Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 12/27/14 For personal use only.
756
C.4N. .I. M I C R O B I O I .
VOL..
24. 1978
sensitivity and the use ofsensitivity tests in tuberculosisconKONNO, K.. R . K u R % ~ I , \ N Nand . K. T . B I R D . 1957. T h e trol programmes. Bull. W . H . O . 41: 21-43. met;lbolism of nicotinic acid in Mycobacteria. A method for E ~ r ~ u L., s , B. B. D I E N ~ Iand , L. G R E E N B E R G1960. . The use of ~1ifferenti;itingtllhercle bacilli of human origin from other. Mycobi~cteria,Am. Rev. Tuberc. Pulm. Dis. 75: 529-537. p - n i t r o - a - > ~ c e t y l : ~ n i i n @ - h y h x y - p r o p i o p h e o e (NAP) in the differentiation of Mycobacter.ia. Am. Rev. Resp. Dis. 81: MEISSNER, G.. and K. H . SCHRODER.1975. Relationship between Myc.ohtrctc~ri~~tt~ sittritrc, and M j ~ c o h o c t o ~ i rIrtrhcl~rci. m 759-760. GAIL. G . L . 1976. Atypical Mycobacteria in a t ~ ~ b e r c u l o s i s Am. Rev. Resp. Dis. 111: 196-200. hospital. Can. Med. Assoc. J . 114: 612-614. T S U K A M U R ~M. I , 1965. Differentiation of Mycob:rcteria by susceptibility to hydrozylamine and 8 - a z a g ~ ~ u n i nJe.. Bacterial. G R O H M A N NR. , 1977. Klinische ~ l n d epidemiologische Becle~rtungdes Myc.ohc~cto.it~ttr(11.i11ttr.Zentl.albl. Bakteriol. 90: 556-557. Parasitenlid. Infektionskr. Hyg. Abt. I: Orig. Reihe A. 238: tr1hrrc.111o.si.s. Am. 1974. Niacin negative Mycohcrctori~~,,r Rev. Resp. Dis. 110: 101-103. 503-573. K t s r t r . D. G., V. D. A B B O II . i111dG . P. K U B I C A1967. . DifferTSUKAMURA M., . and S . T S U K A M U R A 1964. . Differentiation of r r ~ t ~ by ential identification of Mycobactel-ia. 11. Subgr.oups o f g r o ~ ~ p s Mycohoctc~rirr,,r trrbercr11osi.s and M ~ ~ c o h t r c t c ~ r iho1.i.5 I 1 and Ill (Runyon) with different clinical signific:~nce.Am. p-nitrobenzoic acid susceptibility. Tubercle, 45: 64-65. Rev. Resp. Dis. 95: 1041-1052. W A Y N E L. , G . 1966. Classification and identification of Mycobacteria. 111. Species within group 111. Am. Rev. Resp. Dis. KONNO,K. 1956. New chernic:~lmethod todifferentiate human type tubercle bacilli from othel- Mycobacteria. Science. 124: 93: 919-928. 985.
A simple improved method to stain Thiobacillus M . R O D R I G U E Z - L E I V AA N D S. PICHUANTES Ctrtl~olicUtri~.rrsityc?J'Clrile, I~~.stitrrte c?J'Biologicc~l Sc.ie~lce.s,Lohorc~toryofMicrohiology trtrd I ~ r r ~ r ~ r ~ t r oSal~titrgo, l o g ~ , Clrile
Accepted March 15. 1978 1978. A simple improved method tostnin TlriobncilR O D R I G U E Z - L E I VM.. A , and S . PICHUANTES. 111s.Can. J . Microbiol. 24: 756-757. Best stains are obtained by immersion of the fixed smear in 1.5-2%acid fuchsin at pH 3.2-3.6. RODRIGUEZ-LEIVA, M., et S . PICHUAN-IXS. 1978. A simple improved method to stain Tlriohacil111s.Can. J . Microbiol. 24: 756-757. n fix6 dans 1.5 B 2% d e Les meilleurs colo~xtionssont obtenues par I'immersion d ' ~ ~frottis fi~shineacidifite h un pH d e 3.2 i 3.6. [Traduit par l e j o u ~ n a l ]
Introduction method was optimal: A heat-fixed smear of the There is an increased interest in the thiobacilli. organism, as free from sulfur 01- inorganic salts as particularly Tlziohacillrrs tkrrooxititrns, because of possible, is prepared and immersed for 24 h in a its use in the solubilization of minerals. The or- Coplin jar containing 1.5-2.0% acid fuchsin adganisms as agroup are diffic~lltto stain. especially if justed to pH 3.2-3.6. The slide is removed, rinsed, one uses crystal violet or the gram stain (Waksman dried, and examined in oil immersion. The fixed smear method of staining seems to be and Joffee 1922). However, it has been found that staining with acid fuchsin at a pH of 3.2 to 3.6 considerably better than our attempts to stain susprovides an excellent stain. Cells are more clearly pensions of the organisms in wet cell suspensions. visible with this method than with other more clas- We have worked mostly with an organism idensical procedures. The organisms stain dark red and tified as T. ferrooxidans, strain R2, isolated from a the polar bodies, characteristic of these organisms, Chilean copper mine (Gonzales et ctl. 1974), grown are clearly visible (Fig. 1). Of various concentra- in liquid media 9-K (Silverman and Lundgren 1959) tions and exposure times studied, the following at 28°C with agitation on a reciprocal shaker for5 to
NOTES
757
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 12/27/14 For personal use only.
10 days. Cell suspensions essentially free of precipitate were prepared as described by Beck (1960). The method provides a better visualization ofthe organism so that one can use the Breed type direct count (Breed 191 1: Harrigan and McCance 1966), for at least a rough estimate of the nunibersof cells. Acknowledgement This work was aided by a grant from the Catholic University research funds.
#
F I G . 1. Tliiohtrcillrrs fc~rroositltrtrs strain R2 from 10 days growth in 9-K r n e d i ~ ~atm2g°C, stained with 2%acid fuchsin, pH 3.2-3.6. Cells are stainetl ved. Some cells show polal. botlies (arrow) ch;rr;~cteristicof these bacteria. x 900.
79: 502-50. B n t t ~ R. , S. 19 I I . T h e determination of the number of bacteria in milk by tlirect microscopic esaminirtion. Zcntlxlbl. Bakteriol. Pal-[ 11. 30: 337. GONLALES.C . . H. M A ]U K A N A . illid I . M ~ K I : .1I1 . 1974. AisIirmiento d e Tliiohcrc~illrr.s,fi~rroo.~i~I~~~i.s en zonir miner:\ antlina. Rev. Latinoam. Microbiol. 16: 163-167. H A R K I G A NM. . T.. ant1 M. E. MCCANCE.1966. Laboratory methods in microbiology. 1st ed. Academic P~.ess.London and New York. S I L V E R M A M. N . l'.. illid I). G. L U N I I G K ~ 1959. N . S t ~ ~ d i e s othe n c hemo:~~ltotrophic bac terium Fc~rrohtrc~illrrs ,fi,rroo.~itloti.\. I. r e secul-ing An improved medium > ~ n21d harvesting p r o c e d ~ ~ folhigh cell yields. J. Bacteriol. 77: 642-647. W A K S M A NS. . A , , illid J . S. JOFFEE. 1922. Mic~.oorgi~nisnis concerned in the oxidation of sulflrr in the soil. 11. Tlriohocillrrs !liioo~t~itltrtr.s. a new sulfi~r-osidizingorganismisolated from the soil. J . Bacterial. 7: 239-256.
Denitrification in Rhizobium R. M. ZABLOTOWICZ Dc~ptrr!ttrrti! ofSoil crtril Etit~irotrt~ieti!crIS(.ietrcc~,Utii~.ersi!yof'Cal(f'ortiitr. Hil,c,t..side, CA, U.S.A. 92521
D. L. ESKEW Deptrr!t?ietr! of'Biolog.v, Utritfe,:si!y of'Ccrl~fi)t.tiicr.R i ~ ~ ~ r . s i CA, t l c ~ ,U.S.A. 92521 AND
D. D. FOCHT Deptrr!t?rc~tr!of'Soil ertrtl G~~~irotitt~otr!trl Scietrce, Utril~ersityofCcrl~fe)rtiitr,Ril.e,:sidc, CA, U.S.A. 9252 I Accepted March 9. 1978 ZABL(YI'owlCz, R. M.. D. L . ESKEW.and D. D. FOCHT.1978. Denitrification inH1ri:obirrttr. Can. J . Microbiol. 24: 757-760. Thirty-three strains of R1ii:obirrtn were examined for their reduction of nitrate under nnoxic conditions. Three patternsofdissimilatory nitrate reduction were observetl: ( I ) reduction to N 2 0 and Nz (denitrification), (2) reduction to and subsequent accumulation of NO, (nitrate respiration). (3) no reduction. Strains of R . j(rpotriurtri and the cowpen miscellany displayed all three R . plroscoli, and R . trjfolii did not retluce nitrate by types, while strains of H. legr~tt~it~oscrrrrt~i, dissimilatory means. T h e protluction and subsequent metabolism of N,O was considelrrbly different among the denitrifying strains: in some instances, N,O was a transient intermediirte, while in others, it continued to accumulate during the incubation period.
756
C.4N. .I. M I C R O B I O I .
VOL..
24. 1978
sensitivity and the use ofsensitivity tests in tuberculosisconKONNO, K.. R . K u R % ~ I , \ N Nand . K. T . B I R D . 1957. T h e trol programmes. Bull. W . H . O . 41: 21-43. met;lbolism of nicotinic acid in Mycobacteria. A method for E ~ r ~ u L., s , B. B. D I E N ~ Iand , L. G R E E N B E R G1960. . The use of ~1ifferenti;itingtllhercle bacilli of human origin from other. Mycobi~cteria,Am. Rev. Tuberc. Pulm. Dis. 75: 529-537. p - n i t r o - a - > ~ c e t y l : ~ n i i n @ - h y h x y - p r o p i o p h e o e (NAP) in the differentiation of Mycobacter.ia. Am. Rev. Resp. Dis. 81: MEISSNER, G.. and K. H . SCHRODER.1975. Relationship between Myc.ohtrctc~ri~~tt~ sittritrc, and M j ~ c o h o c t o ~ i rIrtrhcl~rci. m 759-760. GAIL. G . L . 1976. Atypical Mycobacteria in a t ~ ~ b e r c u l o s i s Am. Rev. Resp. Dis. 111: 196-200. hospital. Can. Med. Assoc. J . 114: 612-614. T S U K A M U R ~M. I , 1965. Differentiation of Mycob:rcteria by susceptibility to hydrozylamine and 8 - a z a g ~ ~ u n i nJe.. Bacterial. G R O H M A N NR. , 1977. Klinische ~ l n d epidemiologische Becle~rtungdes Myc.ohc~cto.it~ttr(11.i11ttr.Zentl.albl. Bakteriol. 90: 556-557. Parasitenlid. Infektionskr. Hyg. Abt. I: Orig. Reihe A. 238: tr1hrrc.111o.si.s. Am. 1974. Niacin negative Mycohcrctori~~,,r Rev. Resp. Dis. 110: 101-103. 503-573. K t s r t r . D. G., V. D. A B B O II . i111dG . P. K U B I C A1967. . DifferTSUKAMURA M., . and S . T S U K A M U R A 1964. . Differentiation of r r ~ t ~ by ential identification of Mycobactel-ia. 11. Subgr.oups o f g r o ~ ~ p s Mycohoctc~rirr,,r trrbercr11osi.s and M ~ ~ c o h t r c t c ~ r iho1.i.5 I 1 and Ill (Runyon) with different clinical signific:~nce.Am. p-nitrobenzoic acid susceptibility. Tubercle, 45: 64-65. Rev. Resp. Dis. 95: 1041-1052. W A Y N E L. , G . 1966. Classification and identification of Mycobacteria. 111. Species within group 111. Am. Rev. Resp. Dis. KONNO,K. 1956. New chernic:~lmethod todifferentiate human type tubercle bacilli from othel- Mycobacteria. Science. 124: 93: 919-928. 985.
A simple improved method to stain Thiobacillus M . R O D R I G U E Z - L E I V AA N D S. PICHUANTES Ctrtl~olicUtri~.rrsityc?J'Clrile, I~~.stitrrte c?J'Biologicc~l Sc.ie~lce.s,Lohorc~toryofMicrohiology trtrd I ~ r r ~ r ~ r ~ t r oSal~titrgo, l o g ~ , Clrile
Accepted March 15. 1978 1978. A simple improved method tostnin TlriobncilR O D R I G U E Z - L E I VM.. A , and S . PICHUANTES. 111s.Can. J . Microbiol. 24: 756-757. Best stains are obtained by immersion of the fixed smear in 1.5-2%acid fuchsin at pH 3.2-3.6. RODRIGUEZ-LEIVA, M., et S . PICHUAN-IXS. 1978. A simple improved method to stain Tlriohacil111s.Can. J . Microbiol. 24: 756-757. n fix6 dans 1.5 B 2% d e Les meilleurs colo~xtionssont obtenues par I'immersion d ' ~ ~frottis fi~shineacidifite h un pH d e 3.2 i 3.6. [Traduit par l e j o u ~ n a l ]
Introduction method was optimal: A heat-fixed smear of the There is an increased interest in the thiobacilli. organism, as free from sulfur 01- inorganic salts as particularly Tlziohacillrrs tkrrooxititrns, because of possible, is prepared and immersed for 24 h in a its use in the solubilization of minerals. The or- Coplin jar containing 1.5-2.0% acid fuchsin adganisms as agroup are diffic~lltto stain. especially if justed to pH 3.2-3.6. The slide is removed, rinsed, one uses crystal violet or the gram stain (Waksman dried, and examined in oil immersion. The fixed smear method of staining seems to be and Joffee 1922). However, it has been found that staining with acid fuchsin at a pH of 3.2 to 3.6 considerably better than our attempts to stain susprovides an excellent stain. Cells are more clearly pensions of the organisms in wet cell suspensions. visible with this method than with other more clas- We have worked mostly with an organism idensical procedures. The organisms stain dark red and tified as T. ferrooxidans, strain R2, isolated from a the polar bodies, characteristic of these organisms, Chilean copper mine (Gonzales et ctl. 1974), grown are clearly visible (Fig. 1). Of various concentra- in liquid media 9-K (Silverman and Lundgren 1959) tions and exposure times studied, the following at 28°C with agitation on a reciprocal shaker for5 to
NOTES
757
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 12/27/14 For personal use only.
10 days. Cell suspensions essentially free of precipitate were prepared as described by Beck (1960). The method provides a better visualization ofthe organism so that one can use the Breed type direct count (Breed 191 1: Harrigan and McCance 1966), for at least a rough estimate of the nunibersof cells. Acknowledgement This work was aided by a grant from the Catholic University research funds.
#
F I G . 1. Tliiohtrcillrrs fc~rroositltrtrs strain R2 from 10 days growth in 9-K r n e d i ~ ~atm2g°C, stained with 2%acid fuchsin, pH 3.2-3.6. Cells are stainetl ved. Some cells show polal. botlies (arrow) ch;rr;~cteristicof these bacteria. x 900.
79: 502-50. B n t t ~ R. , S. 19 I I . T h e determination of the number of bacteria in milk by tlirect microscopic esaminirtion. Zcntlxlbl. Bakteriol. Pal-[ 11. 30: 337. GONLALES.C . . H. M A ]U K A N A . illid I . M ~ K I : .1I1 . 1974. AisIirmiento d e Tliiohcrc~illrr.s,fi~rroo.~i~I~~~i.s en zonir miner:\ antlina. Rev. Latinoam. Microbiol. 16: 163-167. H A R K I G A NM. . T.. ant1 M. E. MCCANCE.1966. Laboratory methods in microbiology. 1st ed. Academic P~.ess.London and New York. S I L V E R M A M. N . l'.. illid I). G. L U N I I G K ~ 1959. N . S t ~ ~ d i e s othe n c hemo:~~ltotrophic bac terium Fc~rrohtrc~illrrs ,fi,rroo.~itloti.\. I. r e secul-ing An improved medium > ~ n21d harvesting p r o c e d ~ ~ folhigh cell yields. J. Bacteriol. 77: 642-647. W A K S M A NS. . A , , illid J . S. JOFFEE. 1922. Mic~.oorgi~nisnis concerned in the oxidation of sulflrr in the soil. 11. Tlriohocillrrs !liioo~t~itltrtr.s. a new sulfi~r-osidizingorganismisolated from the soil. J . Bacterial. 7: 239-256.
Denitrification in Rhizobium R. M. ZABLOTOWICZ Dc~ptrr!ttrrti! ofSoil crtril Etit~irotrt~ieti!crIS(.ietrcc~,Utii~.ersi!yof'Cal(f'ortiitr. Hil,c,t..side, CA, U.S.A. 92521
D. L. ESKEW Deptrr!t?ietr! of'Biolog.v, Utritfe,:si!y of'Ccrl~fi)t.tiicr.R i ~ ~ ~ r . s i CA, t l c ~ ,U.S.A. 92521 AND
D. D. FOCHT Deptrr!t?rc~tr!of'Soil ertrtl G~~~irotitt~otr!trl Scietrce, Utril~ersityofCcrl~fe)rtiitr,Ril.e,:sidc, CA, U.S.A. 9252 I Accepted March 9. 1978 ZABL(YI'owlCz, R. M.. D. L . ESKEW.and D. D. FOCHT.1978. Denitrification inH1ri:obirrttr. Can. J . Microbiol. 24: 757-760. Thirty-three strains of R1ii:obirrtn were examined for their reduction of nitrate under nnoxic conditions. Three patternsofdissimilatory nitrate reduction were observetl: ( I ) reduction to N 2 0 and Nz (denitrification), (2) reduction to and subsequent accumulation of NO, (nitrate respiration). (3) no reduction. Strains of R . j(rpotriurtri and the cowpen miscellany displayed all three R . plroscoli, and R . trjfolii did not retluce nitrate by types, while strains of H. legr~tt~it~oscrrrrt~i, dissimilatory means. T h e protluction and subsequent metabolism of N,O was considelrrbly different among the denitrifying strains: in some instances, N,O was a transient intermediirte, while in others, it continued to accumulate during the incubation period.
