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A Simple Spectrophotometric Method to Determine Flocked Swab Absorption as an
Alternative to Complex Research Methods.
BHARAT GANDHI1*, TONY MAZZULLI1, 2
1
Department of Microbiology, Mt. Sinai Hospital and University Health Network, and
2 University of Toronto, Toronto, Canada
ABSTRACT (revised) Previous studies evalua6ng absorp6on characteris6cs of whole ﬂocked swabs have focused on research methods such as (1) scanning electron microscopy (SEM), (2) radioac6ve tracer (RT), (3) zeta poten6al (ZP) determina6on, as well as (4) water and protein absorbance. Based on the CLSI M40A document Roll Plate method, we conducted experiments to validate the performance of spectrophotometric op6cal density (OD) determina6on of bi-‐component HydraFlock swabs (Puritan Medical Products) and nylon ﬂocked swabs (Copan Diagnos6cs Inc.) following removal of the swabs that had been immersed in 100 uL of standardised bacterial inocula in a micro6tre plate for ~15 seconds. 12 ﬂocked swabs of each type immersed in serially diluted 1:10 suspensions were tested in triplicate. To visually compare the diﬀerence in volumes, a second set of experiments were conducted which consisted of adding 25 uL of red dye to each well aZer the ﬁrst determina6on was recorded. Finally, a third set of experiments to visually compare the absorp6on of 100 uL of semi-‐
viscous lactophenol co\on blue (LPCB) dye (diluted 1:1 in water) were performed. RESULTS: Optical Density determination
Concentra6on Avg OD Range The OD method and visual assessment methods used in this study were not consistent with Copan’s claim that its nylon-‐ﬂocked swabs can absorb 142 uL. The OD method is rapid, less tedious and requires less training and expensive equipment than the SEM, ZP or RT methods. However the results of the OD method are in agreement with the results of the SEM and ZP methods. The OD method, the red dye visual assessment and the LPCB dye uptake experiments clearly showed that the HydraFlock swab has a greater propensity to absorb ﬂuids than Copan’s nylon ﬂocked swab. (Blank average OD -0.0006)
Concentra6on Avg OD Range Pur 10 5 -‐0.0039 -‐0.001 to -‐0.005 Cop 10 5 +0.026 +0.012 to +0.045 Pur 10 4 -‐0.0019 -‐.0.000 to -‐0.005 Cop 10 4 +0.025 +0.014 to +0.036 Pur 10 3 -‐0.0036 -‐0.000 to -‐0.007 Cop 10 3 +0.024 +0.009 to +0.032 Red dye visual assessment: Copan swabs appeared to have more excess ﬂuid than the Puritan swabs. LPCB dye absorbance test: The Puritan swab buds were ~90% blue whereas Copan swabs were ~60% blue. Conclusions: The OD determina6ons for HydraFlock and Copan ﬂocked swabs compared to the blank demonstrated that there is excess inoculum aZer absorp6on with Copan’s ﬂocked swab but not with Puritan’s swabs. The OD method appears to be consistent with the 100 uL volume recommended in the CLSI M40A document for valida6on of swab transport systems. The other absorp6on method appears to saturate the en6re swab with 1.0 mL of water or serum (protein) and requires sophis6cated instrumenta6on and training to perform as does the SEM, RT and ZP methods. Rows 1, 3, 5, 7 Copan. Rows 2,4,6,8: Puritan (LeZ) 1-‐3: Puritan. (Right) 4-‐6: Copan METHODS Fresh Prevotella melaninogenica ATCC 25845 isolate was suspended in sterile saline and adjusted to ~1.0 McFarland turbidity standard. From this working suspension, three 1:10 serial dilu6ons were prepared to produce ~1.5×105, ~1.5×104 and ~1.5×103 colony forming units/mL. Using an Eppendorf pipe\e and one disposable pipe\e 6p, 100uL volumes of 103, 104 and 105 suspensions were transferred into 24 wells each of a round bo\om micro6tre plate, in columns #4 to #12 star6ng with the lowest concentra6on. Columns #1 to #3 were blank. Each brand of swabs were placed in alternate wells and removed aZer ~ 15 seconds of absorp6on star6ng with Copan’s E–swab followed by Puritan’s bi component ﬂocked swabs. 1. Upon comple6on of the absorp6on process, the micro6tre plate was placed on the spectrophotometer reader set at 430nm (primary ﬁlter) and 630nm (secondary ﬁlter). The reader automa6cally agitated the plate twice before producing an op6cal density printout. 2. AZer the OD reading, the second stage of the evalua6on consisted of adding 25 uL of carbol fuchsin (red) dye in all 96 wells aZer. This was a visual comparison to assess the diﬀerence in excess inoculum by means of color aZer absorp6on. 3. The third experiment consisted of having three ﬂocked swabs of each brand absorb 100 uL of a slightly viscous lacto phenol co\on blue dye diluted 50% in water. The degree of capillary ac6on and uptake of liquids by the swab bud head were used to assess the diﬀerence in absorp6on. CONCLUSIONS 1. OD determina6ons for HydraFlock and Copan ﬂocked swabs rela6ve to the blank clearly suggest there is excess inoculum aZer absorp6on with the Copan ﬂocked swab but not the Puritan swab. The diﬀerence is highly signiﬁcant p <0.05 using a two sample comparison of means. 2. The OD method is consistent with the 100 uL volume the CLSI M40A document recommends for valida6on of swab transport systems, while the other absorp6on method saturates the en6re swab with 1.0 mL of water or serum (protein) and requires sophis6cated instruments and training to perform as does SEM, RT and ZP methods. 3. The results of the OD method and visual assessment methods used in this study clearly diﬀer from the 142 uL volume absorbed by Copan’s nylon –
ﬂocked swabs reported in the literature. 4. The OD method is rapid, less tedious and requires less training and expensive equipment than the SEM, ZP or the RT methods. 5. However results of the OD method are in agreement with those of SEM and ZP methods. 6. The OD method, the red dye visual assessment and the LPCB dye uptake experiments indicate that the HydraFlock swab has a greater propensity to absorb ﬂuids than Copan’s nylon ﬂocked swab. 7. Furthermore the Puritan bi-‐component ﬂocked swabs are designed to provide a greater surface area and therefore absorb more liquid. 1. 
2. 
Madhusudhan K, Turner JC, Harry KH (2013). Comparison of physical characteris6cs and collec6on and elu6on performance of clinical swabs. African Journal of Microbiology Research. 31: 4039-‐4048. Nugent w, Russell MJ, Beck S, Leonardi GP (2007). Evalua6on of the physical and diagnos6c nature of swabs. Poster presenta6on at 23rd Annual Clinical Virology Symposium. Clearwater Beach, FL Apr 29. 3.  Human RP, Jones GA (2006). New concept for transpor6ng clinical material on ﬂocked swabs in liquid Amies medium. Poster presenta6on at American S ociety for Microbiology symposium. 4.  Clinical and Laboratory Standards Ins6tute (2003) Quality Control of Microbiological Transport Systems Approved Standard M40A. Wayne, PA: CLSI,2003. This study was funded by the kind support of Puritan Medical Products C., LLC, Guilford, ME, USA.

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