# Bacterial growth in poly(vinyl alcohol) hydrogel with nutrients and indicator

Mongkol Techakasikornpanich, Duangporn Polpanich, Jaturavit Pantakitcharoenkul, Piyada Na-Nakorn, Yodsathorn Wongngam, Tienrat Tangchaikeeree, Patcharapan Suwannin, Abdelhamid Elaissari, Kulachart Jangpatarapongsa

► To cite this version:

Mongkol Techakasikornpanich, Duangporn Polpanich, Jaturavit Pantakitcharoenkul, Piyada Na-Nakorn, Yodsathorn Wongngam, et al.. Bacterial growth in poly(vinyl alcohol) hydrogel with nutrients and indicator. Scientific Reports, In press, 10.1038/s41598-026-35916-5. [https://doi.org/10.1038/s41598-026-35916-5](https://doi.org/10.1038/s41598-026-35916-5)

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# Bacterial growth in poly(vinyl alcohol) hydrogel with nutrients and indicator

Received: 18 February 2024

Accepted: 8 January 2026

Published online: 13 January 2026

Cite this article as:

Techakasikornpanich M., Polpanich D., Pantakitcharoenkul J. et al. Bacterial growth in poly(vinyl alcohol) hydrogel with nutrients and indicator. *Sci Rep* (2026). https://doi.org/10.1038/s41598-026-35916-5

Mongkol Techakasikornpanich, Duangporn Polpanich, Jaturavit Pantakitcharoenkul, Piyada Na-Nakorn, Yodsathorn Wongngam, Tienrat Tangchaikeeree, Patcharapan Suwannin, Abdelhamid Elaissari & Kulachart Jangpatarapongsa

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

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1 Bacterial Growth in Poly(vinyl alcohol)

2 Hydrogel with Nutrients and Indicator

3 Mongkol Techakasikornpanich¹, Duangporn Polpanich², Jaturavit

4 Pantakitcharoenkul¹, Piyada Na-Nakorn¹, Yodsathorn Wongngam², Tienrat

5 Tangchaikeeree¹, Patcharapan Suwannin¹,³, Abdelhamid Elaissari³, and

6 Kulachart Jangpatarapongsa¹*

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1. Center for Research Innovation and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.

10 2. National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand.

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3. Universite Claude Bernard Lyon 1, ISA, UMR 5280, CNRS, 5 rue de la Doua, 69100 Villeurbanne, France.

*Corresponding author: E-mail: kulachart.jan@mahidol.ac.th

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# Abstract

Bacterial culture has traditionally been a widely used method for isolating and detecting bacteria, particularly in the food industry. However, the conventional culture method is now considered outdated, time-consuming, and labor-intensive. To overcome these limitations, a dried thin film medium known as Petrifilm has been developed for various bacterial culture applications. In this study, a poly(vinyl alcohol) (PVA) hydrogel was developed as an innovative approach for thin film bacterial culture. This hydrogel, incorporating bromothymol blue (BB) and tryptic soy broth (TSB), referred to as BBTH, was synthesized in different thicknesses using freeze-thaw techniques. Comprehensive hydrogel characterization was performed to determine the optimal hydrogel for bacterial culture. The suitability of BBTH for bacterial cultivation was validated using *Escherichia coli* ATCC25922 and *Staphylococcus aureus* ATCC25923, representing Gram-
negative and Gram-positive bacteria, respectively. The results demonstrated
the successful synthesis of BBTH hydrogel in five different thicknesses.

Among these variants, 2-BBTH (thickness 0.50 ± 0.01 mm) exhibited the most
favorable properties, including ideal thickness, high transparency, water
absorption, clear representation of bacterial growth, and colony formation.

Compared to the conventional method using tryptic soy agar (TSA), 2-BBTH
yielded comparable results, with observable bacterial colonies at the same
concentration. This study highlights the potential of BBTH as a promising
alternative for developing innovative commercial bacterial culture platforms.

**Keywords:** Polyvinyl alcohol, Hydrogel, Thin-film culture, and Bacterial
culture
46 **Introduction**

Conventional bacterial culture, together with colony count and
bacterial identification, has been used in clinical and related laboratories for
several decades ¹. The bacterial culture media are generally used in either
solid (agar) or liquid (broth) form for bacterial detection ²⁻⁴. However, since
the concentration of bacterial contamination in the food sample is unknown
and rapid results are required, several dilution titers are usually prepared on
the standard agar plates, a process which is time-consuming and requires a
large area in an incubator. Although novel detection techniques have been
developed for rapid and sensitive detection of foodborne pathogenic bacteria
⁵, bacterial enrichment and isolation is still needed. To solve this issue, thin
film bacterial cultures, which had dehydration and rehydration properties,
were developed. The use of such dry thin films, has reduced the preparation
process in the laboratory, made it more user friendly, and reduced the area
of incubator needed ⁶,⁷. Several years after development of Petrifilm by 3M,
thin films were developed by several companies as an alternative method for
culture of bacteria and fungi, especially in food and environmental safety
settings ⁷. These thin films have been composed mainly of bacterial nutrients,
cold water-soluble gelling agents, and sensing indicators that were spread
and dried on plates ⁸. Most of the development of thin film culture methods
has been by private companies, and the composition of some components is
secret, while the preparation processes may be complex. In addition, these
products are more expensive than conventional media.

Hydrogel, a three-dimensional structure containing high amounts of
water, has been applied in several research fields, including drug delivery,
wound healing, wound dressing, cell engineering, surface coating, and so on.
The type of polymer used gives distinct properties to the synthesized
hydrogel. Poly(vinyl alcohol) (PVA) is a water-soluble polymer which is
produced from hydrolysis of polyvinyl acetate ⁹,¹⁰. Due to its biocompatibility,
non-toxicity, hydrophilicity, chemical stability, mechanical strength, and
biodegradability, PVA has been used in many applications. Physical
crosslinking through the formation of hydrogen bonds between the hydroxyl groups of PVA (due to multiple freeze-thaw cycles) yields a mechanically strong and stable hydrogel<sup>11,12</sup>. These properties of PVA have led to multiple applications of bacterial immobilization: the hydrogel as a matrix for bacterial adhesion and growth, a long-term bacterial carrier<sup>13-15</sup>, a basement for synthetic films used for food packaging<sup>16-18</sup>, and as a hydrogel for wound dressing. Tryptic soy broth (TSB), and bromothymol blue (BB) were also used in our preparation. TSB is a common bacterial nutrient, widely used as an enrichment medium for bacteria. BB, a pH indicator with three-color ranges which represent the acidic, neutral and alkaline conditions<sup>19-22</sup>, is famously used as an indicator in bacterial culture media and for bacterial biochemical identification<sup>23-25</sup>.

This study demonstrated a novel hydrogel, synthesized from PVA, TSB and BB (called BBTH) for bacterial culture. The synthesized hydrogel was characterized by swelling ratio, Fourier transform infrared spectroscopy (FTIR) in Attenuated Total Reflectance (ATR) mode, scanning electron microscopy (SEM) to analyze the swelling properties, surface, chemical composition, and its surface morphology. The thickness of the hydrogel was varied to find the optimal thickness, which is suitable for bacterial growth and colony formation. To ensure the application of BBTH, bacterial culture was performed and compared with the conventional bacterial culture using TSA. This study provides a model for developing an alternative bacterial culture method using a PVA hydrogel.

## Results and Discussion

### Characterization of BBTH

BB- and TSB-containing PVA hydrogel, called BBTH, was successfully synthesized with different volumes of the dissolved solution to optimize the thickness of the formed hydrogel. The dissolved PVA solutions (volumes 1-5 mL) were poured into plastic petri dishes, and named 1-BBTH, 2-BBTH, 3-BBTH, 4-BBTH, and 5-BBTH, respectively. After the freeze-thawing process,
the thickness of the BBTHs as measured by vernier calipers was 0.16 ± 0.01, 0.50 ± 0.01, 0.94 ± 0.01, 1.80 ± 0.01, and 2.10 ± 0.01 mm for 1- to 5-BBTH, respectively (Table 1). Due to the differences in hydrogel thickness, the samples varied in transparency, ranging from clear in 1-BBTH (high transparency), a slight turbidity in 2-BBTH and 3-BBTH (average transparency), and high turbidity in 4-BBTH and 5-BBTH (low transparency) (see Supplementary Information Figures S1A-S1E). Surface morphology of the prepared BBTHs was observed using SEM, with an example given in Figure S1F-S1J. The SEM image of 1-BBTH demonstrated thin, flat, and smooth film with many small pores (Figure S1F). Interestingly, 2-BBTH, 3-BBTH, 4-BBTH, and 5-BBTH, had rough surfaces, probably due to formation of fiber linkages via physical crosslinking (Figure S1G-S1J) 12,26.

<table><thead><tr><th>Sample</th><th>Hydrogel thickness (mm)</th><th>Transparency</th><th>Swelling ratio at 30 min (g/g)</th></tr></thead><tbody><tr><td>1-BBTH</td><td>0.16 ± 0.01</td><td>High</td><td>5.76</td></tr><tr><td>2-BBTH</td><td>0.50 ± 0.01</td><td>Average</td><td>3.16</td></tr><tr><td>3-BBTH</td><td>0.94 ± 0.01</td><td>Average</td><td>2.78</td></tr><tr><td>4-BBTH</td><td>1.80 ± 0.01</td><td>Low</td><td>2.17</td></tr><tr><td>5-BBTH</td><td>2.10 ± 0.01</td><td>Low</td><td>2.16</td></tr></tbody></table>

The swelling phenomenon was studied to estimate the water uptake ability of hydrogel, an important property of this application because hydrogels with high swelling ratios should rapidly absorb sample. Each BBTH was dried using a freeze-dryer. The dried hydrogels were weighed and then immersed in DI water for 0, 30, 60, 90, and 120 min to examine water absorptivity. The swollen BBTH hydrogels were weighed, and swelling ratios
calculated using equation (1) (see Methods). The results are shown in **Figures S2A and S2B**, and **Table 1**. The swollen weight and swelling ratios significantly increased after immersing the individual BBTHs for 30 min. Subsequently, the swollen weight and swelling ratios remained constant (**Figures S2A and S2B**), due to the hydrophilic surface and capillary pores of the hydrogels<sup>27</sup>. The highest swelling ratio at 30 min was that of 1-BBTH, which may have been due to its having the highest void fraction as demonstrated by this gel's minimal shrinkage post-drying compared to that of other formulations<sup>28</sup>. The water absorption of 2-BBTH and 3-BBTH was quite similar (due to their similar thickness). The 4-BBTH and 5-BBTH formulations demonstrated lower swelling ratios due to their greater thicknesses, reflecting lower water absorptivity over a given time. These finding demonstrated that increases in hydrogel thickness contributed to structural shrinkage, and reduced the void fraction of hydrogels<sup>28</sup>. These results indicated that thin hydrogels demonstrated rapid and more-water absorption, while thicker hydrogels reduced water absorption, resulting in reduced swelling ratios<sup>13</sup>. The high swelling capacity of this hydrogel is an important property because it represents the ability to absorb liquids or aqueous samples into the hydrogel<sup>29,30</sup>.

ATR-FTIR was carried out to investigate the differing chemical compositions of plain PVA hydrogel, BB-containing PVA hydrogel, TSB-containing PVA hydrogel, and BBTH. The results are shown in **Figure 1**.
<figure><img src="image_2.png" /><figcaption>**Figure 1** FTIR spectra of plain PVA, BB-containing PVA, TSB-containing PVA and 5-BBTH.</figcaption></figure>

The pattern of peaks is similar in each sample. In these spectra, the absorption peak around 3265 cm⁻¹ refers to the stretching vibration of non-bounding O-H in the hydrogel. The absorption peak around 2940 cm⁻¹ reflects the stretching vibration of C-H. The peak around 1655 cm⁻¹ represents the C-C stretching vibration, the peak around 1416 cm⁻¹ refers to the C-O stretching vibration, and the peak around 1327 cm⁻¹ is due to the C-H stretching vibration. The absorption peak around 1141 cm⁻¹ demonstrates C-H stretching vibration, related to PVA crystallinity. C-O associated with C-C stretching vibration was anti-symmetric with O-C-C vibration, as reflected in the absorption peak around 1086 cm⁻¹. The peaks around 914 and 833 cm⁻¹ refer to the stretching vibrations of O-H and C-O, respectively ¹³,³¹. These
results indicate the normal pattern of PVA hydrogels, which have
hydrocarbon compounds and hydroxyl groups in their chemical structures
$^{32,33}$.

To compare the chemical composition of each study sample, plain PVA
hydrogel was used as a negative control. The increases in intensity of O-H, C-
C, C-H and C-O related peaks observed in spectra of BB-containing PVAs
possibly originated from the presence of BB. Similarly, in TSB-containing
PVAs, the intensity of O-H, C-C, C-H, and C-O related peaks were increased
due to the organic compounds in TSB (papain digest of soybean peptone,
pancreatic digest of casein, and dextrose). With 5-BBTH, the spectrum
demonstrated a high intensity of peaks due to the combination of PVA, BB
and TSB (composed of hydrocarbon compounds, hydroxyl, amine and amide
groups in the hydrogel $^{34-37}$), as shown in **Figure 1**. ATR-FTIR spectra of 2-
to 5-BBTH are presented in **Figure S3**. The peak of transmission was
unmeasurable in 1-BBTH, due to the hydrogel being thin and transparent and
unable to reflect the infrared used in the measurement. The peak intensity
increased in direct relation to the thickness of hydrogel because of the
hydrogel's matrix. Moreover, the intensities of O-H, C-H, C-C, and C-O peaks
may indicate the presence of PVA, TSB and BB in the hydrogel which support
the bacterial growth.

**Bacterial cultures on BBT-Hydrogel**

*E. coli* and *S. aureus* were inoculated on the 1- to 5-BBTH and PVA+TSB formulations and then incubated overnight at 37 °C. Sterile PBS was used as a negative control. On PVA+TSB, the bacterial colonies were not distinctly visible to the naked eye. In contrast, in the presence of BB or in the BBTH formulation, the colonies were clearly observable. These results highlight the advantage of incorporating BB into the BBTH hydrogel to facilitate visual detection of bacterial growth. **Figures 2** shows *E. coli* and *S. aureus* cultured on each type of BBTH. Sterile PBS was inoculated on 1-BBTH, 2-BBTH, 3-BBTH, 4-BBTH, and 5-BBTH (**Figures 2A-2E**). On 1-BBTH, neither bacteria
grew, as shown in **Figures 2F and 2K**. This may have been because of the thin hydrogel matrix and its high surface porosity, resulting in the depletion of water content during incubation. Hydrogel dehydration causes unsuitable conditions for bacterial growth, and may stop bacterial replication due to water loss and bacterial shrinkage ³⁸. In contrast, both bacteria grew and formed colonies on 2-BBTH and 3-BBTH, and a color change of the BBTH was clearly observed (**Figures 2G-2H and 2L-2M**). This color change of BBTH occurred since the bacteria utilized nutrients from the TSB, including glucose, amino acids, and short peptides during bacterial growth and proliferation. Glucose is utilized as the first nutrient for bacterial metabolism, which can be converted to pyruvate and then to intermediate compounds in the TCA cycle. However, due to the small amount of glucose in TSB, bacteria use amino acids as alternative carbon sources for cell proliferation and cell metabolism. *S. aureus* and *E. coli* can express the genes to produce enzymes (deaminase and dehydrogenase) to catabolize protein and amino acids. Amino acids and short peptide in TSB are used to derive essential intermediate compounds in the TCA cycle of bacterial metabolism, such as pyruvate, 2-oxoglutarate, and oxaloacetate ³⁵⁻³⁷. Due to the catabolism of amino acids and short amino peptides, ammonia (the byproduct of deamination), is released ³⁵,³⁹. Ammonia is an alkaline compound that changes the environment from neutral to alkaline, changing the color of BB in BBTH. In contrast, 4-BBTH and 5-BBTH both exhibited bacterial growth, as indicated by color change, compared with the negative control (**Figures 2I-2J and 2N-2O**); however, no visible colonies were observed. This is because of the high thickness of the hydrogel, which makes it difficult for the bacteria to adhere to the BBTH ⁴⁰. These results indicated that fabrication of the BBTHs was successful for bacterial culture. Moreover, the results also showed that 2-BBTH had an optimal thickness of hydrogel (0.50 ± 0.01 mm) for application. This range of thicknesses yielded suitable transparency, which contributed to the visibility of colony formation, as demonstrated in **Figures 2G and 2L**. Therefore, 2-BBTH was chosen for further experiments.
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<figure><img src="image_3.png" /><figcaption>**Figure 2** Sterile PBS (A-E), *E. coli* ATCC25922 (F-J), and *S. aureus* ATCC25923 (K-O) were inoculated on 1-BBTH, 2-BBTH, 3-BBTH, 4-BBTH and 5-BBTH.</figcaption></figure>

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**Comparison of bacterial culture on BBTH and TSA**

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To compare bacterial cultures on 2-BBTH and conventional TSA culture medium. 0.5 McFarland of *E. coli* ATCC25922 and *S. aureus* ATCC 25923 were prepared and then diluted in ten folds to give the range of dilution titers 1:10⁰ to 1:10⁶. Each dilution of each bacteria was inoculated on 2-BBTH and TSA. Sterile PBS, used as a negative control for both bacteria, was also inoculated on 2-BBTH and TSA. The results of each concentration of *E. coli* and *S. aureus* are shown in **Figures 3, S4, and 4, S5**, respectively. The negative controls illustrated no bacterial growth on TSA (**Figures 3a, S4a, and 4a, S5a**) nor on BBTH, and no color changes were observed (**Figures 3A, S4A, and 4A, S5A**). The color of BBTH changed when the bacteria grew on the hydrogel, seen in **Figures 3B-3E, S4B-S4E, and 4B-4E, S5B-S5E**,
when compared with negative controls, represented in **Figures 3A, S4A, and 4A, S5A**. At low dilution titers of both bacteria (1:10⁰ to 1:10²), only the color change on BBTH was observed, without any visible colony formation, seen in **Figures 3B-3D, S4B-S4D, and 4B-4D, S5B-S5D**, respectively. The results were similar to bacterial culture on TSA, which were inoculated with the same dilution titers of bacteria. The non-isolation of colony on the agar, is shown in **Figures 3b-3d, S4b-S4d, and 4b-4d, S5b-S5d**. This suggested that high concentrations of bacteria may cause their non-isolation. In contrast, with high dilution titers (1:10³ to 1:10⁶) of both bacteria, color change of BBTH, and colony formation on the hydrogel were observed (**Figures 3E-3H, S4E-S4H, and 4E-4H, S5E-S5H**, respectively). In comparison, the same dilution titers inoculated on TSA, demonstrated similar results with visible colony formation on the agar, as shown in **Figures 3e-3h, S4e-S4h, and 4e-4h, S5e-S5h**, respectively. To further investigate the efficacy of BBTH, the number of colonies was experimentally determined by colony count when inoculated with the same bacteria compared to TSA.

**Table 2** demonstrates the number of colonies of *E. coli* and *S. aureus* inoculated on 2-BBTH and TSA. The statistical analysis using the paired T-Test exhibited a non-significant difference in the number of colonies (P-value > 0.05). These results confirmed that the efficacy of the prepared 2-BBTH was comparable to TSA. However, when comparing the size of colonies grown on 2-BBTH and TSA, the former were smaller than the colonies on TSA. This was likely due to the effect of spatial area, defined as the distance between bacterial colonies, which affects colony size on the medium plate. Due to the different diameter of 2-BBTH (60 mm) and TSA (90 mm), the bacteria grown on 2-BBTH had a smaller spatial area than those grown on TSA. The shorter distance between bacterial colonies, likely caused reduced colony expansion on 2-BBTH and led to a reduction of the colony size on the 2-BBTH plate ⁴¹⁻⁴³.

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<figure><img src="image_4.png" /><figcaption>**Figure 3** Comparison of *E. coli* ATCC25922 cultured on 2-BBTH and TSA, seen in **A-H** and **a-h**, respectively. **A** and **a** show the sterile PBS culture on 2-BBTH and TSA, respectively, which had no colony growth and no color change. **B-H** represent each dilution of 0.5 McFarland of *E. coli* ATCC25922 cultured on 2-BBTH, from dilution titer 1:10⁰ to 1:10⁶, respectively. **b-h** represent each dilution of 0.5 McFarland of *E. coli* ATCC25922 cultured on TSA, from dilution titer 1:10⁰ to 1:10⁶, respectively.</figcaption></figure>

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<figure><img src="image_5.png" /><figcaption>**Figure 4** Comparison of *S. aureus* ATCC 25923 cultured on 2-BBTH and TSA, seen in **A-H** and **a-h**, respectively. **A** and **a** show the sterile PBS culture on 2-BBTH and TSA, respectively, which had no colony growth and no color change. **B-H** represents each dilution of 0.5 McFarland of *S. aureus* ATCC 25923 cultured on 2-BBTH, from dilution titer 1:10⁰ to 1:10⁶, respectively. **b-h** represent each dilution of 0.5 McFarland of *S. aureus* ATCC 25923 cultured on TSA, from dilution titer 1:10⁰ to 1:10⁶, respectively.</figcaption></figure>

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<table><caption>**Table 2** Comparison of colony count of *E. coli* and *S. aureus* on BBTH and TSA</caption><thead><tr><th rowspan="2">Bacteria</th><th colspan="2">Colony Count (CFU/mL)</th></tr><tr><th>TSA (mean ± SD)</th><th>BBTH (mean ± SD)</th></tr></thead><tbody><tr><td><b><i>E. coli</i> ATCC</b><br>25922</td><td>(4.69 ± 2.00)×10<sup>7</sup></td><td>(3.40 ± 0.33)×10<sup>7</sup></td></tr><tr><td><b><i>S. aureus</i> ATCC</b><br>25923</td><td>(1.52 ± 0.01)×10<sup>8</sup></td><td>(1.58 ± 0.24)×10<sup>8</sup></td></tr></tbody></table>

**Conclusion**

This study successfully demonstrated the formulation of a PVA hydrogel containing bromothymol blue and tryptic soy broth, referred to as BBTH. The hydrogel was prepared in various thicknesses by pouring different volumes of the solution, ranging from 1 to 5 mL. These variants were designated as 1-BBTH, 2-BBTH, 3-BBTH, 4-BBTH, and 5-BBTH, respectively. Characterization of each BBTH variant, along with evaluation of their ability to support bacterial growth, indicated that 2-BBTH had the most suitable thickness. This variant exhibited favorable swelling properties, optimal transparency, and effectively supported bacterial growth, colony formation, and induced a visible color change of the indicator within the hydrogel. Compared to the conventional culture method using TSA, the synthesized 2-BBTH yielded comparable results in terms of bacterial growth. At low dilution titers (1:10⁰ to 1:10²), isolated colonies were not observed, likely due to the high concentration of bacterial cells. In contrast, at higher dilution titers (1:10³ to 1:10⁶), isolated colonies were clearly visible using both methods. These findings confirmed the successful formulation of BBTH and its ability to support bacterial growth and isolated colony formation on the hydrogel. The newly developed BBTH, designed as an innovative bacterial culture method, shows strong potential for use as a culture medium in clinical and other laboratory applications.
319 **Materials and Methods**

320 **Materials**

321 In this study, 99% hydrolyzed poly(vinyl alcohol) (PVA; M<sub>w</sub> 89,000-322 323 98,000 g/mol) was purchased from Sigma-Aldrich, (USA). TSA and TSB were 324 purchased from Merck, (Germany). Bromothymol blue (BB) was purchased 325 from BDH Laboratory Supplies, (England). Plastic petri dishes (diameter 60 326 mm) were purchased from Biofil, (China). *Staphylococcus aureus* ATCC25923 327 and *Escherichia coli* ATCC25922 were provided by the Department of Clinical 328 Microbiology and Applied Technology, Faculty of Medical Technology, 329 Mahidol University.

329

330 **Formulation of BBTH and PVA hydrogels**

331 BB- and TSB-containing PVA hydrogel (BBTH) were formulated by 332 dissolving the mixture of PVA (10% w/v) and TSB (3% w/v) in distilled water, 333 pH 5.5. The mixture was stirred for 5 min and then heated at 180°C with 334 continuous stirring for 60 min or until the solution became clear. After that, 335 0.2% v/v of BB solution (0.01 g/mL) was added to the mixed solution. This was 336 continuous stirred and heated for 30 min or until homogenous. The solution 337 was poured into plastic petri dishes, varying the volume to obtain differing 338 thicknesses (1, 2, 3, 4, and 5 mL). The hydrogels were formed by repeating 339 three cycles of freezing at -20 °C for 18 h and thawing at 4 °C for 6 h. The 340 processed hydrogels were kept at 4 °C for further experiments.

341 Plain PVA, BB-containing PVA, and TSB-containing PVA hydrogels were 342 prepared following the same protocol used for the BBTH formulation. For 343 each control hydrogel, the omitted composition was replaced with deionized 344 (DI) water to maintain the total volume of the mixture. The prepared mixtures 345 were cast into plastic Petri dishes and subjected to freeze-thaw for 3 cycles, 346 consisting of freezing at -20 °C for 18 h and thawing at 4 °C for 6 h. The 347 resulting hydrogels were stored at 4 °C until further characterization.

348

349 **Hydrogel characterization**
The hydrogels with five different volumes were triplicate measured to determine their thickness using vernier calipers. Then they were dried using a freeze-dryer (COOLSAFE SCANVAC, Sweden). The dried hydrogels were weighed and then immersed in DI water (15 mL) for 30, 60, 90, and 120 min. The swollen hydrogels were weighed again, and the swelling ratio was calculated from the triplicate data, using equation (1).

$$ \text{Swelling ratio} = \frac{W_s - W_d}{W_d} \qquad (1) $$

Where $W_s$ and $W_d$ are the swollen and dried weights of the hydrogel (g), respectively. The statistical analysis was conducted by a paired T-test with a P-value less than 0.05 using SPSS Statistics 18 (USA).

The chemical composition of the dried hydrogels was determined using Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) (Nicolet iS50, Thermo Scientific, USA). The samples were measured for 64 scans, in the range of 400-4,000 cm⁻¹.

The dried hydrogels were cut into small pieces and placed on the stub. The samples were coated with gold using a sputter-coater (Q150R S Plus, Quorum, Germany), and the surface morphology was observed using a scanning electron microscope (Versa 3D Dual beam, FEI, USA)

## Bacterial preparation

*Escherichia coli* ATCC25922, and *Staphylococcus aureus* ATCC 25923 were cultured overnight on TSA at 37 °C. Isolated colonies of both bacteria were suspended in TSB and incubated overnight at 37 °C. The bacteria were centrifuged at 3,000 g for 10 min and washed with sterile 1X PBS buffer pH 7.4. Both bacteria were adjusted to a concentration of 0.5 McFarland.

## Bacterial culture on the fabricated hydrogels

The prepared bacteria, following a previously established protocol, were diluted in 10-fold serial dilutions from 10⁰ to 10⁶. The diluted bacteria were spread on TSA and the BBTHs of various thicknesses. Then they were
incubated overnight at 37 °C. Colony formation on BBTH was observed and compared to that on conventional bacterial culture using TSA. Colony count was performed on the plate that provided a bacterial colony of around 30 to 300 colonies. The number of colonies was calculated by equation (2)

$$ \text{Number of colony (CFU/mL)} = \frac{n \times df}{v} \qquad (2) $$

Where n refer to the number of colonies on the optimal colony count. df refer to the dilution factor that provides the optimal number of colonies and v refer to the volume that inoculates the bacteria.

## Data Availability

All data generated or analyzed during this study are included in this published article.

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543
544 Acknowledgements

545 This research project has been funded by Royal Golden Jubilee Ph.D.
546 program, National Research Council of Thailand (NRCT) (NRCT5-RGJ63012-
547 132), and France Embassy to MT, Mahidol University (Fundamental Fund:
548 fiscal year 2026 by National Science Research and Innovation Fund (NSRF))
549 to KJ, Research/Franco-Thai Mobility Programme/PHC-SIAM 2025-2026
550 (53181WJ) to KJ, AE, DP, MT. The National Nanotechnology Center
551 (NANOTEC), National Science and Technology Development Agency
552 (NSTDA), has supported an instrument for the characterization of BBTH.
553 Department of Clinical Microbiology and Applied Technology, Faculty of
554 Medical Technology, Mahidol University has supported the bacteria and
555 indicators. The authors sincerely thank to Dr. Arther E. Brown for English
556 language proofing.

557
558 Author contribution
559 MT, DP, JP, AE, and KJ have designed the experiment of hydrogel
560 synthesis and characterization. MT, PN, TT, PS, and KJ have helped to design
561 the protocol for bacterial testing. YW has helped to prepare and characterize
562 the hydrogel, including dried hydrogel, FTIR, and SEM. MT, DP, AE, and KJ
563 have drafted an article and revised it critically, and final approval of the
564 version to be published.

565
566 **Competing interests**

567 The authors declare no competing interests.

568
569 **Additional information**

570 **Supplementary information** accompanies this paper at

571
572 **Correspondence** and requests for materials should be addressed to KJ.

573
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579