modified in order to enhance its accuracy by Stepanovic et al.¹¹ Crystal violet is a basic dye that can bind to negatively charged surface molecules and polysaccharides in the extracellular matrix.¹² Crystal violet can stain both living and dead cells in addition to the biofilm matrix, and hence it is poorly suited to evaluate killing of biofilm cells.¹³

The traditional method for quantification of adherent micro-organisms is the measurement of their viability by plating on different agar plates after disruption by ultrasonication or vigorous agitation.¹⁴ This technique has the advantage of determining the number of active bacteria. However, it is both labor- and time-consuming. Moreover, certain species may be selected in case of multispecies biofilms.^15,5 Furthermore, many bacteria are not culturable despite being viable.¹⁶

MATERIALS AND METHODS:

Bacterial strains

Pseudomonas aeruginosa (23 isolates) isolated from intensive care unit patients in Zagazig university Hospitals by endotracheal aspiration.

Detection of biofilm production by the tube method

Biofilm formation capability was tested following the method of Stepanovic et al.¹¹ The test strains were inoculated, each into 2 ml tryptone soya broth supplemented with 1% glucose (TSB_glu) in Falcon tubes, and the turbidity was adjusted to be equivalent to a 0.5 McFarland standard. TSB_glu tubes were included as negative controls. After overnight incubation at 37ºC, the content of each tube was carefully aspirated with a pipette. Two mL of 0.25% safranin were added to each tube for 1 minute for staining the tubes, which were then decanted and inverted without washing. The tubes were examined for biofilm production following overnight standing at room temperature. The presence of a stained film on the inner wall of the tube was indicative of biofilm formation. The biofilm formation was estimated as negative (0), weak (+), moderate (++), or strong (+++).

Detection of biofilm production by the spectrophotometric method

Quantification of biofilm was performed according to Stepanovic et al.¹¹ Suspensions of the test strains were made, each in 5 ml of TSB_gluin sterile Falcon tubes and their optical density was adjusted to match a 0.5 McFarland standard. TSB_glu tubes were included as negative controls. Following incubation for 24 hours at 37 ºC; the tubes were aspirated, and washed thrice times with sterile physiological saline. Non-adherent bacteria were removed by vigorous shaking and adherent ones were by addition of 99% methanol for 15 minutes. The tubes were then decanted, air-dried, and stained with 2% Hucker crystal violet for 5 minutes. Excess stain was washed by running tap water and the tubes were left to dry. For elution of the dye bound to the adherent cells, 33% (v/v) glacial acetic acid was used, and the Optical density (OD) was measured at 570 nm using Spectrophotometer (UV-1800 Shimadzu, Japan). The test strain was considered non-biofilm forming (when OD ≤ ODc), weak biofilm forming (when OD > ODc, but ≤ 2x ODc), moderate biofilm forming (when OD>2x ODc, but ≤ 4x ODc), and strong biofilm forming (when OD> 4x ODc). The cut-off OD (ODc) was calcualted as equivalent to the mean OD of the negative control+3 times standard deviations. The test was made in triplicate and repeated three times, and the data were averaged.

Detection of biofilm production by the viable count method

For quantitative assay of biofilm formation, the viable count method described by Černohorská and ¹⁷ was used. The experiments were done in 96-wells polystyrene microtiter plates with round bottoms. For each test strain, an inoculum with optical density equivalent to a 0.5 McFarland standards was prepared in TSB_glu. The inoculated medium was distributed in 75 μL aliquots in the wells of microtiter plates, and the plates were incubated for 24 hours at 37 °C. Microtiter wells were washed three times with phosphate-buffered saline (PBS) under aseptic conditions to remove unattached bacteria and dried in an inverted position. After addition of 100 μL PBS to each well, the biofilms were disrupted by sonication for 5 minutes using ultrasonic bath (Julabo Labortechnik GmBH D-77960 Seelbach, Germany). After serialdilution of the bacterial suspensions in sonicated samples in PBS, the viable counts were determined. The viable counts were expressed as the log₁₀ of colony forming units (CFU/well). The experiment was repeated three times and the results were averaged.

Statistical Analysis

One way ANOVA was used to compare the results of the viable count method. One way ANOVA was performed using GraphPad Prism version 5.04 for Windows, GraphPad Software, La Jolla, CA, USA. P values < 0.05 were considered significant.

RESULTS:

Assessment of biofilm formation

All isolates were found to be biofilm-forming (Table 1). The tube and the spectrophotometric results were strongly correlated and were different from those of the viable count method. The percentage of isolates showing different degrees of biofilm formation by tube and the spectrophotometric method are shown in Figure 1, while those of the viable count method are shown in Figure 2. According to the results of the viable count method, the isolates were grouped into three groups. Group A, which showed high viable counts, included thirteen (56.5%) biofilm forming isolates. Group B, which showed lower counts, included seven (30.4%) biofilm forming isolates. Group C that showed the least viable counts, included three (13%) biofilm forming isolates. Using one way ANOVA test, significant differences in log₁₀viable counts (P value < 0.05) were observed between groups A and B, groups A and C, and groups B and C.

Table 1. Comparison between tube, spectrophotometric and viable count methods for assessment of biofilm formation.

Isolate’s No.

Biofilm forming capacity obtained by

Tube method*

Spectrophotometric method

(Mean OD at 570 nm)

Viable count method

(Log₁₀mean CFU/well)

PA1

PA2

PA3

PA4

PA5

PA6

PA7

PA8

PA9

PA10

PA11

PA12

PA13

PA14

PA15

PA16

PA17

PA18

PA19

PA20

PA21

PA22

PA23

+++

8.73

7.50

7.59

8.67

8.68

7.92

8.88

6.95

5.97

7.86

6.33

8.58

6.36

6.00

8.69

7.02

7.23

6.95

4.78

6.30

5.16

7.40

6.89

+, weak; ++, moderate; +++, strong*

Figure 1. Assessment of biofilm formation by the tube and spectrophotometric methods.

Figure 2. Assessment of biofilm formation by the viable count method.

DISCUSSION:

Biofilm formation ability can be examined in different ways such as the tube method, the spectrophotometric method, the viable counting method, and the scanning electron microscopy.^{10, 18-20}

In this study, biofilm formation was assessed qualitatively by the tube method, and quantitatively by the spectrophotometric method and the viable count method. All clinical isolates of P. aeruginosa were found to be biofilm forming. The results of the tube and spectrophotometric methods were strongly correlated. For both tests, sixteen isolates (69.6%) demonstrated strong biofilm forming ability; two isolates (8.7%) were moderate, while five isolates (21.7%) were weak. Discrepancies in results of the two methods were noted only with two isolates (8.7%). The tube test is simple and easy to perform, however, as a qualitative method based on visual inspection, it lacks objectivity. Oliveira and Cunha²¹ stated that the tube method was reliable for determination of biofilm production in routine use. However, some investigators reported that the tube method could detect strong biofilm formation easily, while it was inadequate to determine weak biofilm formation.²² On the other hand, the current data indicate that the spectrophotometric method is a reliable and practical method for quantitative determination of biofilm formation, and it is better than the tube test related to accuracy and objectivity.

On the other hand, our data show that the viable count method was weakly associated with either of the tube and spectrophotometric methods. High viable counts were recorded for biofilms formed by thirteen isolates (56.5%), all of which but one demonstrated strong biofilm forming ability by both the tube and the spectrophotometric methods. This isolate showed moderate biofilm forming capacity by the spectrophotometric method. Lower viable counts were found for biofilms formed by seven isolates (30.4%) including three strong biofilm forming isolates, two moderate biofilm forming isolates, and two weak biofilm forming isolates as determined by the tube method, and four strong biofilm forming isolates, one moderate biofilm forming isolate, and two weak biofilm forming isolates by the spectrophotometric method. The least viable counts were obtained for biofilms formed by three isolates (13%), all of which were weak biofilm forming by the tube and the spectrophotometric methods. The difference observed between the viable count method and crystal violet assay-based methods may be due to the staining of the matrix material, dead cells and viable cells by crystal violet for both of the tube and spectrophotometric method, while viable cells are the only ones detected by the viable count assay.^13,14 The tube and spectrophotometric methods give no information about biofilm viability as in the viable count method. However, the viable counting method may have the disadvantages of the difficulty of the dissociation of the biofilm clumps into single-cell suspensions for plate counting and the destruction of some cells.^23,24

In accordance with our results, Nagaveni et al.²⁵ and Černohorská and Votava²⁶ reported good correlation between the results of the tube test and the spectrophotometric microtiter plate test. Using 11 P. aeruginosa isolates, Nagaveni et al.²⁵ studied the biofilm formation by the tube and the spectrophotometric microtiter plate methods. All isolates were found to be biofilm forming. For both tests, 5 (45.4%) isolates showed high biofilm forming capacity, 3 isolates (27.3%) showed moderate biofilm forming capacity, while weak biofilm forming capacity were shown by 3 (27.3%) isolates. Černohorská and Votava²⁶ reported that 20 out of 39 (51.3%) P. aeruginosa isolates obtained from patients hospitalised at the Intensive Care Unit of St. Anna’s Faculty Hospital in Brno, Czechia, were biofilm positive as demonstrated by the tube and spectrophotometric microtiter plate methods. In another study, Černohorská and Votava¹⁷ found that among 31 P. aeruginosa isolates, 17 (55%) isolates were biofilm positive by using the tube test. Using the spectrophotometric microtiter plate method, Zhao and Liu²⁷ showed that among twenty P. aeruginosa strains isolated from respiratory samples, 15% of isolates were weak biofilm producers; 50% were moderate biofilm producers, while 35% were strong biofilm producers.

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Received on 25.09.2012

Modified on 02.10.2012

Accepted on 20.10.2012

Research J. Science and Tech. 4(5): September –October, 2012: 181-184