1. Introduction

This protocol describes a procedure for detection and quantitative determination of microbial contamination in a nanoparticle preparation. Nanoparticle samples, along with controls, are spread on the surface of agar and growth of bacterial colonies is monitored after 72 hr of incubation. Additionally, to detect autotrophic and oligotrophic bacteria which grow in media containing low nutrient levels and may contaminate cell culture after passing rich medium agar plate test, the same nanoparticle samples are spiked into RPMI and incubated up to 7 days. The intended purpose of this assay is to avoid introduction of microbial contamination into in vitro cell cultures and in vivo animal studies utilizing the test-nanomaterial, as microbial contamination will confound the results of these tests. The second goal of this test is to determine bacterial identity in samples found to be positive. The ID results can be helpful in determining the source of contamination. This assay is not intended to certify the nanomaterial as sterile.

2. Reagents, Materials, and Equipment

Note: The NCL does not endorse any of the suppliers listed below; these reagents were used in the development of the protocol and their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted. Please note that suppliers may undergo a name change due to a variety of factors. Brands and part numbers typically remain consistent but may also change over time.

2.1.

Reagents

2.1.1.

Sterile PBS (Sigma, D8537)

2.1.2.

Agar (Sigma, 05040-1KG)

2.1.3.

Tryptic Soy Broth (EMD Millipore 1054595000 or Fischer Scientific DF0370-08-4)

2.1.4.

RPMI 1640 (ThermoFisher, 11875101)

2.1.5.

Test nanomaterial

2.1.6.

Buffer used to reconstitute test nanomaterial

2.1.7.

Sodium Hydroxide (NaOH) (Sigma, S2770)

2.1.8.

Hydrochloric acid (HCl) (Sigma, H9892)

2.2.

Materials

2.2.1.

Pipettes, 0.05 to 10 mL

2.2.2.

Sterile pipets, 1-10 mL

2.2.3.

Sterile tubes, 5 mL

2.2.4.

Petri dishes

2.2.5.

Bacterial spreader

2.3.

Equipment

2.3.1.

Incubator at 37°C

2.3.2.

Vortex

2.4.

Bacterial ID and strain typing

NCL outsources this procedure to Charles River Lab. The CRL Accugenix technology is used for strain typing. More details about this technology and the service can be found here: http://www​.criver.com​/products-services/rapid

3. Reagent Preparation

3.1.

Sodium Hydroxide

Prepare from concentrated stock by dilution into sterile water to make a 0.1 N final concentration solution.

3.2.

Hydrochloric Acid

Prepare from concentrated stock by dilution into sterile water to make a 0.1 N final concentration solution.

3.3.

Tryptic Soy Broth (TSB)

Components of this media are:

Bacto^™ Tryptone (Pancreatic Digest of Casein) 17.0 g/L

Bacto Soytone (Peptic Digest of Soybean Meal) 3.0g/L

Glucose (=Dextrose) 2.5 g/L

Sodium Chloride 5.0 g/L

Dipotassium Hydrogen Phosphate 2.5 g/L

pH 7.3 ± 0.2

This is supplied as a liquid but can also be purchased as a powder. If you use liquid media, it does not require any additional manipulation. Powdered media must be reconstituted in water. In this case, please follow the instructions from the manufacturer of the powdered media. Sterilize the media you prepare from powdered formula 15 min at 121°C. Cool to room temperature and either use fresh or store in refrigerator.

3.4.

Tryptic Soy Broth Agar

Components of the TSB agar are:

Bacto^™ Tryptone (Pancreatic Digest of Casein) 17.0 g/L

Bacto^™ Soytone (Peptic Digest of Soybean Meal) 3.0g/L

Glucose (=Dextrose) 2.5 g/L

Sodium Chloride 5.0 g/L

Dipotassium Hydrogen Phosphate 2.5 g/L

Agar 15 g/L

This is prepared by dissolving 15g of agar in 1L of TSB media described in section 3.3 or from the commercial powdered formula containing all components in a dry form. After addition of agar to the liquid media, the solution is heated to boiling while stirring to dissolve the powder. The solution is then autoclaved for 15 minutes at 121°C to sterilize. After slight cooling, the TSA is poured into petri dishes (10 mL per 10 cm dish) and allowed to solidify at room temperature. The plates can be used freshly prepared or stored at 4°C. Note: TSA or TSB agar are also available from ThermoFisher, ATCC and other commercial vendors.

4. Preparation of Controls

4.1.

Negative Control (NC)

Use sterile PBS or water as a negative control. The negative control is acceptable if no colony forming units (CFU) are observed upon completion of the test.

4.2.

Positive Control (PC)

For the positive control, use bacterial cultures (e.g. ATCC #25254) at a dilution which will allow at least 10 CFU/mL.

4.3.

Inhibition Control

To assess whether nanoparticles can inhibit bacterial growth, a positive control sample at the same final dilution as described in section 4.2 is spiked into the test nanoparticle sample. For example, spike 22 CFU per 2.2 mL of nanoparticle solution at a given dilution and use 1 mL for seeding into a paddle as described in section 6 below. The final inhibition control contains the same concentration of nanoparticles as nanoparticle-unspiked sample and the same concentration of bacteria as in the positive control (10 CFU/mL).

It is important to note, this control will only estimate anti-bacterial properties relevant to bacteria present in the given positive control. The anti-bacterial properties of a nanoparticle formulation are often specific to a given strain or type of bacteria and depend on nuances in the bacterial cell wall organization and other biological processes within bacterial cells. Therefore, if the analysis of antibacterial activity is of interest, it is advisable to perform such analysis using bacterial strains that are relevant to the expected type of anti-bacterial activity of the nanoparticle formulation. For example, if a nanoparticle is prepared to inhibit E. coli growth, then relevant strains of E. coli should be used. If such information is not available, conducting a test using a panel of bacteria representative of gram-positive and gram-negative bacteria is recommended.

5. Study Samples

The assay requires 5 mL of the test nanoparticle in its final formulation. The concentration of nanoparticles in this formulation is case-specific. Samples are typically tested at stock and at several serial 1:10 dilutions, i.e., no dilution, 1:10, 1:100, 1:1000. When the final formulation is not available, for example when a test nanomaterial is received from a commercial supplier in a form not intended for biomedical applications, prepare a stock solution at a concentration of 1 mg/mL. The weight information can refer to either active pharmaceutical ingredient or total construct; it can also represent total metal content or other units. Such information is specific to each nanoparticle and should be recorded to aid result interpretation.

Test nanoparticles should be reconstituted in sterile PBS, water or in appropriate vehicle. If vehicle is a buffer or media other than water or PBS, the vehicle control should be included in the test. The pH of the study sample should be checked using a pH microelectrode and adjusted with either sterile NaOH or HCl as necessary to be within the pH range 6-8. If NaOH or HCl are not compatible with a given nanoparticle formulation, adjust pH using a procedure recommended by the nanomaterial manufacturer. To avoid sample contamination from microelectrode, always remove a small aliquot of the sample for use in measuring the pH.

6. Experimental Procedure

7. Interpretation of Results and Acceptance Criteria

8. Abbreviations

CFU

colony forming units

HCl

hydrochloric acid

NaOH

sodium hydroxide

PBS

phosphate buffered saline

MIC

minimal inhibitory concentration

This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.

*

address correspondence to: vog.hin.liam@aniram

Neun BW, Cedrone E, Potter TM, Dobrovolskaia MA, NCL Method STE-2.4: Detection of Bacterial Contamination using Tryptic Soy Agar Plates and RPMI Suspension Tests for the Purposes of Determining Bacterial ID. https://ncl.cancer.gov/resources/assay-cascade-protocols DOI: 10.17917/86Y9-3Y16