## 1 Introduction  
Tetrazolium salts are used to evaluate the antibacterial effect of phytochemical extracts [1]. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is the most common tetrazolium salt used in bioassays [2]. It can be used in colorimetric assays to assess the viability and proliferation of cells [3, 4] or microorganisms [5, 6]. MTT is converted into a purple water-insoluble formazan crystal (Figure 1) by the mitochondrial dehydrogen-genases of living bacterial cells. Therefore, the in vitro cell viability can be tested using the MTT colorimetric assay [7]. The MTT colorimetric assay has been used to show the antibacterial properties of several phytochemicals [8, 9].  
Figure 1: The chemical structure of formazan.  
Phenolic compounds (anthocyanins, phenolic acids, stilbenes, flavanols, and tannins) make up the majority of phytochemicals in plant crude extracts [10; 11]. Phenolic compounds have shown strong UV light absorption due to the \(\pi\)-conjugated systems with hydroxyl-phenolic groups [12]. Most phenolic compounds have intense absorption bands in the 280 nm and 320 nm regions. Some phenolic compounds have shown a wide-UV-light absorption range from 250 to 600 nm [13; 14]. Flavonoids and anthocyanins are two phenolic compounds that have a wider range of UV absorption (250-600 nm) due to the complex molecular structures and the multiple conjugated double bonds [15; 16; 17]. Crude plant extracts not only contain phenolic compounds, but also have several other chemical components (multi-vitamins, proteins, alkaloids, and others) [18; 19]. These components can potentially interfere with the absorbance measurement. For example, proteins can absorb light at various wavelengths due to the amino acid residues with aromatic rings [20]. In addition, the colour intensity, tonality, and molecular structures of compounds can all influence the UV light absorption ranges of crude extracts [13]. Crude extracts with darker colours or intense tonality may absorb more strongly at specific wavelengths, making it more challenging to accurately measure absorption. Therefore, in MTT colorimetric assays, the tested crude extracts may have overlapping UV light absorption ranges with formazan. Furthermore, floating bacterial cells also have significant absorption in the wavelength range used to assess formazan concentrations after treating with MTT (usually 30 min) [21]. The cells can scatter or absorb light, leading to inaccurate readings [22]. These interferences can reduce the accuracy of the final colorimetric assessment used to determine the phytochemical antibacterial effect. To the best of our knowledge, few reports have tried to remove the interference of plant crude extracts and bacterial suspensions within MTT colorimetric assays.  
Avocado seed extract expresses good antibacterial activity against many species of bacteria (_E. coli, P. aeruginosa, E. faecalis, P. vulgaris,_ and S. aureus_) [23; 24]. The current study used New Zealand (NZ) 'Hass' avocado crude ripe seed extracts (RSE) as a crude plant extract example to explore a developed MTT colorimetric assay. The aim of this study was to find a method to remove the interference of background colours (broth and plant extracts), and bacterial cells before formazan microplate testing while ensuring accurate and reproducible results. With this modified method, the aim was then to increase the accuracy of formazan measurement in phytochemical antibacterial results.