact between the horizontal shaft and the gel surface, to which it would adhere owing to surface tension. The shaft must be straight and in the axis of the handle.
2. The platinum marking needle This forms the locating pits in the gel surface on each side of an isolated organism, by means of which the site can readily be seen during dissection
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of the block. The V shaped heating element (Fig. 5 ) is made from two 2.7 cm lengths of 40 s.w.g. platinum wire, welded to 24 s.w.g. platinum wires as supports. At the apex of the V, one thin wire is hooked around the second wire 2 mm from its free end and the joint is welded, the terminal 0.5 mm
FIG.5. The platinum marking needle: A, the heating element; B, the supporting platinum wires; C, the copper leads; D, the glass tubes.
of the single wire being bent vertically downwards to form the marking tip. The platinum needle can be mounted in one of two ways(a) A simple mount, suitable for occasional use consists of two soft-glass tubes, 4 mm external diameter, bent at right angles. The thick supporting wires of the platinum element are first silver-soldered to 24 s.w.g. copper leads and the platinum wires are then sealed into the ends of the glass tubes (Fig. 6). The vertical ends of the tubes pass through a cork, E, pressed firmly into the nosepiece of a manipulating microscope, whose focusing movements are used to form the pits in the gel surface. Connection is made from the copper leads to a 3 V a.c. supply through push switches S1 and Sz (Fig. 7), with variable
FIG.6. A simple glass mount for the marking needle. The cork, E, fits into the objective thread of a microscope nosepiece. The electrical leads, F, pass through the cork.
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resistors R1 and R2,respectively, in series. R1 is adjusted to bring the element to red heat on closing S1,the tip being sterilized by conduction. R2 is adjusted to warm the element sufficiently to melt a pit in the gel surface when SZ is closed, without causing the gel to boil.
FIG.7. Heating circuits for the platinum marking needle.
(b) A permanent mount interchangeable with the microneedle holder in the nosepiece attachment is shown in Fig. 8. The steel rods, A and B, carrying the platinum element, are electrically insulated from the stem C, which enters the nosepiece attachment. This mount can with advantage be fitted with adjustable pneumatic dipping and traversing movements and can itself be mounted on a rigid geometric slide replacing the second microscope. The unit may then be withdrawn from the optic axis and rapidly replaced in centre. Also, the locating pits can be formed, as described below, on each side of the isolate while the glass microneedle remains in situ and with the x 40 objective in position, thus greatly reducing the time taken.
FIG.8 A permanent mount for the platinum marking needle interchangeablewith the microneedle holder in the nosepiece attachment. Electrical connections are made to the rods A and B at D.
3, Isolation procedure During inoculation, the sterile surface of the agar block is protected from bacterial aerosols, created by the inoculating loop, by a sterile metal shield
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covering all but a strip 2 mm wide at one end. With a 1 mm loop, an inoculum from a just visibly turbid suspension of the organisms is streaked across the upper surface of the gel at the exposed end. The fluid is rapidly absorbed, leaving the organisms on the gel surface and the shield is then removed. The block is covered with the sterile Perspex cell, the slide is placed on the mechanical stage of the observing microscope, and the margin of the inoculum is located by dark-ground illumination at x 150 magnification
FIG.9. Isolation of a selected organism by trailing across the agar gel surface with an angulated glass microneedle. The arrows indicate the direction of movement of the gel.
(obtained by the use of a x 10 objective with the x 40 phase-contrast condenser annulus). The microneedle, mounted in the carrier on the nosepiece of the manipulating microscope, is carefully directed into the access slot of the Perspex cell by sliding the manipulating microscope on the bench, taking care to clear the coverglass above and the gel below. The tip is located as it enters the field and is centred first with the x 10 and then with the x 40 objective, while it is poised above the gel surface. By operation of the coarse-focusing movement, the needle tip is lowered until it is just above the gel surface. A selected, well-isolated organism is brought by means of the stage controls to lie below the needle tip and the latter is then lowered, with the fine-focusing movement, to touch the gel, when the organism floats in the exuded water (Fig. 9). By means of the stage controls, the organism is then carried rapidly over the gel surface, while it and the needle tip remain centred in the field. If the organism escapes from the needle, it is located by following in reverse the needle track, which is visible as a bright line on the
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gel, until the organism is found. The needle tip must be raised above the gel during reverse movement. When the isolate has been carried at least 5 mm from the margin of the inoculum, its position is defined by melting two shallow pits in the gel surface 1 mm apart, one above and one below the organism, with the sterile platinum marking needle. The x 10 objective is used unless the pneumatic device for controlling the platinum needle is available, when the x40 phase-contrast objective can be used and the marking carried out in the presence of the glass microneedle, with great saving of time. Further isolations, up to a total of six, are then made on the same block and should be staggered (Fig. 10a) to facilitate dissection of the gel.
FIG.10. (a). A completed agar block showing the routes taken by six isolates trailed in turn with an angulated microneedle from the inoculum. (b). An agar block premarked with 24 isolation sites for use with a microloop. In both parts of this Fig., the shaded areas are those tested for sterility, the stippled areas carry the inoculum and the broken lines indicate the dissection of the gel.
The concentration of agar must be adjusted, after experiment, for each batch prepared. If too high, the organism will not follow the microneedle: if too low, the excess water exuded around the needle tip will cause displacement of adjacent organisms. 4. Dissection of the block and culture of the organisms The completed block, on the supporting slide, is transferred to the stage of a stereoscopic dissecting microscope of magnification x 5. Aerial contamination is excluded (1) by a cabinet enclosing the stage with a roof of
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plate glass immediately below the objectives, and preferably also containing a low-pressure mercury-vapour lamp to sterilize the contained air before use, and (2) by a sterile plastic shield resting on the stage around the slide, with an access slot for the dissecting knives and a central aperture, sealed by a coverglass, below the objectives. The agar block is dissected with sterile, sharp, triangular stainless-steel knives made from safety razor blades soldered into brass handles (Fig. 11).
FIG.11. A knife used in dissectionof the agarblock. Below, the tip enlarged.At A, a portion of a stainless-steel safety razor blade is soft-soldered into the brass rod. The site of the inoculum is first separated and is removed with a platinum scoop. The adjacent section of the gel, A (Fig. lOa), is then isolated and transferred to the optimal recovery medium. Unless this is shown to be sterile, the isolations are not valid. The remainder of the block is then systematically dissected and small portions of the gel, each with a pair of locating pits, and, therefore, an organism, are transferred in turn to separate tubes of the medium for incubation. Single organisms require an optimal medium and optimal conditions of reaction, oxidation-reduction potential and temperature,if a highproportion of the isolates are t o p o v e viable. An advantage of this method is that the medium can be fluid or solid, clear or opaque, depending on the nature of the organism. If solid, the small block of agar is deposited with the platinum scoop on to the medium, pits downwards, the isolate being trapped between the gel surfaces and growth appearing around the margins of the block if the organism is viable.
F. Isolation with a microloop on pre-marked sitea This method has the advantage of great speed and is of especial value when many isolates of the same strain are required. The microloop (Fig. 12) is formed in a high-power microforge at the tip of a needle shaft prepared as for an angulated microneedle. The loop is made from a terminal glass
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filament 1.0-1-5pm diameter and measures internally from 3 x 14pm to 14 x 8pm, depending on the size and the number of organisms to be carried. The loop, which is elliptical in shape, must lie in the plane of the gel surface. For maximum speed, the sites for 24 isolations are punched in the surface of an agar block by a group of 48 steel needle points, mounted in an enclosed sterile punch. The sites, at 3 mm intervals (Fig. lob), are in
FIG.12. The tip of a glass microloop in relation to the agar gel surface. A, the shaft; B, the terminal glass filament;C, the loop lying in the plane of the gel surface, which is stippled; D, the clearance between the shaft and the gel.
register with the readings of the mechanical stage of the observing microscope. After inoculation of one end of the upper surface of the block, selected organisms are picked up with the microloop, to the interior of which they adhere firmly by surface tension when raised above the gel. For the isolation of pure strains, organisms are carried singly in the smallest practicable loop to their respective isolation sites, and are there discharged by slight vibration imparted to the bench while the loop is in contact with the water exuded from the gel surface. For multiple isolations from an already pure strain, 6 or 12 organisms are picked up successively by a larger loop, which is then lowered on to the first site. By slight vibration of the bench, one or more organisms are ejected from the loop and remain on the gel surface as the loop is raised. If more
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than one has been discharged, the excess organisms art again picked up, leaving a single isolate between the pits. The remaining sites are treated in the same way, the whole block of 24 isolations being completed in less than 8 min with a suitable microloop. Dissection of the block and transfer to the culture medium is carried out as for the angulated microneedle technique. In the case of large spores, such as fungal spores, a large microloop will readily pick up the spore, but difficulty may be experienced in releasing it. This can be overcome by using a loop with a strong supporting filament and sinking the loop below the surface of the gel. As the agar rises through and
FIG.13. (a)-(d). Four stages in the deposition of a fungal spore on the agar surface. The vertical arrows indicate movement of the microloop : the horizontal arrows indicate simultaneous movement of the gel. Diagrammatic-not to scale.
around the loop, the spore is carried on the surface of the gel. The microloop is then extracted from the gel by an oblique motion obtained by simultaneous operation of the fine adjustment of the manipulating microscope and of the mechanical stage controls of the observing microscope (Fig. 13). Sterilization of microinstruments cannot be effected by heat. Immersion in chromic-sulphuric acids cleaning solution, followed by washing in sterile water and in sterile ethanol, yields a clean, sterile instrument.
G. Evidence for the reliability of the methods The possibility of spreading of the organisms from the inoculation site along the surfaces of the block of agar is excluded by the demonstration of
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the sterility of the section of the block between the inoculum and the isolation sites. Such a spread of the organisms never occurs on correctly prepared blocks, except when due to carelessness in assembly. The possibility of displacement of isolates from their marked sites by water exuding from the agar during dissection of the gel does not occur provided that sharp knives are used for the dissection. This can be proved in two ways(a) Microscopical evidence. After dissection of a block with 24 isolates, followed by transfer of each portion to a second slide, each isolate can be found microscopically between its locating pits. (b) Cultural midme. Frequently it is found that each of 24 bacterial spores isolated on the same agar block gives rise to growth on transfer to a culture medium, This can only occur if each spore has been transferred to the medium on its alIotted portion of the gel. This test is only valid in cases in which all the isolates are viable. REFERENCES Dickinson, S. (1926). Ann. Bot., 40,273-274. Feinberg, J. G. (1956). Nature, Lond., 178,1406. de Fonbrune, P. (1949). “Technique de micromanipulation,” p. 130. Masson, Paris. Gardner, A. D. (1925). J. Path. Bact., 28, 189-194. Holdom, R. S.,and Johnstone, K. I. (1967). J. gen. Microbiol., 46, 315-319. Johnstone, K. I. (1943). J. Path. Bact., 55, 159-163. Johnstone, K. I. (1953). J. gen. Microbiol., 9,293-304. Koblmiiller, L.O.,and Vierthaler, R. W. (1933). Zentbl. Bakt. Purusitkde, Abt I, Orig., 129,438-446. Malone, R. H. (1918). J. Path. Bact., 22, 222-223. OIS~OV, J. (1922). J. Bad., 7,537-549. Reyniers, J. A.,and Trexler, P. C . (1943). In “Micrurgical and Germ-free Methods” (Ed. J. A. Reyniers), pp. 8-10. C . C.Thomas, Springfield, Illinois. Topley, W. W. C., Bamard, J. E., and Wilson, G. S. (1921). J. Hyg., Cumb., 20, 221-226.
The Isolation and Cultivation of Single Organisms K. I.
JOHNSTONE
Department of Bacteriology, The School of Medicine, Leeds, England I. Introduction . . 11. A Review of the Chief Methods Devised for Single-organism Culture A. Selection of an isolated organism on a randomly inoculated nutrient agar gel B. Formation of droplets with a micropipette . . C. Isolation by micromanipulation on an agar gel surface . D. Destruction of all organisms except the one selected
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111. The Agar Block Dissection Techniques . A. The agar gel . . B. Casting of blocks of gel * C. Optical equipment . . D. Use of a second microscope as micromanipulator . . E. Isolation with a simple angulated microneedle and location of . the isolates . F. Isolation with a microloop on pre-marked sites . . G. Evidence for the reliability of the methods References
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I. INTRODUCTION The cultivation of bacteria from single organisms is especially valuable in the purification of strains, since the only absolute criterion of the purity of a culture is the certainty that it has been derived from the progeny of a single organism. Failure to apply this criterion may lead to much waste of effort in research, especially in regard to the clostridia, apparently pure strains of which may carry latent contaminants for long periods. All strains upon which research is to be based should therefore be rigorously purified at the outset. The study of the properties of individual organisms, as distinct from those of bacterial populations, is a little-explored field, requiring a rapid and accurate method of isolation to enable batches of isolates to be tested, e.g., the heat resistance of bacterial spores.
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Because of the current belief that single-organism culture is necessarily tedious and involves the use of costly and complex apparatus, the technique is often shunned. However, the methods available range from those requiring only the simplest apparatus readily improvised in the laboratory, to those requiring elaborate micromanipulators and ancillary apparatus. The former are only suitable when a few isolations are to be made at rare intervals. When many isolations are required, more complex equipment is necessary to minimize fatigue of the operator. In all cases, it is essential that the method be absolutely reliable and that adequate controls are used to establish the validity of the isolations. For multiple and rapid bacterial isolations under sterile conditions, it is essential that the microinstrument should be readily removable from the sterile chamber to facilitate interchange of culture materials and should be as readily replaceable. Many of the types of micromanipulator now available are unnecessarily complex for this work and the agar gel dissection methods here described in detail do not require a conventional instrument. A simple type of microforge is, however, a necessity for the satisfactory production of microinstruments.
11. A REVIEW OF THE CHIEF METHODS DEVISED FOR SINGLE-0 RGANI SM CULTURE
A. Selection of an isolated organism on a randomly inoculated nutrient agar gel This method, employing a block cut from a lightly-inoculated nutrient agar plate (Orskov, 1922) and improved by the use of a cast agar block (Gardner, 1925), requires only the simplest apparatus, which can be assembled at very slight cost in the laboratory. The disadvantages are that (a) the method of location of the isolate by drawing a pattern of scratches on the glass slide is tedious, (b) observations on the growing microcolony may extend over many hours, (c) the subculture of the microcolony is a blind operation and (d) aerial contamination of the exposed gel may occur.
B. Formation of droplets with a micropipette 1. At an &-glass interfuce (Mulone, 1918) Using a second microscope as manipulator, microdroplets are deposited on the lower surface of a sterile coverglass and are searched for those containing only single organisms. Such droplets are each drawn into separate sterile micropipettes, the tips of which are broken off in nutrient broth tubes. The advantage lies in the use of the x 100 oil-immersion objective, which is not possible in methods operating above the air-gel interface. The
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disadvantages are (a) the difficulty in excluding the presence of a second organism at the margin of each droplet, (b) the rapid drying of the minute droplets and (c) the sacrifice of a micropipette for each organism isolated.
2. In an oil chamber (a2 Fonbrune, 1949) The coverglass forms the roof of a chamber filled with sterile liquid paraffin and droplets are formed between the glass and the paraffin by means of a micropipette, being examined at intervals until multiplication of a single organism is observed. Such a droplet is removed using a fresh sterile pipette and is cultured. The advantages are (a) the use of the x 100 oil-immersion objective, (b) the complete elimination of drying of the droplets and (c) the ease with which the successive cell divisions may be observed. The disadvantages are that (a) the charged pipette must traverse the paraffin seal during formation of the droplets and the possibility exists of contamination of the oil and subsequent contamination of the subculturing pipette, (b) a clear fluid medium alone can be used and (c) the conditions are anaerobic, and strictly aerobic organisms may not grow. The method is more suitable for observations on the process of multiplication in silu, without recovery of the resulting microcultures.
3. On a mechanically propelled Cellophane strip (Reyniers and Trexler, 1943) Microdroplets are formed mechanically on the lower surface of a strip of Cellophane, which then passes through the microscope and is taken up on a spool. When a droplet is recognized as containing a single organism, it is cut out on a disc of Cellophane by a micro-fly cutter and falls direct into a culture tube. The method attains the highest degree of mechanization, but relies on the recognition of droplets containing single organisms and requires specialized and complex apparatus. C. Isolation by micromanipulation on an agar gel sudace 1. Below the air-gel interfme (Dickinson, 1926) Using the lower surface of a coverglass coated with a thin layer of nutrient agar, enclosed to reduce drying of the gel, selected organisms are carried in turn across the agar surface in the water column formed by contact of a vertical needle tip with the gel. The advantages are that (a) the isolates are taken to sterile portions of the gel remote from the inoculum, (b) the organisms can be cultured in situ, or portions of the agar may be cut out carrying isolates and cultured separately, (c) the x 100 oil-immersion objective can be used and (d) aerial contamination is excluded. The disadvantages are that (a) there is no rapid and positive method of location of the isolates, their positions being marked with ink on the upper surface of
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the coverglass, (b) manipulations below the air-gel interface are difficult to carry out and (c) the necessarily thin layer of agar renders dissection of the gel tedious, especially if many isolations are required.
2. Above the air-gel interface (a) Technique of Koblmuller and Vierthaler (2933).The technique involves the use of a metal needle on a nutrient gel, and cultivation of the isolates in situ. Selected organisms on the upper surface of a nutrient agar gel are drawn in turn to isolated sites on the sterile surface by means of a metallic needle inclined at an angle to the surface. The selected organism floats in the water exuded from the gel around the needle tip. The isolates are cultivated on the agar surface. The advantage lies in the greater ease of manipulation on the upper surface of the gel. The disadvantages are that (a) the method of location of the isolates by ink marks is tedious, (b) a metal microneedle is less easy to prepare than one of glass and (c) a dry objective must be used.
(b) Technique ofJohnstone (2943,2953). The technique involves the use of a glass needle and positive location of the isolates on a non-nutrient gel, which is subsequently dissected. Selected organisms are carried across the upper surface of a cast block of clarified non-nutrient agar gel, with an angulated glass microneedle. The location of each isolate is defined by melting two pits in the gel surface, the organism lying between them. The isolates are cultivated by dissection of the gel, as shown by the pairs of pits, and transfer of portions, each carrying a pair of pits and therefore an organism, to separate tubes of medium. The advantages are that (a) a glass microneedle is used which is readily prepared, (b) manipulations are carried out on the plane upper surface of the gel and are readily observed, (c) the method of location is positive and can be made rapid by mechanical means and (d) any type of culture medium may be used for cultivation of the isolates. The disadvantage, in common with other methods operating above the air-gel interface, is that immersion objectives cannot be used. (c) Technique of Holdom and Johnstone (2967).The technique involves the use of a glass microloop on a non-nutrient gel with pre-marked isolation sites and subsequent dissection of the gel. The cast block of agar gel is pre-marked with 24 isolation sites in a sterile punch. With a glass microloop, prepared in a high-power microforge, selected organisms are carried rapidly above the gel to the isolation sites and are there ejected from the loop on to the agar surface. Dissection of the gel and cultivation of the isolates follow. The advantages are that (a) the isolations are carried out rapidly and (b) when many isolations of the same
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strain are required, the microloop can be charged with 6-12 selected organisms which are deposited singly on the isolation sites, with great saving of time. The disadvantage is that the microloop must be prepared in a high-power microforge, but such a loop will serve for several thousand isolations.
D. Destruction of all organisms except the one selected This method (Topley et al., 1921)is unique, in that the selected organism, located in a gelatin film under a quartz coverglass, is protected by a globule of mercury on the coverglass from ultraviolet radiation, which kills all other organisms in the film. After incubation, the resulting microcolony is subcultured. The advantage lies in the simple apparatus required. The disadvantages are that (a) careful controls are necessary to establish the validity of each experiment, especially in the presence of spores, (b) incubation is at 25°C and (c) the method is not suitable if many isolations are required. 111. THE AGAR BLOCK DISSECTION TECHNIQUES A. The agar gel The gel must be free from both living and dead bacteria, the latter being abundant in most samples of agar and causing confusion during micromanipulation. They are removed by clarification of a fluid 2.0% (w/v) aqueous solution of New Zealand agar with 2.0% (w/v) of Hyflo Super Cel diatomaceous earth (Koch-Light Laboratories Ltd, Colnbrook, Buckinghamshire) at 60°C for 4 days, with gentle inversion of the bottle twice daily (Feinberg, 1956). This is followed by filtration through Hyflo Super Cel sandwiched between layers of paper pulp and yields a gel free from bacteria, when autoclaved. The reaction should be pH 7.0-7-4. No nutrients are added, since multiplication of the organisms is not desired during manipulations, and a batch of agar can be stored in sealed ampoules for several years, being used for manipulation of a great variety of bacteria. After trial with the microinstrument, the concentration of agar is lowered, by adding water, to the optimum for the method of isolation used. B. Casting of blocks of gel A clean 7.6 x 2.5 cm glass slide is separated from a clean 7.6 x 3.8 cm slide by two glass strips 2.0 mm thick to form a casting cell (Fig. l), the slides being held in apposition to the strips by wire paper clips and the whole sterilized by dry heat in a Petri dish. Diamond-ruled lines on the outer surfaces of the cell serve as guides in sectioning the agar slab. Clarified agar at 100°C is pipetted into the casting cell at 60°C and, when set, the excess of gel beyond the margin of the smaller slide is removed with
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a sterile scalpel. The slides are drawn apart with a sliding motion and the gel, which remains adherent to one slide, is cut into blocks 2.5 x 1.3 cm and 2.0 mm thick. Each block is lifted with the back of the scalpel blade and is
F I ~1.. A glass casting cell for the agar gel. The portion occupied by the gel is
stippled.
0
I
I
5 1
cm
FIG.2. The Perspex cell and coverglass for protection of the agar block during manipulations. A, the access slot for the microinstrument, or marking needle; B, the coverglass.
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deposited on a separate sterile 7.6 x 3.8 cm slide, enclosed in a Petri dish with a Duralumin humidifying trough containing glass wool moistened with distilled water. A t all stages it is essential that thegel beprotectedfrom desiccation, as otherwise the plane upper surface will be &formed and the concentration of agar will rise, producing a surface unsuitablefor micromanipulation. Protection of the agar block from airborne contamination and from desiccation during manipulation is provided by a sterile Perspex cell (Fig. 2), which rests on the supporting slide around the block and carries a 5.5 x 2.5 cm No. 0 coverglass. For optical system 1, the lower surface of the coverglass is 0.9-1.0 mm above the gel surface, but for system 2, this height can be greatly increased, thus allowing more working space for the needle tip. A slot, A, (Fig. 2) gives access to the shaft of the microneedle, a second slot at the opposite end of the cell giving access to the platinum marking needle.
C. Optical equipment Phase-contrast microscopy renders earlier methods, including darkground illumination, obsolete. Positive phase contrast shows bacteria clearly on the gel surface as dark objects against a green background, except for the mature spore, which appears bright before germination. The gel surface appears faintly mottled. Two systems may be used.
1. A refroacting phase-contrast objective x 40 The numerical aperture (N.A.) must be at least 0.65, and the working distance 1.0 mm, excluding the coverglass thickness. This has the mechani-
cal disadvantage of a very confined space for operation of the microneedle and also of condensation on the lower surface of the coverglass, requiring a warm-air jet playing on the upper surface of the glass to disperse the water droplets. With a high-absorption phase-plate, the Parachromatic objective x 40 (Watson) gives good resolution.
2. A Dyson long-working-distance phase-contrast objective x 40 (VicRers Instruments) Incorporating both reflecting and refracting units, this overcomes both mechanical and thermal disadvantages with a working distance of 12.8 mm, but the N.A. is reduced to 0.57 and the light intensity must be greatly increased to compensate for losses by reflection, with possible lethal action on sensitive organisms. The phase-contrast condenser must also have a long working distance, to operate through the slide and a 2 mm thickness of agar gel. The illuminant should be of high intensity with a cooling trough, followed by a Chance’s ON 20 heat-absorbing filter, and a yellow-green filter to improve optical performance.
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D. Use of a second microscope as micromanipulator A second microscope on a smooth and rigid bench surface contributes the sensitive vertical movement to the microinstrument through its fine adjustment. A nosepiece attachment (Fig. 3) is screwed firmly into the objective thread and the bar A is clamped by the screw B so as to project towards the axis of the observing microscope. This bar is bored to receive the microneedle holder, or the platinum marking needle, as required. Centration of the microneedle tip in the field of the observing microscope is effected by
FIG.3. The nosepiece attachment (left), the microneedle holder (centre) and the microneedle handle (right). C, the r.m.8. objective thread; D, the clamping screw for the microneedle holder; E, the clamping screw for the microneedle.
sliding the manipulating microscope on the bench by finger pressure from both hands applied to the foot of the instrument, while movement of the tip is observed in the field. Movement in the horizontal plane is obtained by means of the mechanical stage of the observing microscope, which carries the agar block on the sterile slide and therefore gives motion in two dimensions to the gel surface relative to the needle tip, which remains centred in the field.
E. Isolation with a simple angulated microneedle and location of the isolates The selected organism is carried across the sterile gel surface in the minute pool of water that exudes from the gel around the glass microneedle where it is in contact with the gel. The isolate is under constant observation during its passage and the presence of a second organism of comparable size cannot be
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overlooked. This method is especially suitable for pure strain isolations, when only a limited number is required.
1. The angulated microneedle Soft-glass rod, 6 mm diameter, is drawn out by hand to 0.9-1.0 mm diameter thus forming the handle of the microneedle. The thin rod is again drawn out after being heated in a minute coal-gas flame burning at the tip of a glass capillary tube, to form the needle shaft 3.8 cm long and 0.15 mm diameter at the free end (Fig. 4a). The tip (Fig. 4b) is formed in a low-power
FIG.4. (a). An angulated glass microneedle. A, the handle; B, the shaft; C, the tip. (b). The tip of the microneedle enlarged, showing its spatial relation to the coverglass, D, and the agar gel surface, E. The distance F is the clearance of the shaft above the gel.
microforge (Johnstone, 1953) and consists of a portion tapering rapidly from 150 pm to 20 pm at an angle of 60" to the axis of the shaft, and a point inclined at 30" to the same axis, tapering rapidly to 1.5 pm at its tip. The clearance of approximately 400 p m is essential to avoid contact between the horizontal shaft and the gel surface, to which it would adhere owing to surface tension. The shaft must be straight and in the axis of the handle.
2. The platinum marking needle This forms the locating pits in the gel surface on each side of an isolated organism, by means of which the site can readily be seen during dissection
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of the block. The V shaped heating element (Fig. 5 ) is made from two 2.7 cm lengths of 40 s.w.g. platinum wire, welded to 24 s.w.g. platinum wires as supports. At the apex of the V, one thin wire is hooked around the second wire 2 mm from its free end and the joint is welded, the terminal 0.5 mm
FIG.5. The platinum marking needle: A, the heating element; B, the supporting platinum wires; C, the copper leads; D, the glass tubes.
of the single wire being bent vertically downwards to form the marking tip. The platinum needle can be mounted in one of two ways(a) A simple mount, suitable for occasional use consists of two soft-glass tubes, 4 mm external diameter, bent at right angles. The thick supporting wires of the platinum element are first silver-soldered to 24 s.w.g. copper leads and the platinum wires are then sealed into the ends of the glass tubes (Fig. 6). The vertical ends of the tubes pass through a cork, E, pressed firmly into the nosepiece of a manipulating microscope, whose focusing movements are used to form the pits in the gel surface. Connection is made from the copper leads to a 3 V a.c. supply through push switches S1 and Sz (Fig. 7), with variable
FIG.6. A simple glass mount for the marking needle. The cork, E, fits into the objective thread of a microscope nosepiece. The electrical leads, F, pass through the cork.
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resistors R1 and R2,respectively, in series. R1 is adjusted to bring the element to red heat on closing S1,the tip being sterilized by conduction. R2 is adjusted to warm the element sufficiently to melt a pit in the gel surface when SZ is closed, without causing the gel to boil.
FIG.7. Heating circuits for the platinum marking needle.
(b) A permanent mount interchangeable with the microneedle holder in the nosepiece attachment is shown in Fig. 8. The steel rods, A and B, carrying the platinum element, are electrically insulated from the stem C, which enters the nosepiece attachment. This mount can with advantage be fitted with adjustable pneumatic dipping and traversing movements and can itself be mounted on a rigid geometric slide replacing the second microscope. The unit may then be withdrawn from the optic axis and rapidly replaced in centre. Also, the locating pits can be formed, as described below, on each side of the isolate while the glass microneedle remains in situ and with the x 40 objective in position, thus greatly reducing the time taken.
FIG.8 A permanent mount for the platinum marking needle interchangeablewith the microneedle holder in the nosepiece attachment. Electrical connections are made to the rods A and B at D.
3, Isolation procedure During inoculation, the sterile surface of the agar block is protected from bacterial aerosols, created by the inoculating loop, by a sterile metal shield
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covering all but a strip 2 mm wide at one end. With a 1 mm loop, an inoculum from a just visibly turbid suspension of the organisms is streaked across the upper surface of the gel at the exposed end. The fluid is rapidly absorbed, leaving the organisms on the gel surface and the shield is then removed. The block is covered with the sterile Perspex cell, the slide is placed on the mechanical stage of the observing microscope, and the margin of the inoculum is located by dark-ground illumination at x 150 magnification
FIG.9. Isolation of a selected organism by trailing across the agar gel surface with an angulated glass microneedle. The arrows indicate the direction of movement of the gel.
(obtained by the use of a x 10 objective with the x 40 phase-contrast condenser annulus). The microneedle, mounted in the carrier on the nosepiece of the manipulating microscope, is carefully directed into the access slot of the Perspex cell by sliding the manipulating microscope on the bench, taking care to clear the coverglass above and the gel below. The tip is located as it enters the field and is centred first with the x 10 and then with the x 40 objective, while it is poised above the gel surface. By operation of the coarse-focusing movement, the needle tip is lowered until it is just above the gel surface. A selected, well-isolated organism is brought by means of the stage controls to lie below the needle tip and the latter is then lowered, with the fine-focusing movement, to touch the gel, when the organism floats in the exuded water (Fig. 9). By means of the stage controls, the organism is then carried rapidly over the gel surface, while it and the needle tip remain centred in the field. If the organism escapes from the needle, it is located by following in reverse the needle track, which is visible as a bright line on the
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gel, until the organism is found. The needle tip must be raised above the gel during reverse movement. When the isolate has been carried at least 5 mm from the margin of the inoculum, its position is defined by melting two shallow pits in the gel surface 1 mm apart, one above and one below the organism, with the sterile platinum marking needle. The x 10 objective is used unless the pneumatic device for controlling the platinum needle is available, when the x40 phase-contrast objective can be used and the marking carried out in the presence of the glass microneedle, with great saving of time. Further isolations, up to a total of six, are then made on the same block and should be staggered (Fig. 10a) to facilitate dissection of the gel.
FIG.10. (a). A completed agar block showing the routes taken by six isolates trailed in turn with an angulated microneedle from the inoculum. (b). An agar block premarked with 24 isolation sites for use with a microloop. In both parts of this Fig., the shaded areas are those tested for sterility, the stippled areas carry the inoculum and the broken lines indicate the dissection of the gel.
The concentration of agar must be adjusted, after experiment, for each batch prepared. If too high, the organism will not follow the microneedle: if too low, the excess water exuded around the needle tip will cause displacement of adjacent organisms. 4. Dissection of the block and culture of the organisms The completed block, on the supporting slide, is transferred to the stage of a stereoscopic dissecting microscope of magnification x 5. Aerial contamination is excluded (1) by a cabinet enclosing the stage with a roof of
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plate glass immediately below the objectives, and preferably also containing a low-pressure mercury-vapour lamp to sterilize the contained air before use, and (2) by a sterile plastic shield resting on the stage around the slide, with an access slot for the dissecting knives and a central aperture, sealed by a coverglass, below the objectives. The agar block is dissected with sterile, sharp, triangular stainless-steel knives made from safety razor blades soldered into brass handles (Fig. 11).
FIG.11. A knife used in dissectionof the agarblock. Below, the tip enlarged.At A, a portion of a stainless-steel safety razor blade is soft-soldered into the brass rod. The site of the inoculum is first separated and is removed with a platinum scoop. The adjacent section of the gel, A (Fig. lOa), is then isolated and transferred to the optimal recovery medium. Unless this is shown to be sterile, the isolations are not valid. The remainder of the block is then systematically dissected and small portions of the gel, each with a pair of locating pits, and, therefore, an organism, are transferred in turn to separate tubes of the medium for incubation. Single organisms require an optimal medium and optimal conditions of reaction, oxidation-reduction potential and temperature,if a highproportion of the isolates are t o p o v e viable. An advantage of this method is that the medium can be fluid or solid, clear or opaque, depending on the nature of the organism. If solid, the small block of agar is deposited with the platinum scoop on to the medium, pits downwards, the isolate being trapped between the gel surfaces and growth appearing around the margins of the block if the organism is viable.
F. Isolation with a microloop on pre-marked sitea This method has the advantage of great speed and is of especial value when many isolates of the same strain are required. The microloop (Fig. 12) is formed in a high-power microforge at the tip of a needle shaft prepared as for an angulated microneedle. The loop is made from a terminal glass
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filament 1.0-1-5pm diameter and measures internally from 3 x 14pm to 14 x 8pm, depending on the size and the number of organisms to be carried. The loop, which is elliptical in shape, must lie in the plane of the gel surface. For maximum speed, the sites for 24 isolations are punched in the surface of an agar block by a group of 48 steel needle points, mounted in an enclosed sterile punch. The sites, at 3 mm intervals (Fig. lob), are in
FIG.12. The tip of a glass microloop in relation to the agar gel surface. A, the shaft; B, the terminal glass filament;C, the loop lying in the plane of the gel surface, which is stippled; D, the clearance between the shaft and the gel.
register with the readings of the mechanical stage of the observing microscope. After inoculation of one end of the upper surface of the block, selected organisms are picked up with the microloop, to the interior of which they adhere firmly by surface tension when raised above the gel. For the isolation of pure strains, organisms are carried singly in the smallest practicable loop to their respective isolation sites, and are there discharged by slight vibration imparted to the bench while the loop is in contact with the water exuded from the gel surface. For multiple isolations from an already pure strain, 6 or 12 organisms are picked up successively by a larger loop, which is then lowered on to the first site. By slight vibration of the bench, one or more organisms are ejected from the loop and remain on the gel surface as the loop is raised. If more
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than one has been discharged, the excess organisms art again picked up, leaving a single isolate between the pits. The remaining sites are treated in the same way, the whole block of 24 isolations being completed in less than 8 min with a suitable microloop. Dissection of the block and transfer to the culture medium is carried out as for the angulated microneedle technique. In the case of large spores, such as fungal spores, a large microloop will readily pick up the spore, but difficulty may be experienced in releasing it. This can be overcome by using a loop with a strong supporting filament and sinking the loop below the surface of the gel. As the agar rises through and
FIG.13. (a)-(d). Four stages in the deposition of a fungal spore on the agar surface. The vertical arrows indicate movement of the microloop : the horizontal arrows indicate simultaneous movement of the gel. Diagrammatic-not to scale.
around the loop, the spore is carried on the surface of the gel. The microloop is then extracted from the gel by an oblique motion obtained by simultaneous operation of the fine adjustment of the manipulating microscope and of the mechanical stage controls of the observing microscope (Fig. 13). Sterilization of microinstruments cannot be effected by heat. Immersion in chromic-sulphuric acids cleaning solution, followed by washing in sterile water and in sterile ethanol, yields a clean, sterile instrument.
G. Evidence for the reliability of the methods The possibility of spreading of the organisms from the inoculation site along the surfaces of the block of agar is excluded by the demonstration of
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the sterility of the section of the block between the inoculum and the isolation sites. Such a spread of the organisms never occurs on correctly prepared blocks, except when due to carelessness in assembly. The possibility of displacement of isolates from their marked sites by water exuding from the agar during dissection of the gel does not occur provided that sharp knives are used for the dissection. This can be proved in two ways(a) Microscopical evidence. After dissection of a block with 24 isolates, followed by transfer of each portion to a second slide, each isolate can be found microscopically between its locating pits. (b) Cultural midme. Frequently it is found that each of 24 bacterial spores isolated on the same agar block gives rise to growth on transfer to a culture medium, This can only occur if each spore has been transferred to the medium on its alIotted portion of the gel. This test is only valid in cases in which all the isolates are viable. REFERENCES Dickinson, S. (1926). Ann. Bot., 40,273-274. Feinberg, J. G. (1956). Nature, Lond., 178,1406. de Fonbrune, P. (1949). “Technique de micromanipulation,” p. 130. Masson, Paris. Gardner, A. D. (1925). J. Path. Bact., 28, 189-194. Holdom, R. S.,and Johnstone, K. I. (1967). J. gen. Microbiol., 46, 315-319. Johnstone, K. I. (1943). J. Path. Bact., 55, 159-163. Johnstone, K. I. (1953). J. gen. Microbiol., 9,293-304. Koblmiiller, L.O.,and Vierthaler, R. W. (1933). Zentbl. Bakt. Purusitkde, Abt I, Orig., 129,438-446. Malone, R. H. (1918). J. Path. Bact., 22, 222-223. OIS~OV, J. (1922). J. Bad., 7,537-549. Reyniers, J. A.,and Trexler, P. C . (1943). In “Micrurgical and Germ-free Methods” (Ed. J. A. Reyniers), pp. 8-10. C . C.Thomas, Springfield, Illinois. Topley, W. W. C., Bamard, J. E., and Wilson, G. S. (1921). J. Hyg., Cumb., 20, 221-226.