I REVIEW OF FUNDAMENTAL DEVELOPMENTS IN ANALYSIS
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I Microbiological Assays I I 1 [
S. H. HUTNER, Haskins laboratories, New York 17, N. Y. AMADEU CURY, Institute of Microbiology, University o f Brazil, Rio de Janeko, Brazil HERMAN BAKER, Department of Chemistry, Mt. Sinai Hospital, New York 29, N. Y.
assays apply when a microbe responds to a metabolite for which physical and chemical determinations are neither sensitive nor specific enough; fruitful applications usually betoken intimacy between chemist and biologist. Nevertheless, we here address the chemist who, needing a microbiological assay, but only as one tool among others, has to arrange his own equipment and thinking to use or develop an assay method, After outlining the principles underlying present assays, we discuss how to develop new ones. The thesis is that chemist and microbiologist (and, increasingly, clinician) will be making greater and more reasonable demands on each other. Gavin’s exposition of analytical microbiology (76-78) includes biocidal materials, the subject of a recent book (87)). Our account centers on procedures exploiting positive growth responses. The broadest scope of microbiological assays is defined by the unity of biochemistry: all creatures use much the same assortment of elements and lowmolecular compounds. There are exceptions-e.g., steroid and epinephrine hormones seemingly play no part in the essential metabolkm of microbeswhich is to say these hormones cannot yet be assayed microbiologically, although such assays are badly needed, ICROBIOLOGICAL
STATISTICS AND BASAL MEDlA
Microbiological assays obey the canons of analytical chemistry. The central reagent being alive does not alter matters-e.g., recoveries are done as one test of accuracy. If a culture medium, the basal medium, supports no growth minus a substance and full growth with it, the plot of growth us. concentration of substance should be linear over an appreciable range of concentrations if inhibitors are absent. Concentration of unknown is obtained as the ratio of the slopes of the linear portions of the curves of unknown and known or the ratio of the intercepts on the Y axis when concentration is the abscissa and growth the ordinate. To shorten arithmetic, growth should
be expressed in units linearly proportional to increments in mass organismse.g. (absorbance optical density), not per cent transmission, when growth is measured turbidometrically. Sample calculations are in many of the books and papers cited here. The many papers on microbiological assays which exalt precision while being casual about accuracy moved Hamilton (98) to assert: “His [Hamilton’s] credo was that a bio-assay was only good when the growth response was linearly proportional to the concentration of the substance to be estimated and that logarithmic plots of bio-assays were mathematical smoke screens calculated consciously or unconsciously to conceal a variety of variables that could not be measured. Patently, if one Euglena required 4800 molecules of vitamin BI2, two Euglenas required 9600 molecules, three Euglenas. . .etc., then this uncomplicated relationship was linear. Any departure from linearity a t the upper end of the response curve indicated intrusion of other variables and a t this point the validity of the assay ended. He believed this generalization to hold no matter what kind of response was used-e.g., acid production by lactobacilli; it was precisely at the point where acid production impeded growth or other limiting factors came into play that these assays lost validity. I n certain amino acid assays, it was true that unidentified factors bent the curves away from linearity, affecting both the curves for the amino-acid standards and the assayed amino-acids in simplified materials such as protein hydrolyzates in approximately equal measure. But he was inclined to be more suspicious of the results when complex materials were assayed, where there was less reason to suppose that the variables encountered evenly affected both standard and assayed material. He was not satisfied that an assay was suitable for general use until the response curve was linear from the origin. The region near the origin was especially informative because not only did it indicate the extent of carry-over and background contamination, but it also clearly brought out imperfections in the basal medium. Lag phase phenomena and induction periods, showing up as concavities in the curve, were signs that the medium was not complete enough for a clean-cut
response to :t single metabolite. These inadequacies in the basal medium tended to be blotted out by higher concentrations of natural materials and might allow a misleading linearity of response. These remarks applied only to bio-assays1 in liquid media.” In a few assays linearity extends over a 50-fold or greater range of concentration; it is then occasionally convenient to plot gron t h us. log concentration of unknown. I n discusidng mathematics in biology, Hamihon et al. (99) note that statistics generate neither specifications nor criteria. So many variables enter into developing an assay that biochemical and microbiological knomledge and intuitions are the real guides. Tests of an a.ssay method should include runs with a t least one other organism having a different nutritional pattern and specificity towards the kind of compound being assayed; for obvious reasons the rat and chick are used where they need the same compound. Routine checking against physical or chemical methods is seldom practicable except for standards. Fixation on barely adequate basal media and preoccupation with one organism have inspired prodigies of precision, while accuracy, ofhen good enough for purified materials, remained unsatisfactory for natural crudes such as cells or blood. To lend concreteness to these dicta, some vicissitudes of the Euglena assay for vitamin BIZ will be sketched. This example WBS selected because every general situation in microbiological assays is represented in the story of Blz and, as with other bioassays, its popularity fluctuates with changing biochemical knowledge. The first basal medium of Eugglena contained as main fuel and carbon sources (substrates) butyrate and glutamatc (1.24). The linear growth response extended over only a &fold range of co~centrationof Bl2 and only up to an aksorbance -1.0; the assay took about 10 days and there was a bad drift when natural materials low in B1s were assayed-i.e., growth was appreciably increased by materids other than BIZ. With better substratesmalic and glutamic acids at lower p H VOL. 30,
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(123) (substrates had been the main limiting factors coming into play after Bla, and they had been supplied unpredictably by natural materials)linearity held up to absorbances of 1.8, and over a 10-fold concentration range; drift lessened, and growth was substantially complete in 8 days. More assays could now be completed in one run without preliminary ranging. Later, Euglena responded to cobalamins wholly inactive for higher animals (pseudo BIZ’S) and which sometimes made up nearly all the cobalamins in fermentations intended to supply BIZ for animal and human consumption. Which to do: devise preliminary separations of “true” from pseudo B12)s or seek a more specific assay organism? As others mere studying separations, we intensified studies of Ochromonas malharnensis; in preliminary runs it had given values parallel to chick and rat assays (121). The Euglena method, notwithstanding this, was entering into wide use because of its sensitivity, and other features (described later), for measuring Bl2 in tissue fluids and fresh water bodies where pseudo B12)s did not seriously interfere or where an ultrasensitive response approaching total cobalamins was wanted. It was worth the effort to remedy some shortcomings of the Eugleiza method encountered by others. The next basal medium mas enriched with sucrose, glycine, and succinic and aspartic acids. I n 7 days, cultures reached absorbances of 8 or higher, linearity held to absorbances of 4 and even ti, through a 100-fold or greater concentration range, and the pH during growth remained constant, eliminating a precipitation of serum proteins bothersome in assays of blood. Also a more vigorously heterotrophic Euglena was used (117, 214). New complaints were voiced: Some samples of blood serum low in BIZ, and which therefore had to be used a t not more than 1 to 10 or 1 to 20 dilution, showed a bad drift upwards-i.e., the sera contained still another growth stimulant. Unpublished work with Ross (218) permits hope that this drift will be eliminated by putting lactic acid in the medium, making the assays some hours faster. Adding nutrients stepwise to a basal medium, each bettering growth, can go on indefinitely, up to the osmotic tolerance of the organism. In effect, one gives the organism prefabricated building blocks, bypassing cellular syntheses that are successivelimiting factors for growth; as one synthesis is bypassed and so removed as a bottleneck, another comes into play. A point can be reached where growth is better (faster or finally greater) in the new chemically defined basal medium than in the initial medium supplemented with crude natural materials. Natural
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materials added to the new chemically defined medium may then promote still better growth. Where to halt this microbiological .4chilles in chasing the tortoise? If diverse crude natural materials supplied at levels nearing the limits the organism can tolerate do not increase find growth >lo%, the basal medium is seasonably satisfactory. Actually, stimuhnts in natural materials are nearly always diluted out, except when an :issay is pushed to the limit of sensitiirity in assaying lowpotency materials. Then we were informed that a new Escherichia coli inutant (86) is a t least twice as sensitixo as the mutant previously used. 0 ther microbiological Blz assays, descr bed later, have special advantages. One Blz assay can be checked against :mother. There are otkcr incentives for improving basal media. With media that support heavy grAwth, error from background contamin2tion in chemicals and glassmare is prc portionately reduced. Furthermorp, tho inoculum culture need not be drastically depleted; hence vigor is improl cd. Heavier inocula can be used which favors uniformity and speeds growth, and aseptic washing of inocula (all weptic operations are hazardous in d(1ing assays!) can be omitted. Although blanks may be higher, experimer la1 error is decreased. APPARATUS AND GENERAL TECHNIQUE
This review is :ittempted as a guide to literature and procedure, not a compendium of methods. It also looks a t routine practicee that bear on the practicability of microbiological assays. Pipets. Microbiological assays require an abundEuce of pipets; they get hard use. lVith ordinary care they wear out by mechanical and chemical abrasio 1 rather than breakage. The thicker the walls and harder the glass the bett :r, as graduations can be incised deeplir without weakening the pipet; the deeper the incisions the longer graduation:; stay readable. Graduations should e.ctend to a base line (Llohr type) and have a permanent filling. Ilimble amber-filled pipets are excellent. Lime-&tss pipets quickly etch into uselessuess. Experience has shown the followitig sizes to be useful: 1.0-ml. graduated in l/looth’s; and 2-, 5-, and 10-ml. graduated in For cleaning borosilicate pipets, household detergents ara satisfactory. Less alkaline, special 1:tboratory detergents are useful for glassware which is washed by hand or whick is dirty with resistant material-e.g. dried blood. For inoculating cultures some workers prefer hypoder iiic syringes to pipets. The 5- and 10-nil. Luer-Lok type is sturdy. They arc? sterilized disassembled in tubes, like pipets. For assays
on a very large scale, bacteriological pipet ting machines-e .g., those made by Baltimore Biological Labs. and Sorvall-are useful and call for very little dexterity. Nonsterile pipets for making up solutions are not plugged with cotton. They are stored on Lane pipet racks. After use, pipets are immersed tip down in detergent contained in polyethylene pails, gathered in a stainlesssteel basket (Technicon) ; the basket is plunged into detergent in a stainlesssteel cylinder (Metalsmiths, Orange, N. J.), set on hot plate. The vertically held pipets are subjected to violent convection currents as the detergent is brought to a boil. The carrier basket is then rinsed in an automatic device (Technicon) and the pipets are allowed to drain dry while warmed. A compact automatic pipet washer (Virtis Co.) is available. Pipets for aseptic operations-e.g., for inoculation of assay media-are cotton .plugged and autoclaved in individual borosilicate glass tubes open a t both ends. One end of this tube is crimped to retain a permanent plug of borosilicate glass wool. The head end has a cotton plug. After autoclaving, the damp pipets and tubes dry on standing overnight. The cotton plugs for tubes are used over and over. This procedure eliminates a tedious wrapping of individual pipets in paper and avoids the great risk of contamination when many pipets are used from the same container. Sterilization by dry heat wastes fuel and fouls pipets with decomposition products of cotton. Long-staple nonabsorbent cotton is sold by the Rock Cotton Co., Janesville, JJ7is. If there is much condensation on autoclaving, materials being sterilized are covered with towels. Sealable Glassware. Screw-capped borosilicate glass tubes (Corning or Ilimble) are recommended for maintenance of stock cultures: Media dry out more slowly, and the tubes are practically mite-proof and easier to manipulate than cotton-plugged tubes. Caps with rubber liners (commercially available) screw tight enough for liquid cultures to be mailable. Liquid basal media, usually made up 2X or more final strength, are conveniently stored in borosilicate glass-stoppered bottles to retain volatile preservative. Frozen bottle stoppers (a very common hindrance) can be safely unfrozen by running a flame around the neck of the bottle, Use of glass heating-tapes for unfreezing joints is recommended (169). The polyethylene standard-taper stoppers now available may well be less prone to freeze. Amber-glass containers should be used for media containing light-sensitive compounds such as riboflavin and pteridines. Contact of solutions with cotton should be avoided; l i e most natural materials, cotton is
freighted with appreciable amounts of such growth factors as biotin ( I n ) , thiamine, p-aminobenzoic acid, and folic acid (22.4). Flasks. Aerobic organisms have seldom been used as assay organisms but are likely to become much more widely used (see section on assay organisms). If one wishes to avoid the use of shakers or roller-tube apparatus, thin layers of media are necessary. Kimble micro-Fernbach flasks are excellent but expensive; they have a broad, heavy base and so are less liikely to tip over; a simple technique serves for handling them aseptically (118). The Kimble Co. makes glass caps for these flasks, for assay of organisms grown photosynthetically. A technique based on large tubes is described in the section on ciliates. A shaker with Lucite racks (New Brunswick Scientific Co., New Brunswick, N. J.) has been made for the Ochromonas Blz assay. Cleaning Glassware. Small items of glassware can be cleaned by immersing them in detergent solution brought to a boil in aluminum or stainless-steel pots. Chromic-sulfuric slurry is used for burning away cotton plugs jammed in the necks of pipets. The pipets now available with constricted necks eliminate this annoyance. Chromicsulfuric mixture is dangerous for routine use and works poorly on grease. The polyethylene pipet baskets now made are not intended to withstand boiling water. The availability of heat-resistant polyethylene raises the hope that pipet baskets will be made of it, decreasing pipet handling. If glassware is cleaned on a large scale, an investment is justified in more ehborate and flexible equipment such as that made by Heinecke Instrument Corp., 2035 Harding St., Hollywood, Fla. The Amherst automatic tube-rinser saves labor if many tubes are used. Water. Rinsing glassware in ordinary distilled or glass-distilled water is commonly advised. Metal pipes give off iron, copper, and other potentially inhibitory heavy metals. Heavy-metal toxicity may be eliminated by adding water-soluble chelating agents such as hydroxyethylethylenediaminetriacetic acid and the like to basal media which are supplemented with minimal concentrations of the essential heavy metals to compensate for sequestering by the chelators (12285). It is worth the effort to design media which embody a large margin of safety in this respect; the analyst then is less a t the mercy of heavy-metal contaminations of nutrient chemicals and of assay samples-e.g., a marine alga 10-6.M cupric was inhibited by 2 ion but only by 5 X lO-31lf of the copper EDTA complex (2.40). The solicitude about the purity of water
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contrasts oddly with the faith in the purity or uniformity of nutrient chemicals. Chelated media have served to rear fastidious algae (100). Chlorinated water seems harmless for cleaning purposes and has the advantage of tending to destroy growth factors retained on glassware or present in the water (121). Water purified by industrial-scale ion exchangers is cheaper than distilled water and is probably satisfactory for nearly all assays. Various kinds of plastic pipe are being developed; some are already satisfactory for cold rrater, allowed by building codes, and should yield a clearer tap water. Hot-water tanks are apt to be corroded and give off dangerous amounts of heavy metals; aluminum or glass-lined steel tanks provide much cleaner water. Distilled water is used for solutions and culture media, Present commercial laboratory-scale metal stills waste fuel and water because of skimping on heat exchange; it would be economical in the end to employ efficient condensers, such as those devised for preparation of fresh water from sea water. A compact electrically heated still (Loughborough Glass Co., Ltd., Loughborough, Leicestershire, England) has a well-baffled glass condenser and so may be expected to yield a very pure water. The condenser might be replaceable by one of Vycor, quartz, Teflon, or high-temperature polyethylene, should an even purer water be desirable-e.g., when assaying trace elements. Baird & Tatlock also makes all-glass stills. Little use seems to have been made in the United States of water condensed from house steam; devices (Townson & Mercer) for making a good product in this way are advertised in British journals. Polyethylene carboys are excellent for storing mater; unlike glass, polyethylene keeps water nutrient-free. Sterilization. Sometimes directions for steam sterilization of assay media read “10 pounds for 10 minutes.” This is unsafe. It may work for acidic assays that employ media and such fast-growing organisms as Streptococcus faecalis and most lactobacilli, which quickly make much acid and so kill off contaminating organisms other than lactobacilli. Thirty minutes a t 118’ to 121’ C. kills resistant spore formers and drives off volatile preservative. Some new gas, electric, and steam autoclaves have automatic safety and timing devices, making them safer and simpler to use. Autoclaves operating off house steam may be useless in summer or when power plants undergo repair. For small scale work, domestic pressure cookers are fairly satisfactory, but before long they need tinkering with gaskets or wingbolts; they are a poor substitute for an autoclave. Bacteriological autoclaves are versatile;
operations t Tat call for prolonged refluxing a t 100’ C. or lower go faster in the autoclave; this situation arises when samples must be hydrolyzed. An autoclave’s performance should be checked with a thermometer set in the exhaust line (air is heavier than steam) and Ey such devices as temperature-sensitive crayons or sealed tubes of approprixtely melting crystalsi.e., succinic anhydride (TAT indicator, Baltimore Biological Laboratories). Volatile Preservative. Fluids awaiting antilysis and stock solutions may be protected against microbial action by a preservative removable upon steam sterilization. Toluene, sometimes used, is a fine food for some common h l drocarbon-oxidizing bacteria. Our Erst volatile preservative (119), a mixture (by volume) of 1 part of o-fluxotoluene, 1 part of 1,2dichloroetharie, and 2 parts of n-butyl chloride, waE superseded by a mixture in which the expensive fluorotoluene (boiling point 114’ C.) was replaced by chlorobenzene (boiling point 132’ C.), The preservative is removed on autoclaving by the steam-distillation effect and d spensed from a dropping bottle into sll thermostable nutrient solutions, including solutions of inorganic salts One must assume that microbes will grow in any solution thermodynamically capable of supporting life; they will multiply appreciably in such nutritional deserts as distilled wrter stored in glass bottles (28285). Rigorous chemical asepsis is demanded in work with such nearly uniquitous and potent growth factors as biotin, ljI2, and p-aminobenzoic acid (28285); fingerprints can transfer enough of these vitamins to raise blanks appreciably. For working with thermolabile metabolites, a low-boiling preservative (one removable on gentle warming) wo d d be exceedingly useful. Tube Racks. Durable large-capacity racks for tubes wider than 12 mm. are not catalogued by laboratory supply houses. Aluminum and stainless steel withstand autoclaving; galvanized, tin- or cadmium-plated steel racks soon rust to pieces, Heavy stainlesssteel wire racks are made to order by the Norwich Wire Works (agent, W. W. Brett, 570 Cedar Lane, Teaneck, N. J.), Large aluminum racks for tubes up to 13 mm. in diameter for assay purposes are sold by the H. &I. Chemical C o , 1754 22nd St., Santa Monica, Calif. Brass, copper, and aluminum racks made for serological tests have holes which can be drilled larger to accommodate tubes up to 16 mm. in diameter. Tube Closures. Ordinary test tubes are sat sfactory for assays with such facultative anaerobes as lactobacilli. Boi*osilicate straight-sided tubes with sandblasted areas for VOL. 30, NO. 4, APRIL 1958
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labeling are now available. They can be autoclaved if covered with several thicknesses of thoroughly laundered cotton towels. However, if a singlewalled autoclave is used, there is likely to be much condensation, and it may be safer to use individual closures such as glass (Kimble), aluminum (A. S. Aloe Co.), or stainless-steel (17'1) (Belco Glass, Inc., Vineland, N. J.) caps. Jj7e have not tried plastic caps with stainless-steel clips, just on the market (Precision Plastic & Die Co., 8720 North End Ave., Oak Park 37, Mich.). Stock Cultures and Special Sterilization Procedures. An assay procedure should include information on how to keep the culture the longest safe time between transfers and, if possible, should include simple tests for purity. Stock cultures should be maintained in a t least two sublines, each on different media and in a different place. Otherwise, a thermoregulator sticking in an incubator (a common cause of disaster), a refrigerator failing in summer, an insufficiently autoclaved batch of conservation media, or contamination during transfer, may lose the culture. Stock-culture media especially should be carefully autoclaved. Media in screw-capped tubes should be sterilized with the caps slightly loosed to admit steam, allowed to cool to room temperature in the closed autoclave, and the caps tightened immediately when the tubes are removed. Past cooling augments the intake of dust entrained in air; if air comes in slowly, taking say, 5 hours to reach equilibrium, the met walls of the autoclave efficiently trap dust. To lower the microbial count of air a t the inoculating bench, it is highly advantageous, especially in cities, to mount an ultraviolet lamp above eye level; Hanovia lamps are excellent. Such lamps make ozone (poisonous!) and therefore the inoculating area must be well ventilated but not draughty. Floors should be waxed often to trap dust. If contaminations are serious, despite no obvious break in asepsis, counts of organisms in air by exposure of plates of nutrient agar, or of any standard agar for molds, will generally run down the trouble, usually an activity raising dust. Moldy materials such as Neurospora cultures are autoclaved before discarding. Most bacteria keep well in the lyophilized (frozen and dried) condition ($263). Lyophilization equipment is available from laboratory supply houses. Little is known about how long the bacteria used for analytical purposes stay alive after simple freezing. Yeasts and molds on agar slants kept well for 9 months at -20" C. (58). Many new domestic refrigerators have large freezing compartments, and freezer 852
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chests have become cheaper. More attention to stori ig cultures frozen is therefore warranl ad. Prolonged viability while frozen depends largely on preventing crystallization of intracellular water. The widely practiced artificial insemination of livestock has led to much work on 13 ophilization of sperm. Some of this infxmation is relevant to the maintenanc:: of large microorganisms such as protozoa, as shown by success, among others, in lyophilizing Trichoinoiias foetu 5 by sperm technique (143); glycerol (E to 10%) added to a serum medium tillowed survival as long as 128 days if the cultures were brought to -20' C. over a period of 7 hours; there wm no advantage in The authors constorage a t -76". clude that while ihe technique appears promising it s e e m unlikely that a standard procedure will do for all protozoa. Freeze-drying is reported to maintain well tb: stability of ETemotkecium ashbyi, t , yeast employed industrially for prc duction of riboflavin ( I l l ) . The best method was to lyophilize actively sporulating cultures after freezing therri a t -68" C. Miscellaneous Gadgetry. Individually trivial, collectively they eliminate much fuss. If an assay cills for dispensing a uniform volume of the same solution to many culture vessels, the Aupette (Clay-Adams, In;!.) is handy for volumes from 1.0 to 10 ml., depending on the syringe inseIted. For large scale dispensing, the br deriological pipetting machines already mentioned can be used. Wire loops for inoculation are often of Nichrome or Chromel. They burn out and must bi: replaced frequently. Platinum wire ( 2 2 or 24 gage) alloyed with 5y0 iridium (American Platinum Works, Sewark, N. J.) is stiffer than pure platinum, la:& many times longer than nickel-chroriium, and is generally worth the cost. Torsion balances sensitive to 2.0 mg. suffice for nearly all weighings. Compounding ilijredients of culture media are discuct.ed in the section on the preparation :)f basal media as dry mixes. Some working standards of vitamins -e.g:, folic a&--are kept as frozen solutions. Thich - walled borosilicate glass ignition lubes do not crack; ordinary tubes o1ten do. For writing 011 glass, crayons that withstand autodaving lessen confusion. Dixon ?hano pencils stand autoclaving bett tir than heat-resistant crayons from sur ply houses. MEASUREMENT OF GROWTH
Let us recall tliiit growth factors were identified by simple means in the classical studies of I.~voff, Knight, Pildes,
and the Wisconsin school. Visual estimations often sufficed. Simple techniques-e.g., placing tubes of yeasts in liquid media in front of cards with ruled India ink lines, then scoring growth by the distinctness in discerning the lines (264)-furthered important studies. Qualitative or somewhat quantitative tests (a favorite phase of Beijerinck's), or limit tests-e.g., does a microorganism make practical amounts of an antibiotic or growth factor? -can often be scored very simply. Before buying expensive specialized instruments, one may mind a working rule for exploratory experiments: what's worth doing is worth doing poorly; many a valuable exploration, one suspects, was aborted by counsels of instrumental perfection. Turbidimetric Methods. Recently reviewed (7'8),these methods are discussed only briefly. They have come into favor because of their speed and accuracy; they are also much more sensitive than corresponding agarplate methods, as to be expected from the hindered diffusion of nutrients in solid media. Use of the Lunietron photoelectric colorimeter has been detailed (87). Any sensitive photoelectric instrument which has a linear output is suitable. Beer's law is not obeyed by suspension having an absorbance >1.0 or so; divergence is bad with microbes the size of Euglena (103). I n such cases, cultures are diluted until Rayleigh scattering is negligible. It is nevertheless convenient to use instruments sensitive enough to measure absorbances as high as 3.5 without having to dilute; this is especially useful with bacteria. It is convenient to graph for each assay organism correct absorbances plotted against readings, the correct value being derived by extrapolation to infinite dilution. m e use a Welch Densichron equipped with a home-made light-source and cuvette-holder. A red-sensitive light probe has worked well without filters. Photometers with very sensitive photomultiplier tubes (American Instrument Co., Silver Spring, hfd.) should be equally effective. A simple device permits rapid flushing of cuvettes for wholesale determinations with the Bausch & Lomb Spectronic 20 photometer (979). Gravimetric Methods. These have seldom been used, except for Neurospora. When a n organism grows in flakes, the culture fluid remaining clear, one simply filters off the growth on a tared Gooch crucible having a filter-paper disk. To increase filtering speed, a layer of diatomaceous earthe.g., Celite-is placed on the disk. After drying, the crucibles may be weighed on a torsion balance. If this becomes routine, estimations can be speeded with a direct-reading balance.
P a d or Paper-Disk Methods. These are coming increasingly into use, above all for large-scale work. Because the methods have been reviewed (7‘7, 87), attention is called to only a few papers. Julies and Williams (131) in their detailed description of pad assays for Biz, point out that the paper disks containing the standard amounts of B I ~ can be prepared in advance. Their photograph of a pad-plate assay brings out another sufficiently appreciated advantage: Such assays provide a crude but informative index of molecular weight. Thus methionine (molecular weight, 149) produces a wide diffuse zone of growth as compared with the sharp smaller zone of B12(molecular weight, 1490). In using an organism such as Escherichia coli 113-3, which responds both to cobalamins and methionine, one can then tell a t a glance whether a natural material has enough methionine to interfere. This applies to distinguishing responses to deoxyribosides and vitamin Blz by Lactobacillus leichinannii. A large scale plate technique (50 to 150 zones) has been described (142, 229); details are given for riboflavin assay (zone reading with the help of needle-pointed vernier calipers) (228), and penicillin assay by means of the sensitive Sarcina lutea ($50). Sarcina subjlava ATCC 7465 is recommended for assay of bacitracin (269). This implies that the sarcinas and other saprophytic cocci may have unappreciated virtues for assay of antibiotics; a comprehensive study of thcm for this end is overdue. The demand for new antibiotics creates a need for very sensitive assay methods for determining blood levels of antibiotics. Sterilization of Heat-Unstable Materials. It is occasionally necessary t o assay heat-unstable materials. One way is to pipet the sample from a selfsterilizing solution, such as 70 to 95% alcohol, and hope that the solvent is diluted enough by the basal medium t o be nontoxic. Another way, applicable to microbes able to grow well in or on agar, is to add the compound as a solution or suspension in acetone (230) to a disk, and let it dry before applying to the agar. Samples of solution may be sterilized by filtration through Seitz filters. These are easier to clean than glass filters and are far less fragile. The filter pad may absorb appreciable solute. Seitz filter holders are plated with silver, an exceedingly toxic metal; nevertheless we have not heard of trouble from this source. Stainlesssteel Seitz filters are now available (Scientific Glass Co., Bloomfield, N. J.). Seitz filter pads, an asbestos composition, may add magnesium, calcium, and iron to filtrates. This offers no difficulty with media fortified with chelating
agents as described earlier. Some Seitz pads had 0.004 y of biotin per pad (17). Membrane filters would probably serve well. Bioautographs. This powerful analytical tool is the microbiological counterpart of the radioautographic techniques indispensible in studying photosynthesis and intermediary metabolism, Bioautograph technique is an extension of pad-plate technique. A paper chromatogram is pressed onto an agar plate of the basal medium seeded with the assay organism. Wherever the growth factor is on the paper, a zone of growth will appear. Instead of a strip chromatograph, an entire sheet can be pressed to the agar, which permits more efficient resolutions of complex mixtures-e.g., combined chromatographic and ionophoretic separations, a technique which separated very similar congeners of the B1? family, reviewed by Porter (196). Aseptic operations on this large scale are difficult. This has not hitherto been serious as quick-growing organisms Strep. faecalis, have been used-eg., other lactobacilli, and E. coli. By incorporating antibiotics into basal medium, slowgrowing organisms should be usable-e.g., in assays by means of fungi and protozoa, antibiotics (chloramphenicol or one of the tetracyclines) should keep down bacteria. Conversely, nystatin mill suppress molds without harming bacteria. Actidione suppresses the saprophytic molds which are among the commonest contaminants; it should be possible to find a concentration which will not seriously inhibit assay organisms other than Neurospora. Inhibitors of yeast (19) should be tried if yeasts are troublesome contaminants. Answers to special contamination problems can probably be found in the extensive literature on diagnostic microbiology. Information on effective antibiotic combinations is also coming from studies of the presurgical sterilization of the bowel. For assay organisms which are strongly reducing, detection of zones of growth is eased by incorporation in the basal medium of tetrazolium salts, which form bright insoluble dyes on reduction. Thus, tetrazolium chloride helps bring out Strep. faecalis (1SO) and E. coli (84) spots in bioautographs. Some assay organisms are so aerobic that they grow only on the surface. To achieve uniform seeding, the organism can be sprayed on the agar, much as a reagent is applied to a paper chromatogram; the conidia of fungi have thus been applied (73). Many bacteria have difficulty growing from small inocula, and so small zones may be missed. The use of cysteine as a reducing agent allowing growth of small inocula of Group A streptococci (79)is probably
applicable to lactobacilli and other organisms. Dry Mixes for Basal Media. pH. Some basal niedia are complicated and laborious to compound and they are likely to become ever more so. Also, liquid media are bulky and may need refrigeration; few chemical laboratories have enough refrigerators. One may get around this by making basal media as dry mixes and then weighing out only enough for a day’s or week’s work. Most mixes keep well a t room temperature except those containing thiamine, tj,rosine, or phenylalanine in simple mictures. Shelf life is much extended in rrozen dry mixes; freezers also desiccate. Wide-mouthed heatresistant polyethylene bottles are excellent for storage. Hygroscopic ingredients are avoided-e.g., calcium carbonate and basic magnesium carbonate are wed instead of the chlorides. For the sake of complete, rapid dissolving of Ghe mix, the ingredients should be adjusted to yield a solution
