MORECOVERY: A Swab-Based Surface Sampling Protocol Incorporating a Nutrient-Free Resuscitation Step for the Detection of Clinically Relevant Gram-Negative Pathogens in the Viable but Non-Culturable State

Materials and reagents

Reagents

1. Tryptic soy agar (TSA) (Becton, Dickinson and Company, catalog number: 212305)

2. M9, minimal salts, 5× (Merck, catalog number: M6030)

3. Calcium chloride (CaCl₂) (Merck, catalog number: C7902)

4. Magnesium sulfate (MgSO₄) (Merck, catalog number: M2643)

Solutions

1. Tryptic soy agar (TSA) (see Recipes)

2. Resuscitation buffer (RB) (see Recipes)

Recipes

1. TSA

To prepare 1 L of TSA, suspend 40 g of dehydrated TSA culture medium in 1 L of distilled (type II) water in a suitable flask or beaker under magnetic stirring and heat with agitation until completely dissolved. Sterilize by autoclaving at 121 °C for 15 min. After cooling to ~45–50 °C, aseptically pour into sterile Petri dishes (25 mL per plate) and allow to solidify.

Note: Commercially prepared TSA plates, purchased by many companies, may be used as an alternative.

2. Resuscitation buffer (RB)

Before preparing the RB, stock solutions of 5× M9 minimal salts solution, CaCl₂ (500×, 10 g/L), and MgSO₄ (500×, 60 g/L) should be made in advance. To prepare 1 L of M9 minimal salts solution (5×), dissolve 56.4 g of dehydrated powder in approximately 800 mL of ddH₂O in a suitable flask or beaker under magnetic stirring until completely dissolved. Adjust the final volume to 1 L with ddH₂O and sterilize by autoclaving at 121 °C for 15 min. The M9 basal salts solution contains KH₂PO₄ (15 g/L), NaCl (2.5 g/L), Na₂HPO₄ (33.9 g/L), and NH₄Cl (5 g/L) [11]. The sterile M9 minimal salts solution (5×) can be stored at 4 °C for up to 6 months. Before use, inspect for precipitation or signs of contamination. The pre-mixed M9 minimal salts are also available from Merck (see Reagents).

For the CaCl₂ stock solution (500×, 10 g/L), dissolve 0.20 g of CaCl₂ in 20 mL of ddH₂O in a 50 mL tube and sterilize by 0.2 μm filtration or by autoclaving. Similarly, prepare the MgSO₄ stock solution (500×, 60 g/L) by dissolving 1.20 g of MgSO₄ in 20 mL of ddH₂O in a 50 mL tube, followed by sterilization through 0.2 μm filtration or by autoclaving. The sterile CaCl₂ and MgSO₄ stock solutions are stable and can be stored for extended periods at room temperature, when properly sealed; however, storage at 4 °C is recommended for long-term use.

The required volume of RB depends on the number of surfaces to be sampled. Considering that each swab contains 2 mL of RB, 1 L of RB is sufficient for 500 swabs. For RB preparation, combine the appropriate volumes of sterile stock solutions in a sterile container under aseptic conditions. As an example, to prepare 200 mL of RB, mix 40 mL of 5× M9 minimal salts solution, 400 μL of 500× CaCl₂ stock solution, and 400 μL of 500× MgSO₄ stock solution, then adjust the final volume to 200 mL with sterile ddH₂O. Mix thoroughly to ensure homogeneity.

When needed, fresh preparation of RB from sterile stock solutions under aseptic conditions is recommended to avoid potential contamination. However, RB remains stable for up to five weeks at room temperature, and it must be used at room temperature to ensure optimal handling conditions and to avoid any potential thermal shock to stressed bacterial cells.

Laboratory supplies

1. Sterile nylon swabs (FLOQSwabs, Copan, catalog number: 519C)

2. Sampling templates (10 × 10 cm) (Copan, catalog number: T2905)

3. Sampling templates (5 × 4 cm) (Copan, catalog number: T2906)

4. Sterile Petri dishes (Sarstedt, catalog number: 82.1472)

5. Sterile disposable spreader (Sarstedt, catalog number: 86.1569.005)

6. Disposable sterile 10 mL syringe (Biosigma, catalog number: 050840)

7. Syringe filters 0.2 μm (Sarstedt, catalog number: 83.1826.001)

8. 1.5 mL tubes (Sarstedt, catalog number: 72.690.001)

9. 15 mL conical tubes (Sarstedt, catalog number: 62.554.001)

10. 50 mL conical tubes (Sarstedt, catalog number: 62.547.255)

11. 200 μL pipette tips (Sarstedt, catalog number: 70.3030)

Equipment

1. Vertical laminar flow hood (Bioair, model: TopSafe 1.2)

2. Vortex (Merck, catalog number: Z258423)

3. Microbiological incubator (Merck, catalog number: CLS6754)

4. Magnetic stirrer with heating plate (Carl Roth, catalog number: XT23.1)

5. Pipette P200 (Gilson, catalog number: FA10005M)

6. Refrigerator (4 °C) (Miele, catalog number: KFN 7795C)

7. Analytical balance (Merck, catalog number: Z742879)

8. Autoclave (ASAL, catalog number: 29960018)

9. Nitrile gloves (Merck, catalog number: Z677272)

10. Felt marker, waterproof (Sarstedt, catalog number: 95.954)

Procedure

A. Preparation of sampling materials

1. Prepare the RB.

2. Dispense 2 mL of RB into the sterile swab tube under aseptic conditions to preserve its sterility.

3. Keep nylon swabs sterile until use.

B. Surface sampling and incubation of the swab

1. Label all swab tubes with sample ID and date.

2. Select the area to be sampled.

3. If the area of interest is flat, a sampling template should be used. The sampling template may be either 100 cm² (10 × 10 cm) or 20 cm² (5 × 4 cm) (Figure 1), depending on the surface to be sampled. The sampled area should be representative of the surface being sampled. For irregular surfaces, the sampled area should be approximated by decomposing it into simple geometric shapes in order to estimate its surface area as accurately as possible.

Figure 1. Materials and equipment for the MORECOVERY surface sampling protocol and method for surface sampling. (A) Materials and equipment: (1) Sterile plastic tube containing the nylon swab soaked with RB; (2–3) 10 × 10 cm and 5 × 4 cm sampling templates; (4) TSA plate; (5) disposable spreader for inoculation; and (6) P200 pipette with disposable tip. (B) Surface sampling procedure. For flat surfaces, swab the sampling area using a horizontal zig-zag pattern followed by a vertical zig-zag pattern, performing at least 10 strokes in each direction, to ensure sampling of the whole surface.

4. Keep the swab tube upright and check that the RB is at the bottom; if necessary, gently shake the tube downward so that all the liquid gathers at the bottom of the tube.

5. Gently press the swab tip against the inner wall of the tube to eliminate any excess RB, ensuring the swab remains damp and uniformly soaked with the solution.

6. Hold the swab handle between the thumb and forefinger. For flat surfaces, perform sampling by swabbing horizontally and vertically, carrying out at least 10 strokes in each direction. For small or hard-to-reach areas, ensure that the entire defined location is sampled, including crevices, gaps, joints, and other surface connections.

7. Transfer the swab back into the tube containing RB. Make sure the tube containing the swab is properly closed so that the swab remains submerged in RB until analysis.

8. If appropriate, include additional swabs to be used as field negative controls (i.e., swabs that are not used for surface sampling but are processed alongside the samples to monitor potential contamination).

Note: Samples should be transported to the laboratory at room temperature within 8 h. Ensure that storage and transport temperatures do not exceed 37 °C. Refrigerated conditions are not required, as the RB does not contain carbon sources that could support bacterial growth and potentially affect the accurate assessment of contamination.

C. Resuscitation of VBNC bacterial cells and plating

1. Incubate the tube containing the swab at 37 °C for 24 h with shaking at 200 rpm.

Critical: This step corresponds to the resuscitation phase, during which VBNC cells may regain culturability. Continuous shaking at 200 rpm during the resuscitation step is recommended to ensure adequate oxygenation and homogeneous contact between the cells and the RB. During incubation, swab tubes should preferably be positioned at a 45° angle on the shaker to facilitate mixing and aeration.

2. Following incubation, vortex the tube for 1 min prior to plating.

Critical: Vortexing for 1 min is essential to promote the release of bacterial cells from the swab fibers into the RB. Pipette 100 μL of the sample (presumptive bacterial cell suspension in RB) onto TSA agar plates and spread evenly using a sterile disposable spreader. Operate under a laminar flow hood to minimize the risk of contamination.

Note: Alternative culture media may be used according to analytical requirements. When the aim is the detection of a specific pathogen, selective media appropriate for the isolation of the target microorganism may also be employed, in addition to TSA.

3. Incubate plates at 37 °C for up to 72 h.

Note: Since this method can be applied to the detection of specific microorganisms, incubation time and temperature may be adjusted according to the growth requirements of the target organism.

D. Colony-forming unit (CFU) quantification and data analysis

1. Count the number of CFUs on each plate.

2. Calculate the bacterial load on the sampled surface, expressed as CFU/cm² (Ns), using the following formula:

N s = N × D A

where:

N = number of CFUs on the plate.

A = sampled surface area (cm²).

D = the inverse of the plated fraction (e.g., if 100 μL out of 2 mL are plated, this corresponds to 1/20 of the sample, so D = 20).

Example results obtained with the MORECOVERY protocol, which integrates the resuscitation step into the sampling procedure, are shown in Figure 2. In particular, Figure 2A shows the CFU counts of representative Gram-negative bacteria (A. baumannii, Enterobacter cloacae, K. pneumoniae, and P. aeruginosa) before and after the resuscitation step in a simulated contamination experiment. The increase in bacterial recovery ranges approximately from 10 to 10⁴, depending on the test species. Figure 2B shows the results of a field test in which different surfaces (laboratory bench, classroom desks, and washbasin) were sampled, demonstrating that the resuscitation step can markedly enhance bacterial detection, with up to a one-million-fold increase of CFU recovery in some cases.

Figure 2. Resuscitation of viable but non-culturable (VBNC) cells by the MORECOVERY protocol improves bacterial detection [colony-forming unit (CFU) counts] on environmental surfaces under controlled laboratory conditions and in field tests. (A) Representative strains of Gram-negative bacteria were air-dried for 1 week on a glass surface. After desiccation, cells were suspended in RB and incubated at 37 °C under shaking for 24 h for resuscitation. Culturability was expressed as CFU recovery, as determined before desiccation, after 1-week desiccation, and after 24-h resuscitation in RB at 37 °C. The grey area indicates the limit of detection (LoD), corresponding to 2.7 × 10² CFU. Data are the mean ± standard deviation (error bars) of three independent experiments. Red asterisks indicate statistically significant differences between samples before desiccation and after desiccation. Green asterisks indicate statistically significant differences between samples after desiccation and after resuscitation. Statistically significant differences (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were determined using the unpaired t-test. (B) Microbial contamination of laboratory benches, classroom desks, and washbasins, determined before (T₀) and after 24-h resuscitation in RB at 37 °C (T₁). Six samples were collected and analyzed for each surface type (modified from [7]). (C) Representative photographs of Petri dishes inoculated with samples before (T₀) and after resuscitation (T₁).

E. Test report

Write a test report including the following information: date, identification of the sampling location, culture media and incubation conditions used, and the results obtained, expressed in relation to the sampled surface (including its size or designation). The report should also include details of any incidents or relevant deviations that may have influenced the test results.

Validation of protocol

This protocol has been used and validated in the following research article:

• Visaggio et al. [7]. ESKAPE Gram-negative bacteria escape culture-based detection upon desiccation on abiotic surfaces. Microbiol Spectrum. 14(3): e03357–25. https://doi.org/10.1128/spectrum.03357-25 (Figures 1 and 3)

General notes and troubleshooting

General notes

1. This protocol markedly increases the detection of Gram-negative bacteria, as it enables the recovery of both culturable cells and bacteria in the VBNC state. By contrast, the detection of some Gram-positive bacteria is not or only marginally improved by the resuscitation step [7].

2. This protocol can be adapted to different applications wherever monitoring of microbial contamination is required. When applied in settings where potentially pathogenic microorganisms may be present, appropriate institutional biosafety procedures should be followed, including BSL-2 practices when applicable.

Troubleshooting

Problem 1: Bacterial contamination below the limit of detection.

Possible cause: The swab is immersed in 2 mL of RB, from which 100 μL are plated for CFU counting. This results in a limit of detection (LoD) of 20 CFU per swab, corresponding to 10 CFU/mL, since only one-twentieth of the total sample volume is plated. Thus, contamination levels below the threshold of 20 CFU per sampled area cannot be detected.

Solution: For surfaces with expectedly low microbial contamination, or when detection of single bacterial cells is critical, sensitivity can be maximized by centrifuging the sample and plating the whole pellet originating from the 2-mL sample.

Problem 2: Plate overgrowth due to high bacterial contamination.

Possible cause: The sampled surface has a high bacterial load, leading to overgrowth and coalescence of colonies on the agar plate, which prevents accurate colony counting and reliable CFU quantification.

Solution: Perform serial ten-fold dilutions of RB prior to plating to obtain countable colonies on agar plates and ensure accurate quantification of the bacterial load.

Acknowledgments

Conceptualization, C.S., D.V., F.I., M.L., P.V.; Investigation, C.S., D.V.; Writing—Original Draft, C.S., D.V., M.L.; M.B.; Writing—Review & Editing, F.I., P.V.; Funding acquisition, D.V., P.V.; Supervision, P.V., F.I.

This work was supported by the Excellence Departments grant (art. 1, comma 314-337 Legge 232/2016) from the Italian Ministry of University and Research (MUR) to the Department of Science, Roma Tre University; the PRIN 2022 grant (CUP F53D23000860001) from MUR to D.V.; the Rome Technopole, PNRR grant (M4-C2-Inv. 1.5 CUP F83B22000040006).

The protocol was adapted from and validated as described in [7]. Early evidence of the ability to resuscitate the Gram-negative bacterium A. baumannii after incubation in RB was reported in [8]. The graphical overview was created using BioRender.

Competing interests

A patent application entitled “Metodo per la quantificazione di microrganismi patogeni su superfici abiotiche” was filed on June 19, 2025 (application number 102025000014596), and extended as a European patent (application number 26185781.7) on June 17, 2026. The authors have no additional conflicts of interest.

Ethical considerations

No ethical considerations apply to this study.

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