Skip to content
Research Article Open access CC BY 4.0

Bacterial Quantification in Different Plating Methodologies

Kawana Nasser Machado, Marise Fonseca dos Santos, Robson Fernando Missio, Eliane Cristina Gruszka Vendruscolo

Journal of Advances in Biology & Biotechnology · pp. 964–973 · Published 3 Aug 2024

10.9734/jabb/2024/v27i81217

Abstract

Determining the number of bacterial cells in a product is a fundamental procedure in the inoculant industry, as it ensures its quality. The current legislation in Brazil recommends the spreading technique as the standard procedure to count viable cells per gram or milliliters (mL). However, in order to carry out these analyses, there is a considerable material demand. Hence, the objective of this study was to test an alternative methodology, the microdrop technique, and determine whether it mitigates costs and material demands, with the added benefit of being faster and more accurate for operators. To carry out the experiment, 8 treatments were made in both saline solution (SS) and saline solution (0.9%) + Tween 80 (0.1%), at different concentrations, using a homemade inoculant as inoculum. After plating, bacterial growth was conducted in a growth chamber for 4–6 days. A parameter of 30–300 colonies per plate was used to count colony-forming units (CFU). In addition, contaminant tests were performed for Tryptic Soy Agar (TSA) bacteria and saprophytic fungi (Sabouraud). Dilution samples (105) and inoculum from commercial packages were evaluated by Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR). Despite the microdroplet presenting higher CFU values when compared to the spreading technique, the dilutions did not alter its order of magnitude (107 CFU.mL-1). The use of the microdrop method, when combined with Tween 80 (0.1%), increased the unitary CFU value, providing a cost-effective method for counting viable Azospirillum brasilense cell numbers compared to standard culture media.

Microbiological parameters microorganisms quality control

References (22)

  1. 1 Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013) [DOI]
  2. 2 How to optimize the drop plate method for enumerating bacteria [DOI]
  3. 3 Microbial inoculants: reviewing the past, discussing the present and previewing an outstanding future for the use of beneficial bacteria in agriculture [DOI]
  4. 4 Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples [DOI]
  5. 5 A method for high throughput determination of viable bacteria cell counts in 96-well plates [DOI]
  6. 6 Effects of Tween 80 on Growth and Biofilm Formation in Laboratory Media [DOI]
  7. 7 Validation of drop plate technique for bacterial enumeration by parametric and nonparametric tests.
  8. 8 Viable but Nonculturable and Persister Cells Coexist Stochastically and Are Induced by Human Serum [DOI]
  9. 9 Revisiting soil bacterial counting methods: Optimal soil storage and pretreatment methods and comparison of culture-dependent and -independent methods [DOI]
  10. 10 Interrelation between Tween and the membrane properties and high pressure tolerance of Lactobacillus plantarum [DOI]
  11. 11 PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk [DOI]
  12. 12 The smaller, the better? The size effect of alginate beads carrying plant growth-promoting bacteria for seed coating [DOI]
  13. 13 Inhibitory effects of polysorbate 80 on MRSA biofilm formed on different substrates including dermal tissue [DOI]
  14. 14 Propidium monoazide–quantitative polymerase chain reaction (PMA-qPCR) assay for rapid detection of viable and viable but non-culturable (VBNC) Pseudomonas aeruginosa in swimming pools [DOI]
  15. 15 Shelf Life of Azospirillum brasilense in Alginate Beads Enriched With Trehalose and Humic Acid [DOI]
  16. 16 DNA-based methodologies for the quantification of live and dead cells in formulated biocontrol products based on Pantoea agglomerans CPA-2 [DOI]
  17. 17 Azospirillum brasilense viable cells enumeration using propidium monoazide-quantitative PCR [DOI]
  18. 18 The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network [DOI]
  19. 19 Methods to quantify Bacillus simplex-based inoculant and its effect as a seed treatment on field-grown corn and soybean in Brazil [DOI]
  20. 20 Tween 80 Improves the Acid-Fast Bacilli Quantification in the Magnetic Nanoparticle-Based Colorimetric Biosensing Assay (NCBA) [DOI]
  21. 21 Physical impaction injury effects on bacterial cells during spread plating influenced by cell characteristics of the organisms [DOI]
  22. 22 Evaluation of Tween 80 incorporated media to increase pathogen isolation from peritoneal fluid of CAPD patients at Hospital Kuala Lumpur.

Cited by 4

Antibacterial Activity of CuO Nanoparticles, Ethanolic Extract of Amphipterygium adstringens, and Their Combination Against Multidrug-Resistant Bacteria

Rosa Ruvalcaba-Ontiveros, Hilda Esparza-Ponce, Reyna Reyes-Martínez · International Journal of Nanomedicine · 2025

Showing 1 of 4 known citations — external sources report more than can currently be individually listed.

Article metrics

Real usage data collected on this platform.

23

Page views

0

PDF downloads

4

Outbound clicks

4

Citations

Views over time

Views by country

Approximate, from request IP at view time — not citizenship or institution. Countries with fewer than 5 views are grouped as "Other".

Traffic sources

Referring site, by host.

Views and downloads exclude known bots/crawlers. Citations combines this platform's own DOI-resolved index with each external source's own reported total — see Cited by above for individually listed citing works. Last refreshed 0 seconds ago.