Accurate enumeration of specific Bacillus strains in probiotic formulations is challenging for both research and industry. Conventional plate counts cannot reliably distinguish strains, causing inconsistencies in viability assessment and potentially affecting product quality and applications. The present study addresses this limitation by developing a viable count method tailored for the selective enumeration of Bacillus spores in a blend of three proprietary strains: Bacillus coagulans LBSC, Bacillus subtilis PLSSC, and Bacillus clausii 088AE based on optimized pH and antibiotic selection pressures. Spore counts on standard and selective media correlated strongly for LBSC (P=0.4736), PLSSC (P=0.3387), 088AE (P=0.4721). Colony morphology, microscopy, biochemical testing and 16S rRNA sequencing eliminated potential ambiguities.

The method met the validation criteria of specificity, precision, intermediate precision, linearity and accuracy for working range of blends with concentrations 0.36 to 36×10⁹ CFU/g (0.12 to 12×10⁹ CFU/g of each Bacillus strain in the blend). Specificity showed no growth of non-target bacteria. Precision and intermediate precision was < 10% RSD and a statistically significant linear regression model confirmed linearity (R²>0.99, F>1000, p<0.001). Accuracy was demonstrated with no significant difference in the means of theoretical and observed spore count (P-value>0.05).

This innovative approach provides a simple guiding solution to the limitations of current enumeration techniques and a valuable contribution to probiotic research for enhancing the application of Bacillus-based probiotics.

• Selective

• Bacillus

• Probiotic

• Enumeration

• Validation

• Blends

Dixit Y, Bhingardeve N, Inamdar A, Saroj D (2026) Development of a Method for Enumeration of Individual Bacillus Strains (Bacillus coagulans LBSC, Bacillus subtilis PLSSC, Bacillus clausii 088AE) from Mixed Blends. J Hum Nutr Food Sci 11(2): 1207.

P: Probability value; CFU: Colony Forming Units; g: Gram; 16S rRNA: 16S Ribosomal Ribonucleic Acid; RSD: Relative Standard Deviation; R2: Coefficient of Determination; F: F-statistic 

The global market offers an extensive variety of probiotic products in the form of dietary supplements and functional foods, reflecting the increasing recognition of their health-promoting effects [1]. For exerting their beneficial effects on host health, these preparations should contain a sufficient amount (1×109 CFU per day or per food serving) of the indicated living microbes [2]. The rigorous quality control by probiotic manufacturers is imperative and include identification and enumeration of living organisms, and determination of microbial purity [1]. Most of the studies and publications on probiotics are focused on the beneficial properties exhibited by probiotic microbes. A relatively low number of studies analyze the real-time composition of probiotic formulations that consumers find on the market

Most probiotic studies focus on single microbial strains, with fewer investigations into the effectiveness of mixed probiotic strains. Although limited, available research suggests that multi-strain probiotics tend to be more effective than individual strains. The health benefits offered include, but are not limited to, alleviating gastrointestinal issues and chronic allergic diseases, improving immunity, restoring a balanced gut ecosystem, reducing inflammation [3].

Several lactic acid bacteria (LAB) belonging to the Lactobacillus, Bifidobacterium and Streptococcus genera are traditionally used as probiotics, constituting the vast majority of commercial products [4-6]. Bacteria belonging to the genus Bacillus are attracting growing interest because they can be delivered as spores, which remain stable under the hostile conditions of the gastrointestinal tract [1]. Bacillus probiotics are particularly noted for their ability to improve digestion, enhance immune function, and provide therapeutic benefits in preventing and treating various diseases [7]. Bacillus species such as B. coagulans, B. subtilis, B. clausii, B. licheniformis, B. velezensis are commonly used in probiotic formulations [8, 9].

One of the challenges in the study and application of Bacillus probiotics lies in the accurate enumeration of specific strains when present in a mixture. The gold standard for probiotic enumeration is agar pour plate method. Many probiotic strains form colonies that look very similar on standard culture media. In addition, in mixed culture, fast-growing strains overgrow slow ones. Hence, this conventional method often fails to distinguish between different Bacillus strains from a blend leading to inaccuracies in assessing each strain’s viability and concentration. This can impact both research outcomes and the quality control of commercial probiotic products.

The current study addresses this issue by developing a modified viable count method tailored for the selective enumeration of Bacillus spores in mixed cultures. The focus is on three specific strains: Bacillus coagulans LBSC, Bacillus subtilis PLSSC and Bacillus clausii 088AE. By introducing targeted modifications to standard analytical procedure, we aim to selectively enumerate each Bacillus strain within a mixed culture, ensuring precise and reliable results. Selective enumeration method overcomes the previously mentioned limitations of the conventional method. As each strain grows only on its respective selective medium, issues arising due to similar colony characteristics and overlapping growth time are eliminated. Further, the modified enumeration method was subjected to rigorous validation and statistical analysis to confirm its accuracy, reproducibility, and suitability for practical applications. This approach not only enhances the reliability of probiotic research but also supports the development of high-quality Bacillus-based probiotic products by providing a tool for assessment.

This study presents an innovative method for the selective enumeration of Bacillus spores in mixed probiotic formulations. By overcoming the limitations of existing techniques, we contribute to the advancement of probiotic research by providing a guideline to design strain specific enumeration protocols.

Media and Chemicals

Nutrient media used for microbiological analysis, including buffered peptone saline (MH1275), Nutrient Agar (M001) and PNY Medium (M835), bacteriological agar (GRM026), MR-VP broth (LQ082), nitrate broth (M439), methyl red indicator (I007), and α-napthylamine solution (R009) were purchased from HiMedia Laboratories Ltd. (Mumbai, India). Sulphanilic acid (15354) was procured from SRL Laboratories. Antibiotics used in the present study namely ceftazidime (FORTACEF, ceftazidime for injection IV, Troikaa pharmaceuticals Ltd.) and cefixime (ZIFI 200 tablet, FDC ltd.) were procured from local market. Spray dried preparations of Bacillus coagulans LBSC, Bacillus subtilis PLSSC, and Bacillus clausii 088AE and their blends were provided by Advanced Enzyme Technologies Ltd. (India).

Procedure to enumerate individual Bacillus strain using standard media

Ten g of spray dried preparations was suspended in sterile 90 mL of buffered peptone saline and mixed thoroughly under magnetic stirring at 1000 rpm for 15 min. This stock was further ten-fold serially diluted and appropriate dilutions that would give 30-300 colony forming units (CFU) on the plate were selected. Spore activation and pour plating was carried out separately for each Bacillus strain [10-13]. Bacillus coagulans LBSC was analyzed by spore activation at 75 °C for 30 min followed by pour plating using sterile PNY agar and incubating at 37 °C for 48 h. Bacillus clausii 088AE was analyzed by spore activation at 70 °C for 15 min followed by pour plating using sterile nutrient agar and incubating at 37 °C for 24-48 h. For Bacillus subtilis PLSSC, spores were activated at 70 °C for 15 min and pour plated using sterile nutrient agar; after drying the agar surface, it was carefully overlaid with sterile 1% bacteriological agar (to prevent swarming of colonies and allow counting) and plates. were incubated at 37 °C for 24 h. Uninoculated or blank media served as sterility control (three plates each batch).

Procedure to enumerate individual Bacillus strain using newly designed selective media

Preparation of sample dilutions and spore activation remained same for selective enumeration and was carried out as described earlier. The activated spore dilutions were pour plated with 1 mL of sample dilution using the newly designed selective media (Table 1.0). Selective medium for Bacillus coagulans LBSC was prepared by first formulating PNY medium according to the manufacturer’s instructions. Following autoclaving under standard conditions, the medium was cooled to approximately 45 °C, and its pH was adjusted to 4.5 ± 0.1 using a predetermined volume of 1 N HCl. Immediately thereafter, 3.0 mL of a 100 ppm ceftazidime stock solution was added to 200 mL of medium, mixed thoroughly, and poured into plates preloaded with 1 mL of the sample dilution. Selective media for Bacillus clausii 088AE and Bacillus subtilis PLSSC were prepared in the same manner in line with the selective parameters mentioned in (Table 1). Uninoculated or blank media served as sterility control (three plates each batch).

Table 1: Newly designed media for selective enumeration of individual Bacillus strains

PNY: Peptone-Nutrient-Yeast; µg: MicroGram; mL: Mililitre; h:Hour

Selectivity of newly designed media and comparison of spore count with standard media

Spores of individual Bacillus strain (individual spray dried preparations of 120×109 CFU/g LBSC; 200×109 CFU/g PLSSC and 100×109 CFU/g 088AE) were enumerated using standard and newly designed selective media. Further, spore counts (CFU/g) obtained for six replicates were analysed by T-test.

Selective enumeration of individual Bacillus strain from blend

Once the selectivity of the new media for individual spray dried preparations was established, ability of these media to selectively grow and enumerate individual strain from blend was determined. A blend of spray dried preparations of three probiotic Bacilli namely Bacillus coagulans LBSC, Bacillus subtilis PLSSC, and Bacillus clausii 088AE corresponding to 3.6×109 total CFU/g; 1.2×109CFU/g each was used for analysis (Figure 1).

Figure 1: Flow diagram for selective enumeration of the three Bacilli from blend/mixtures

The blend was dry mixed to ensure sample homogeneity. Sample dilutions and spore activation was carried out as described earlier and were pour plated with 1 mL of sample dilution in the respective sterile selective medium (Table 1). Post incubation, confirmation of the selectivity of the medium was done by phenotypic and microscopic observation of the colonies. Phenotypic observation involved observation of colony characteristics such as shape, margin, opacity and pattern. Microscopic observation was done using phase contrast microscope (Olympus BX43) to observe typical morphology such as long slender rods or short thick rods, etc. Specific biochemical tests namely methyl red and nitrate reduction were performed for Bacillus coagulans LBSC and Bacillus clausii 088AE [14]. Further confirmation was carried out using 16SrRNA sequencing of a single pure colony from each selective medium. Uninoculated or blank media served as sterility control (three plates each batch).

Validation of selective enumeration method

Once the selectivity of newly designed media was established qualitatively and quantitatively in triplicate, the selective enumeration method was subjected to method validation [15]. Specificity was studied by analyzing blank matrix of the blends on the selective media. Precision was determined by calculating RSD (%) for analysis performed by single operator on a single day in triplicate over 5 concentration levels. Intermediate precision was determined by calculating RSD (%) for analysis performed by two operators on different days in triplicate over 5 concentration levels. RSD (%) was calculated using formula:

Relative standard deviation (%)=(Standard deviation for triplicate analyses) ×100/(Mean for triplicate analyses)

Linearity was determined for a working range of concentrations [0.12 to 12 ×109 CFU/g of each Bacillus strain in the blend of 0.36 to 36 ×109 CFU/g (total)] for which suitable level of precision was obtained. Finally, accuracy was determined for the working range of concentrations by comparing the means with theoretical concentration. Briefly, blends of five different concentrations-0.12, 0.60, 1.2, 6.0, 12.0 ×109 CFU/g of each Bacillus strain in the blend of 0.36, 1.8, 3.6, 12, 36 ×109 CFU/g total respectively-were prepared and analysed. Observed count and theoretical count means were compared using paired t-test. Regression model was also used additionally to confirm the accuracy. Data was processed using Prism 10 version 10.4.1 software.

The health benefits of probiotics are usually specific to the species and often even to the particular strain [2,16]. Therefore, the correct identification of microorganisms contained in commercial probiotic formulations is of crucial importance [17]. Further, accurate enumeration of the strains poses another challenge in the quality control and product development of probiotic formulations. For assessing the total viable count of probiotic formulations, the plate count method currently represents the gold standard applied in probiotic industry for quality controls [18-21]. In culture-dependent analyses, enumeration of different strains in multi-strain formulations can be critical. To overcome this limit, selective media are commonly used to discriminate strains with different metabolic features [1]. Selective growth can be achieved with the use of culture media with specific nutrients, pH and/or antibiotics to inhibit growth of non-desired micro organisms [22].

While lot of studies and literature are available for selective enumeration of LactoBacillus and Bifidobacterium in formulations containing their consortia [22-24], there are no reports on selective enumeration of probiotic products containing mixtures of Bacilli. Most studies with multi-strain Bacillus based probiotic products report total spore counts of the product [8,9]. This study is an attempt to overcome this limitation and device simple method without requirement of high-end sophisticated technology and expertise, for selective enumeration of individual Bacillus strains in commercial probiotic formulations containing their mixtures.

In the present study, a new medium was designed for spore count of each Bacillus strain based on its antibiotic and pH sensitivity data. The innate property of antibiotic resistance or sensitivity of each of these probiotics was exploited to develop the strain differentiation media/ conditions. Strain specific antibiotic sensitivity data and optimization trials were performed to select the concentration of antibiotics. PNY agar and nutrient agar were selected as the growth medium for LBSC and 088AE, PLSSC respectively based on the previously carried out growth optimization studies. Bacillus clausii 088AE is an alkaliphilic bacteria and acidic pH is not suitable for its growth. Thus, growth of Bacillus clausii 088AE gets inhibited at pH 4.5-5.0. Bacillus subtilis PLSSC is sensitive to both ceftazidime and cefixime while Bacillus coagulans LBSC is sensitive to cefixime but does not get inhibited with 1.5 µg/mL of ceftazidime. PNY agar (pH 4.5±0.1) supplemented with 1.5 µg/mL of ceftazidime did not allow Bacillus clausii 088AE to grow due to low pH while ceftazidime inhibited Bacillus subtilis PLSSC, thus making it selective for Bacillus coagulans LBSC. Nutrient agar supplemented with 5 µg/mL of cefixime was selective for Bacillus clausii 088AE as cefixime inhibited both Bacillus coagulans LBSC and Bacillus subtilis PLSSC. Similarly, nutrient agar (pH 5.0±0.1) allowed growth of only Bacillus subtilis PLSSC and inhibited Bacillus clausii 088AE due to low pH while Bacillus coagulans LBSC did not proliferate due to higher generation time.

Selectivity of newly designed media and comparison of spore count with standard media

Initially, selectivity of the newly designed media was verified using spray dried preparations of individual Bacillus strain. We found that newly designed selective media allowed growth of only one Bacillus strain as per the selectivity. Spore count obtained for spray dried preparations of individual Bacillus using standard medium (served as control) and newly designed selective medium (Figure 2) was same.

Figure 2: Comparison of spore count obtained for spray dried preparations of individual Bacillus coagulans LBSC, Bacillus subtilis PLSSC, Bacillus clausii 088AE using standard (control) and selective media. T-test indicated no statistical difference for 6 replicates performed.

As analysed by T-test, there was no significant difference in the mean spore count obtained for Bacillus coagulans LBSC (P-value=0.4736), Bacillus subtilis PLSSC (P-value=0.3387), Bacillus clausii 088AE (P-value=0.4721) using standard (control) and selective media. Thus, spore count obtained for selective media correlated well with the standard media with no statistical difference as indicated by P-value>0.05. This confirms that the newly designed selective media are suitable for reliable quantification of the three Bacillus strains without affecting the spore count when compared to the standard medium.

Selective enumeration of individual Bacillus strain from blend

Once the suitability of selective media was established for individual spray dried preparations, the same was investigated for blend. Bacillus coagulans LBSC, Bacillus subtilis PLSSC, and Bacillus clausii 088AE were enumerated using selective media from a blend of three, corresponding to theoretical count of 3.6×109 total CFU/g (1.2×109CFU/g each Bacillus). We found good correlation between the theoretical and observed spore count (Figure 3).

Figure 3: Observed spore count for Bacillus coagulans LBSC, Bacillus subtilis PLSSC, Bacillus clausii 088AE analysed from blend with theoretical count of 1.2×109CFU/g each.

To ensure only single and desired species is able to grow on the respective selective medium, confirmation was done considering colony morphologies, microscopic examination of colonies, biochemical testing and 16S rRNA sequencing to avoid any ambiguity arising due to selective pressures. Bacillus coagulans LBSC selectively grown on PNY agar (pH 4.5, supplemented with 1.5 µg/mL ceftazidime) showed typical colony morphology (circular with opaque centre). Microscopic observation of colony revealed long rods for LBSC from individual spray dried preparations as well as blend (Supplementary Figure 1a, b). Methyl red and nitrate reduction test gave LBSC specific results i. e. MR positive and nitrate reduction negative, thus confirming selectivity of the medium (Supplementary Figure 2a,b). The 16SrRNA sequencing of pure colony showed 100% homology with sequence as per GenBank accession no. KX355750.

Bacillus subtilis PLSSC grew selectively on nutrient agar (pH 5.0) giving colonies with a rough surface and microscopy as short thick rods, typical of PLSSC (Supplementary Figure 1c, d). The 16SrRNA sequencing of pure colony showed 100% homology with sequence as per GenBank accession no. CP031129. PLSSC does not have distinct or unique biochemical character to differentiate it from LBSC and 088AE. In addition, PLSSC has a very short generation time and grows within 20-24 h on the plate. While 088AE and LBSC take beyond 24 h to show up as a colony on the plate. This eliminated the need to have any further test (apart from colony characters and 16S rRNA) to confirm PLSSC on its selective medium.

Bacillus clausii 088AE also showed typical colony morphology (irregular margin) and microscopy as very long rods (Supplementary Figure 1e, f). Any discrepancy with LBSC was eliminated with biochemical tests MR and nitrate reduction which gave negative and positive results respectively, as typically seen for 088AE (Supplementary Figure 2a,b). The 16SrRNA sequencing of pure colony showed 100% homology with sequence as per GenBank accession no.CP031128.

Validation of selective enumeration method for individual Bacillus from blend

Finally, 5 blends of varying spore count were analysed to validate the selective enumeration method on five method validation parameters as per ICHQ2R2 guidelines [15].

Specificity

Specificity of the method was validated using blank matrix to show that none of the non-target bacteria are able to grow on the newly designed media. Absence of any growth from blank matrix demonstrated that the newly designed media are specific for the target bacteria. Additionally, only the desired strain grew on the respective selective medium. This was confirmed by observing only single type of colonies, biochemical tests and 16SrRNA sequencing when blend or mixture of three Bacillus strains was analyzed.

Precision

Precision of the method expresses closeness of agreement between a series of measurements obtained LBSC PLSSC 088AE from multiple samplings of the same homogeneous sample under the prescribed conditions. We carried out precision at repeatability and intermediate precision levels. Repeatability of the method was confirmed (Figure 4a) with RSD (%) less than 10 for three Bacillus strains at five concentration levels (Table 2) when performed by single operator on a single day in triplicate. Intermediate precision was obtained for analyses performed by two operators on different days in triplicate (Figure 4a,b). We found RSD (%) to be less than 10 (Table 2). RSD (%) below 10 indicated good precision and falls within the acceptable limit.

Table 2: Precision and intermediate precision of the selective enumeration method 

RSD: Relative Standard Deviation; CFU: Colony Forming Units; g: Gram

Figure 4: Scatter plot for precision and intermediate precision of the selective enumeration method. a) Spore count CFU/g obtained for 5 concentrations of blends performed by operator 1 on a single day indicates precision b)Spore count CFU/g obtained for 5 concentrations of blends performed by operator 2 on a different day indicates intermediate precision together with (a).

Working range and linearity

The method was evaluated over a working range of 0.12–12 ×10? CFU/g per Bacillus strain, corresponding to 0.36–36 × 10? CFU/g for the total blend. The linearity of the method was evaluated over this range for the three Bacilli. A simple linear regression analysis showed an excellent correlation between theoretical and observed spore counts, with a coefficient of determination (R²) above 0.99. The regression model was statistically significant (F >1000, p < 0.001), confirming a strong linear relationship (Table 3).

Table 3: Best fit values for simple linear regression model

R2: Coefficient of Determination; F: F-statistic; P value: Probability value

Additionally, for LBSC, a slope of 1.03 (95% CI: 0.929–1.13) and an intercept of 5.56 × 10? (95% CI: –5.58 × 10? to 6.69 × 10?) with an R² of 0.997 was obtained. Since the confidence intervals for the slope and intercept included 1 and 0, respectively, the method demonstrated no significant proportional or constant bias and exhibited excellent linearity across the tested range. Similarly, for PLSSC, regression analysis yielded a slope of 1.10 (95% CI: 1.06–1.14) and an intercept of –4.47 × 10? (95% CI: –2.69 × 10? to 1.80 × 10?) with an R² of 1.00. While the intercept was not significantly different from zero, the slope was greater than one, indicating a consistent ~10% proportional overestimation yet within the acceptable limit. For 088AE, regression analysis gave a slope of 1.06 (95% CI: 1.01–1.10) and an intercept of –7.28 × 10? (95% CI: –3.65 × 10? to 2.20 × 10?), with an R² of 0.999. The intercept was not significantly different from zero, while the slope was greater than one, indicating a proportional overestimation of ~6% again within acceptable limit.Overall, the method demonstrated excellent linearity across the tested range for all the three strains. Residuals were randomly distributed around zero without systematic deviation or increasing variance, supporting the assumptions of linearity (Figure 5). No significant outliers were observed. These results demonstrate that the method exhibits robust linearity across the selected working range, suitable for quantitation.

Accuracy

Accuracy of the method was evident based on regression model and paired t-test results. No significant difference in the means of theoretical and observed spore count was found for the three Bacilli with P-value>0.05. Slope of regression model- close to 1.0 showed good match between theoretical and observed count indicating accuracy. The method demonstrated mean recoveries ranging from 88% to 112% across the tested range, corresponding to bias values of –12% to +11% (Table 4).

Table 4: Recovery and bias percentage across the working range

CFU: Colony Forming Units; g: Gram

These values fall within the generally accepted limits of ±15% for microbiological enumeration methods, confirming acceptable accuracy.

The method demonstrated acceptable levels of precision, accuracy, and linearity over the working range of 0.12 × 10? CFU/g to 12 × 10? CFU/g for individual Bacillus strains. This range was selected to adequately cover the spore counts typically present in commercial formulations. The validated selective enumeration method was successfully applied for the analysis of commercial formulations, namely BioSEB Trio™ and ProbioSEB Duo™ (data not shown).

The development of an agar plate count method for the enumeration of a single Bacillus strains from a complex microbial blend comprising LBSC, PLSSC and 088AE as an example, is a significant advancement in selective enumeration techniques for Bacilli. This method’s uniqueness lies in its targeted application for proprietary Bacillus strains, which are widely recognized for their industrial and probiotic potential [25,27]. This is the first instance where a selective plate count method has been developed specifically for selected Bacillus species in a mixture, offering a critical guiding tool for microbiologists and researchers in the field. The method is accurate, reproducible and specific, filling a crucial need for the controlled assessment of Bacillus strains in both research and applied settings. The present work using a mixture of LBSC, PLSSC and 088AE as a model shall form a guide in quantification and viability assessment of probiotics in multi-strain formulae with an understanding of the properties of the individual strains in the blend.

Authors are grateful to Mr. V.L. Rathi and Mr. M. Kabra at Advanced Enzyme Technologies Ltd., India for their support and guidance.

Conflict of Interest: All authors are employed by Advanced Enzyme Technologies limited and declare no competing interests.

1. Mazzantini D, Calvigioni M, Celandroni F, Lupetti A, Ghelardi E. Spotlight on the Compositional Quality of Probiotic Formulations Marketed Worldwide. Front Microbiol. 2021; 12: 693973.
2. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014; 11: 506-514.
3. Chapman CM, Gibson GR, Rowland I. Health benefits of probiotics: are mixtures more effective than single strains? Eur J Nutr. 2011; 50: 1-17.
4. Williams NT. Probiotics. Am J Health Syst Pharm. 2010; 67: 449-458.
5. Hanchi H, Mottawea W, Sebei K, Hammami R. The Genus Enterococcus: Between Probiotic Potential and Safety Concerns-An Update. Front Microbiol. 2018; 9: 1791.
6. Zheng J, Wittouck S, Salvetti E, Franz CMAP, Harris HMB, Mattarelli P, et al. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol. 2020; 70: 2782-2858.
7. Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, Panwar H. Bacillus As Potential Probiotics: Status, Concerns, and Future Perspectives. Front Microbiol. 2017; 8: 1490.
8. Ramlucken U, Ramchuran SO, Moonsamy G, Jansen van Rensburg C, Thantsha MS, Lalloo R. Production and stability of a multi-strain Bacillus based probiotic product for commercial use in poultry. Biotechnol Rep (Amst). 2020; 29: e00575.
9. Elleithy EMM, Bawish BM, Kamel S, Ismael E, Bashir DW, Hamza D, et al. Influence of dietary Bacillus coagulans and/or Bacillus licheniformis-based probiotics on performance, gut health, gene expression, and litter quality of broiler chickens. Trop Anim Health Prod. 2023; 55: 38.
10. Speck ML. Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, DC. 1984.
11. Lim PY. Microbiological procedure: Spore forming bacteria count. In: Hasegawa H, Miwa K. (ed) Laboratory Manual on Analytical Methods and Procedures for Fish and Fish Products, 1992; 2nd edn. Marine Fisheries Research Department, Southeast Asian Fisheries Development Center, Singapore. 1-11.3
12. Leuschner RG, Bew J, Cruz A, Adler A, Auclair E, Bertin G, et al. Enumeration of probiotic bacilli spores in animal feed: interlaboratory study. J AOAC Int. 2003; 86: 568-575.
13. International Standards Organisation [ISO]. Animal feeding stuffs. Isolation and enumeration of presumptive Bacillus spp. 2009; ISO Standard BS EN 15784:2009.
14. Harley JP, Prescott LM. Laboratory exercises in microbiology. McGraw-Hill, New York, USA. 2002.
15. Guideline, IHT. Validation of analytical procedures Q2 (R2). ICH, Geneva, Switzerland. 2023.
16. Vitetta L, Coulson S, Thomsen M, Nguyen T, Hall S. Probiotics, et al, oxidative stress and strain specificity. Gut Microbes. 2017; 8: 311-322.
17. Araya M, Morelli L, Reid G, Sanders M, Stanton C, Pineiro M, et al. Joint FAO/WHO Working Group report on drafting guidelines for the evaluation of probiotics in food.World Health Organization, Food and Agriculture Organization of the United Nations, London, Canada. 2002.
18. International Standards Organisation [ISO] Milk Products–Enumeration of Presumptive LactoBacillus acidophilus on a Selective Medium–Colony-Count Technique at 37 °C. 2006; ISO 20128: 2006 (IDF 192: 2006).
19. International Standards Organisation [ISO] Milk Products – Enumeration of Presumptive Bifidobacteria – Colony Count Technique at 37 °C. 2010; ISO 29981:2010 (IDF 220:2010).
20. Davis C. Enumeration of probiotic strains: Review of culture-dependent and alternative techniques to quantify viable bacteria. J Microbiol Methods. 2014; 103: 9-17.
21. Jackson SA, Schoeni JL, Vegge C, Pane M, Stahl B, Bradley M, et al. Improving End-User Trust in the Quality of Commercial Probiotic Products. Front Microbiol. 2019; 10: 739.
22. Chiron C, Tompkins TA, Burguière P. Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotic products. J Appl Microbiol. 2018; 124: 572-584.
23. Byrd PM, Fallico V, Tang P, Wong C. Novel microaerobic agar plate method delivers highly selective and accurate enumeration of probiotic lactobacilli in freeze-dried blends containing bifidobacteria. J Microbiol Methods. 2022; 195: 106451.
24. Shah NP. Probiotic bacteria: selective enumeration and survival in dairy foods. J Dairy Sci. 2000; 83: 894-907.
25. Dixit YM, Bhingardeve NN, InamdarAS, Saroj D. In-depth functional 

    characterization of Bacillus subtilis PLSSC revealing its robust probiotic attributes. J Hum Nutr Food Sci. 2024; 12: 1182.

26. Dixit Y, Kanojiya K, Bhingardeve N, Ahire JJ, Saroj D. Identification and characterisation of antimicrobial compound produced by probiotic Alkalihalobacillus clausii 088AE. Braz J Microbiol. 2023; 54: 1737-1743.
27. Maity C, Gupta PhD AK, Saroj DB, Biyani A, Bagkar P, Kulkarni J, et al. Impact of a Gastrointestinal Stable Probiotic Supplement Bacillus coagulans LBSC on Human Gut Microbiome Modulation. J Diet Suppl. 2021; 18: 577-596.