Treatment of Candida Albicans Italicize : Assessing the Efficacy of Two Popular Home Remedies

Treatment of Candida albicans[italicize]: 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 theLactobacillus acidophilus bacteria found in unpasteurized yogurt. These home remedies were predictedhypothesizedto inhibit C. albicans growth byeither byinducing apoptosis(On what? Be more specific.) 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 isbecomes moreinhibited(you are measuring inhibition on a gradient and increasing acidity is already inhibiting Candida growth.) as the pH of its environment becomes more acidic. Sterilized chadswithcontaining deionized (DI—can abbreviate) water, apple cider vinegar, or Lactobacillus acidophilus were placed(?) on the agar 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)(And the other plates?).

* What was your hypothesis? The goal of your experiment seems unclear.

Introduction

Candida albicans is a naturally occurring fungus in the human(only in humans?)mouth and vagina and is the most common human fungal pathogen (Dai et al.[italicize]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 (oral does not include the throat)and yeast infections in the vagina(infections in the mouth and throat are also yeast infections)[Overgrowth of C. albicans can cause superficial mucosal infections in the mouth, throat, and vagina]. (Needs transition)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 likesuch asLactobacillus acidophilus,which is commonly found in yogurt (Jeavons, 2002; Pinto et al., 2008). (Start new paragraph)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.(This is repetitive) Both vinegar and Lactobacillus work by lowering the vaginal pH down to itsanideal level to inhibit C. albicans growth. It has been foundPrevious experiments demonstratedthat after exposure to Lactobacilli bacteria, C. albicans cells died off due to a lack of metabolic activity (Kohler et al., 2012).What may have caused this lack of metabolic activity? In another recent study regarding apoptotic stimuli, C. albicans cells induced programmed cell death(C. albicans induced apoptosis on themselves? Apoptosis is usually induced by an outside factor. What mechanisms were involved?)after being exposed to 60mM acetic acid for 3 hours (Dai et al. 2012). Our study aims(use third person!) to support that Lactobacillus acidophilus and apple cider vinegar are two differentaffordable and highly effective treatments to reduce C. albicans growth.(Is this your hypothesis? If so, it is vague and needs to be written in proper hypothesis format. Which one do you predict to have a higher inhibition potential?)

Methods and Materials

Researchers used DifcoTM Potato Dextrose agar formula obtained in the Saddleback College Biology Lab to fill 40 sterile Petri Dishes(does not need to be capitalized) (passive voice).[Forty sterile petri dishes were filled with DifcoTM Potato Dextrose agar.] The DifcoTM Potato Dextrose consisted of 4.0g potato starch (from infusions), 20.0 g dextrose, and 15.0g of agar. (Combine these two sentences)

During the process of making the agar, once it(what?)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(What flasks? How many total?) 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 drop-wise until goal pH was attained. (Try to be more concise. This is a bit confusing. The process of making the agar does not need to be explained in too much detail.)

The agar was autoclaved,(why autoclave?)and thenallowed to coolcooled 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(why the difference? Which is your control and which is the experimental? Elaborate). Plates were prepared aseptically in order to prevent growth of unwanted microorganisms on the C. albicans Petri dishes(how?). 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.Why did they have to be refrigerated for so long?This information is all extraneous.

The potato dextrose plates were inoculated to produce a lawn of C. albicans[C. albicans was spread onto the agar plates to form a lawn.] (The main focus should be on the C. albicans, not on the agar plates).Sterilized chads (how many on each?)were immediately placed onontothe inoculated plates and treated with apple cider vinegar, Lactobacillus acidophilus, or deionized water(how were they treated?). (Elaborate on these groups. Which are the controls and which are the experimentals?)All of the plates were inoculated with C. albicans(Repetitive). 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(youmean negative? DI water should give a negative result)control(This should have been explained in the beginning). The ten plates set to a pH of 4 were then divided into three partssections, 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 incubateincubated for 24 hours at 37ºC in a normal aerobic atmosphereconditions.

After the growth period, the plates were inspected for clear zones surrounding the sterile chads with apoptotic stimuli(How so?). 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…to be analyzed using ANOVA. Once all measurements were made, all data were was 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.

** You do not need three paragraphs explaining how you prepared your agar plates if your main focus was on the Candida.

**Elaborate more on your experimental procedure. How would your experiment lead to the results you were expecting?

**The information was all over the place and difficult to read in places. Information needs to be arranged in a more logical order.

Results

Each zone of inhibition was measured in reticle units, converted to millimeters, and recorded to run a data analysis.(Already explained in the procedure)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)[Consider revising to: Neither…nor… showed any inhibition].On average, plates(The plates are not what’s being treated, the Candida is)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). Results ofplates set to a pH of 4 and treated with only deionized water, another positive control, were not shown to be significantly different (higher or lower?) 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(why were the sample sizes unequal?), thereby increasing the likelihood that chance affected the results[that the results were affected by chance (passive voice)].

**Refer to your figure!! Cite in passage.

Figure 1: Mean Clear Zone of Inhibition of Candida albicans growth based on the which type of stimuluswas 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.This should be shortened, since most of it was already stated above.

Discussion

In this study, only one sample group was able to inhibit a significant amount of C. albicans growth. Plates(please refrain from saying “plates” too often, they are not the main focus of your experiment, the Candida is the main focus)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 (Ramsdaleet al.(italicize), 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(how?).

Other studies have shown that Lactobacilli can also inhibit growth(vague), but our results(third person!) did not support this (Jeavons, 2003)(Why not?).Under normal conditions, Lactobacillusareis 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, Lactobacillusproduces 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.Finally Third, and possibly most importantly(this is argumentative),Lactobacilluscan strongly adhere to the vaginal epithelial cells and compete with pathogens for receptor-binding sites. (Why are these mechanisms significant? Expand on this)

In this study, inhibition of growth was expected for Lactobacilli as well as apple cider vinegar, but Lactobacillus was not able to significantly inhibit growth of C. albicans. There are a number of possible explanations as to why Lactobacillus did not sufficiently inhibit growth(repetitive). In this study, C. albicans plateswas were only treated once with each stimulus, whereas individuals treating Candida overgrowth would most likely repeat treatments multiple times over the course of a few days(How may this have affected your outcome?). Why else?Therefore, we(again, use third person) cannot conclude [It cannot be concluded]that Lactobacilluswould not be able to inhibit growth of C. albicans, but this study(We already know you are referring to “this study.” Also, use passive voice) supports that apple cider vinegar is more able to inhibit C. albicans growth.—What is the significance?

**Discuss your results more. Explain what your results mean rather than what other research says about your experiment.

** What can future research indicate?

** If apple cider vinegar really is a more effective inhibitor of Candida albicans, what can we say about its future use?

Literature Cited

Dai, B., Wang, Y., Li D., Xu, Y., Liang, R., Zhao, L., Cao, Y., Jia, J., Jiang, Y. 2012. Hsp90 Is Involved in Apoptosis of Candida albicans by Regulating the Calcineurin-Caspase Apoptotic Pathway(underline or quotations). PLoS ONE 7(9): e45109. doi:10.1371/journal.pone.0045109

Jeavons, H. S. 2003. Prevention and Treatment of Vulvovaginal Candidiasis Using Exogenous Lactobacillus. Journal of Obstetric, Gynecologic and Neonatal Nursing 32(3): 287-296.

Kohler G.A., Assefa S., Reid G. 2012. Probiotic Interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the Opportunistic Fungal Pathogen Candida albicans. Infectious Diseases in Obstetrics and Gynecology 2012. doi:10.1155/2012/636474

Pinto T., Neves A., Leao M., Jorge A., 2008, Vinegar as an Antimicrobial Agent for Control of Candida spp. In Complete Denture Wearers. Journal of Applied Oral Science 16(6): 365-90.

Ramsdale, M., Selway, L., Stead, D., Walker, J., Yin, Z., Nicholls, S., Crowe, J., Sheils, E., and Brown, A. 2008. MNL1 Regulates Weak Acid-induced Stress Responses of the Fungal Pathogen Candida Albicans.Molecular Biology of the Cell 19: 4393-4403.

Vediyappan, G., Dumontet, V., Pelissier, F., and d’Enfert, C. 2013. Gymnemic Acids Inhibit Hyphal Growth and Virulence in Candida Albicans.PLoS ONE 8(9): 1-11.

**At least 10 sources needed

Review Form

Department of Biological Sciences

Saddleback College, Mission Viejo, CA 92692

Author (s):______Jackie Mitchell, Kim Lehmann, and Karina Gadea______

Title: Treatment of Candida albicans: Assessing the Efficacy of Two Popular Home Remedies

Summary

Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.

The purpose of this paper was to determine the efficacy and effectiveness in using apple cider vinegar and Lactobacillus in yogurt to inhibit Candida albicans. Candida albicans is an opportunistic yeast infection that can result when the pH of the mouth or vagina is too high or when the normal microbial flora is absent. These remedies were also hypothesized by previous studies to induce apoptosis on Candida cells. The authors compared the efficacies of these two home remedies by culturing Candida albicans in the presence of either apple cider vinegar or Lactobacillus, using DI water as a control, at two different pH levels. The level of inhibition was measured by zones of inhibition around sterilized chads placed on a lawn of Candida. The results of this experiment showed greater inhibition at a lower pH, and that the apple cider vinegar was most effective in inhibiting Candida albicans. Data also showed that the only significant difference was between the water and apple cider vinegar group. Thus, the results of the experiment did not support the previous studies that supported that Lactobacillus was the most effective in inhibiting Candida albicans.

General Comments

Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.

The strength of this paper was structure. Each of the sections followed a logical order. The experiment setup was also strong and they authors referred to many sources to support their research. Unfortunately, the weaknesses of the paper are serious and in need of work. Overall, the writing seems rushed. Almost all of the sections are unfocused and lacks flow and organization, and some of the important procedures and results were unclear. Although the topic of the research was interesting, the authors struggled to convey their research in a way that was professional and intriguing to the reader.

Technical Criticism

Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.

This paper was a final versionX This paper was a rough draft

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