Lactophenol Cotton Blue (LPCB) adalah pewarna standar yang digunakan di
laboratorium mikologi untuk mengidentifikasi jamur patogen seperti Candida
albicans dan Aspergillus fumigatus Namun, LPCB mengandung bahan kimia yang
berbahaya bagi lingkungan dan cenderung mahal. Bunga telang (Clitoria ternatea
L.) mengandung pigmen antosianin dalam kadar tinggi, menjadikannya alternatif
pewarna alami yang potensial; akan tetapi, warnanya sering kali tidak stabil akibat
faktor lingkungan seperti pH dan paparan cahaya. Penelitian ini bertujuan untuk
mengetahui pengaruh penambahan asam askorbat (vitamin C) terhadap stabilitas
warna ekstrak bunga telang serta mengevaluasi efektivitas kualitas pewarnaannya
sebagai alternatif LPCB untuk analisis mikroskopis Candida albicans dan
Aspergillus fumigatus. Penelitian eksperimental ini menggunakan berbagai
konsentrasi asam askorbat 0,5%, 0,7%, dan 1,0

Lactophenol Cotton Blue (LPCB) is a standard stain used in mycology laboratories
to identify pathogenic fungi such as Candida albicans and Aspergillus fumigatus,
however, LPCB contains environmentally hazardous chemicals and is relatively
expensive. Butterfly pea flowers (Clitoria ternatea L.) contain high levels of
anthocyanin pigments, making them a potential natural alternative stain; yet, their
color is often unstable due to environmental factors such as pH and light exposure.
This study aimed to determine the effect of adding ascorbic acid (vitamin C) on the
color stability of butterfly pea flower extract and to evaluate its staining quality as
an LPCB alternative for the microscopic analysis of Candida albicans and
Aspergillus fumigatus. This experimental study utilized ascorbic acid
concentrations of 0.5%, 0.7%, and 1.0% within the butterfly pea flower extract.
Physical and chemical stability were assessed by measuring absorbance (via UV￾Vis spectrophotometry at wavelengths of 500–640 nm) and pH on days 1, 3, 5, and
8 of storage. Staining quality was quantitatively evaluated based on color intensity
(Mean Gray Value) using ImageJ software and statistically analyzed via One-Way
ANOVA using SPSS. The addition of ascorbic acid (vitamin C) proved effective in
maintaining the extract\'s acidic condition, thereby preserving the stability of the
flavylium cation structure (blue color). A copigmentation mechanism efficiently
increased absorbance values up to the third day, with the 0.7% concentration
yielding the most intense blue color. ImageJ analysis revealed that the butterfly pea
flower extract with 0.7% ascorbic acid provided the best staining for Aspergillus
fumigatus hyphae and spores on the first day, achieving an intensity value of 66
nearly equivalent to the LPCB control (value of 67). However, One-Way ANOVA
statistical analysis indicated that the addition of ascorbic acid (vitamin C) did not
have a significant effect on Aspergillus fumigatus (p = 0.881 > 0.05) due to high
variability in the morphological density of the fungal filaments. Conversely, a
significant effect was observed in Candida albicans preparations (p = 0.025 < 0.05)
due to very low data variance, with the 0.5% concentration emerging as the most
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stable formula. Butterfly pea flower extract supplemented with ascorbic acid
demonstrated good color stability attributed to a co-pigmentation mechanism and
successfully maintained an acidic pH during storage. This formula proved visually
highly effective at staining Aspergillus fumigatus structures, thereby showing great
potential as a safe and eco-friendly microscopic fungal stain for use in mycology
laboratories.
Keywords: Ascorbic acid, Butterfly pea flower, Candida albicans, Aspergillus
fumigatus, Anthocyanins, ImageJ