SCANNING VOL. 9999, 1–4 (2014) © Wiley Periodicals, Inc.

Scanning Electron Microscopy of Bacteria Tetrasphaera duodecadis E. ARROYO,1 L. ENRI´QUEZ,2 A. SA´NCHEZ,3 M. OVALLE,4 AND A. OLIVAS4 1

PCeIM, Centro de Nanociencias y Nanotecnologı´a-UNAM, Ensenada, B.C., Mexico Lababoratorio de Ecologı´a Molecular, Facultad de Ciencias Marinas-UABC, , Ensenada, B.C., Mexico 3 CICESE, Ensenada, B.C., Mexico 4 Centro de Nanociencias y Nanotecnologı´a-UNAM, Ensenada, B.C., Mexico 2

Summary: This study reports the characterization of the Tetrasphaera duodecadis bacteria and the techniques used therein. In order to evaluate the morphological characteristics of the T. duodecadis bacteria scanning electron microscope (SEM) was used throughout its different growth stages. These microorganisms were grown in vitamin B12 broths with 1% tryptone, 0.2% yeast extract, and 0.1% glucose. The turbidimetric method was employed for the determination of bacterial concentration and growth curve. The SEM results show small agglomerates of 0.8  0.05 mm during the lag phase, and rod-like shapes during the exponential phase with similar shapes in the stationary phase. SCANNING 9999:XX–XX, 2014. © 2014 Wiley Periodicals, Inc. Key words: scanning electron microscopy, Tetrasphaera duodecadis, vitamin B12, growth curve, freeze-dryer

Introduction First described by Lochhead in 1958, the Arthrobacter duodecadis bacterium has a crucial need of vitamin B12 for its growth (Lochhead and Thexton, ’51). It was not until the year 2006, when Takashi et al., reclassified A. duodecadis as Tetrasphaera duodecadis, based on the genotypic and chemotaxonomic studies that were made in order to clarify its taxonomic position by means of phylogenetic analysis based on 16S rRNA gene sequences (Ishikawa and Yokota, 2006; Nguyen et al., 2011).

Conflicts of interest: None. Address for reprints: Eurydice Arroyo, PCeIM, Centro de Nanociencias y Nanotecnologı´a-UNAM, CP 22860, Ensenada, B.C., Mexico. E-mail: [email protected] Received 11 May 2014; Accepted with revision 15 July 2014 DOI: 10.1002/sca.21154 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).

The vitamin B12 performs a key role as a coenzyme in the synthesis of DNA, synthesis of neurolipids, and cellular growth. The deficiency of this vitamin can result in megaloblasts (abnormal cell growth, anemia) and longterm deficiency leads to nerve degeneration and irreversible neurological damage (Robert and Brown, 2003). Traditional quantification methods of vitamin B12 within the human body are expensive and require long spans of time, which makes their use impractical for a quick diagnosis that would allow adequate treatment in case of deficiency. An alternative for detection of this vitamin could be for electrochemical methods based on biosensors. One method that has been reported is a microbial biosensor where they used Escherichia coli 215 and an oxygen electrode. However, the response time was 2 h, and E. coli is not specific to vitamin B12 (Isao et al., ’87). The potential use of T. duodecadis, a bacterium that specifically catabolizes vitamin B12, is to be a component for a microbial sensor that can quantify this molecule. Until now there is no reported information on its growth rate, morphology, and nutritional requirements. It is necessary to obtain data reliable to assess the feasibility and potential of this species as biological recognition element in a biosensor, which is the goal of this study. In the interest of establishing the bacterial growth curve, the optic density (OD) of the culture media can be determined by means of turbidimetry and the N number of cells per cubic centimeter through direct microscopic count (Zwietering et al., ’90; Madigan and Brock, ’91). A calibration diagram was constructed that expresses the relation between these two parameters. This diagram shows that the data set was closely linear (OD ¼ 1.73 N  108; r2 ¼ 0.9857) at the investigated concentration range of up to 2.2  107 cells/cm3 with a turbidimetric determination relative error of no more that 5%. The turbidimetric method is preferred over the Rosebrough et al. (’51) spectrophotometric method, as the latter has a relative error of 20–50%, an execution time of close to 90 min, and a nonlinear standard curve. Although commercially available since 1965, scanning electron microscopes (SEM) have mainly been used in the industrial sciences, whereas in microbiology,

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to this day, their use have been limited to the study of surface structures of foraminifera (Bartlett, ’67), actinobacteria (Davies and Williams, ’67), and soil microorganisms (Gray, ’67), among other (Klainer and Betsch, ’70; Thomas et al., ’83; Franson et al., ’84). Other applications for such studies have been partially  limited due to the low resolution of about 100 A currently available, which is thought to be insufficient in order to characterize the surface morphology of the majority of known bacteria (Gray, ’67). In this study, we reported the morphology of the T. duodecadis and its growth curve.

Materials and Methods Development of the Bacterial Culture

Here we used the Gram-negative T. duodecadis bacteria. These bacteria were obtained from the American Type Culture Collection (ATCC) No. 13347. The medium and culture conditions used were: 1% tryptone, 0.2% yeast extract, 0.1% glucose, 0.1% MgSO4, 0.5% NaCl, and 1 ppm vitamin B12 in distilled water (pH 7.0) at 26˚C stirred at 250 rpm.

Growth Curve Method

For the characterization of the growth parameters, 5 mL of a pre-inoculum culture was taken and seeded in 50 mL of broth, incubating at 26˚C for 24 h. A subculture was then prepared which was incubated under the same conditions. From the latter cultivation, 5 mL of the culture were taken every 2 h and five serial dilutions were made (1:10), taking 10 mL of these which were then spread on plates and incubated at 26˚C. The growth was monitored every 2 h to record the optical density (OD) at 620 nm on a UV–Vis spectrophotometer (HACH-DR5000) and by counting colony forming units (CFU).

with window for detection of light elements. The current used was 15 kV electron beam using a tungsten filament. The operating pressure of the team was 1  105 Torr, high vacuum. The working distance of samples was 15 mm.

Results and Discussion Figure 1 shows the OD values obtained during the first 80 h of bacteria growth. Three main phases of the growth curve were clearly observed: (1) Lag phase (0–16 h) when bacteria start to grow, (2) log or exponential phase (16–60 h) when population doubles every 20 min, and (3) stationary phase (60–80 h) when the growth become dynamic. The growth curve allows us to know at which moment to use the bacteria in the biosensor, which is the optimal time of bacterial activity (Fig. 1) that we consider to be at half of its exponential growth. The relationship between the OD readings and the number of viable cells obtained from direct counting on plates (OD vs. CFU  106), is described by the following equation: y ¼ 62.3x þ 0.0132. The value r2 ¼ 0.97 of the calibration curve, indicated a good approximation and made reliable to determine the bacterial concentration from the OD readings of the bacterial medium cultures. The morphological characteristics of the T. duodecadis bacterium were evaluated by means of SEM. The presented micrographs of Figure 2 were made in order to define the morphology during its growth stage: (a) Lag phase presents size coccus 0.8  0.05 mm, (b) exponential phase shows the shapes of small rods with lengths of approximately 3.5  0.5 mm, (c) marked zone magnification within (b), and (d) stationary phase with rods measuring 1.4  0.5 mm in length. The micrographs of lag phase shown coccus around WO3, it was not possible to get a micrograph of higher magnification, since the

Scanning Electron Microscopy Method

After a 4-h fixation in 2.5% glutaraldehyde on 0.1 M phosphate-buffered saline (PBS), the sample was washed four times with PBS, followed by two rinses with distilled water, then dehydrated in ethanol solutions of 60%, 70%, 80%, 90% and three times in 100%, for 10 min each. Afterwards, they were dried in a freeze-dryer for a period of 48 h. They were then allowed to warm to room temperature overnight (Castillo et al., 2005). The specimens were then examined with a Jeol JSM5300 SEM integrated with an energy analyzer for electron scattering Thermonoran, SuperDry II model

Fig 1.

Growth curve of the Tetrasphaera duodecadis bacteria.

E. Arroyo et al.: SEM T. duodecadis

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Fig 2. SEM micrographs of the Tetrasphaera duodecadis bacteria in growth phases: (a) Lag phase 5,000, (b) lag phase 7,500, (c) exponential phase 3,500, (d) exponential phase 7,500, (e) stationary phase 3,500, and (f) stationary phase 7,500.

image is overloaded by static more than the others, in fact the W was added and it helped to a better quality. This study is a clear indication that SEM techniques can be used for microbiology studies. The obtained resolution is more than adequate in order to study the surface morphology of microorganisms. SEM has

certain advantages over other techniques, which in turn give us better details about the morphology, topography, and size of the studied microorganism. These data, which appertain to the visual shape of the organism, can also be corroborated through the use of a conventional microscope.

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Acknowledgments E. C. Arroyo would like to thank the CONACyT scholarship, as well as the DGAPA IN108613-2 project for their support. We would also like to thank Israel Gradilla for his technical assistance.

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Ishikawa T, Yokota A. 2006. Reclassification of Arthrobacter duodecadis Lochhead 1958 as Tetrasphaera duodecadis comb. nov. and emended description of the genus Tetrasphaera. Int J Syst Evol Microbiol 56:1369–1373. Klainer AS, Betsch C. 1970. Scanning-beam electron microscopy of selected microorganisms. J Infect Dis 121:339–343. Lochhead H, Thexton G. 1951. Vitamin B12 as growth factor for soil bacteria. Nature 167:1034–1035. Madigan TD, Brock MT. 1991. Biology of microorganisms. 6th edition. Englewood Cliffs, NY: Prentice-Hall, Inc. Nguyen HTT, Le VQ, Hansen AA, Nielsen JL, Nielsen PH. 2011. High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems. FEMS Microbiol Ecol 76: 256–267. Robert COH, Brown D. 2003. Vitamin B12 deficiency. J Am Geriatr Soc 67:979–986. Rosebrough OH, Farr NJ, Randall AL, Lowry RJ. 1951. Protein measurement with the follin phenol reagent. J Biol Chem 193:265–275. Thomas JM, Noble M, Costerton JW. 1983. Examination of the morphology of bacteria adhering to peritoneal dialysis catheters by scanning and transmission electron microscopy. J Clin Microbiol 18:1388–1398. Zwietering MH, Jongenburger I, Rombouts FM, Van’T Riet K. 1990. Modeling of the bacterial growth curve. Appl Environ Microbiol 56:1875–1881.

Scanning electron microscopy of bacteria Tetrasphaera duodecadis.

This study reports the characterization of the Tetrasphaera duodecadis bacteria and the techniques used therein. In order to evaluate the morphologica...
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