The MTT antibacterial assay is an important method in natural antibacterial component discovery. Researchers can use the MTT antibacterial assay to quickly and efficiently evaluate the antibacterial activity of natural compounds. The aim of this research was to find a method to reduce background colour and bacterial cell interference when using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to study the antibacterial effect of phytochemicals. This study used NZ ‘Hass’ ripe avocado seed extract as an example to develop a silica gel column chromatography method that could combine with the MTT assay to remove the background colour and bacterial cells before the formazan measuring stage. This method is particularly suitable for MTT-based antibacterial inhibition studies when the tested phytochemical extracts have an interfering colour.

Avocado production is a growing industry in New Zealand. Processing of avocado generates considerable waste that may have biological value

‘Hass’ avocado (Persea americana Mill) by-products (peel, and seed) are rich sources of potential antimicrobial agents. The current study used thiazolyl blue tetrazolium bromide (MTT) as a colour indicator to evaluate the antibacterial capacities of ‘Hass’ avocado by-product extracts from China, Chile, and New Zealand. MTT reagent can react with mitochondrial dehydrogenases in living bacterial cells to produce formazan. The number of colony-forming units (CFU) of Staphylococcus aureus were linearly correlated with formazan content. The current study used a formazan–CFU standard curve to evaluate the antibacterial properties of ‘Hass’ avocado by-product extracts by monitoring the CFU of S. aureus after 12 h treatment with the by-product extracts. The results showed that there was a significant (p < 0.05) difference in S. aureus survival if the ‘Hass’ avocado by-product extracts were recovered from mature unripe fruit or mature ripe fruit derived from the three countries. Chilean ripe seed extracts resulted in the lowest bacterial survival percentage (2% ± 0.98) after 12 h of treatment. Chilean ripe seed extracts had the highest S. aureus inhibition activity among all the extracts. In addition, there was a linear correlation between the total polyphenol content (TPC) and S. aureus (r = 0.546, p < 0.0010) bacterial survival percentage. Therefore, the current results lead us to consider that the main antioxidant polyphenol compounds of avocado by-product extracts are also the main compounds required to provide antibacterial activity inhibiting bacteria growth.