Development of new G media for culture of Flavobacterium columnare and comparison with other media

    Development of new G media for culture of Flavobacterium columnare and comparison with other media Dana X. Gao, Patricia S. Gaunt PII: DOI: Reference:

S0044-8486(16)30241-1 doi: 10.1016/j.aquaculture.2016.05.006 AQUA 632134

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

22 March 2016 2 May 2016 5 May 2016

Please cite this article as: Gao, Dana X., Gaunt, Patricia S., Development of new G media for culture of Flavobacterium columnare and comparison with other media, Aquaculture (2016), doi: 10.1016/j.aquaculture.2016.05.006

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ACCEPTED MANUSCRIPT Development of new G media for culture of Flavobacterium

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columnare and comparison with other media

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Dana X. Gao* and Patricia S. Gaunt

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Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, P.O.

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Box 197, Stoneville, Mississippi, 38776, United States

*Corresponding author at: Phone: (662) 686-3307; Fax: (662) 686-3568; email:

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[email protected]

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ACCEPTED MANUSCRIPT Abstract Each component of existing agar and broth media was compared based on observations

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of Flavobacterium columnare growth. Newly formulated G media resulted in the fastest

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proliferation of F. columnare cells in broth and yielded fully developed colonies on inoculated

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agar. In the formulation of the G media, consideration was given to factors such as Sodium chloride and antibiotics, which affect bacterial growth. Ingredients were added or subtracted

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from existing media, with the resulting formulation of 3 broths (Gb1, Gb2, Gb3) and 3 agars (Ga1, Ga2, Ga3). F. columnare culture on G agar media could be confirmed within 24 h for disease

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diagnostics. Because of the rapid growth of the bacterium, colony counts were performed after 24 h, avoiding the merging of adjacent colonies at 48 h. In G broth, the absorbance reached 0.3-

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0.5 within 24 h, indicating ≥3×108 CFU/ml in broth. In addition, no clumps formed in G broth, which ensured a uniform distribution of F. columnare cells compared to existing broths. The G

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media are superior for F. columnare culture because of the rapid uniform growth of the bacterium compared to existing media.

Statement of Relevance

The newly developed G media significantly support faster growth of Flavobacterium columnare in broth and on agar surface than that with the existing media. The formulations of the broth media are focused on the elimination of bacterial cell clumping and quick cell proliferation, and to maximize the colony development of Flavobacterium columnare cultured on agar surface when the antibiotic of neomycin presented. The ingredients and their concentrations were thoroughly investigated and the reasons that new media are better than existing media are explained and discussed.

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ACCEPTED MANUSCRIPT Abbreviation YE: yeast extract

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MH: Mueller Hinton

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MHB: Mueller Hinton broth

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MHA: Mueller Hinton agar CAMHB: cation adjusted Mueller Hinton broth

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CAMHA: cation adjusted Mueller Hinton agar

MBHI: modified brain heart infusion

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TYES: tryptone yeast extract salt

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SCA: selective cytophaga agar

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BHI: brain heart infusion

-N: neomycin

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FCGM: Flavobacterium columnare growth medium

Ga: G agar medium

Gb: G broth medium Abs: absorbance(s)

Key words: Columnaris disease; Flavobacterium columnare; nutrient and chemical constituents; media comparison; salinity; optimal bacterial growth

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1. Introduction

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Columnaris disease, caused by the Gram-negative bacterium Flavobacterium columnare,

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infects more than 36 species of cultured and wild fish (Plumb, 1999) and causes significant

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disease losses in freshwater aquaculture worldwide (Bernardet, 1989; Wagner et al., 2002; Pulkkinen et al., 2010). Although the disease was first described in warm water fish in 1922

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(Davis, 1922), the bacterium was not isolated on media until 22 years later. In fact, F. columnare

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does not grow on or in commercially available media such as Mueller Hinton Broth (MHB) and Agar (MHA), Cation-adjusted Mueller Hinton Broth (CAMHB), brain heart infusion (BHI or

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modified (MBHI) or tryptic soy agar. Selective Cytophaga agar (SCA) (Ordal and Rucker, 1944),

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composed of a low nutrient agar, was the first medium developed that supports, albeit slow, F. columnare growth and was later modified (Anacker and Ordal 1955, 1959 I & II). As the

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nutrients and minerals for supporting F. columnare growth, a number of tryptone, peptone, yeast extract, yeast infusion, beef extract, fish extract, casein, casitone, gelatin, amino acids, hemoglobin, sodium acetate, sodium citrate, sodium bicarbonate, sodium chloride, barium chloride, calcium chloride, potassium phosphate and ferric sulfate had been tested, modified or omitted (Garnjobst, 1945; Chase, 1965; Shieh, 1980; Liewes et al., 1982; Bullock et al., 1986; Holt, 1988; Farmer, 2004). Commercial MH media were diluted (Fijan and Voorhees, 1969), and neomycin and polymyxin B were first added to SCA medium for culture purification from bacterially contamination (Fijan, 1969); All of these media are summarized in Tables 1 and 2. Song et al. (1988) compared SCA, tryptone yeast extract, tryptone yeast infusion, Chase, Shieh and Liewes broths and concluded that Shieh and Liewes broths result in a higher absorbance than other media with improved cell yields. In addition, Shieh medium supports a 4

ACCEPTED MANUSCRIPT shorter incubation time (Shieh, 1980; Song et al, 1988). Farmer (2004) evaluated SCA, Shieh, Hsu-Shotts, diluted MHA and FCGM agar with or without neomycin and polymyxin B. In all

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media without antibiotics, F. columnare colonies were poorly isolated from contaminating

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bacteria; with antibiotics, better growth was found on SCA and Shieh agar than on Hsu-Shotts, diluted MH, or FCGM. In addition, FCGM broth supported faster growth and higher yields of

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cells than SCA, Shieh, Hsu-Shotts, diluted MHB and TYES broths.

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The Clinical and Laboratory Standards Institute (CLSI, 2006) recommends 1:7 diluted MHB or MHA for antibiotic susceptibility testing of fastidious bacteria such as F. columnare.

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MH diluted at a ratio of 1:5 showed significantly better growth compared to a 1:7 dilution for determining the antibiotic susceptibility of F. columnare (Darwish et al., 2008), and 1:5 diluted

(Gieseker et al., 2012).

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CAMHB has been adopted for standardized antimicrobial susceptibility testing methods

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When F. columnare is examined in a fish disease diagnostic laboratory, its unique morphological characteristics that distinguish it from other bacteria are readily apparent: 1) scrapes from clinical lesions on the gills, skin or mouth yield bacterial cells amassing together in a “haystack” formation; 2) post-incubation, cultures of F. columnare adhere tightly to the agar surface and are nearly impossible to dislodge from culture plates; 3) the cells bind together and form ring-like colonies around the broth surface and adhere to the inner wall or bottom of culture flasks; 4) F. columnare cells form clumps in suspension. Upon the formation of such clumps, the cells are no longer evenly distributed, which makes cell colony counting or cell concentration estimation inaccurate. Furthermore, the clumps are not easily segregated into single cells, even with vortexing for minutes even hours. As an external pathogen, these characteristics that allow

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ACCEPTED MANUSCRIPT the bacterium to adhere to its host without being dislodged most likely increase its chances of survival on a host and in a fish pond environment.

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Various water bacteria also attach to the surface of the fish body, particularly on lesion

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areas; thus, contamination occurs when culturing external lesions on a solid agar medium. To inhibit culture contamination, antibiotics such as neomycin and polymyxin B (Fijan, 1969) had

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been introduced into solid agar media to reduce the overgrowth of water bacteria. Decostere et al.

and suggested tobramycin as a replacement.

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(1997) proposed that the neomycin and polymyxin B in Shieh agar inhibit F. columnare growth

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The objective of this project was to focus on the problematic issues associated with F. columnare culture in existing media, i.e., clump formation, antibiotic usage, salinity that inhibits

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bacterial growth, and slow growth rate and to develop new media to eliminate these problems.

2. Material and methods

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2.1. Bacterial isolates

F. columnare ATCC 49513 and two isolates (S06-227 and S07-531), obtained from the aquatic animal diagnostic laboratory under the College of Veterinary Medicine of Mississippi State University were used in these studies. The isolates were confirmed as F. columnare by PCR (Gaunt et al., 2010).

2.2. Comparison of G media with existing media The growth of F. columnare in different broths was assessed. All inocula and cultures were incubated at 27°C. Each medium was prepared with the ingredients listed in Tables 1 and 2. For media that did not specify a solid form, 10 g agar was added to 1000 ml broth. 2.2.1. Test 1

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ACCEPTED MANUSCRIPT SCA (Anacker and Ordal, 1959-I), 1:5-diluted MHB, Shieh, FCGM and initial G broth, which is salt reduced from 5 g to 0.5 g of FCGM, were compared. Fifty milliliters of each broth

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was inoculated with 50 µl of three isolates separately cultured in diluted MH broth. After mixing, the initial cell number for each isolate was estimated by the 10-fold dilution method; the colony

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counts were 4.1×104 CFU/ml, 7.5×104, and 3.1×104 for ATCC 49513, S07-531, and S06-227,

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respectively. All cultures wereplaced in an incubator and sampled in triplicate at 24, 48, 72, and

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96 h for absorbance (Abs) measurement. The optical density (OD) at a wavelength of 600 nm (OD600) was measured using a Genesys™ 10 series spectrophotometer. Each broth alone was

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used as an Abs blank. Each Abs measurement of the three samples in each medium was recorded, and the mean of triplicate readings was used for comparison graph(s). Bacterial clumping in the

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broths was observed before measuring the Abs. 2.2.2. Test 2

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The same method as used in test 1 was performed with SCA, 1:5-diluted MHB, 1:5diluted CAMHB, Shieh, TYES, FCGM and G1 broth (G1-b); Abs was measured at 24, 48 and 72 h. The three isolates were cultured in Shieh broth and incubated for 72 h before pipetting (100 µl) into all seven broths (10 ml). The initial cell number of the three isolates was estimated as 12×106 CFU/ml. 2.2.3. Test 3 The following agar media were prepared with the ingredients listed in Tables 1 and 2: SCA, 1:5-diluted MHB, 1:5-diluted CAMHB, Shieh, TYES and FCGM with 10 gram agar in 1000 ml broth. G1-a was used for comparison. All agar media were prepared in duplicate, with or without 0.5% neomycin. Three-day cultures of each isolate, ATCC49513, S06-227 or S07-531, in 1:5-diluted MHB were serially diluted using 1:5 MHB as the diluent with the 10-fold dilution

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ACCEPTED MANUSCRIPT method to a concentration containing between 10-500 cells per 100 µl. One hundred microliters of isolate was dispensed onto each agar plate, and a sterile culture loop was used to gently spread

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the inoculum across the plate surface. The plates were incubated for 24 to 96 h or longer until all

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colonies had fully developed from each individual cell. The colony numbers on each plate were counted, and the color and shape of colonies on each type of medium were compared. With

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duplicates of the seven types of media with or without neomycin, a total of 28 plates were

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examined for each isolate.

The colony number, shape and color of isolate S07-531 on SCA, 1:5 MHA, 1:5 CAMHA,

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Shieh, TYES, FCGM, Ga-1, Ga-2 and Ga-3 with and without neomycin (-N) were photographed for comparison after 2 days of incubation at 27°C.

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2.3. Statistical comparisons of broth and agar media In test 1, the absorbance for each isolate was noted at four time points (24, 48, 72 and 96

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h) for each of the five broths. At each time point, the Abs values of three isolates in five media were measured, for a total of 12 sets of readings (4 time points of 5 media×3 isolates) in test 1. In test 2, the Abs values of three isolates in seven media were measured at three time points (24, 48, and 72 h), for a total of 9 sets (3 time points×3 isolates) of readings. To compare differences among media, each set of three isolates in test 1 at 48 h was arbitrarily selected for multiple statistical analyses by ANOVA and Fisher‟s protected least significant difference procedure (Steel et al., 1997) (SAS Institute, Cary, North Carolina). In test 3, multiple statistical analyses for colony counts on seven media with or without neomycin were also performed as described above. 2.4. The formulation of G media 2.4.1. Initiation of medium development

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ACCEPTED MANUSCRIPT While using Shieh, FCGM or diluted MH broths, clumps and slow multiplication of cells occurred. Thus, to achieve the maximal growth of F. columnare, different formulations of media

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were investigated. Sodium chloride was the first ingredient noted because when saline was used

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to dilute F. columnare cells from concentrated broth culture onto Shieh agar, colony counts were reduced to zero compared with Shieh broth as the diluent, which yielded >100 colonies with

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same dilution of 1:100000 from the same original culture. Commercial MH, tryptic soy agar and

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BHI media that contain 5 g of sodium chloride (NaCl) do not support the growth of F. columnare. Coincidently, vital cultures of F. columnare isolates or colony counts were poor on FCGM agar

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plates, which contain 5% NaCl plus additional cations of sodium contributed from sodium bicarbonate and sodium citrate as well as anion chloride from calcium chloride, and cell

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multiplication in FCGM broth was initially inhibited. Thus, it was hypothesized that NaCl limits the growth of this bacterium. The initial G agar and broth were modifications of FCGM media

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and contained only 0.5 g of NaCl or approximately 10% of the original formulation. The initial G broth and agar were prepared and tested with isolates for growth. In broth, F. columnare cells grew faster with no clumps than in the original FCGM broth, but colonies still formed poorly on agar. Therefore, further investigation of each ingredient was performed. 2.4.2. Broth

All ingredients of FCGM were individually tested by adding one at a time at different doses. After autoclaving, each broth was inoculated with F. columnare isolates and incubated for 48 h before Abs measurement. Using 13 ml snap-top sterile plastic tubes, 5 ml of each broth was pipetted into each tube, and 200 µl of frozen isolate was added and mixed; the tubes were placed in a 27°C incubator. Tryptone was added at 4.0, 8.0 or 10.0 g/l. After Abs comparison, the broth that provided the higher Abs was selected. YE was added to the selected tryptone at a

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ACCEPTED MANUSCRIPT concentration of 0.8, 1.2, or 0.4 g/l. After the optimal tryptone and YE concentrations were selected, calcium chloride (CaCl2.2H2O)/magnesium sulfate (MgSO4·7H2O) at a ratio of 1:2 was

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added at concentrations of 1.48/1.5, 0.74/1.0, 0.37/0.5 or 0.074/0.10 g/l to achieve Ca2+/Mg2+ concentrations of 400/200, 200/100, 100/50 or 20/10 mg/l, respectively. Sodium chloride was

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repeatedly tested at a level of 0.5 or 1.0 g/l, along with CaCl2.2H2O and MgSO4·7H2O. Sodium

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citrate was later added at a concentration of 2.0, 1.0, 0.5 or 0.0 g/l after the

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CaCl2.2H2O/MgSO4·7H2O and NaCl were selected. As essential nutrients, according to Chase (1965) and Shieh (1980), potassium phosphate monobasic (KH2PO4) and dipotassium phosphate

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(K2HPO4) were added at concentrations of 0.2, 0.1 or 0.05 g/l. The higher Abs and fewer clumps were the primary concerns for the development of optimal media.

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Because 1:5- or 1:7-diluted MHB or diluted CAMHB support F. columnare growth, diluted BHI or MBHI might also do so. Media of MHB and BHI or MBHI contain 5 g/l NaCl.

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As the NaCl concentration is proportionally diluted when all ingredients are diluted, dilutions at 1:5, 1:10 and 1:20 were tested with F. columnare isolates to assess whether BHI or MBHI could be an alternative medium. The Abs values of the three isolates incubated in diluted BHI and MBHI broths were measured and compared for optimal cell growth. After many comparisons focusing on higher cell density and fewer clumps, the final formulas were established as three broths (G1-b, G2-b and G3-b).

2.4.3. Agar Using Difco™ agar for the solid growth support, different components were added gradually in a manner similar to the broth testing. The initial formulations were as follows: 10 g/l agar only, served as a blank; agar with 10.0, 8.0, 4.0, 3.0, or 2.0 g/l tryptone; agar with 3.0, 1.0 or

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ACCEPTED MANUSCRIPT 0.4 g/l YE in selected doses of tryptone. The optimal permutations of the two ingredients were assessed by the rapidity of F. columnare colony development. After the best concentrations of

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tryptone and YE were selected, CaCl2.2H2O/MgSO4·7H2O at concentrations of 0.74/1.0, 0.37/0.5

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and 0.074/0.10 g/l was separately added to assess the impact on colony growth. Next, NaCl (0.5 g/l) and KH2PO4 (0.1 or 0.05 g/l) were added to compare their impact on colony development.

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BHI and MBHI diluted 1:5, 1:10 and 1:20 with 10 g/l agar were also tested for colony

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development. The combination of tryptone with diluted MBHI instead of YE was also tested. Gelatin is present in Garnjobst‟s (1945), Liewes et al (1982), Hue-Shotts‟ medium (Bullock et al,

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1986) and in BHI media (Tables 1 and 2), and according to Griffin (1992), this component is among the six ingredients important for F. columnare identification. To ensure its growth-

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supportive effect, gelatin at 1.0 or 3.0 g/l in 10 g agar was assessed with the three isolates. Based on multiple cross-comparisons of rapid development of F. columnare colonies and counts for

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extended periods, the final formulas were established as three agars (G1-a, G2-a and G3-a), paired with three broths (G1-b, G2-b and G3-b). For agar plates containing antibiotics, 0.5% of 10 mg/ml neomycin (Sigma) was added after autoclaving at ≤50°C. 2.4.4. Testing G broths and G agars After G broths and G agars established, three F. columnare isolates were tested with three broths under stirring condition, and thirty F. columnare isolates were tested with G agars with neomycin to ensure the universal usage. Other bacteria such as E. coli, usually used for quality control purpose, were also tested for the negative growth there for the G media are selective for culturing F. columnare only. 2.5. Parameter measurements of media and chemicals

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ACCEPTED MANUSCRIPT To understand and explain the reasons that commercial media inhibit F. columnare growth, total nitrogen as the primary nutrient and the total salinity as a possible limiting factor

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were measured.

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BBL™ CAMHB powder, BHI powder, MBHI powder, Bacto™ tryptone, peptone, YE, gelatin powder, and Difco™ agar (all from Becton, Dickinson and Company, Sparks, MD) were

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examined for total nitrogen (%) using an Elementar vario MAX cube Protein Analyzer, for pH

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using a Thermo Orion Model210 pH Meter and for salinity (‰ or ppt) using an Oaklon Multiparameter PCSTester™ 35. NaCl, CaCl2∙2H2O, MgSO4∙7H2O, KH2PO4 and sodium citrate (from

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Sigma-Aldrich, Inc., St. Louis, MO) were measured for salinity. Fresh water collected from fish farming ponds at Delta Research and Experiment Center

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in Stoneville, MS, was also assayed for nitrogen content, pH, and salinity. One liter of fish pond water was autoclaved and freeze-dried, yielding 0.2809 g organic dry matter, which was

3. Results

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analyzed for total nitrogen using an Elementar vario MAX cube Protein Analyzer.

3.1. Broth comparison

Test 1 is summarized in Figures 1-1, 1-2 and 1-3. The Abs values of the three isolates in SCA were the lowest, and only S06-227 barely reached 0.100 at 96 h. Shieh and 1:5 MHB had similar effects on the growth of the three isolates (Abs96h>0.150), except for S07531(Abs96h>0.100) in Shieh broth. The Abs24h values of the three isolates in FCGM were low compared to that in Shieh and 1:5 MHB but exceeded both in the following days (Abs96h 0.3700.450). The Abs of the three isolates in initial G broth was the highest overall (Abs96h 0.4000.500). ANOVA analysis at P-0.01 is shown in Table 4. Letters of a, b, c, or d represent the

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ACCEPTED MANUSCRIPT differences among media. For isolate ATCC49513, initial G broth (a) resulted in the highest Abs and significantly differed from the other four media (Abs48h 0.302); FCGM (b), at second highest,

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also significantly differed from the others; Shieh (c) and diluted MHB (c) showed no difference;

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and SCA (d) was the lowest, with significance. For S06-227, G broth (a) was significantly higher than the other media (Abs48h 0.261); FCGM (ab) was between G and Shieh, with no significant

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difference among these three media; Shieh (bc) was between FCGM and diluted MHB, with no

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significant difference; diluted MHB (bc) showed no significant difference between Shieh and SCA; SCA did not significantly differ from diluted MHB but significantly differed from the

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other three media, with the lowest Abs. For S07-513, G medium (a) significantly differed from the other four media, with the highest Abs48h of 0.231; FCGM (b) significantly differed from the

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other four media, at second highest; Shieh (cd) showed no difference between diluted MHB and SCA; diluted MHB (c) significantly differed from the other media, except for Shieh; and SCA (d)

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significantly differed from the other media, except for Shieh. In test 2, (Figures 2-1, 2-2 and 2-3), the Abs24h of the three isolates in all broths, except for S07-531 in FCGM, reached 0.050 and above. In G1 broth, the Abs24h values of the three isolates were between 0.100-0.180. The Abs values of the three isolates in SAC and 1:5 MHB did not differ much (Abs72h 0.090-0.0.140); the Abs of these isolates in Shieh and 1:5 CAMHB were similar (Abs72h 0.160-0.180); TYES was slightly better than the other 4 media for all three isolates (Abs24h 0.100 and above and Abs72h ~ 0.200). Overall at last, G1and FCGM broths resulted in the highest absorbance (Abs72h 0.200-0.230 for FCGM and Abs72h 0.250-0.300 for G1 broth).

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ACCEPTED MANUSCRIPT Both tests ended at either 72 or 96 h, and the Abs values of the three isolates in SAC, 1:5 MHB, 1:5 CAMHB and Shieh were approximately 0.200; values were higher in TYES, FCGM,

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initial G and G1 broths.

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3.2. Clump observation

Before measuring the absorbance, each sample was pipetted into a cuvette and observed

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for clump formation under laboratory lighting. Only SCA and 1:5 CAMHB showed no clumps

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for all samplings. When the Abs values were greater than 0.250, although SCA, 1:5-diluted MHB, CAMHB or Shieh typically never reached that high, traces of cell swirls could be found

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after pipetting the samples from culture tubes into cuvettes, delaying Ab measurement until the cells appeared to be evenly distributed. The cultures in broths that were static in the incubator for

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a few days would form large chunks or lumps floating on the top of Shieh, TYES, FCGM or G broth. The inner wall of the container would be covered with ring-like colonies surrounding on

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the edge of a surface. Although vortexing reduced some of these clumps or broke them into smaller pieces, not all of the clumps in Shieh and TYES broths could be dispersed. Loose clumps that later developed (after two days) in FCGM, initial G and G1 broths could be dispersed by vortexing, and those broths became slightly slimy upon thickening. 3.3. Agar comparison

Table 5 presents the average colony counts for the three isolates on seven agar media with or without neomycin and statistical analyses of multiple comparisons (7×2 factors) at a Pvalue of 0.01. For each isolate, the colony counts should be in a similar range because the cells were transferred to the agar plates from the same dilution tube. For ATCC49513, colony counts on SCA and Shieh showed no significant difference (a); SCA with neomycin (-N), Shieh-N and G1-a-N (ab) also exhibited no difference from SCA,

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ACCEPTED MANUSCRIPT Shieh and TYES (b) and G1-a (b); 1:5 CAMHA and FCGM were identical (c), significantly lower; 1:5 CAMHA-N (cd) was between 1:5 CAMHA and FCGM and 1:5 MHA (d); and 1:5

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MHA-N, TYES-N and FCGM-N were identical (e). Only SCA, Shieh and G1-a resulted in

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similar colony numbers on both agars with or without neomycin. In reality, colony counts can vary within certain levels from the same dilution, and the statistical analyses presented here are

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more sensitive than the test itself. Nonetheless, the differences in medium type and antibiotic

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effect on colony development were obvious for this particular isolate. This particular test is merely one of many mini tests with a similar trend: diluted MHA or FCGM with neomycin

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always yields fewer colonies or zero than Shieh.

From Table 5, the differences for isolates S06-227 or S07-513 are also presented with

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letters of a-h or a-d. FCMG-N increased the differences. The sizes of colonies on agar plates vary greatly and are relatively larger on agar without

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neomycin. The colonies spread irregularly as large as the space allows or remain restricted as a core. Colonies on SCA can merge, which hampers precise colony counting. The colors of the colonies depend on the type of medium rather than the isolate itself. Based on test 3, the lower colony counts on 1:5 MHA, TYES and FCGM neomycin agar plates suggest that neomycin somewhat inhibits the development of single cells to colonies. Colony counts on FCGM plates were conducted after incubating the cultures for 6 days, and the colonies were tiny. Larger colonies on agar media without neomycin also indicate inhibition by the antibiotic. The lack of colonies developed on FCGM might be caused by high concentration of sodium chloride, chemicals or high dose of tryptone as a nutrient. Test 3 also suggests that 1:5 MHA, TYES and FCGM are not optimal or universal media for culturing various isolates. In

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ACCEPTED MANUSCRIPT practical for columnaris disease diagnostics, neomycin must be added to agar media to reduce the contamination of water and airborne bacteria for archiving pure cultures.

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3.4. G broth formulation

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Tryptone at a dose of 10 g/l makes vague broth after autoclaving, which could be confused with contamination. Compared with 4.0 g/l tryptone for cell growth, 8.0 g/l tryptone

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yielded higher Abs of cells. Thus, 8.0 g/l tryptone was chosen for a clear broth and higher cell

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density. YE at levels of 0.4 to 0.8 g/l or 1.2 g/l were added to 8.0 g/l, and Abs values among the three F. columnare isolates were compared; the highest Abs was at 0.4 g/l YE and the lowest at

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1.2 g/l YE. With CaCl2.2H2O/MgSO4·7H2O at doses of 0.37/0.5 or 0.074/0.10 g/l added in, clumps appeared and were distributed throughout the broth. However, clumps were

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tremendously reduced at high doses of 1.48/1.5 and 0.74/1.0 g/l, so 0.74/1.0 g/l

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CaCl2.2H2O/MgSO4·7H2O which provide 200 mg/l Ca2+ and 100 mg/l Mg2+ was chosen. Sodium

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chloride at both 0.5 and 1.0 g/l further eliminated clumps so 0.5 g/l was selected. As sodium citrate at 2.0, 1.0 or 0.5 g/l added in, any dose of it neither increased Abs nor reduced clumps so this chemical was totally eliminated. Potassium phosphate, monobasic or dibasic, was added last to the already formulated medium to provide K and P ions for cell growth. Dipotassium phosphate at doses of 0.2 g or 0.1 g immediately caused a milky turbidity by reacting with the chemicals already in the broth; thus, it was eliminated. Potassium phosphate monobasic at doses of 0.2, 0.1 or 0.05 g/l neither changed the pH nor affected the Abs and clumps. In the case of a possible need to maintain natural characters or gene expression (no evidence found in the literature), 0.10 g/l KH2PO4 (0.1 g/l) was added to provide 28.7 mg/l K ions and 22.8 mg/l P ions. As tryptone is processed from milk, all essential minerals such as calcium should be included, and known chemicals were added to ensure the proper quantities of essential minerals.

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ACCEPTED MANUSCRIPT The first broth (G1-b) was composed of 8.0 g tryptone, 0.4 g YE, 0.74 g/0.5 g CaCl2.2H2O/MgSO4·7H2O, 0.50 g NaCl and 0.10 g KH2PO4 (Table 6). The pH (6.81), which

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was lowered by YE (pH 5.94, Table 3), could be adjusted to 7.0-7.4 by adding a few drops of 1

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N NaOH. Based on the testing(s) of all three isolates, a pH slightly lower than 7 did not affect cell proliferation.

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Compared with all dilutions of BHI or MBHI broths (1:5, 1:10 and 1:20), the Abs (0.405)

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of S06-227 was highest in 1:5 MBHI and lowest (0.095) in 1:20 BHI. The Abs values of ATCC49513 were zero at 24 h in 1:5 BHI and MBHI, and 0.109 in 1:10-diluted MBHI. Clumps

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did not form in any of the 1:5 BHI and MBHI cultures. The Abs values of S06-227 and S07-531 in 1:5 MBHI within the first 24 h fell in the range of 0.1–0.3 under static conditions and between

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0.30–0.50 under stirring conditions. The diluted MBHI (7.6 - 3.8 g/l) is labeled as G3-b in Table 6.

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Because MBHI contains various nutrients from brain and heart infusion, animal tissues and casein (Table 2) and has a higher pH (7.4) than that (5.94) of YE (Table 3), 1.0 g/l MBHI was used to replace 0.4 g/l YE to enhance tryptone broth to formulate G2-b (Table 6). Two hundred fifty milliliters of G1-b, G2-b and G3-b was tested for 24 h under stirring conditions at 27°C with the ATCC49513, S06-227 and S07-513 isolates with 0.5 ml 48 h culture in 1:5 MHB. The Abs values were 0.428 (5.1×108 CFU/ml from colony count), 0.454 (8.3×108 CFU/ml) and 0.500 (1.3×109 CFU/ml) for G1-b; 0.340, 0.455 and 0.549 for G2-b; and 0.000, 0.401 and 0.402 for G3-b (7.6 g/l), 0.110 in 3.8 g/l for ATCC49513, respectively. Any sensitive isolates like ATCC 49513 could be cultured in diluted MBHI from 1:10 to 1:20. The medium G3-b is a choice only when other media are not available. 3.5. G agar formulation

17

ACCEPTED MANUSCRIPT Without any added nutrient, no colony or positive culture developed on agar surfaces inoculated with the three isolates for 48 to 72 h. Adding 10.0, 8.0, 4.0, or 2.0 g/l tryptone and

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later adding 3.0 g/l to agar yielded colonies with various shapes and colors. Regardless of the

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isolate tested, taller colonies with a hard core formed with 10 or 8 g/l tryptone, whereas flat and widely spread colonies were found at 2 g/l. Large and quickly spreading colonies appeared on 4

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g/l tryptone agar without neomycin, similar shapes to TYES agar. With neomycin, cultures on 10,

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8 or 4 g/l tryptone agar were either negative or fewer colonies grew compared with the same dose of tryptone without neomycin, showing the same pattern as FCGM and TYES neomycin

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plates (8 g/l tryptone for FCGM and 4 g/l for TYES, Table 5). Agars with 3.0 and 2.0 g/l tryptone with 0.5% neomycin were fully supportive of colony formation tested with all three

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isolates. Thus, 3 g/l tryptone was selected.

Cultures on YE agar were positive after 3 days at doses of 0.4 and 1.0 g/l and after 4 days

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at 3 g/l. With a mini test using isolate S05-531, the colony count on 3 g/l YE was 130 compared to with neomycin, which yielded 18 colonies. Because YE is low in pH (5.94, Table 3) and because a pale culture became positive after 3 days, the lower dose, 0.4 g/l, was selected with 3 g/l tryptone. Similar pattern occurred in broth formulation as mentioned above. When adding CaCl2.2H2O/MgSO4·7H2O at a level of 0.74/1.0, 0.37/0.5 or 0.074/0.10 g/l, cultures of all isolates were positive. Agar plates of 0.74/1.0 g/l or 0.37/0.5 g/l CaCl2∙2H2O/MgSO4∙7H2O and neomycin inhibited colony formation or spreading, similar to

FCGM and TYES neomycin agar plates (0.74/1.0 g/l or 0.5/0.5 g/l, Table 1). Adding 0.074 g/0.10 g calcium and magnesium, for achieving 20 mg/l calcium and 10 mg/l magnesium ions, similar to CAMHB (Table 2), thick cultures developed quickly within 24 h, and colony counts were very close with or without neomycin.

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ACCEPTED MANUSCRIPT Adding 0.5 g/l NaCl to tryptone, YE and CaCl2∙2H2O/MgSO4∙7H2O reversed the effect of colony formation: colonies were inhibited instead of spreading. Thus, NaCl was eliminated from the agar

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formulation.

Since Chase (1965) and Shieh (1980) mentioned that potassium and phosphorus are

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essential mineral to the bacterium, to provide 14.4 mg/l K ions and 11.4 mg/l P ions, 0.05 g/l

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KH2PO4 was added to the above ingredients for cell growth, with no obvious positive or negative

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effect on colony formation or growth speed.

G1-a was composed of 3 g/l tryptone, 0.4 g/l YE, 0.074 g/l CaCl2∙2H2O, 0.10 g/l

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MgSO4∙7H2O and 0.05 g/l KH2PO4 (Table 6).

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Gelatin in agar without neomycin supported very weak growth of cells at 1.0 g/l and no

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growth at 3.0 g/l. The cultures on agar were faint in color and barely detectable. Gelatin contains the highest amount of nitrogen (16.5%) compared to other compounds but has a low pH (4.89;

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Table 3); the acid might hinder F. columnare growth.

One-fifth-diluted BHI and MBHI (7.6 g/l) with 10 g agar resulted in no growth of the three isolates, with or without neomycin, whereas one-tenth-diluted BHI and MBHI (3.8 g/l) supported colony development for S06-227 and S07-531 but not ATCC49513. With neomycin, colonies were restricted or absent. Colonies on 1.9 g/l BHI or MBHI (1:20 dilution) grew well with or without neomycin. Overall, MBHI notably supported faster colony development than BHI for the three isolates. After testing the cultures of the three isolates, 2.0 g/l MBHI was chosen (G3-a). Because this medium was more supportive than YE, 1 g/l MBHI was selected to replace 0.4 g/l YE to formulate G2-a. KH2PO4 was omitted. Table 6 summarizes the three pairs of G media.

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ACCEPTED MANUSCRIPT Thirty F. columnare isolates collected from various regions of Mississippi, Florida, Alabama, Georgia, Tennessee, and Louisiana during the years 2007 to 2013 were cultured with

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the newly developed G broths and neomycin agars to assess the suitability of universal use. All cultures were positive within 24 h on G1-a, G2-a and G3-a, and their Abs values in broths of G1-

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b or G2-b were ≥0.200 after 24 h under static conditions at 27°C. The fish pathogens

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Edwardsiella tarda, Edwardsiella ictaluri and Aeromonas hydrophila, which were archived from

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diseased fish cultures from our diagnostic lab, and quality control bacterium Escherichia coli ATCC25922 were also tested with neomycin G agar media to ensure negative growth. No single

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colony of those bacteria grew on G agar neomycin plates. 3.6. Parameter analyses

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As shown in Table 3, the total salinities (‰) of the media that inhibit F. columnare growth were measured: 7.98 (CAMHB), 7.16 (BHI) and 7.69 (MBHI). FCGM is 6.63, and

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casein acid hydrolysate in CAMHB or MHB contains approximately 36.5% salt (http://www.mpbio.com/detailed_info.php?family_key=02101291), for 6.39 g of 17.5 g. This number is close to the measurement of the total salinity of CAMHB, which also includes Ca and Mg. Sodium chloride is the primary contributor to the salinity of those media, by at least 5‰ (Tables 1 and 2). After a dilution of one to five (1:5), the salinity contributed by salt of CAMHB, MHB and MH agar is proportionally diluted to 1.28, which is close to the measurement of 1.36. All broth media that support F. columnare growth have salinities approximately 2.0 or under, and agar media that support F. columnare growth have salinities mostly under 1.0. In contrast, the 1:5-diluted CAMHB and MH agars, with 1.36 salinity, could not optimally support colony development (Table 5). This fact might explain why saline at 0.85% (8.5 ‰) salt killed cells when used as a diluent and why FCGM (6.63) inhibited cell proliferation early in incubation.

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ACCEPTED MANUSCRIPT However, after F. columnare cells adapted to high salinity, the cells exhibited tolerance to salt up to 6.63‰ with rich nutrients, still allowing the cells to proliferate.

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The total nitrogen of CAMHB was calculated as 1.76 g/l. BHI contains primarily

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pancreatic digest of gelatin and less animal tissue than MBHI; MBHI contains pancreatic digest of casein from milk as well as more animal tissue. The calculated concentrations of nitrogen

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were 3.8 g/l for BHI and 4.03 g/l for MBHI, which are higher than that of CAMHB. Perhaps the

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higher nitrogen concentration explains why diluted MBHI was better than diluted MHB at supporting F. columnare growth.

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The salinities of the G broths were 1.94 (Gb-1), 2.05 (G2-b) or 1.51 or 0.77 (G3-b), and the nitrogen concentrations were 1.11, 1.08 or 0.81-0.41 g/l. For G1, G2 or G3, the primary

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contributors to salinity were calcium chloride (0.546), magnesium sulfate (0.390) and sodium chloride (0.5; Table 3), and that for Gb-3 was mainly sodium chloride (1.0 g/l after 5-fold

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dilution, Table 2). The salinities of agars Ga1, Ga2, or Ga3 were 0.633, 0.627 or 0.589, and the nitrogen doses were 0.44, 0.51 or 0.21, respectively. The salinities of SCA, 1:5 CAMHB, Shieh, TYES or FCGM were 0.204, 1.36, 0.418, 0.822 and 6.63, and the nitrogen concentrations were 0.12, 0.35, 0.83, 0.58 and 1.15, respectively. The salinities of SCA and pond water (0.204 and 0.192) were very close. SCA broth supports the slow growth of F. columnare cells due to the low concentrations of nutrients and minerals. F. columnare cells could remain viable for at least a month (unpublished data). Fresh water was tested because it is the environment where F. columnare dwells without any aquatic animals as the hosts. Notably, YE and gelatin contain higher amounts of nitrogen than the other compounds but do not better support F. columnare cells growth, particularly at the higher concentration (3 g/l). The low pH (5.94 for YE and 4.89 for gelatin) or perhaps the lack of other essential

21

ACCEPTED MANUSCRIPT nutrients and minerals (low salinities also indicated low ions in YE [0.053] or in gelatin [0.02]) may be responsible. All F. columnare culture media with yeast extract, except for Shieh (pH

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increased by 0.1 g K2HPO4), were slightly low in pH (6.77- 6.89) but did not appear to be a

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problem. The pH could be adjusted to 7.2-7.4 by adding a few drops of 1 N sodium hydroxide if necessary.

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4. Discussion

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4.1. Antibiotic effect

One of the characteristics that Griffin (1992) identified for F. columnare is its tolerance

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to neomycin and polymyxin B on Hsu-Shotts agar medium. However, according to our tests, the bacterial cells were killed either completely or partially by neomycin on FCGM, TYES and

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diluted MHB neomycin agar media. Thus, Griffin‟s conclusion of bacterial tolerance to neomycin is conditional and only suitable for media that contain relatively low doses of nutrients

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and low salinity such as SCA, Shieh, even G agars. Hsu-Shotts agar medium that Griffin tested with is low in nutrients and no chemicals (Table 1). Whether the slow development of colonies on 1:5 MHB, TYES and FCGM neomycin agar is caused or inhibited by the increased toxicity of neomycin in combination or reacting with high chemical (salinity≥0.822) or nutrient concentrations (tryptone≥8 g or nitrogen≥1.07, Table 3) is unclear. Decostere et al. (1997) reported that the combination of neomycin (0.5%) and polymyxin B (10 IU/ml) together in Shieh agar reduced F. columnare cell counts by 103-104 CFU/ml compared to tobramycin (0.1%) alone while using phosphate-buffered saline (PBS) as the diluent for 10-fold serial dilutions. However, whether the reduced cell counts were caused by neomycin and polymyxin B or by PBS is unknown. With our several years of experience using Shieh medium with neomycin, we have never had any problem with colony growth, and Shieh agar provides the quickest growth

22

ACCEPTED MANUSCRIPT compared to diluted MH agar and FCGM. Notably, Shieh agar only has a shelf life of two months, i.e., colony counts will decrease if Shieh agar plates are used after two months. The PBS

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or saline used as diluent from our experience do kill F. columnare cells. And the notice of their

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effect initiated new media creation.

4.2. Studies of salinity and pH related to media and columnaris disease

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Bernardet (1989) added NaCl at 0.5, 1.0, 2.0 or 3.0% to Ordal‟s broth and found that

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more than 0.5% (salinity 5‰) NaCl inhibited the growth of all 9 strains of F. columnare. Shamsudin and Plumb (1996) treated Hsu-Shotts broth with 0.5, 1.0, 2.0 or 3.0% NaCl and

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adjusted the pH from 5 to 11. Of a total of 11 isolates, 2 grew in 0.5% salt Hsu-Shotts broth and none above 0.5%; 4 of 11 grew at pH 6 and 2 at pH 10. The tolerance of F. columnare to salt

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was 0.5%. The pH measurement of YE and gelatin in the present study indicate that they are not suitable ingredients for culturing F. columnare because of the low pH of 5.94 or 4.89

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respectively (Table 3). The pancreatic digest of gelatin in commercial BHI medium was replaced by casein in MBHI (Table 2). Adding 0.5 g/l salt to G agar media inhibited colony development. Altinok and Grizzle (2001) studied the effects of salinities on F. columnare infection of euryhaline and freshwater stenohaline fish and found that after acclimation to 1.0, 3.0 or 9.0‰ salinities with F. columnare infection, only fish that acclimated to 1‰ salinity exhibited mortality. The in vitro growth of bacteria was significantly higher at 1.0 or 3.0 ‰ than in control Hsu-Shotts medium at 0.3‰, and bacterial adhesion was reduced with increasing salinity, which could explain the lower mortality of fish at higher salinities. Suomalainen et al. (2005) treated rainbow trout with low pH and salt for columnaris disease. In in vitro trials, 99.9% F. columnare cells were reduced after exposure of pH 4.6 for 15 minutes, and 98-100% cells were eliminated

23

ACCEPTED MANUSCRIPT within 1 h with pH 5.0 or 4% salt. With such conditions and challenged with F. columnare, the mortality rate of rainbow trout was lower than the control group.

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Regardless of whether high salinity and low pH are effective for treating columnaris

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disease, they are effective at eliminating F. columnare cells in vitro. Of note, the study of Altinok and Grizzle demonstrated reduced adhesion of bacteria in in vitro trials with increasing

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salinity. This finding may provide evidence to support that the addition of 0.5-1.0 g/l salt and

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sufficient calcium chloride and magnesium sulfate reduces or eliminates clumps in G media.

5. Conclusion

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F. columnare is considered a fastidious and slow-growing bacterium. All existing media have problems, either slow growth (SAC and Diluted MHB and CAMHB agar), clumping in

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broth (diluted MHB, Shieh, TYES or FCGM), antibiotic restrictions for colony development on agar (diluted MHB, TYES or FCGM) or complicated preparation (Shieh, Lewis or Chase).

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Indeed, perfect broth-agar pair exists. Due to the clumps formed in broth and the antibiotics in agar, achieving an optimal culture medium of both broth and agar with same ingredients and doses for this particular bacterium is impossible. Nonetheless, focusing on those factors, G media provided the most suitable conditions for the optimal growth of F. columnare. Adequate cell growth without clumping can be achieved within 24 h in G broths, and notable positive cultures on agar within 24 h will shorten the diagnostic time.

Acknowledgments We thank Penny Lucas for measuring the total nitrogen of the medium samples and Larry McIntyre for measuring the salinities of the media and chemicals and for culturing the isolates on G media. The cost from the beginning to the end of formulating media was funded with Catfish Health Initiative with the number of 310354 18202 0210000 371180. 24

ACCEPTED MANUSCRIPT References

Chondrococcus columnaris. J. Bacteriol. 70(6), 738-741

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Anacker, R.L., Ordal, E.J., 1955. Study of bacteriophage infecting the myxobacterium

Serological Typing. J. Bacteriol. 78, 25-32

II.

Bacteriocins. J. Bacteriol. 78, 33-40

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I.

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Anacker, R.L., Ordal, E.J., 1959. Studies on the myxobacterium Chondrococcus columnaris.

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Altinok, I., Grizzle J.M., 2001. Effects of low salinities on Flavobacterium colunmare infection

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of euryhaline and freshwater stenohaline fish. J. Fish Dis. 24, 361-367 Bernardet, J.F., 1989. „Flexibacter columnaris‟: first description in France and comparison with

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bacterial strains from other origins. Dis. Aquat. Organ. 6, 37-44

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Bullock, G.I., Hsu, T.C., Shotts, E.B., 1986. Columnaris disease of fishes. United States Fish and Wildlife Service Fish Disease Leaflet 72: 9. Kearneysville, West Virginia.

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Chase, J. M., 1965. Nutrition of Some Aquatic Myxobacteria. Master Thesis (75 pages), University of Washington Seattle, p. 15 Clinical and Laboratory Standards Institute, 2006. Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated From Aquatic Animals: Approved Guideline. CLSI document M42-A. CLSI. Wayne, PA Clinical and Laboratory Standards Institute, 2006. Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals: Approved Guidelines. CLSI document M49-A. CLSI. Wayne, PA Casein Acid hydrolysate: http://www.mpbio.com/detailed_info.php?family_key=02101291; http://www.neogen.com/Acumedia/pdf/ProdInfo/7229_PI.pdf Davis, H.S., 1922. A new bacterial disease in freshwater fishes. United States Bureau of 25

ACCEPTED MANUSCRIPT Fisheries Bulletin 38, 261-280 Darwish, A.M., Farmer B.D., Hawke J.P., 2008. Improved method for determining antibiotic

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susceptibility of Flavobacterium columnare isolates by broth micro-dilution.

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J. Aquat. Anim. Health 20, 185-191

Decostere, A., Haesebrouck F., Devriese L.A., 1997. Shieh medium supplemented with

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tobramycin for selective isolation of Flavobacterium columnare Flexibacter columnaris)

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from diseased fish. J. Clin. Microbiol. 35, 322-324

Gaunt, P.S., Gao, D., Sun, F., Endris, R., 2010. Efficacy of Florfenicol for Control of Mortality

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caused by Flavobacterium columnare Infection in Channel Catfish. J. Aquat. Anim. Health 22, 115-122.

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Garnjobst, L., 1945. Cytophaga columnaris in pure culture: A myxobacterium pathogenic to fish. J. Bacteriol. 49, 113-128

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Gieseker, C.M., Mayer, T.D., Crosby, T.C., Carson, J., Dalsgaard, I., DArwish, A.M., Gaunt, P.S, Gao, D.X., Hwsu, H-M., Lin, T.L., Oaks, J.L., Pyecroft, M., Teitzel, C., Somsiri, T., Wu, C.C., 2012. Quality control ranges for testing broth microdilution susceptibility of Flavobacterium columnare and F. psychrophilum to nine antimicrobials. Dis. Aquat. Org. 101, 207-215

Griffin, B.R., 1992. A simple procedure for the identification of Cytophaga columnaris. J.Aquat. Anim. Health 4, 63-66 Farmer Bradley, 2004. Improved Methods for the Isolation and Characterization of Flavobacterium columnare. Thesis, Louisiana State University, p. 16-17 Fijan, N.N., 1969. Antibiotic additives for the isolation of Chondrococcus columnaris from fish. Appl. Microbiol. 17, 333-334

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ACCEPTED MANUSCRIPT Fijan, N.N., Voorhees, P.R. 1969. Drug sensitivity of Chondrococcus columnaris. Vet. Arhiv 9-10, 259-267

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Hawke, J., Khoo, L., 2004. Infectious Diseases in Biology and Culture of Channel Catfish.

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Craig Tucker and John Hargreaves. Elsevier. New York, USA. p. 397-404. Holt, R.A., 1988. Cytophaga psychrophila, the causative agent of bacterial cold-water disease

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in salmonid fish., PhD. Dissertation, Oregon State University, Corvallis, Oregon.

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Liewes, E.W., Van Dam, R.H., Vos-Maas, M.G., Bootsma, R., 1982. Presence of antigen sensitized leukocytes in carp (Cyprinus carpio L.) following bath immunization against

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Flexibacter columnaris. Vet. Immunol. Immunopathol. 3, 603-609 Ordal, E.J., Rucker, R.R., 1944. Pathogenic myxobacteria. Society of experimental biology

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and medicine proceedings 56, 15-18 Plumb, J.A., 1999. Health maintenance and microbial diseases of cultured fish: Principal

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microbial diseases. Iowa State University Press, Ames, Iowa Chapter 14: catfish bacterial diseases, 181-209

Pulkkinen, K., Suomalainen, L, R., Read, A.F., Ebert, D., Rintamaki, P., Valtonen, E.T., 2010. Intensive fish farming and the evolution of pathogen virulence: the case of clomunaris disease in Finland. Proc R Soc Lond B Biol Sci 277: 593-600 Prescott, Harley and Klein‟s Microbilogy, 7th edition 2007, ISBN: 0072992913 Shamsudin, M.N., Plumb, J.A., 1996. Morphological, biochemical, and physiological characterization of Flexibacter columanris isolates from four species of fish. J. Aquat. Anim.Health 8, 335-339 Shieh, H.S., 1980. Studies on the nutrition of a fish pathogen, Flexibacter columnaris. Microbios Lett. 13, 129-133.

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ACCEPTED MANUSCRIPT Suomalainen, L.R., Tiirola, M., Valtonen, E.T., 2005. Treatment of columnaris disease of rainbow trout: low pH and salt as possible tools? Dis Aqual Org 65, 115-120

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Song, Y.L., Fryer J.L. and Rohovec J.S.1988. Comparison of six media for the cultivation of

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Flexibacter columnaris. Fish Pathol. 23 (2), 91-94.

Steel, R.G., Torrie, J.H., Dickey, D.A., 1997. Principles and procedures of

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statistics: a biometrical Approach 3rd edition. McGraw-Hill, New York.

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Wagner, B.A., Wise, D.J., Khoo, L.H., Terhune, J.S., 2002. The epidemiology of bacterial

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diseases in food-ize channel catfish J. Aquat. Anim. Health 14, 263-272

Table 1. Existing media (Unit: g/l).

Ingredients Tryptone Yeast extract Beef extract Sodium acetate

SCA 1944

SCA 1955

SCA 1959-I

SCA 1959-I

SCA 1959-II

2.5-5.0

0.5 0.5 0.2 0.2

0.5 0.5 0.2 0.2

4.0

8.0

Yeast infusion

30.0

Tween 80 NaCl

1.0

Peptone

0.06 mole

Gelatin Fish extract

Garnjobst 1945 5 (for test 3)

10 or 2.5 (for Test 1 or Test 2) Not quantified Not quantified

28

ACCEPTED MANUSCRIPT

4.0

9.0

0.0

7.0

Chase

Shieh

Liewes

0.5 0.01

4.5 0.015 2.04

Hsu-Shotts 2.0 0.5

TYES 4.0 0.4

5.0 4.0

4.5 10.0

BaCl2∙7H2O KCl

0.12

K2HPO4

0.03

0.1

KH2PO4

0.02

0.05

CaCl2∙2H2O MgSO4∙7H2O

0.004 0.012

0.0067 0.03

FeSO4∙7H2O

0.00002

0.01

5.0

3.0

0.12

0.04

0.5 0.5

0.74 1.0

7.0

1.5 7.5

D

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0.03

0.001

TE

0.05

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NaHCO3 Sodium citrate Agar (for solid medium)

FCGM 8.0 0.8

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0.015 2.04

PT

5.0-9.0

SC

Ingredients Tryptone Yeast extract Sodium acetate NaCl Peptone Casein (acid hydrolyzed) Casitone Gelatin

5 ml (for Test 4)

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Hemoglobin Agar (for solid medium)

9.0

15.0

Table 2. Ingredients in MH and BHI broths and with 1:5 dilutions (Unit: g/l).

Ingredients

Brain heart infusion Peptic digest of animal tissue NaCl Dextrose Starch Pancreatic digest of gelatin Pancreatic digest of casein Na2HPO4 Beef extract Casein acid hydrolysate* Ca2+(mg/l) Mg2+ (mg/l) Agar

Total weight

BHI 6 6 5 3

MBHI 3.5 15 5 2

1:5 MBHI 0.7 3 1 0.4

MHB

1:5 MHB

CAMHB

1:5 CAMHB

1.5

0.3

1.5

0.3

3 17.5

0.6 3.5

3 17.5 20-25 10-12.5

21

10** 4.4

0.6 3.5 4.0-5.0 2.0-2.5 10** 4.4

14.5 2.5

37

10 2.5

38

2

10** 7.6

29

22

ACCEPTED MANUSCRIPT *Casein acid hydrolysate contains 36.5% NaCl according to the manufacturer; MHB, MHA and CAMHB contain 6.39 g/l salt.

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**Difco™ agar was added to the broth to prepare solid agar plates.

Table 3. Total nitrogen, salinity and pH measurements of media and chemicals. Media/chemicals

Nitrogen %

g/l in water

CAMHB

7.98

22.0

BHI

10.01

MBHI 1:5 CAMHB Diluted MBHI (or G3-b) G3-a Peptone YE Shieh Tryptone SCA TYES FCGM

10.60

15.63 10.12 13.4

Salinity

pH

1.76 (22×7.98%)*

7.27

7.38

38.0

3.80

7.16

7.27

38.0 4.40 7.60-3.8 2.0 5.00 0.40 5.0 P**+0.5 YE 8.00 0.5 T***+0.5 YE 4.0 T+0.4 YE 8.0 T+0.8 YE

4.03 0.35 0.81-405 0.21 0.78 0.04 0.83 1.07 0.12 0.58 1.15

7.69 1.36 1.51-0.77 0.378 0.236 0.053 0.418 0.445 0.204 0.822 6.63

7.39 7.32 7.39 7.62 7.04 5.94 7.26 7.02 6.77 6.80 6.89

30

Nitrogen (g/l)

ACCEPTED MANUSCRIPT 8.0 T+0.4 YE 3 T+0.4 YE

1.11 0.44

1.94 0.633

6.81 6.88

G2-b G2-a Fish pond water KH2PO4 NaCl

8 T+1 MBHI 3 T+1 MBHI 0.281 dry matter 0.05 0.50

1.08 0.51 0.0056

2.05 0.624 0.192 0.023 0.537

6.99 7.15 8.51 0.49 5.92

0.546

5.94

0.390 0.755 0.026

6.01 8.60 8.34

0.02 0.0

4.89

0.74

MgSO4∙7H2O Sodium citrate Agar

0.5 1.50 10.0

16.47

1 1000 ml

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Gelatin Distilled water

0.15

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CaCl2∙2H2O

SC

2.01

PT

G1-b G1-a

0.02 0.16

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*The numbers were calculated based on the previous 2 columns. ** P represents peptone.

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*** T represents tryptone

Table 4. Mean absorbance and standard deviation values at 48 h for three isolates cultured in five broths. Means within a column followed by different letter differ (P≤0.01). Medium

ATCC49513

S06-227

S07-513

SCA

0.005 d

0.061 c

0.037 d

DMH

0.097 c

0.069 bc

0.090 c

Shieh

0.106 c

0.116 b

0.064 cd

FCGM

0.161 b

0.150 ab

0.132 b

31

ACCEPTED MANUSCRIPT Initial G

0.302 a

0.261 a

0.231 a

0.006

0.011

0.008

Pooled SE*

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*SE represents standard error

Table 5. Mean colony count for three isolates cultured on seven agar media. Means within a column followed by a different letter differ (P≤0.01). Medium

ATCC49513

S06-227

S07-513

SCA

144.5 a

114.0 d

137.5 a

SCA-N

133.5 ab

104.0 ef

132.0 ab

1:5 MHA

72.5 d

133.5 a

135.5 a

1:5 MHA-N

8.0 e

124.5 bc

132.0 ab

1:5 CAMHA

90.0 c

103.5 f

128.5 b

1:5 CAMHA-N

83.0 cd

94 g

133.0 ab

32

ACCEPTED MANUSCRIPT 146.5 a

119.0 cd

120.0 c

Shieh-N

133.5 ab

112.5 de

129.5 b

TYES

120.5 b

112.5 de

134.0 ab

TYES-N

0.0 e

101.0 fg

FCGM

96.0 c

129.5 ab

132.0 ab

FCGM-N

0.0 e

0.0 h

0.0 d

G1-a

125.5 b

118.0 cd

132.5 ab

G1-a-N

137.0 ab

Pooled SE

5.71

Two-way ANOVA P-value for 3 isolates

PT

Shieh

NU

SC

RI

133.0 ab

120.5 c

2.89

1.95

Medium

Antibiotic

Medium Antibiotic

<0.01

<0.01

<0.01

AC CE P

TE

D

MA

118.0 cd

Table 6. Developed G media (Unit: g/l). G media Tryptone YE MBHI

Pair 1 G1-b 8.0 0.4

G1-a 3.0 0.4

Pair 2 G2-b 8.0

G2-a 3.0

1.0

1.0

CaCl2 ∙2 H2O

0.74

0.074

0.74

0.074

MgSO4∙7H2O NaCl

1.0 0.50

0.10

1.0 0.50

0.10

KH2PO4 Agar Distilled water (ml)

0.10

0.05 10 1000

0.10

1000

Neomycin (10 mg/ml)

0.5 ml

33

1000

10 1000 0.5 ml

Pair 3 G3-b

7.6-3.8

1000

G3-a

2.0

10 1000 0.5 ml

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Analyses of each medium Ca2+ (mg/l) 200 20 200 20 2+ Mg (mg/l) 100 10 100 10 Nitrogen (g/l) 1.11 0.44 1.08 0.51 Total salinity 1.74 0.633 1.85 0.642 pH* 6.81 6.88 6.99 7.15 *pH can be adjusted by adding a few drops of 1 N NaOH to 7.2 if necessary.

Figure 1-1. ATCC49513 growth in five broths (test 1).

34

0.81-0.41 1.51-0.755 7.39

0.21 0.378 7.62

ACCEPTED MANUSCRIPT Absorbance with five broths for isolate ATCC49513

PT

0.600

SC

RI

0.400

0.300

0.200

NU

Absorbance at OD600 nm

0.500

0.000 SCA

1:5 MHB

MA

0.100

Shieh

FCGM

AC CE P

TE

D

Five broths at 24, 48, 72, and 96 h

Figure 1-2. Growth of isolate S06-227 in five broths (test 1). 35

G

ACCEPTED MANUSCRIPT Absorbance with five broths for isolate S06-227 0.500 0.450

PT RI

0.350 0.300

SC

0.250 0.200 0.150

NU

Absorbance at OD600 nm

0.400

0.100

0.000 SCA

1:5 MHB

MA

0.050

Shieh

FCGM

G

AC CE P

TE

D

Five broths at 24, 48, 72 and 96 h

Figure 1-3. Growth of isolate S07-531 in five broths (test 1).

36

ACCEPTED MANUSCRIPT Absorbance with five broths for isolate S07-531 0.450

PT

0.350

RI

0.300

SC

0.250 0.200 0.150

NU

Absorbance at OD600 nm

0.400

0.100

MA

0.050 0.000 SCA

1:5 MHB

Shieh

FCGM

G

AC CE P

TE

D

Five broths at 24, 48, 72 and 96 h

Figure 2-1. Growth of ATCC49513 in seven broths (test 2). 37

ACCEPTED MANUSCRIPT Absorbance with different broths for isolate ATCC49513 0.350

PT RI

0.250

SC

0.200 0.150

NU

Absorbanc at OD600 nm

0.300

0.100

MA

0.050 0.000 1/5 MHB

1/5 CAMHB

Shieh

TYES

D

SCA

AC CE P

TE

Seven broths at 24, 48 and 72 h

Figure 2-2. Growth of isolate S06-227 in five broths (test 2).

38

FCGM

G

ACCEPTED MANUSCRIPT Absorbance with different broths for isolate S06-227

0.300

PT RI

0.200

SC

0.150 0.100 0.050 0.000 1/5 MHB 1/5 CAMHB

Shieh

MA

SCA

NU

Absorbanc at OD600 nm

0.250

TYES

FCGM

AC CE P

TE

D

Seven broths at 24, 48 and 72 h

Figure 2-3. Growth of isolate S07-531 in five broths (test 2). 39

G

ACCEPTED MANUSCRIPT Absorbance with different broths for isolate S07-531

PT

0.300

SC

RI

0.200

0.150

NU

0.100

MA

0.050

0.000 SCA

1/5 MHB 1/5 CAMHB

Shieh

TYES

TE

D

Seven broths at 24, 48 and 72 h

AC CE P

Absorbance at OD600 nm

0.250

40

FCGM

G

ACCEPTED MANUSCRIPT Highlights Three pairs of broth and agar media for culturing Flavobacterium columnare were developed.

PT

Within 24 h the bacterial culture on agar plates was confirmed and > 3 × 108 CFU/ml cells in

RI

broths were achieved.

SC

The clumps were eliminated so the colony counts were more accurate and reliable for all

AC CE P

TE

D

MA

NU

research purposes.

41