JOURNAL OF BACTERIOLOGY, May, 1966Copyright @ 1966 American Society for Microbiology

Method of Temperature-Gradient Incubation andIts Application to Microbiological Examinations

TOSHITAKA NAKAEFaculty of Agriculture, Tohoku University, Sendai, Japan

Received for publication 8 February 1966

ABSTRACTNAKAE, TOSHITAKA (Tohoku University, Sendai, Japan). Method of temperature-

gradient incubation and its application to microbiological examinations. J. Bac-teriol. 91:1730-1735. 1966.-An apparatus for incubating organisms by means of a

temperature-gradient system under steady-state conduction of liquid was devised.This "temperature-gradient bath" consists of a cylindrical temperature-gradient com-partment mounted between two circulating water compartments which are regulatedseparately at constant temperature, the upper compartment being warmer than thelower compartment. A series of different, constant temperatures in the temperature-gradient compartment can be established simultaneously. Water is a suitable heat-transfer liquid for this purpose. Incubation tubes containing solid media are insertedvertically into the temperature-gradient bath and incubated. Visible growth of theorganism appears in a definite portion of the agar column, corresponding to its tem-perature range for growth. The apparatus was successfully applied to the rapid andsimultaneous estimation of maximal, minimal, and optimal temperatures for micro-bial growth, as well as for the simultaneous isolation of psychrophiles, mesophiles,and thermophiles.

It is well understood that the activities oforganisms are conditioned by their environmentaltemperatures. Organisms are usually incubatedat a constant temperature for their isolation andfor determining their biochemical activities.There are, however, many troublesome and time-consuming tests for the isolation of psychrophilic,mesophilic, and thermophilic organisms, and forthe determination of maximal, minimal, andoptimal temperatures for the growth or metabolicactivities of organisms. A means of overcomingthese difficulties is to incubate the organisms in atemperature-gradient system in which a series ofdifferent incubation temperatures can be estab-lished and maintained simultaneously.

Little information is available on the applica-tion of such a temperature-gradient system tobacteriology. Oppenheimer and Drost-Hansen(3) introduced a polythermostat consisting of analuminum bar in which a temperature gradient isproduced. Bacteriological liquid media were

placed in vertical wells along the temperaturegradient in order to measure optimal temperaturefor growth of a sulfate-reducing bacterium. Thisapparatus was modified by Landman, Bausum,and Matney (2), who devised temperature-gradient aluminum plates for the study of micro-

bial growth on solid media through continuoustemperature ranges, or in liquid media at finelygraded temperatures. The apparatus is charac-terized by the use of metal as a heat-transfermedium; the disadvantages are that a specialmetal mold with proper insulation is required andthat it is impossible to observe cultural changesduring incubation. The use of a transparent liquidas the heat-transfer medium should be advanta-geous for overcoming these difficulties.The object of this paper is to describe an

apparatus for incubating organisms in a tempera-ture-gradient system maintained in a steady stateby heat conduction in a liquid. Experiments arereported to show that this method is applicable tomicrobiological tests relating to the temperatureof incubation.

MATERIALS AND METHODS

Apparatus. The apparatus for temperature-gradientincubation is shown in Fig. 1. The main part of theapparatus, the "temperature-gradient bath" (Fig.la), which is made of transparent polyethylene, con-sists of a cylinder, 20.5 cm in diameter, divided intothree horizontal compartments. Water is pumpedthrough the upper and lower compartments at se-lected constant temperatures, the former being always

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FIG. 1. Apparatus for temperature-gradient incubation. (A) Main part of the uninsulated apparatus with incuba-tion tubes; (B) the complete apparatus, which consists of cold-water source (a), insulated apparatus (b), antd hot-water source (c) .

warmer than the latter. Hot or warm water is circu-lated from a Taiyo Thermounit (Taiyo Kagaku KogyoCo., Tokyo, Japan) into the upper compartment, andcold water is circulated from a Sharp Cool-Unit(Hayakawa Denki Kogyo Co., Osaka, Japan) into thelower compartment, as shown in Fig. 1B. The tem-perature-gradient compartment is 27.5 cm deep, andhas six mercury-in-glass thermometers inserted hori-zontally through rubber stoppers at different levels inthe bath. Five inlets for inserting incubation tubes areinstalled so as to pass through the upper compartmentwithin 5 cm of the center. The outer surface of thetemperature-gradient bath may be either uninsulatedor insulated with a 50-mm layer of glass fiber, Micro-wool (Nippon Glass Fiber Co. Ltd., Mie, Japan),protected by cardboard. The temperature-gradientcompartment is filled with a transparent liquid to actas the heat-transfer medium. An overflow tube at-tached to a small cylinder slightly above the gradientcompartment allows for expansion and ensures thatthe compartment remains full. After a stable tempera-ture gradient has been established, the incubationtubes are carefully inserted into the temperature-gradient bath through the inlets. The incubation isthen started.

Incubation tubes. Two types of incubation tubeswere used for the temperature-gradient incubation.For anaerobic incubation, a pipette-type tube (2 or 3mm, internal diameter, and about 40 cm long) wasused; for aerobic incubation, a roll tube (7 or 9 mm,internal diameter, and about 40 cm long) was used.A melted-agar medium containing the organisms to betested was either drawn into the pipette by suction, orwas spread so as to be 1 mm thick on the inside sur-face of the roll tube. In the latter case, 2 to 3 ml of themelted-agar medium was poured into the roll tube,which was then held horizontally and rolled on thetable. The medium can be solidified in 1 to 2 min.

Rubber stoppers were tightly applied to both ends ofpipettes and to the necks of roll tubes.

Microbiological examinations. An experiment wascarried out to determine maximal, minimal, andoptimal temperatures for the growth of five differentorganisms by means of the temperature-gradient incu-bation. In another experiment, a temperature-gradi-ent bath was used for the simultaneous isolation ofpsychrophilic, mesophilic, and thermophilic bacteria.

RESULTSTemperature distribution in the temperature-

gradient bath. Two heat-transfer media, distilledwater and 95% glycerol, were selected for theexperiments. Running water at either 65 or 67.5 Cwas introduced into the upper water compartmentand, at 2 C, into the lower compartment. Theentire apparatus was placed in a constant temper-ature (25 C) room.The times required to reach constant tempera-

tures at the six points on the center axis of theuninsulated temperature-gradient bath were 8 and9 hr when water and glycerol, respectively, wereused. Corresponding values were 12 and 14 hrwhen the temperature-gradient bath wasinsulated.The relationship between depth and tempera-

ture in the temperature-gradient bath, when theupper compartment is at 67.5 C, is shown inFig. 2. The temperature curves for the uninsulatedtemperature-gradient bath exhibit a large regionof moderate temperatures, favorable for meso-philic organisms, between narrow regions of highand low temperatures. When the temperature-gradient bath was insulated, both liquids showed

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deviation in temperature was found at any pointon the cross section. Therefore, the five incubationtubes which are located within 5 cm of the axis ofthe temperature-gradient bath have identicaltemperature distributions.

Temperature distribution in the incubation tube.A sterile nutrient agar medium was used for thisexperiment. The concentration of the agar wasincreased to 2.5%/ to prevent melting due to thehigh temperature in the upper portion of theincubation tube. The melted-agar medium waseither enclosed in the pipette or spread on theinside surface of the roll tube, which was theninserted into the temperature-gradient bath.

Mercury-in-glass or thermister probe ther-5 10 15 20 25 27.5 mometers were used for measuring the tempera-

DEPTH cm tures at various depths in the nutrient agar in the

lationship between the depth in the tem- incubation tubes. When water was used as therent bath and the temperature of the heat- heat-transfer medium at a gradient temperatureum at the center axis in the bath. Tem- of from 60 to 6 C, the time required for thepper and lower water compartments were nutrient agar to reach a constant temperature atrespectively. Room temperature was 25 C. all depths was less than 10 min for the pipette andfer media, water (0) and 90% glycerol less than 13 min for the roll tube. After the steady?d. Solid lines are from apparatus with state was established, the temperature of the agarken lines, without insulation. was almost identical to the curve shown in Fig. 2.

Simultaneous determination of maximal,ight lines. Although glycerol gives minimal, and optimal temperatures for microbials, water is more economical and growth. Five strains from our laboratory collec-,e. In either case, a definite stable tion, Streptococcus lactis 527, Bacillus subtilisgradient can be established under the 6010, Brevibacterium linens BL-1, Aspergillusescribed. oryzae Chosen B, and Penicillium candidum FC-1,)erature was measured at several were used as test organisms. Temperaturerom the axis of the uninsulated relationships of these organisms were previously-gradient bath at each of the six determined by means of the conventional methodr locations. The temperature did not based on the comparative observation of micro-a radial direction up to a radius of 7.5 bial growth in agar-stab or on agar-streakwater or glycerol. When the tempera- cultures at different fixed temperatures.t bath was insulated, no appreciable Temperature-gradient incubation was con-

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FIG. 2. Reiperature-graditransfer mediperatures of u,67.5 and 2 C, i

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distances frtemperaturethermometeifluctuate in a

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FIG. 3. Cultural changes ofStreptococcus lactis in anaerobic (pipette-type) incubationi tubes during temperature-gradient incubation at 57.5 to 6 C. Glucose-bouillon-agar containing methylene blue was used as solid medium. (A)Before incubation; (B) incubated for 10 hr; (C) incubated for 24 hr.

FIG. 4. Growth zones offive organisms in incuibation tubes under temperature-gradient incubation at 60 to 6 C.(A) Streptococcus lactis incubated for 48 hr in a pipette-type tube with glucose-bouillon-agar containing methyleneblue; (B) Bacillus subtilis incubated for 16 hr in a roll tube with glucose-bouillon-agar; (C) Brevibacterium linensincubated for 24 hr in a roll tube with glucose-bouillon-agar; (D) Aspergillus oryzae incubated in a roll tube withCzapek agar for 4 days; (E) Penicillium candidum incubated in a roll tube with Czapek agarfor 5 days.

FIG. 5. Growth behavior ofsingle cultures isolated from a combined culture of S. cremoris and S. thermophilusunder temperature-gradient incubation at 60 to 6 C. Glucose-bouillon-agarcontaining metlhylene blue was used assolid medium. (A) Two growth zones with methylene blue-reducing zones of the combined culture; (B) growth zonewith methylene blue-reducing zone of a single culture of S. thermophilus isolated from the upper growth zone ofincubation tube A; (C) cultural change of a single culture of S. cremoris isolated from the lower growth zone ojtube A. Upper portion of tube C shows the methiylene blue-reducing zone, which contains no growth.

FIG. 6. Growth of psychrophilic, mesophilic, and thermophilic bacteria isolated from a raw-milk sample bytemperature-gradient incubation at 60 to 6 C for 48 hr. (A) Growth zone of the organisms in a roll tube containingglucose-bouillon-agar inocuilated with 1% raw milk; (B) growth zone ofa psychrophilic bacterium isolatedfrom thepoint corresponding to 15 C of tube A; (C) growth zone of a mesophilic bacterium isolated from the point corre-sponding to 38 C of tube A; (D) growth zone ofa thermophilic bacterium isolated from the point corresponding to50 C of tube A.

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ducted, with water as the heat-transfer medium,at a temperature of either 57.5 to 6 C or 60 to 6 C.The strains were previously cultivated in glucose-bouillon (bacteria) or in Czapek broth (fungi).One drop of the broth culture was uniformlymixed with 5 ml of a melted glucose-bouillon-agar of Czapek agar, with or without 0.025 mgof methylene blue, and the mixture was thenintroduced into the incubation tube in the man-ner previously described.The typical development of the growth zone of

S. lactis, showing the reduction of methylene blue,is illustrated in Fig. 3. The optimal temperaturefor growth can be determined by measuring thepoint where the growth zone, or methylene blue-reducing zone, first becomes visible (Fig. 3B).Furthermore, the minimal and maximal tempera-tures, or the temperature range for growth for adefinite period of incubation, can be determinedsimultaneously (Fig. 3C). Although another zoneof reduction of methylene blue appeared in theupper high-temperature portion of the incubationtube, owing to a shortage of oxygen, it wasdistinguished from the reduction zone due tobacterial growth because the latter is alwaysaccompanied by bacterial colonies.Growth zones of the five strains used were

observed after various periods of incubation (Fig.4). The temperature relationships of the pigment-producing strains were easy to determine. Theoptimal temperatures, and the temperature rangesover a definite period of incubation, were de-termined (Table 1). The values were similar to,but more accurate than, the results of the conven-tional method, determined previously.

In further experiments, more accurate de-TABLE 1. Optimal temperatures, and temperature

ranges, for growth of organisms tested*

Optimal Temp rangetemp (C) (C)

Species

A B A B

Streptococcus lactis .... 34 35 11-40 10-40Bacillus subtilis......... 39 40 20-54 20-50Brevibacterium linens.... 28 25 12-44 15-40Aspergillus oryzae. 38 40 13-43 15-40Penicillium candidum.... 27 25 8-34 10-30

* The maximal period of incubation for deter-mining temperature range was 3 days for threestrains of bacteria and 7 days for two strains offungi. (A) Determined by temperature-gradientincubation method; (B) determined by conven-tional incubation method based on comparativeobservation of the growth in agar-stab or on agar-streak cultures incubated at different fixed tem-peratures at intervals of 5 C.

terminations of optimal temperatures for growthof S. lactis, B. subtilis, and A. oryzae were done atgradient temperatures of 20 to 50 C. The valuesdetermined were 34.6, 40.4, and 28.3 C, respec-tively. This indicates that optimal temperaturescan be measured accurately to the first decimalplace with gradient temperatures of 50 to 20 C.

Simultaneous isolation of psychrophiles, meso-philes and thermophiles. In a preliminary experi-ment, three strains of streptococci, S. lactis 527,S. cremoris NC-5, and S. thermophilus 510, wereselected as representative strains having differenttemperature ranges for growth. Equal amounts oftwo or three of the single broth cultures weremixed to make combined cultures, which werethen inoculated into the melted glucose-bouillon-agar containing methylene blue (in a pipette).After temperature-gradient incubation at 60 to6 C for 16 hr, the tube was cut at three positions,corresponding to 25, 38, and 50 C, from whichcolonies were transferred to, and recultivatedseparately in, the same broth as described above.The recultivated broth cultures were againtransferred to the agar medium and incubatedagain in the same temperature gradient. The resultwith two-strain combined cultures of S. cremorisand S. thermophilus indicated that the combinedculture (Fig. SA) can be separated into itscomponent parts (Fig. 5B and 5C) by means oftemperature-gradient incubation. Similarly, threecombined cultures of streptococci were separatedsimultaneously. In this case, however, specialprecautions were required to select the time ofpicking the growing colonies.Another experiment was carried out to separate

psychrophiles, mesophiles, and thermophilies atthe same time from raw milk. Glucose-bouillon-agar, inoculated with 1% raw milk, was incubatedfor 48 hr in a temperature gradient of 60 to 6 C.Strains of psychrophilic, mesophilic, and thermo-philic bacteria were isolated from the pointscorresponding to 15, 38, and 50 C, respectively(in the manner described for the streptococci,except that roll tubes were used instead of pipette-type tubes). The growth behavior and temperaturerelations of the strains isolated were distinctlydifferent (Fig. 6). In further experiments, 1 ml ofraw milk was serially diluted with Ringer solu-tion and mixed with melted glucose-bouillon-agar,which was then placed in a roll tube and incu-bated. It was then possible to estimate, based onthe temperature range of incubation, the numbersof psychrophiles, mesophiles, and thermophilesby separate counting of their colonies.

DIscussIoNThe time required for agar media to reach

constant temperature in the incubation tube is an

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important consideration. In a preliminary experi-ment, the time required for nutrient agar in a petridish to reach a constant temperature, after beingmoved from room temperature (20 C) to anincubator at 38 C, was 17 min, which is longerthan the time required for the same agar in theincubation tube to reach thermal equilibrium. Thetime is therefore considered to be negligible,considering the time of incubation.Another important consideration is the possible

occurrence of thermal diffusion of components inthe agar medium owing to the temperaturegradient, resulting in a concentration gradientalong the incubation tube. To ascertain whetherthermal diffusion may occur in the agar medium,an aerobic incubation tube containing un-inoculated glucose-nutrient agar was placed inthe apparatus for 48 hr and was then cut intoten equal segments; 1 g of each fraction was mixedwith 5 ml of glucose-nutrient broth, and wascultivated with S. lactis. There were no appreci-able variations in acidity among cultures from theten segments. This fact suggests that no apprecia-ble thermal diffusion occurs, owing probably tothe high agar concentration.The temperature-gradient bath has several

advantages in that the incubation tube makescontact intimately with the heat-transfer medium,and also in that the cultural changes duringincubation can be easily observed. Furthermore,the temperature range of incubation and the typeof liquid used as heat-transfer medium can beselected to suit the purpose of incubation.Although water was selected as a suitable heat-transfer medium for the temperature-gradientbath, squalane, which has been used as a heat-transfer medium in a thermo-gradient heater forsublimatography (4), should be another suitablemedium.The optimal growth temperature is believed to

differ from the optimal temperature for otherphysiological activities of the cell (1). The optimal

temperature for some physiological activities maybe precisely determined by setting the temperatureof the apparatus to cover a narrow range aroundthe optimum for growth. In an experiment on thetemperature-gradient incubation of S. lactisinoculated into glucose-bouillon-agar containingbromocresol purple, it was shown that the optimaltemperature for acid production by the cells wassomewhat different from that for growth.

Conventional techniques for estimating thenumber of viable bacteria are limited to de-termining the "viable count" of cultures incubatedat a constant temperature. If the simultaneousestimation of viable psychrophiles, mesophiles,and thermophiles is successfully established bymeans of temperature-gradient incubation, moreaccurate information concerning the bacterio-logical quality of a sample could be obtained.

In conclusion, the apparatus described here isundoubtedly applicable to the determination oftemperature relations of unknown organisms, foridentification, for industrial utilization, and forthe isolation and estimation of viable bacteria.

ACKNOWLEDGMENTSI express thanks to J. A. Elliott, Food Research

Institute, Canada Department of Agriculture, forcritical review of the manuscript. I also thank A.Yamaji for his assistance in obtaining the photo-graphs.

LITERATURE CITED1. BURROWS, W. 1959. Textbook of microbiology, p.

173. W. B. Saunders Co., Philadelphia.2. LANDMAN, O. E., H. T. BAUSUM, AND T. S. MAT-

NEY. 1962. Temperature-gradient plates forgrowth of microorganisms. J. Bacteriol. 83:463-469.

3. OPPENHEIMER, C. H., AND W. DROST-HANSEN.1960. A relationship between multiple tempera-ture optima for biological systems and the prop-erties of water. J. Bacteriol. 80:21-24.

4. SHIBATA, E., AND S. SAITO. 1959. Sublimatography.J. Chem. Soc. Japan 80:604-609.

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