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Laboratory plasticware - Use at your own risk: Suitability of microcentrifuge tubes for spores' analysis of
1Public Health Ontario Laboratories, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Abstract:
Clostridium difficile is a Gram-positive spore forming rod-shaped bacterium which causes mild to severe diarrhea. Spores play a key role in transmission of C. difficile in hospital environment. To investigate ability of spores to stay on fomites and to assess levels of contamination it is essential to prevent loss of spores collected for the analysis. Working with C. difficile spores we noticed a significant loss after vortexing of spore suspensions and investigated if it can be prevented by using a specific brand or type of microcentrifuge tubes. 7 types of microcentrifuge tubes from 3 manufacturers were tested. Spores of three types of C. difficile, NAP1/027, NAP4/014 and NAP7/078 (clinical isolates) were used. C. difficile was grown on Brucella Supplemented Agar for 9 days, spores were collected, washed and density of 3 suspensions was normalized to optical density (OD) 550 0.1 or 0.05. These suspensions (OD 0.1) were used in serial dilutions with 3 experimental conditions - pipetting, vortexing or vortexing in 3% albumin solution and in vortexing experiment when 150 μl were vortexed for 1 min at 1500 rpm per tube and loss of spores was measured by a decrease in dipicolinic acid (DPA) concentration measured by time-delayed terbium fluorescence. Inner surface of the tubes was visualized with microscopy to observe adhered spores. In serial dilution experiment, initial concentration of spores would be underestimated by up to 18X in case of vortexing for NAP1 strain and 9X for NAP4 strain. Presence of 3% albumin significantly decreases this effect but does not eliminate it completely. Comparison of 7 types of tubes shows that a single vortexing for 1 min of diluted spore suspension at concentrations of 1.8 × 107 spores per ml leads to a loss of up to 90% of spores in some tubes. Degree of spores' adhesion varied between brands and types of tubes and between tubes of the same type. In some brands there was a significant variability in adhesion between tubes from the same batch. Microscopy after vortexing shows a film of spores attached to the tube's wall. Adherence could be affected by the type of plastic, additives (plasticizers) used in manufacturing and quality of moulds (e.g."diamond polished"). To identify the most appropriate type of tubes for the experiment it is essential to test it beforehand as not every brand is suitable for this purpose. Using tubes with a high degree of adherence could significantly affect measurement of spores' concentration in serial dilutions, e.g. when quantifying spores production by a specific strain or when limits of detection are measured. Also, sensitivity of commercial tests for detection of C. difficile in clinical specimens can be decreased if an unsuitable type of plastic containers and tubes is used.
Insights
Microcentrifuge tube choice significantly impacts Clostridium difficile spore quantification. Vortexing spore suspensions can lead to substantial spore loss, up to 90%, depending on the tube type.
Area of Science:
- Microbiology
- Infectious Diseases
- Environmental Health
Background:
- Clostridium difficile is a Gram-positive bacterium causing diarrhea, with spores crucial for hospital transmission.
- Accurate quantification of C. difficile spores is essential for assessing contamination and developing effective control measures.
- Previous work indicated significant spore loss during suspension handling, prompting investigation into microcentrifuge tube effects.
Purpose of the Study:
- To investigate the impact of different microcentrifuge tube types on Clostridium difficile spore recovery.
- To determine if specific tube materials or manufacturing processes influence spore adhesion and loss.
- To identify optimal microcentrifuge tubes for reliable C. difficile spore quantification in laboratory settings.
Main Methods:
- Tested 7 types of microcentrifuge tubes from 3 manufacturers using three C. difficile strains (NAP1/027, NAP4/014, NAP7/078).
- Assessed spore loss after pipetting, vortexing, and vortexing in 3% albumin solution using dipicolinic acid concentration.
- Microscopy was used to visualize adhered spores on the inner surface of the tubes post-vortexing.
Main Results:
- Vortexing spore suspensions resulted in significant underestimation of spore concentrations, up to 18X for NAP1.
- Spore loss varied greatly between tube brands and types, with some tubes losing up to 90% of spores.
- Albumin solution reduced but did not eliminate spore loss; microscopy revealed significant spore adhesion to tube walls.
Conclusions:
- The choice of microcentrifuge tube is critical and can significantly affect C. difficile spore quantification.
- Variability in spore adhesion exists even between tubes from the same batch, highlighting manufacturing inconsistencies.
- Pre-testing of microcentrifuge tubes is essential to ensure reliable results in spore quantification and diagnostic sensitivity.