Treatment of Candida Albicans: Assessing the Efficacy of Two Popular Home Remedies
Jackie Mitchell, Kim Lehmann, and Karina Gadea
Department of Biological Science
Saddleback College
Mission Viejo, CA 92692
Candida albicans, an opportunistic and polymorphic fungus, can infect superficial mucosal
membranes in the mouth and vagina, causing oral thrush and yeast infections. Common
home remedies for vaginal yeast infections include apple cider vinegar and the
Lactobacillus acidophilus bacteria found in unpasteurized yogurt. These home remedies
were predicted to inhibit C. albicans growth either by inducing apoptosis or by adhering to
the vaginal epithelium and competing with C. albicans for receptor-binding sites. Potato
dextrose agar was prepared at a pH of 4 and a pH of 7 and inoculated with a streak of C.
albicans. Candida thrives at a neutral pH, and its growth is inhibited as the pH of its
environment becomes more acidic. Sterilized chads with deionized water, apple cider
vinegar, or Lactobacillus acidophilus were placed on the plates. After incubation, clear
zones of inhibition around each sterile chad were measured. Plates set to a pH of 7 and
treated with apple cider vinegar were able to significantly inhibit the growth of C. albicans
(ANOVA, Bonferroni Correction, p<0.00833).
Introduction
Candida albicans is a naturally occurring fungus in the human mouth and vagina and is the most common
human fungal pathogen (Dai et al. 2012). C. albicans is an “opportunistic and polymorphic fungal pathogen” and its
yeast-hyphal transition is one of its key virulence factors, allowing it to invade our tissues, evade macrophages, and
form biofilms (Vediyappan et al., 2013). Overgrowth of C. albicans can cause superficial mucosal infections such as
oral thrush in the mouth and throat and yeast infections in the vagina. Instead of using anti-fungal medications,
which can produce unwanted side effects, there are many common home remedies used to treat and prevent yeast
infections. These include apple cider vinegar and probiotics like Lactobacillus acidophilus, which is commonly
found in yogurt (Jeavons, 2002; Pinto et al., 2008). Vaginal pH ranges from 3.8-4.4 under regular conditions and is
regulated in part by Lactobacilli bacteria. Lactobacilli help to maintain acidity in the vagina by producing lactic
acid.; the acidic environment helps to prevent overgrowth of C. albicans. When vaginal conditions change even
slightly, overgrowth can occur. Both vinegar and Lactobacillus work by lowering the vaginal pH down to its ideal
level to inhibit C. albicans growth. It has been found that after exposure to Lactobacilli bacteria, C. albicans cells
died off due to a lack of metabolic activity (Kohler et al., 2012). In another recent study regarding apoptotic stimuli,
C. albicans cells induced programmed cell death after being exposed to 60mM acetic acid for 3 hours (Dai et al.
2012). Our study aims to support that Lactobacillus acidophilus and apple cider vinegar are two different affordable
and highly effective treatments to reduce C. albicans growth.
Methods and Materials
Researchers used Difco Potato Dextrose agar formula obtained in the Saddleback College Biology Lab to fill
40 sterile Petri Dishes. The Difco Potato Dextrose consisted of 4.0 g potato starch (from infusions), 20.0 g dextrose,
and 15.0 g of agar.
During the process of making the agar, once it became transparent, the pH was adjusted by dropwise addition
of sodium hydroxide and hydrochloric acid. To ensure a proper pH reading, a Thermo Scientific Orion 815600 pH
probe was accurately stabilized and then used to periodically measure the pH of the agar. Three of the flasks were
adjusted to a pH of 7.0, while the fourth flask was adjusted to a pH of 4.0. The NaOH and HCl were added dropwise until goal pH was attained.
The agar was autoclaved and then allowed to cool to room temperature. After the agar was sufficiently cooled,
but not cool enough to solidify, thirty Petri dishes were filled with agar at a pH 0f 7, while the ten remaining dishes
were filled with agar at a pH of 4. Plates were prepared aseptically in order to prevent growth of unwanted
microorganisms on the C. albicans Petri dishes. After pouring, plates were closed and allowed to cool for a period of
ten minutes before being stored upside down to prevent condensation. The plates were left in refrigerated storage for
five days before being inoculated with C. albicans.
The potato dextrose plates were inoculated to produce a lawn of C. albicans. Sterilized chads were
immediately placed on the inoculated plates and treated with apple cider vinegar, Lactobacillus acidophilus, or
deionized water. All of the plates were inoculated with C. albicans. Of the thirty plates set to a pH of 7, ten were
also inoculated with apple cider vinegar to inhibit growth, ten additional plates were inoculated with 10mL of
Lactobacillus on sterilized chads to inhibit growth, and the final ten plates were treated with deionized water, to
serve as a positive control. The ten plates set to a pH of 4 were then divided into three parts, with each section
receiving either apple cider vinegar, Lactobacillus, or deionized water. Each plate at a pH of 4 was inoculated with
one sterile chad of each treatment. The plates were then left to incubate for 24 hours at 37ºC in a normal aerobic
atmosphere.
After the growth period, the plates were inspected for clear zones surrounding the sterile chads with apoptotic
stimuli. The clear zone of inhibition around the sterilized chad was measured; because zones of inhibition were
small, zones were measured in reticle units using a microscope and later converted to millimeters. Once all
measurements were made, all data were inputted into MS Excel (Microsoft Corporation, Redmond, Washington) so
that data analysis could be completed. After the data were compiled, an Analysis of Variance (ANOVA) was
completed, followed by a Bonferroni correction to analyze the inhibition of C. albicans growth depending on which,
if any, apoptotic stimuli was used.
Results
Each zone of inhibition was measured in reticle units, converted to millimeters, and recorded to run a data
analysis. Neither of the positive control tests showed any inhibition of C. albicans growth. Plates set to a pH of 7
and treated with deionized water were negative for inhibition (0.00, n=30) and the plates set to a pH of 4 were
negative for inhibition as well (0.00, n=10). On average, plates at a pH of 7 that were treated with apple cider
vinegar showed a zone of inhibition 0.0525 ± 0.0203 mm (±SEM, n=30), while plates set to a pH of 4 showed
average inhibition zones 0.0175 ± 0.00917 mm (±SEM, n=10). Plates treated with Lactobacillus bacteria showed
less inhibition of C. albicans at a pH of 7 and slightly more inhibition at a pH of 4. Plates set to pH 7 and treated
with Lactobacillus showed average zones of inhibition 0.0183 ± 0.00722 mm (±SEM, n=30), and plates at pH 4 that
were treated with Lactobacillus showed zones of inhibition 0.0200 ± 0.0117 mm (±SEM, n=10). An ANOVA,
followed by a Bonferroni Correction post test, revealed that plates set to a pH of 7 and treated with apple cider
vinegar were significantly different from the plates set to a pH of 7 that were treated with only deionized water.
(ANOVA, Bonferroni Correction, p<0.00833). Plates set to a pH of 4 and treated with only deionized water, another
positive control, were not shown to be significantly different than the plates set to a pH of 7 and treated with apple
cider vinegar due to the pH 4 control test’s small sample size. Although both deionized water control tests showed
no inhibition of C. albicans growth, the pH 4 plates had a smaller sample size, thereby increasing the likelihood that
chance affected the results.
Mean Clear Zone of Inhibition (mm)
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
pH 4pH 7
DI Water
pH 4pH 7
pH 4pH 7
Apple Cider Lactobacillus
Vinegar
Proapoptotic Stimuli Placed on Disk
Figure 1: Mean Clear Zone of Inhibition of Candida Albicans growth based on which type of stimuli was used.
When treated with deionized water, no zones of inhibition were observed for plates set to a pH of 4 (0.00 mm ± 0.00
SEM, n=10) or a pH of 7 (0.00 mm ± 0.00 SEM, n=30). When treated with apple cider vinegar, plates at a pH of 4
showed average zones of inhibition 0.0175 mm (± 0.00917 SEM, n=10) while plates set to a pH of 7 showed zones
an average of 0.0525 mm (± 0.0203 SEM, n=30). Plates treated with Lactobacillus bacteria showed average zones
of inhibition 0.0200 mm (± 0.00117 SEM, n=10) on pH 4 plates and zones 0.0183 mm (± 0.00722 SEM, n=30) on
pH 7 plates. An ANOVA followed by a Bonferroni correction showed that the only significant difference was
between pH 7 plates treated with apple cider vinegar and the plates treated with deionized water.
Discussion
In this study, only one sample group was able to inhibit a significant amount of C. albicans growth. Plates
set to a pH of 7 and treated with apple cider vinegar showed the highest amounts of inhibition as compared to the
ability of Lactobacilli and deionized water to inhibit growth of C. albicans. When treated with apple cider vinegar,
C. albicans cells are exposed to acetic acid, prompting a weak acid stress response. This weak acid stress response
activates the Ras-cAMP pathway, sending signals that accelerate programmed cell death in C. albicans (Ramsdale et
al., 2008). It was discovered that MNL1, a gene found in C. albicans that was previously believed to have no known
function, regulates weak acid stress response. Over time, MNL1 helps C. albicans adapt to weak acid stress
responses, but the short-term exposure to apple cider vinegar in our study was able to inhibit C. albicans growth.
Other studies have shown that Lactobacilli can also inhibit growth, but our results did not support this
(Jeavons, 2003). Under normal conditions, Lactobacilli are naturally present in the human vagina and help to keep C.
albicans in check. Three main mechanisms have been proposed as to how C. albicans growth is regulated by
Lactobacilli (Jeavons, 2003). First, Lactobacilli produce lactic acid as a by-product of its glycogen metabolism,
helping to maintain the vagina’s ideal pH level of 3.8-4.5 and inhibiting the growth of many pathogenic microbes,
including C. albicans. Secondly, Lactobacillus acidophilus produces hydrogen peroxide, further inhibiting microbial
growth. Third, and possibly most importantly, Lactobacilli can strongly adhere to the vaginal epithelial cells and
compete with pathogens for receptor-binding sites.
In this study, inhibition of growth was expected for Lactobacilli as well as apple cider vinegar, but
Lactobacilli was not able to significantly inhibit growth of C. albicans. There are a number of possible explanations
as to why Lactobacilli did not sufficiently inhibit growth. In this study, C. albicans plates were only treated once
with each stimuli, whereas individuals treating Candida overgrowth would most likely repeat treatments multiple
times over the course of a few days. Therefore, we cannot conclude that Lactobacilli would not be able to inhibit
growth of C. albicans, but this study supports that apple cider vinegar is more able to inhibit C. albicans growth.
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