Growth of Desulfovibrio on the Surface of

Transcription

1 APPLIED MICROBIOLOGY, July, 1966 Copyright ( 1966 American Society for Microbiology Vol. 14, No. 4 Printed in U.S.A. Growth of Desulfovibrio on the Surface of Agar Media WARREN P. IVERSON U.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland Received for publication 14 January 1966 ABSTRACT IVERSON, WARREN P., (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.). Growth of Desulfovibrio on the surface of agar media. Appl. Microbiol. 14: Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor. Media used for the cultivation of Desulfovibrio species commonly employ a hydrogen donor such as lactate, mineral salts including sulfates, an iron indicator salt to detect hydrogen sulfide production, and one or more oxidation-reduction (redox) potential lowering agents such as ascorbic acid, cysteine, or thioglycolate (1, 3, 5, 6). Organic materials such as yeast extract and peptone have often been routinely added since the work of Bunker (Abstr. Commun. Intern. Congr. Microbiol., 3rd, p. 64, 1939) and others, who showed that these materials stimulated growth. Techniques for counting these organisms usually employ most probable number determinations in liquid media or colony counts in tubes using semisolid (0.75% or less) agar (6). Attempts by the author and others to grow these organisms on the surface of agar plates of these media have generally resulted in poor and erratic growth. Evidence is presented in this paper to demonstrate that Desulfovibrio will 529 grow very well on the agar surfaces of completely organic media, and that incubation together with inoculated plates of Trypticase Soy Agar (TSA) plus salts medium is necessary for rapid growth on agar surfaces of some commonly used media for the cultivation of these organisms. MATERIALS AND METHODS Organisms. Three strains of Desulfovibrio were used in the initial part of this investigation: a strain of D. desulfuricans (Mid-Continent strain A), isolated from a Texas water flood and used by the American Petroleum Institute (API) for biocide testing; a strain isolated from a corrosion pit in an aluminum alloy; and a strain isolated from a heavily contaminated jet fuel storage tank water bottom. The strain from the water bottom was isolated, while investigating the anaerobic flora, by streaking directly onto the agar (2%) surface of Trypticase Soy Broth (BBL) under a hydrogen atmosphere. Well-isolated colonies, identified as Desulfovibrio, were found to develop. It

2 530 IVERSON APPL. MICRoBIoL. was also found that Mid-Continent strain A, as well as the strain from the corrosion pit isolated in a similar manner, grew quite well on this medium. These three isolates were maintained as pure cultures on the surface of this medium for over 2 years in Brewer Anaerobic Jars under an atmosphere of hydrogen at 28 C. At frequent intervals, they were introduced into API medium and Starkey's medium to test their ability to reduce sulfate. They did not lose this ability during this time. Culture purity was checked by macroscopic observation of streak plates for different colony types and by microscopic observation. Upon transfer, a duplicate plate was incubated aerobically at roon temperature for several weeks to check for aerobic or facultative anaerobic contaminants. The three strains were transferred monthly. All plates of media used in this study were inoculated by the streak-plate method directly from growth on agar surfaces. The Mid-Continent (API) strain was mainly used for these studies. Media. In addition to Trypticase Soy Broth (BBL), I used TSA (BBL), yeast extract (Difco), Starkey's medium (7), API medium (1), TSA plus salts medium (the medium found to be most favorable for surface growth and sulfate reduction), and a mineral salts medium. The TSA plus salts medium consisted of TSA (4.0%) fortified with additional agar (0.5%) to which was added 60% sodium lactate (0.4%, v/v), hydrated magnesium sulfate (0.2%), and ferrous ammonium sulfate (0.05%). The ph was adjusted to 7.2 to 7.4. After autoclaving at 15 pounds of pressure for 15 min, this medium is clear and free from precipitate. The mineral salts medium consisted of dipotassium phosphate (0.2%), monopotassium phosphate (0.05%), ammonium chloride (0.1%), hydrated magnesium sulfate (0.2%), ferrous ammonium sulfate (0.05%), sodium bicarbonate (0.1%), and noble agar (Difco; 2.0%). The ph was 6.8 i 0.1. The ferrous ammonium sulfate was sterilized separately in 10 to 50 mi of distilled water, and the sodium bicarbonate was sterilized by Seitz filtration. The other nitrogen sources tested included Casamino Acids (Difco), N-Z-Amine, Type A (Sheffield Chemical, Norwich, N.Y.), N-Z-Case (Sheffield Chemical), lactalbumin hydrolysate (Nutritional Biochemicals Corp., Cleveland, Ohio), and Trypticase (BBL). The agar concentration used in plating was 2% for all the media to provide a dryer surface. The ph was adjusted to 7.0 to 7.2, when necessary, with sodium hydroxide. All media were sterilized at 15 pounds of pressure for 15 min. Anaerobic cultivation. All plates were inoculated within 1 hr after the agar hardened. This does not appear to be too critical, although the plates should be inoculated within 4 or 5 hr after hardening to prevent saturation with oxygen. To prevent moisture condensation on the petri dish covers, the covers were replaced with sterile absorbent tops until 10 to 15 min after the agar hardened. The plates were then placed in desiccator jars or Brewer jars (plates not inverted), and the atmosphere was replaced with tank hydrogen or tank nitrogen by evacuation (water aspirator) and replacement with the gas three times. Hydrogen sulfide was obtained from small tanks (Matheson Co.) as well as from a generator (H.SO4 + Na2S.9H.O). The jars were incubated at room temperature (21 to 24 C) except where indicated otherwise. RESULTS Growth and sulfate reduction on organic media in a hydrogen atmosphere. Well-isolated colonies of the three strains of Desulfovibrio grew to about 1 mm in diameter on Trypticase Soy Broth plus 2% agar (Fig. 1). They were quite transparent and appeared to be very pale yellow when observed against a dark background. The cells appeared to be quite small, commashaped, and nonmotile. When transferred to FIG. 1. Two colonies (ca. 1-mm diameter) of Desulfovibrio desulfuricans (API strain) on Trypticase Soy Broth plus 2% agar. Plates incubated for 7 days at room temperature in a hydrogen atmosphere. TABLE 1. Growth of Desulfovibrio desulfuricans on Trypticase Soy Broth (TSB) minus its components in a hydrogen atmospherea Medium Relative growthb 7 Days 21 Days TSB (complete) TSB minus Trypticase... TSB minus dextrose TSB minus K2HPO TSB minus NaCIC. 4+ TSB minus Phytone a Petri plates in triplicate. Nine-day-old culture on TSB plus agar plates used as inoculum. b Symbols: - = no growth along streak path; 1+ = slight growth confluent along streak path; 2+ = fair growth confluent along streak path; 3+ = good growth confluent along streak path; 4+ = excellent growth confluent along streak path.

3 VOL. 14, 1966 GROWTH OF DESULFOVIBRIO 531 FIG. 2. Plates of Trypticase Soy Agar plus salts streaked with Desulfovibrio desulfuricans (API strain) after 7 days of incubation in a hydrogen atmosphere. Plate at left immediately after removal from jar and plate at right after exposure to the air for 24 hr. *I FIG. 4. Colonies ofplate shown in Fig. 3 after about 5 to 6 hr of exposure to the air. Downloaded from FIG. 3. Colonies of Desulfovibrio desulfuricans (API strain) on Trypticase Soy Agar plus salts. Plate incubated 10 days at room temperature in an atmosphere of hydrogen and exposed to air for 2 hr. liquid API medium and Starkey's medium, they assumed motility and appeared to be larger; many of them did not separate after division and showed the classical spiral forms. Growth appeared to be very poor or was absent on the surface of a number of routinely used media, including nutrient agar (Difco), Stock Culture Agar (Difco), Heart Infusion Agar (Difco), dextrose-tryptose-agar (Difco), and Anaerobic Agar (BBL), under hydrogen. To determine which of the materials in the Trypticase Soy Broth were essential to the growth of Desulfovibrio, duplicate agar plates of Trypticase Soy Broth and Trypticase Soy Broth minus one of each of its components were streaked with the API strain, incubated under hydrogen, and FIG. 5. Colonies of Desulfovibrio desulfuricans, API strain, (ca. 1-mm diameter) on plate from a 10-7 dilution after exposure to air for 5 days. examined at 7- and 21-day intervals (Table 1). Initially, growth was favored by the eliminatiqn of glucose and K2HPO4 and hindered by deletion of sodium chloride. The essential nitrogenous component in Trypticase Soy Broth appeared to be Trypticase rather than phytone. A few other nitrogenous materials were then compared with Trypticase for their ability to support growth on agar surfaces under hydrogen. As nitrogenous substances, yeast extract > Trypticase > Casamino Acids. Lactalbumin hydrolysate, N-Z-Case, and N-Z-Amine failed to support growth. on November 2, 2017 by guest

4 I532 IVERSON APPL. MICROBIOL. TABLE 2. Growth and sulfate reduction of Desulfovibrio desulfuricans on yeast extract-agar and TSA plus sulfate with and without sodium lactate under nitrogen Medium Growthb Color of colonies Color of agar Deparesson Sulfate reduction Yeast extract (2%) Black Trace Yeast extract (2%) + lactate (4 ml/liter) Gray Blackd + Good TSA Gray Black Good TSA + lactate (4 ml/liter) Gray Black + Excellent amagnesium sulfate (2 g per liter) and ferrous ammonium sulfate (0.5 g per liter) added to all of the media. b After 14 days of incubation; +1 = seven to eight colonies; +2 = three to four large mucoid masses; +3 = many isolated colonies along streak path; +4 = excellent confluent growth along entire streak path. Depression of agar beneath colonies after exposure to air for 5 days. d No general blackening of agar; black areas restricted to agar beneath colony. Since the absence of glucose and K2HPO4 tended to favor growth and the absence of sodium chloride tended to retard growth, TSA seemed to be the best and simplest medium to use because it consists of only Trypticase (1.5 %), phytone (0.5%), sodium chloride (0.5%), and agar (1.5%). Growth on this medium, fortified with agar to 2%, was just as good and even slightly better than on Trypticase Soy Broth plus agar. When magnesium sulfate, sodium lactate, and ferrous ammonium sulfate were added to TSA (TSA plus salts), blackening of the entire medium and excellent growth occurred along the entire streak path. It was also noted that just as good growth and blackening under hydrogen were obtained without the addition of lactate. When plates of this medium were exposed to the air after growth, the black color was replaced by a blue color and then the medium eventually became white (Fig. 2). If a high concentration of ferrous ammonium sulfate was added (0.2%), the plates turned a lightyellow to orange color instead of the normal white color after becoming blue. Duplicate plates of TSA plus salts were streaked with various dilutions of a 12-day-old culture grown on TSA, and, after incubation for 10 days, the plates were examined. One of the plates (10-6 dilution) with 72 colonies is shown in Fig. 3. Upon exposure to air, the colonies themselves became white except for a black halo around each colony at the surface of the agar (Fig. 4). This black halo, which remains upon exposure to air, is not generally found around mass extensive growth or isolated colonies near extensive growth, but only on plates where all the growth is in the form of isolated colonies or small isolated masses of growth formed by coalescence of adjacent colonies. Plates streaked with the next highest dilution (10-v) had four colonies on one plate and five on another. Figure 5 shows the unique character of these colonies after they have been exposed to the air for about 5 days. Each colony is about 1 mm in diameter and has vertical sides and a rounded top very much resembling miniature oil tanks. It was of interest to note that these colonies depressed the agar surface around and under them upon further exposure to the air at room temperature. Growth and sulfate reduction on 2% yeast extract-agar plus magnesium sulfate, ferrous ammonium sulfate, and sodium lactate in the same concentrations as used for the TSA plus salts medium was very poor and usually consisted of a few small black areas along the initial streak path where the inoculum was the heaviest. Growth and sulfate reduction on organic media in a nitrogen atmosphere. To determine whether any materials present in the yeast extract and TSA would act as hydrogen donors, triplicate plates of 2% yeast extract-agar, 2% yeast extract plus sodium lactate (4 ml per liter)-agar, TSA, and TSA plus sodium lactate (4 ml per liter) were streaked with a 6-day-old culture of the API strain. The plates were incubated in four separate jars (one medium to one jar) under a nitrogen atmosphere. After 6 days of incubation at room temperature, yeast extract-agar supported growth in N2 only when lactate was present. Lactate was not required in TSA medium for growth. In subsequent experiments, a few colonies developed on yeast extract-agar under the same conditions, possibly as a result of some hydrogen donor being carried over with the inoculum. To determine growth and sulfate reduction in the above media, magnesium sulfate and ferrous ammonium sulfate were added to the media, and triplicate plates of each medium were inoculated with a 10-day-old culture. The plates of

5 VOL. 14, 1966 GROWTH OF DESULFOVIBRIO 533 yeast extract media were placed in one jar and the plates of TSA media were placed in another jar; both jars contained a nitrogen atmosphere. After 4 days of incubation, the three plates of TSA plus salts were almost completely black with excellent growth. The plates of TSA plus salts without lactate had a few dark colonies, and the other two yeast extract media were devoid of any dark areas. The results after 14 days of incubation are indicated in Table 2. Growth on organic and inorganic media in the presence of inoculated TSA plus salts. It has been noted that somewhat better growth took place on the agar surface of API media and other media used for the routine cultivation of sulfate reducers if freshly inoculated plates of these media were placed in the same chamber under hydrogen with plates of the same media on which a few large colonies had already appeared. It was thought at first that hydrogen sulfide might be the stimulatory factor as Grossman and Postgate (3) had postulated. API medium and yeast extract plus magnesium sulfate (2 g per liter), ferrous ammonium sulfate (0.05%), and sodium lactate (4 ml per liter) were used in the following experiments as being representative of two typical media. Duplicate sets (three plates per set) of inoculated yeast extract-agar plus sulfate and lactate and API agar were prepared. One set was incubated together with three inoculated plates of TSA plus salts under hydrogen. The other set was incubated under hydrogen in the absence of the inoculated TSA plus salts plates. Growth occurred first on the TSA plus salts plates, which showed excellent growth and sulfate reduction (completely black) within 3 days. This was followed by blackening and excellent growth on API agar and on yeast extractagar plus salts (in the same jar) 1 to 2 days later. After 1 week, only three to four small black areas were observed on the yeast extract plus salts medium incubated in the absence of the inoculated TSA plus salts plates. At that time, no black areas were noted on the API medium. A few very small black areas were observed 4 days later (11 days of total incubation time). After 14 days of incubation, all of the plates had turned black. Growth on the yeast extract medium was extensive, but not as good as on the plates in the jar with the TSA plus salts medium plates. Growth on the API medium was poor, and occurred as isolated colonies along the streak path. Growth was also obtained on the mineral salts-agar under hydrogen and maintained for four serial transfers in the presence of inoculated TSA plus salts plates. The growth followed the streak path in the form of very small (<1 mm) isolated colonies, but was not nearly as extensive as on the organic media. Black areas were noted around a few of the colonies, thus indicating their ability to reduce sulfate. The organisms appeared quite motile, and many long spiral forms were present. This is in contrast to those grown on the "dry" agar surface of organic media, where the organisms appear as short curved rods and are nonmotile or sluggishly motile. No growth took place on the mineral salts-agar plates in the absence of inoculated TSA plus salts plates. It seemed of interest to determine whether this stimulation would also occur in the absence of hydrogen. When inoculated plates of yeast extract-agar plus sulfate and lactate were incubated (nitrogen atmosphere) in Brewer jars in the presence of inoculated TSA plus salts plates, growth on the agar was found to be much better than when replicate plates of the same medium were incubated in the absence of TSA plus salts plates. Such stimulation also occurred in yeast extract agar (nitrogen atmosphere) without any added sulfate and lactate. Excellent growth occurred on three plates of yeast extract (2%)-agar incubated with inoculated plates of TSA plus salts medium, and no growth occurred after 7 days of incubation on three plates incubated alone. Hydrogen sulfide (tank) in initial concentrations of 5 and 10% in an atmosphere of hydrogen appeared to inhibit rather than stimulate growth on API and yeast extract plus sulfate and lactate media. Likewise, generated hydrogen sulfide (10%) in a nitrogen atmosphere completely inhibited growth on yeast extract-agar. It appeared, therefore, that H2S was not stimulating growth, and that another volatile substance possibly was involved. To test this assumption, three plates of API medium were incubated (27 C) in an atmosphere of helium with six inoculated plates of TSA plus lactate without any added sulfate (very little, if any, detectable H2S production) in the same jar and separately in another jar. After 7 days of incubation, growth on the plates of API agar incubated in the absence of TSA plus lactate plates consisted of one or two small (<2 mm), dark are4s on two of the plates. No growth was evident on the third plate. The plates of API medium incubated in the same jar with inoculated plates of TSA plus lactate showed as extensive growth as that obtained in the presence of TSA plus salts media (H2S production). The plates of API media were not as black as they were when incubated with the TSA plus salts plates. Most of the blackening was at the edges of the surface growth.

6 534 IVERSON APPL. MICROBIOL. DIscussIoN The excellent growth of the three strains of Desulfovibrio obtained on the surface of agar without any added reducing agents leaves doubt as to their more exacting requirements for anaerobic conditions. Difficulty in the growth of these organisms on the agar surface of standard media for their cultivation seems to be experienced only when sulfate and an indicator salt are present. For example, excellent growth was found to take place on yeast extract-agar alone, in the presence of hydrogen, whereas poor or slight growth occurred on yeast extract-agar with the addition of these two salts or on API agar (yeast extract plus sulfate and other salts including an indicator salt). Since growth occurred on Trypticase soy medium in nitrogen and without any added hydrogen donor such as lactate, some substance in the medium must be providing the hydrogen or electrons, possibly an amino or organic acid. Baars (Thesis, Delft, 1930) observed that sulfatereducing bacteria could oxidize several organic compounds including carbohydrates, organic acids, amino acids, and alcohols. Knowledge of the final reduced compound or compounds in the Trypticase soy and yeast extract media must also await further investigation. Until further analyses of the sulfate content are made, a possibility of small traces of sulfate in these media still exists. Sulfate, however, need not be required for growth, since it has been shown that Desulfovibrio can be grown in a pyruvate medium free from sulfate (4). Since such excellent surface growth was obtained on the Trypticase soy media as compared with the standard media, a comparison by colony count was not made. Microscopic counts of the original inoculum versus colony counts on the Trypticase soy media must be compared with counts in semisolid media and most probable number determinations before any real evaluation can be made. Preliminary evidence has indicated quantitation should be as good as, if not better than, present available methods for counting. Of interest is the observation that, when isolated colonies are growing on TSA plus salts medium and exposed to the atmosphere for several days, the agar around and under the colonies tends to be depressed. The agar is quite soft and appears to undergo some hydrolysis, which may be due to some product or products of sulfide oxidation. The greatly enhanced growth on the agar surface of API medium in a helium atmosphere when in contact with inoculated TSA plus lactate plates, where little if any H2S was apparently produced, suggested that the diffusible material was not H2S but some other substance. The possibility still remains, however, that the volatile material may still be H2S, as it may be stimulatory in exceedingly small concentrations even though high initial concentrations may inhibit. The stimulation of growth on yeast extract-agar, where no growth occurred without H2 or an added hydrogen donor such as lactate, strongly suggested that the diffusible material might be acting similarly to a hydrogen or electron donor. Identification of this volatile substance is in progress. ACKNOWLEDGMENT This investigation was supported by Air Force MIPR RD-123, entitled: "Microbial Contamination of Air Force Petroleum Products." LITERATURE CITED 1. ALLRED, R. G Methods used for the counting of sulfate-reducing bacteria and for the screening of bactericides. Producers Monthly 22: GROSSMAN, J. P., AND J. R. POSTGATE The estimation of sulphate-reducing bacteria (D. desulfuricans). Proc. Soc. Appl. Bacteriol. 16:1. 3. GROSSMAN, J. P., AND J. R. POSTGATE Cultivation of sulphate-reducing bacteria. Nature 171: POSTGATE, J. R Growth of sulphate-reducing bacteria in sulphate-free media. Research (London) 5: POSTGATE, J. R Sulphate reduction by bacteria. Ann. Rev. Microbiol. 13: POSTGATE, J Versatile medium for the enumeration of sulfate-reducing bacteria. Appl. Microbiol. 11: STARKEY, R. L A study of spore formation and other morphological characteristics of Vibrio desulfuricans. Arch. Mikrobiol. 9: