Comparison of visual observation and emission intensity of resazurin for antimicrobial properties of hexane, dichloromethane, methanol and water extracts from a brown alga, Turbinaria ornata

Figures & data

Table 1. Antimicrobial activity of T. ornata extracts against Gram-positive bacteria by disk diffusion method

Extract	(mg/mL)	Zone of Inhibition (mm)
		B. cereus	B. subtilis	S. aureus	S. aureus ATCC 25923	S. epidermidis	S. saprophyticus	E. faecalis
T. ornata Hexane	3	–	–	12.00 ± 0.00	–	–	9.70 ± 0.58	–
	5	8.00 ± 0.00	–	14.00 ± 1.00	–	–	11.00 ± 1.00	–
	10	8.30 ± 1.15	–	15.30 ± 0.58	–	–	14.00 ± 0.00	–
	15	11.60 ± 1.15	–	15.60 ± 1.15	7.30 ± 0.58	–	15.00 ± 0.00	–
	25	13.00 ± 1.41	–	16.00 ± 1.00	7.70 ± 0.58	–	16.30 ± 0.58	–
	50	15.60 ± 0.58	8.50 ± 0.71	17.70 ± 0.58	9.00 ± 1.34	8.30 ± 0.58	18.00 ± 0.00	8.00 ± 0.00
	100	16.00 ± 1.00	9.60 ± 2.08	19.30 ± 0.58	9.70 ± 0.58	9.30 ± 0.58	20.60 ± 0.58	9.70 ± 0.00
T. ornata Dichloromethane	3	7.67 ± 0.58	7.33 ± 0.58	–	–	14.33 ± 0.58	12.00 ± 0.00	6.33 ± 0.58
	5	10.00 ± 0.00	8.33 ± 0.58	–	–	17.00 ± 0.00	13.67 ± 0.00	6.67 ± 0.58
	10	11.33 ± 0.58	11.00 ± 0.00	13.00 ± 0.00	7.67 ± 0.58	19.00 ± 0.00	15.67 ± 0.00	8.00 ± 0.00
	15	14.00 ± 0.00	12.67 ± 1.53	16.67 ± 0.58	13.00 ± 0.00	21.00 ± 0.00	16.00 ± 0.00	8.00 ± 0.00
	25	15.33 ± 0.58	17.00 ± 0.00	17.67 ± 0.58	16.33 ± 0.58	24.00 ± 1.00	17.33 ± 0.00	8.00 ± 0.00
	50	16.00 ± 0.00	18.67 ± 1.53	19.00 ± 0.00	18.67 ± 0.58	26.00 ± 0.00	19.00 ± 0.00	9.00 ± 0.00
	100	16.33 ± 0.58	20.00 ± 1.73	20.67 ± 0.58	19.67 ± 0.58	28.00 ± 0.00	20.00 ± 0.00	9.67 ± 0.58
T. ornata Methanol	3	–	–	–	–	–	–	–
	5	–	–	–	–	–	–	–
	10	–	–	–	–	–	–	–
	15	–	–	–	–	–	–	–
	25	–	–	–	–	–	–	–
	50	8.00 ± 0.00	7.70 ± 0.58	–	7.00 ± 0.00	–	8.00 ± 0.00	–
	100	10.30 ± 0.58	9.00 ± 0.00	8.00 ± 0.00	10.00 ± 0.00	–	8.80 ± 0.29	–
Gentamicin	10 μg/disk	30.80 ± 0.41	25.00 ± 0.00	23.00 ± 0.00	27.00 ± 1.10	31.00 ± 0.89	29.30 ± 0.52	12.30 ± 0.52

Notes: “–” indicates no antimicrobial activity. Gentamicin was used as positive control. 99.5% DMSO was used as negative control where no inhibition was observed. Disk diffusion test was performed in at least duplicates and diameter of zone of inhibitions was expressed as mean ± standard deviation in mm. Zones of inhibition include 6 mm for disk diameter.

Table 2. Antimicrobial activity of Turbinaria ornata extracts against Gram-negative bacteria by disk diffusion method

Extract	(mg/mL)	Zone of Inhibition (mm)
		P. aeruginosa ATCC 27853	Enterobacter spp.
T. ornata dichloromethane	3	–	–
	5	–	–
	10	–	–
	15	–	–
	25	–	–
	50	7.00 ± 0.00	–
	100	7.00 ± 0.00	–
T. ornata methanol	3	–	–
	5	–	–
	10	–	–
	15	–	–
	25	–	–
	50	–	–
	100	–	8.33 ± 0.58
Gentamicin	10 ug/disk	18.20 ± 0.41	20.80 ± 0.41

Notes: “–” indicates no antimicrobial activity. Gentamicin was used as positive control. 99.5% DMSO was used as negative control where no inhibition was observed. Disk diffusion test was performed in at least duplicates and diameter of zone of inhibitions was expressed as mean ± standard deviation in mm. Zones of inhibition include 6 mm for disk diameter.

Table 3. Antimicrobial activity of T. ornata extracts against yeasts by disk diffusion method

Extract	(mg/mL)	Zone of Inhibition (mm)
		C. guilliermondii ATCC 6260	C. parapsilosis ATCC 22019	S. cerevisiae
T. ornata Hexane	3	–	–	–
	5	–	–	–
	10	–	–	–
	15	–	–	–
	25	–	–	9.00 ± 1.41
	50	–	–	9.30 ± 0.58
	100	–	–	10.00 ± 0.00
T. ornata Dichloromethane	3	–	–	–
	5	7.00 ± 0.00	8.00 ± 0.00	10.00 ± 0.00
	10	9.00 ± 0.00	9.00 ± 0.00	13.00 ± 0.00
	15	11.00 ± 0.00	9.55 ± 0.64	17.67 ± 0.58
	25	14.67 ± 1.15	10.00 ± 0.00	21.33 ± 1.15
	50	18.67 ± 2.52	10.67 ± 0.58	29.00 ± 1.41
	100	20.33 ± 2.08	11.33 ± 0.58	32.00 ± 0.00
T. ornata Methanol	3	–	–	–
	5	–	–	–
	10	–	–	–
	15	–	–	–
	25	–	–	–
	50	–	8.00 ± 0.00	–
	100	–	8.50 ± 0.71	–
Amphotericin B	10 ug/disk	32.50 ± 0.84	20.00 ± 0.63	29.70 ± 0.52

Note: “–” indicates no antimicrobial activity. Amphotericin B was used as positive control. 99.5% DMSO was used as negative control where no inhibition was observed. Disk diffusion test was performed in at least duplicates and diameter of zone of inhibitions was expressed as mean ± standard deviation in mm. Zones of inhibition include 6 mm for disk diameter.

Table 4. Antimicrobial activity of T. ornata extracts against bacteria and yeasts using REMA

Micro-organisms	Hexane (mg/mL)		Dichloromethane (mg/mL)		Methanol (mg/mL)		Gentamicin (μg/mL)	Amphotericin B (μg/mL)
	Colorimetric	Fluorometric	Colorimetric	Fluorometric	Colorimetric	Fluorometric	Colorimetric	Colorimetric
B. cereus	>32 ± 0	>32 ± 0	0.125 ± 0	0.125 ± 0	>32 ± 0	16 ± 0	0.5 ± 0	NA
B. subtilis	>32 ± 0	>32 ± 0	0.5 ± 0	0.5 ± 0	>32 ± 0	>32 ± 0	1 ± 0	NA
S. aureus ATCC 25923	>32 ± 0	>32 ± 0	2 ± 0	4 ± 0	>32 ± 0	>32 ± 0	0.67 ± 0.3	NA
S. aureus	>32 ± 0	>32 ± 0	4 ± 0	4 ± 0	>32 ± 0	32 ± 0	0.25 ± 0	NA
S. saprophyticus	>32 ± 0	>32 ± 0	1 ± 0	1 ± 0	>32 ± 0	>32 ± 0	0.06 ± 0	NA
S. epidermidis	>32 ± 0	>32 ± 0	2 ± 0	2 ± 0	NT	NT	0.25 ± 0	NA
E. faecalis	>32 ± 0	>32 ± 0	32 ± 0	32 ± 0	NT	NT	0.5 ± 0	NA
Enterobacter spp.	NT	NT	NT	NT	>32 ± 0	>32 ± 0	0.67 ± 0.3	NA
P. aeruginosa ATCC 27853	NT	NT	32 ± 0	8 ± 0	NT	NT	0.5 ± 0	NA
C. parapsilosis ATCC 22019	NT	NT	>32 ± 0	16 ± 0	>32 ± 0	>32 ± 0	NA	1 ± 0
C. guilliermondii ATCC 6260	NT	NT	>32 ± 0	16 ± 0	NT	NT	NA	0.25 ± 0
S. cerevisiae	>32 ± 0	>32 ± 0	0.5 ± 0	32 ± 0	NT	NT	NA	4 ± 0

Notes: NT indicates not tested. NA indicates not applicable. Gentamicin and Amphotericin B were used as positive controls for bacteria and yeasts/fungi respectively. Tween 80, PBS and DMSO (6.25%) were used as negative controls for hexane, dichloromethane and methanol extracts respectively. REMA was performed in at least duplicates and concentrations were expressed as mean ± standard deviation in mg/mL.

Figure 1. Percentage viability of microbial cells tested using different concentrations of Turbinaria ornata hexane extract in fluorometric REMA.

Figure 2. Percentage viability of microbial cells tested using different concentrations of Turbinaria ornata dichloromethane extract in fluorometric REMA.

Figure 3. Percentage viability of microbial cells tested using different concentrations of Turbinaria ornata methanol extract in fluorometric REMA.

Table

	fREMA_coded	Total
	0.125	0.5	1	2	4	32	>32
cREMA_coded	0.125	1	0	0	0	0	0	0	1
	0.5	0	1	0	0	0	1	0	2
	1	0	0	1	0	0	0	0	1
	2	0	0	0	1	1	0	0	2
	4	0	0	0	0	1	0	0	1
	32	0	0	0	0	0	1	1	2
	>32	0	0	0	0	0	1	13	14
Total	1	1	1	1	2	3	14	23

Table

	Value	Asymp. Std. Error^a	Approx. T^b	Approx. Sig.
Measure of Agreement	Kappa	.712	.123	6.533	.000
N of Valid Cases	23