Mcfarland's Method of Cell Enumeration From Turbidometric Reading

1. Introduction

Microbial concentrations can be determined in diverse ways, including direct counting, plate counting and measurement of light scattering by bacterial cells in a liquid medium. Although these methods are all non-subversive, the last has the reward that information technology is much more expeditious than the other ii. Light passing through a microbial break is partly absorbed by the microbes and subsequently re-emitted in all directions, as a effect, the suspension has a milky appearance under visible light [1]. Measuring the amount of light absorbed (turbidimetry) or scattered (nephelometry) under appropriate conditions allows i to approximate the amount of biomass nowadays in the interruption.

McFarland [two] devised a nephelometer for measuring suspended bacteria based on standards optically mimicking bacterial suspensions and obtained by chemical precipitation. Thus, mixing appropriate amounts of sulphuric acrid (H_twoSO₄) and barium chloride (BaCl_ii) produced known amounts of a fine barium sulphate (BaSO₄) precipitate with the aforementioned calorie-free-scattering capacity equally a suspension of bacterial cells. Although visual comparison is indeed possible, obtaining precise results entails comparing microbial suspensions and McFarland standards via turbidimetric or nephelometric measurements made at wavelengths over the range of 420–660 nm. Some defended commercial instruments have fifty-fifty been designed to provide measurements in 'McFarland units' [3].

This technique has the advantage that the standards are chemical and thus require no incubation, and too that (if visual) no instrument other than one'southward eyesight is required for comparison. However, it can be direct applied simply to Gram-negative bacteria such every bit Escherichia coli because others differ in volume and mass—and hence in their ability to scatter light.

Although McFarland standards currently consist of suspensions of latex or titanium dioxide particles [4,5], which are more stable and longer lived than the one-time precipitates, barium sulphate remains in use for this purpose—in fact, its suspensions accept been shown to remain stable for nearly 20 years if stored in tightly sealed tubes at room temperature in the dark [6].

In microbiology, McFarland standards proceed to be used as reference suspensions for comparison with bacterial suspensions in liquid media for purposes such every bit obtaining antibiograms [seven] and biochemical testing [eight].

The increasing technical improvement and affordability of digital photography hardware and software [9] have promoted their use in quantitative chemical analyses. Today's digital cameras capture images by a low-cal-sensitive flake called 'accuse-coupled device' (CCD), which plays the aforementioned role as photographic pic in conventional (chemic) photography. Each cell in a CCD acts as a low-cal-sensitive private chemical element providing an electrical response to low-cal that can be digitized to build an optical image. Because CCD pixels answer to light intensity—only not to color—reproducing the three master colours the human heart tin can perceive [10] (red, green and blue) requires using a 'color' CCD. Colour digital images are the additive combination of the three colours, which most of the sensors used in digital cameras obtain by superimposing a mosaic of scarlet, dark-green and bluish filters (viz. a Bayer mask) over the pixel array in social club to interpolate colour-related information for each private pixel.

A CCD consisting of eight-fleck pixels tin can answer to 2⁸ = 256 levels of gray ranging from 0 (blackness) to 255 (white). This allows each pixel in the red, green or blue channel in a CCD-captured paradigm to be assigned a numerical value from 0 to 255, which tin be subsequently used for analytical calibration. As a result, a digital photographic camera tin can be used equally an analytical sensor because each captured image provides a vast amount of data [9].

Commercial CCD cameras accept been available for thirty years, and they have been well appreciated and widely used for analytical purposes. Analytical imaging (CCD) methods are increasingly existence used in Raman spectroscopy [xi], chemiluminescence spectroscopy [12] or electron transmission microscopy [13] at inquiry laboratories. This has promoted the evolution of dedicated commercial equipment where analyses are implemented on inductively coupled plasma-atomic emission spectroscopy (ICP-AES) [fourteen], mass spectrometers [15] or fast-browse systems [16]. In biological inquiry, CCDs have been used to capture digital images for UV-fluorescing substances with a diversity of purposes including documentation and quantitative analysis in, for example, electrophoretic separations of nucleic acids and proteins—even in in vivo tests in the latter case [17]. In the biotechnology field, digital prototype analysis techniques have been even used for the in situ label of multiphase dispersions [eighteen] and for the monitoring of activated sludge processes [nineteen]. CCDs have also been used to obtain digital images with a view to quantifying chemical species post-obit separation by thin-layer chromatography (TLC) or high-performance thin-layer chromatography (HPTLC) [xx–23]. However, in spite of their extensive use, the CCDs intended for the analytical work are quite expensive.

On the other paw, nowadays the very low-toll mass-produced digital cameras (based on CCD technology) for the home consumer market place have not yet been widely appreciated and accept scarcely been used for analytical purposes.

These low-cost devices take been employed in various fields, including forensic scientific discipline [24,25], telemedicine and laboratory analyses. In fact, digital cameras take become an constructive, inexpensive alternative to commercially available equipment (scanners) for qualitative and quantitative sparse layer chromatographic assay [26]. Quantitative imaging analysis with a digital camera and the software ImageJ recently proved an effective, affordable option for fluorimetric measurements in teaching laboratories [27]. This software–hardware combination has also enabled the quantitation of haemoglobin and melanin with a view to assess erythema and pigmentation in human skin [10].

Digital images obtained with very cheap CCD cameras known equally 'webcams' accept been used to assess colour changes during acid–base titrations and found to provide results on a par (viz. no pregnant differences at the 95% confidence level) with those of spectrophotometric monitoring [28]. Likewise, a computer monitor has been successfully used as a light source and, a webcam as detector, for purposes such as distinguishing vino samples [29].

Even built-in cameras in mobile phones accept been used in telemedicine to capture and transfer biotesting results obtained past newspaper-based microfluidic devices to quantify glucose and proteins [30].

The method reported in this paper, which is similar to one used in previous work and very recently proposed by one of the authors to quantify analytes absorbing visible lite in aqueous solutions [31,32], uses digital images of a series of McFarland standards in combination with appropriate software (ImageJ) to assign a numerical value to each colour hue (turbidity). Such a value is direct proportional to the concentration of the McFarland standard concerned and can thus be used for scale. The proposed method performs on par with the classical turbidimetric and nephelometric methods for this purpose, but has the advantage that it uses much more attainable and inexpensive hardware (a low-cost mass-produced digital camera for the home consumer market) and software (ImageJ, which is public domain software).

2. Material and methods

2.1. Material

All reagents used were analytically pure unless stated otherwise. Solutions were prepared from water purified past reverse osmosis, de-ionized to eighteen MΩ cm with a Sybron/Barnstead Nanopure II water purification organisation furnished with a fibre filter of 0.2 μm pore size. H₂And then₄ and anhydrous BaCl₂ were obtained from Panreac, both in analytical reagent grade.

Once prepared, the McFarland standards (three ml of each i) were transferred to the wells of an Iwaki 3820-024N polystyrene microplate (their holder for photographing; figure 2) with the assistance of an Accumax VA-900 micropipette.

All photographs were taken with a Nikon Coolpix E995 digital photographic camera and processed with the public domain software ImageJ (windows version) developed by the National Institutes for Health and available for costless download at http://rsbweb.nih.gov/ij.

Lighting was provided by 2 parallel fluorescent strips (Philips Master TL-D 36 West/840) located 1.5 m over the microplate (effigy 1). The diffusing screen was fabricated with white paper filter from ALBET (LabScience), 60 one thousand yard⁻² (in reams) 420 × 520 mm (code RM2504252). The microplate was placed on a piece of black cardboard (NE 30K A4, 180 thou) from Hermanos Cebrián, Spain. Absorbance measurements were made with a Spectronic Genesis 20 UV–vis spectrophotometer, and fluorescence measurements with a Perkin Elmer LS50 luminescence spectrometer equipped with the software FL Due westinLab. The McFarland value for each standard was obtained by a BD CrystalSpec dedicated nephelometer.

Figure 1. Schematic of the organisation for obtaining images. (i) Static support for microplate and photographic camera, (2) black cardboard, (3) microplate containing the McFarland standards, (iv) camera, (5) diffusing screen and (6) fluorescent strips.

2.2. Procedure

All experimental work was performed in five sessions (A–E), involving the operations described below. Solutions containing one per cent (w/v) BaCl₂ (equivalent to 0.04802 mol 50^–1) and one per cent (v/v) H_iiSo_four (equivalent to 0.18010 mol l^–1) were used to prepare a series of McFarland standards and their composition and equivalence in colony forming units (CFU) per millilitre of microbial suspension, which are shown in table 1. The standards were prepared in disposable, thread-cap test tubes.

Table 1. Composition and equivalences of the standards in the McFarland series.

Collapse
McFarland standard no.	one.0% anhydrous BaCl₂ (ml)	i% H₂SO₄ (ml)	estimate bacterial density (×10⁸) (CFU ml⁻¹)
0.five	0.05	nine.95	ane.5
i	0.1	ix.9	3.0
two	0.ii	ix.8	half-dozen.0
3	0.3	9.vii	9.0
four	0.four	9.half-dozen	12.0

Adjacent, the optimum photographic conditions were established (figure i). The supports for McFarland standards were polystyrene microplates on account of their advantageous geometry and transparency. This facilitated the simultaneous capturing of an image of all scale standards nether identical lighting weather. A volume of three ml of each standard was measured with the micropipette and transferred to a plate well. Once an aliquot of all standards was transferred to the plate, then an image was captured and processed with the software ImageJ.

The camera was operated as follows: because illuminating with the camera strobe light (wink) would accept caused reflections on solution surfaces, all lighting was provided by fluorescent strips. This makes the procedure like shooting fish in a barrel to implement at about any laboratory. The consequence of potential reflections of the strips on the solutions was avoided past placing a diffusing screen made with a sheet of white newspaper filter over and around the camera and plate. This setup provided soft lighting; in addition, using the largest aperture (i.due east. the smallest F-number) on the camera lens minimized exposure times, which were set every bit recommended by the camera in gild to avoid besides dark (underexposed) or too light (overexposed) images. The camera was placed 20 cm from the plate, on a static support to ensure reproducible framing and shooting under identical conditions: F/5 as lens discontinuity and 1/2 s as exposure fourth dimension. The plate was placed on a piece of black paper-thin to raise the milky turbidity of the McFarland standards.

Images were processed with the software ImageJ. Operation A > B > C means select command B on carte A and then select subcommand C in command B. Outset, the original colour image (in jpg format, 24 bit, 2048 × 1536 pixels) was split up into three according to its RGB (scarlet, light-green, blueish) values (Image > Colour > RGB split), each split prototype being in jpg format, viii-bit and 2048 × 1536 pixels. The prototype for the greenish aqueduct, which exhibited better linearity than those for the other two, was used to determine the 'grey level' for each standard; this was taken to be the average for a uniform circular area that was selected with the software's drawing tool (mean of 23 000 pixels) in each plate well (figure 2). The influence of the size of the mentioned circular area was studied by testing five unlike sizes: namely (in pixel) 230 000 (maximum allowed past the well size), 100 000, 50 000, 25 000 and 12 500. A linear scale curve was obtained for each tested size. Just the obtained with the maximum tested expanse showed a slope smaller (0.2728) than the others, among which there were no pregnant differences (hateful slope and s.d.: 0.3188 ± 0.0014; CV = 0.four%). This difference could be attributed to the reflections produced by the illumination nigh the internal border of each well, which tin can be easily observed in effigy ii. Finally, a circular area containing 23 000 pixels was selected as the optimum, because it avoids the inclusion of whatever reflection feature in the measured area.

Figure 2. Image of a microplate holding McFarland standards and the circular area used to measure the average grey level by ImageJ. (Channel, eight-bit, 2048 × 1536 pixels.)

Then, each McFarland standard was used to make absorbance (625 nm) and fluorescence measurements (λ _ex = 625, λ _em = 625 nm, both slits being prepare at their minimum value and signal attenuation at 1%) during each working session.

Finally, each solution was assigned a McFarland value by the BD CrystalSpec nephelometer.

iii. Results and discussion

By way of representative example, this section presents and discusses the results of working session C.

As stated in §two.2, the first step in the determinations involved capturing colour images of the McFarland standard series. The images were then processed with the software ImageJ for splitting into the three primary channels, and the light-green ane (G) was stored for subsequent processing as information technology resulted in more linear calibration curves than the reddish (R) and blue channel (B). Each standard was used to mensurate the average grey level in a circular surface area spanning 23 000 pixels (effigy 2). As can be seen from figure three, plotting the results exposed a logarithmic trend in them.

Figure 3. Calibration curve straight obtained with the proposed method.

In club to accurately compare the calibration curves with the turbidimetric (spectrophotometric) and nephelometric (spectrofluorimetric) results, which exhibited a linear tendency, the logarithmic calibration curve was easily converted into a straight line (figure 4) past a log–log plot. Negative ten-values were avoided by adding one unit (+1) to all values. So, the absorbance of each McFarland standard was measured and a calibration curve run from both these results, and those of the spectrofluorimetric and nephelometric measurements of the standards. These operations were all conducted in each working session.

Figure 4. — Effigy 4. Linear transformation of the calibration curve of figure three via a log–log plot.

Table 2 shows the slopes of the calibration curves obtained with the four instrumental techniques used in each session. Following application of Dixon'southward Q benchmark for rejection of outliers—none was in fact detected—averages and their respective standard deviations were calculated and rounded off to the required decimal place by the usual criteria, and the coefficients of variation between slopes of the calibration curves for each technique were obtained (table two).

Tabular array two. Results obtained with the four instrumental techniques compared.

Plummet
	slope of the calibration curve
working session	nephelometer	spectrophotometer	spectrofluorimeter	ImageJ
A	1.492	0.1263	168.ane	0.3200
B	1.539	0.1317	160.4	0.3304
C	1.378	0.1474	128.2	0.3289
D	1.525	0.1424	137.4	0.3107
Due east	ane.521	0.1384	140.iii	0.3450
south.d.	0.07	0.008	17	0.013
boilerplate slope	1.49	0.137	147	0.327
CV (%)	four.7	5.8	11.half-dozen	four.0

4. Conclusions

On the basis of the overall results, the proposed method provides results comparable with those of the conventional turbidimetric and nephelometric methods, with correlation coefficients between calibrations exceeding 0.99 in all cases. This imaging method possesses a high repeatability (CV = 4.0% with n = 5) exceeding even that of the nephelometric method used by the dedicated instrument for measuring McFarland standards (CV = 4.7%, n = v).

The proposed method is operationally simple and inexpensive: in fact, all it requires is a digital camera and a estimator equally measuring instruments, and both are more flexible, accessible and affordable than the conventional spectrophotometers, spectrofluorimeters or dedicated nephelometer typically used for this purpose. In add-on, images tin be readily candy with the public domain software ImageJ, adult by the National Institutes of Health and freely available on the Internet, which is very user-friendly.

In a scenario dominated by increasingly sophisticated and expensive commercial instruments, the proposed method provides an interesting alternative inasmuch as it enables quantitative determinations in instruction laboratories and modest facilities in developing countries, where economic resources for purchasing and maintaining measuring equipment are typically scant.

Acknowledgements

The authors thank the students Mª Isabel Cano Esteban and María Solana Altabella, for their assistance during the experimentation.

Footnotes

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Source: https://royalsocietypublishing.org/doi/10.1098/rsif.2011.0809

Mcfarland's Method of Cell Enumeration From Turbidometric Reading

1. Introduction

2. Material and methods

2.1. Material

2.2. Procedure

iii. Results and discussion

4. Conclusions

Acknowledgements

Footnotes

References

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