BACTERIA SENSITIVE TO NUTRIENT BROTH MEDIUM IN TERRESTRIAL ENVIRONMENTS

Soil Sci. Plant Nutr., 26 (1), 99-107, 1980 BACTERIA SENSITIVE TO NUTRIENT BROTH MEDIUM IN TERRESTRIAL ENVIRONMENTS Hiroyuki OHTA and Tsutomu HATTORI Institute for Agricultural Research. Tohoku University. Sendai. Japan Received July 2. 1979 Effect of dilution of plate counting media were studied on various terrestrial samples. It was noticed that nutrient broth (NB) medium always gave less counts than its 100-fold dilution (DNB). Many isolates on diluted media did not show appreciable growth on NB agar medium. Such isolates whose growth was suppressed in NB medium but not in DNB medium were called "DNB-organisms." Percentages of DNB-organisms among isolates were 58% with Japan Alps soil, 30-40% with river and field samples, and 5-10% with avenue samples. About half the DNB-organisms were gram-negative, 10% were gram-positive, and the remainder were irregularly gram-stained organisms. About 65% of the DNBorganisms from Japan Alps soil and 80% of those from the field soil showed irregularly rodshaped forms. Almost all irregularly gram-stained organisms were also irregularly rod-shaped. Those from river samples were either rod-shaped or filamentous. All the DNB-organisms were non-sporeformers. Additional Index Words: plate count method, nutrient broth medium, halo-sensitive bacteria, organo-sensitive bacteria. Choice of the proper plating medium is a basic problem which has attracted much attention, especially when the plate count method is used to enumerate soil bacteria. It is well known that media containing low concentrations of organic substances (lownutrient media) give higher colony counts than those containing high concentrations of organic substance (high-nutrient media). Lower counts in high-nutrient media were presumed to be due to the rapid overgrowth of certain organisms (1). However, recent studies have shown that high-nutrient media not only allow a rapid overgrowth of certain organisms but also suppress the growth of many soil organisms (2, 3). High-nutrient media are used to investigate physiological and taxonomical characteristics of isolates from aquatic and terrestrial sources as well as animal bodies. Nutrient broth (NB) medium is often used for many kinds of bacteria, but its concentration of organic substances is 16-fold higher than that of albumin (A) medium which is also commonly used for enumeration and study of soil bacteria. HATTORI (3) observed that higher colony counts were obtained by the dilution of NB medium with soil samples, and found many bacteria which were sensitive to NB medium on diluted NB plates. This indicates that many microorganisms which live in the soil have not been noticed in microbiology. 99

100 H. OHTA and T. HATTORI In this study, the effect of dilution of NB medium on the plate count of bacteria was examined with mountain, avenue, field, and river samples. In addition, certain physiological and morphological characteristics of isolates sensitive to NB medium were investigated. MATERIALS AND METHODS Samples. Soil samples were obtained from the Japan Alps (Alpine Lithosol), the wheat field (volcanic ash soil) of the Central Experimental Station in Kitamoto city, and Aobadori Avenue (the soil surrounding street trees and roadside sand) in Sendai city. Water and sand samples were obtained at several stations of the Hirose River. Media. Nutrient broth (NB) medium contained 10.0 g each of peptone (Kyokuto Co., Chuo-ku, Tokyo, Japan) and meat extract (Kyokuto Co.), and 5.0 g of NaCI in 1,000 ml of tap water. Diluted nutrient broth (DNB) medium contained the same composition as NB medium but in the strength of 1/100. Albumin (A) medium contained 1.0 g of glucose, 0.25 g of albumin, 0.5 g of K 2 HP04, 0.2 g of MgS04,7H 2 0, and a trace of Fe2(S04)a in 1,000 ml of deionized water. Diluted albumin (DA) medium had the same composition as A medium in the strength of 1/10. Yeast extract albumin (YA) medium contained 0.1 % (w/v) yeast extract (Daigo Co., Higashiyodogawa-ku, Osaka, Japan) in A medium. Diluted yeast extract albumin (DY A) medium had the same composition as YA medium in the strength of 1/10. The ph value of each medium was adjusted to 7.0-7.2 and each was solidified by adding 10.0 g of agar (Wako Co., Higashi-ku, Osaka, Japan) in 1,000 ml of water for the plate count. Plate count. One gram of a soil sample or I ml of a water sample was dispersed in 10.0 ml of sterilized water by hand shaking for 10 min. After suitable dilutions, 1 ml of suspension was pipetted onto plates. Inoculated plates were incubated at 28 C for 30 days except for NB plates, which were kept for 20 days. Physiological characteristics of isolates sensitive to NB medium. Sensitivity to NaCl and peptone-meat extract was examined by the growth in standing or shaken cultures, to which were added respectively, 0.1, 0.5% (w/v) NaCl, and 0.1, 1.0% (w/v) peptone-meat extract to DNB medium. Growth in each culture was compared with that ofdnb medium as a control for IO-day incubation at 28 C. Growth was followed visually on an arbitrary scale of 0 to 3. Morphological characteristics of isolates sensitive to NB medium. Cell shape and gram-stain were observed on logarithmic and stationary phase cells grown on DNB medium. Huker's modification (4) was employed for gram-stain. As to spore formation of isolates, cultures surviving IO-min heating at 80 C were regarded as sporeformers (5).

Table 1. Plate counts of bacteria in various samples. Samples Medial) NB DNB A DA YA DYA Japan Alps soils 1. (2,670 m") 10. 9±3. 3 3 ) 13.1±2.2 23.6±6.5 17.8±5.1 2. (2, 530m) 5.3±1.1 6.l±2.0 10. 6±3. 1 6. 8±1. 7 3. (2, 150m) 12. 8±4. 4 27. 8±6. 5 26.1±5.8 38. 5±7. 5 4. (1,670 m) 69. 8±10. 2 137. 6±12. 9 210. 8±20. 9 231. 8±41. 9 5. (l,420m) 10.6±0.8 16. 2±2. 9 19.0±4.2 19.9±5.4 Avenue samples Soil I 155±47 402± 18 676±33 520±52 Soil 2 36±3 85±13 216±15 146±16 Sand 1,949±159 2,370±135 2, 789± 118 2,003±158 Wheat field soils 1. (0 to» 82±8 55±7 44±10 75±17 2. (2 t) 73±7 69±1l 61±8 79±14 3. (4 t) 88±12 71±4 75±9 loo± 13 4. (8 t) 92±8 75±31 68±13 77±14 Hirose River samples 1. {water sand 270±160 3,800±570 2. {water 49±14 426±29 sand 3, ooo± I, 000 27, 000±5, 000 27±3 53±6 I, 340±290 4, 830±700 211±41 45l±56 19, OOO±4, 000 56, OOO±6, 000 3. water 290±110 700±130 420±60 980±200 4. water 300±200 I, 200 ± 200 500±100 1,500±400 1) Media: NB, nutrient broth medium; DNB, diluted nutrient broth medium; A, albumin medium; DA, diluted albumin medium; Y A, yeast extract albumin medium; DYA, diluted yeast extract albumin medium. 2) Figures show the altitude of the place at which we obtained samples. 3) Figures indicate the mean of viable counts and the standard deviation. Counts with Japan Alps soils, avenue samples, and wheat field soils were expressed in 106/g of dry soil. Those with Hirose River waters and sands were done in to"/ml and 10 2 /g of fresh samples, respectively. ') Figures show the quantity of farmyard manure per to ares which applied to the plot of field. t:!:i I» 0 rd :::!. I» {Il " ::s ~. :;::. " 0" Z S n... a... t:!:i 0 s- -o -

102 H. OHTA and T. HATTORI RESULTS AND DISCUSSION 1) Plate counts of bacteria in various samples Table 1 gives counts of bacteria in mountain, avenue, field, and river samples which were obtained by using the media NB, A, Y A medium, and their dilutions. DNB medium always gave higher counts than NB medium. In the case of soil samples, counts in DNB medium were between 1.2- to 2.6-fold higher than those in NB medium, but somewhat lower than those in A medium which is usually used for soil bacteria. With river samples, counts in DNB medium were 2.4- to 14-fold higher than those in NB medium. DY A medium also gave higher counts than Y A medium with river and field samples, but DA medium did not always give higher counts than A medium. This seems to suggest that a supplementation of 0.1 % (w Iv) yeast extract to A medium promotes the development of some organisms and suppresses others. 2) Isolation of the bacteria sensitive to N B medium After 30-day incubation, bacterial cells were picked up from colonies in A or DYA medium and grown on DNB semisolid agar medium. Some colonies did not show appreciable growth on NB agar medium. Such bacteria whose growth was suppressed in NB medium but not in DNB medium are called "DNB-organisms." Ratios of DNB-organisms among isolates from various samples are shown in Table 2. It is notable that the ratio varied with different samples. With Japan Alps soil, 58% of isolates were DNB-organisms, but with the avenue samples, the ratio was low (5% and 11%). The ratio of river and field samples was between the values of Japan Alps soil and the avenue samples. HATTORI et al. (6) also observed that the ratio was about 0.5 with paddy soil samples. DNB-organisms were abundant in paddy soil or Japan Alps soil, but scarce in the avenue samples. Table 2. The sensitivity of isolates to full strength nutrient broth (NB) medium. Number of Number of isolates unable Samples Medium ' ) isolates to grow on NB agar Y/X (X) (Y) Japan Alps soils A 65 38 0.58 A venue samples A 89 10 0.11 DYA IDS 5 0.05 Wheat field soils A 28 5 0.18 DYA 90 32 0.36 Hirose River waters DYA 83 28 0.34 sands DYA 42 16 0.38 1) Medium: A, albumin medium; DYA, diluted yeast extract albumin medium.

Bacteria Sensitive to Nutrient Broth 103 3) The sensitivity of DNB-organisms to NaC/ or peptone-meat extract In these experiments 62 DNB-organisms were used. Growth responses of DNBorganisms to NaCI were classified into three types (Fig. I-I). In the case of type A, growth was completely suppressed by the addition of 0.5% (w/v) NaCl. In type C growth was not at all suppressed by the addition of 0.5% (w/v) NaCl. Type B indicates intermediary suppression. Sensitivity to peptone-meat extract was also classified into three types as shown in Fig. 1-2. In the case of type a, growth was completely suppressed by the addition of 1 % (w/v) peptone-meat extract. In type c growth was not affected detrimentally by the addition of 1 % (w/v) peptone-meat extract. In type b growth was suppressed by the addition of 1 % (w/v) peptone-meat extract, but optimum growth was observed with supplementation of 0.1% (w/v) peptone-meat extract. i J\. r-'\ r-o _ j:q 1) 2) 3 A B C iii iii Iii o 0.1 0.5 0 0.1 0.5 0 0.1 0.5.s j"\. ~ Additional concentration of NaCl (%. w Iv) a b c ( ~ o o iii iii iii o 0.1 1.0 o 0.1 l.0 o 0.1 1.0 Additional cncentration of peptone-meat extract (%. w Iv) Fig. 1. Types of sensitivity of DNB organisms to NaCI and peptone-meat extract. 1) Sensitivity to NaCl: Sensitivity to NaCl was examined by noting the growth in the DNB media to which 0.1 and 0.5% (w/v) NaCI were added. Growth in each culture was compared with that of DNB medium as a control for 100day incubation at 28 C. Growth was followed visually on an arbitrary scale of 0 to 2. 2) Sensitivity to peptone-meat extract: Sensitivity to peptone-meat extract was examined by noting the growth in the DNB media to which 0.1 and 1.0% (w/v) peptone-meat extract were added. Growth in each culture was compared with that of DNB medium as a control for IO-day incubation at 28 C. Growth was followed visually on an arbitrary scale of 0 to 3. o

104 H. OHTA and T. HATTORI Table 3. The sensitivity of DNB-organisms to NaCI and peptone-meat extract. Sources Number of isolates belonging to type A-a A-b A-c B-a B-b B-c C-a C-b C-c Total Japan Alps soils 4 3 0 5 2 I 0 0 16 Avenue samples 0 0 0 I 0 0 0 I 3 Wheat field soils 4 0 0 4 0 0 4 2 IS Hirose River waters 2 3 0 0 2 2 5 16 Hirose River sands 3 2 0 0 6 0 0 0 12 Total 13 9 5 IS 3 7 8 62 Table 3 shows the sensitivity of DNB-organisms to NaCl or peptone-meat extract. Most DNB-organisms (87%) were halo- and/or organo-sensitive bacteria. Among six types, A-a and B-b type organisms were most abundant, but A-c and C-a type organisms were scarce. This may indicate that the sensitivity of many DNB-organisms to NaCl is related to sensitivity to peptone-meat extract. 4 ) Morphological characteristics of DN B-organisms Table 4 shows the morphological characteristics of DNB-organisms. About 65% of organisms from Japan Alps soil and 80% of those from the field soil showed irregularly rod-shaped cells which were branched, club-shaped or irregularly curved (Figs. 2 and 3). Those from river samples were both rod-shaped and filamentous (Figs. 4 and 5). About half the DNB-organisms were gram-negative and 10% gram-positive. The remainder were irregularly gram-stained organisms, i.e. some parts of the cell were stained with crystal violet while other parts were unstained. Many irregularly gram-stained organisms were obtained from soil samples, and almost all irregularly gram-stained organisms were irregularly rod-shaped. All DNB-organisms were nonsporeformers. In general, DNB-organisms may be divided into three groups on the basis of the morphological characteristics: Group I are regularly rod-shaped and gram-negative or gram-positive organisms containing organisms from various samples. Group II are filamentous and gram-negative, and composed of organisms from river samples. Group III are irregularly rod-shaped and irregularly gram-stained and composed of soil samples. Hattori (unpublished data) also observed that many DNB-organisms isolated from paddy soils were irregularly rod-shaped and gram-variable organisms. Their results should indicate that many DNB-organisms from paddy soils also belong to Group III. More taxonomic characteristics of DNB-organisms are currently being studied. We are now investigating their physiological characteristics and ecological roles.

Bacteria Sensitive to Nutrient Broth 2.., I 105..1'4, ',eo:- \' ':". '. ; ~ ; :.. ~ I ttl. ~ f..,,... "... ',' " ~ ".\ ~... " Fig. 2, Phase-contrast photomicrograph of Y 19 strain which was isolated from the Japan Alps soil. This strain showed branched rod-shaped cells in DNB medium. Fig. 3. Phase-contrast photomicrograph of H63 strain which was isolated from the wheat field soil. This strain showed irregularly rod-shaped cells in DNB medium.

106 H. OHTA and T. HATTORI '.. '.,./...." I - t Fig. 4. Phase-contrast photomicrograph of Kw6 strain which was isolated from Hirose River water. This strain showed filamentous cells in DNB medium. Fig. 5. Phase-contrast photomicrograph of Kw53 strain which was isolated from Hirose River water. This strain showed filamentous or chain of cells in DNB medium. and occasionally showed swollen or clubbed ends.

1) Cell shape!) Table 4. Bacteria Sensitive to Nutrient Broth 107 Morphological characteristics of DNB-organisms. Sources Cell shape 00 [J o 0 0 ~ ~ ~ Japan Alps soils 0 6 11 0 17 Avenue samples 0 2 0 0 2 Wheat filed soils 0 3 12 0 15 Hirose River waters 7 0 8 16 Hirose River sands 0 5 5 11 Total 23 24 13 61 1) Cell shape were observed on the cells grown on DNB medium. 2) Gram-stain 2 ) Sources Gram-stain Strongly Weakly Irregularly positive positive stained Negative Japan Alps soils 0 4 10 3 17 A ven uesamples 0 0 0 2 2 Wheat filed soils 2 0 10 3 15 Hirose River waters 0 0 4 12 16 Hirose River sands 0 0 10 11 Total 2 4 25 30 61 I) Gram-stain were observed on the cells grown on DNB medium. REFERENCES Total Total 1) WAKSMAN, S.A., Principles of Soil Microbiology, The Williams & Wilkins Co., Baltimore, 1932. p. 14 2) MISHUSTlN, E.N., Association of Soil Microorganisms, Publisher Nauka, Moscow, 1975. p. 69 3) HATIORI, T., Plate count of bacteria in soil on a diluted nutrient broth as a culture medium, Rep. Insf. Agr. Res. Tohoku Univ., 27, 23-30 (1976) 4) CONN, H.J., BARTHOLOMEW, J.W., and JENNISON, M.W., Staining methods in "Manual of Microbiological Methods," Society of American Bacteriologists (M.J. Pelczar ef al.), McGraw-Hili Book Co., New York, 1957. p. 10 5) SATO, K. and USHIGOSHI, A., Experimental methods of soil bacteria in "Dojobiseibutu Jikkenho (Experimental Methods of Soil Microbiology)," Society of Soil Microbiology, Yokendo Co., Tokyo, 1975. p. 43 (in Japanese) 6) HATIORI, T., HATIORI, R., SHIODA, Y., and OHTA, H., Microorganisms in low-nutrient conditions in "Biseibutu no Seitai (Microbial Ecology)," Vol. 6, Japan Scientific Societies Press, Tokyo, 1979. p. 55 (in Japanese)