This work has received funding from the Ministry of Science and Technology, Taiwan [MOST 110-2314-B-006-086-MY3], National Cheng Kung University [K111-B094], [K111-B095], National Cheng Kung University Hospital, Taiwan [NCKUH-11204031], [NCKUMCS2022057].

1. Culturing of C. glabrata
NOTE: A multidrug-resistant C. glabrata (C2-1000907) that is resistant to most antifungal agents, including fluconazole, is used for the experiments. The experimental conditions may need to be adapted to the specific strain, as variations may exist among different strains. All experiments used log-phase Candida grown at 25 &#176;C (mimicking natural infection) for consistency. C. glabrata&#39;s lack of hyphae simplifies quantification compared to C. albicans, which forms hyphae at 37 &#176;C10.
<ol>
	<li>To prepare a log-phase culture of C. glabrata, pick a single colony from an agar plate and transfer it to a glass test tube with 3 mL of sterile yeast peptone dextrose (YPD) medium (see Table of Materials). Incubate for 14-16 h at 25 &#176;C with shaking (155 rpm, 45&#176; angle to increase air transmission to the medium).</li>
	<li>After incubation, dilute the culture with fresh YPD to an OD600 of 0.1 using sterile technique. Incubate at 25 &#176;C for 6 h with shaking at 155 rpm. Verify the log phase of C. glabrata by measuring the OD600. Aim for 0.65-1.00, which corresponds to 1 &#215; 107-1.5 &#215; 107 cells/mL.</li>
</ol>
2. Induction of petite colonies by ethidium bromide, fluconazole and photodynamic therapy
<ol>
	<li>Ethidium bromide petite colony induction
	<ol>
		<li>Adjust a yeast suspension to an OD600 of 0.1 (5 &#215; 106 cells/mL) with YPD to a final volume of 3 mL, then add 30 &#181;L of ethidium bromide stock (10 mg/mL) (see Table of Materials) for a final concentration of 100 &#181;g/mL.</li>
		<li>Incubate the yeast suspension overnight (16-18 h) at 25 &#176;C, 155 rpm, 45&#176; angle. Adjust to an OD600 of 0.65 (1 &#215; 107 cells/mL). Perform a 10-fold dilution series in a 96-well plate by adding 20 &#181;L of the adjusted yeast suspension to 180 &#181;L of PBS per well. This creates six dilutions ranging from 10-1 to 10-5.</li>
		<li>Select 4 dilution factors (e.g., 100, 101, 102, and 103) and plate 3 drops (20 &#181;L) each on 4 quadrants of YPD agar plates (triplicate).</li>
		<li>Incubate plates overnight at 37 &#176;C. Select quadrants with 5-80 colonies from the previously chosen dilutions for triphenyltetrazolium chloride (TTC) staining11 (see step 3). Scan plates at 1200 dpi using a 48-bit full-color optical scanner.</li>
	</ol>
	</li>
	<li>Fluconazole petite colony induction 
	NOTE: To expedite the process, use T3 cells (a mixture of normal and petite cells obtained after 3 RB-PDT treatments; see step 2.3) for fluconazole-induced petite colony formation. This is because standard treatment typically results in slower formation.
	<ol>
		<li>Prepare and adjust a yeast cell suspension as described in step 2.1.1.</li>
		<li>Incubate overnight (16-18 h) at 25 &#176;C. Adjust the OD600 to 0.1 again.</li>
		<li>Transfer 1 mL of the adjusted suspension to a sterile 5 mL tube.</li>
		<li>Dip a sterile cotton swab (15 cm long, with a 0.9 cm x 2.6 cm tip, see Table of Materials) in the yeast suspension, ensuring contact with the tube bottom and twisting to remove excess liquid from the tube wall.</li>
		<li>Prepare the plates in the hood using Mueller-Hinton agar (see Table of Materials).</li>
		<li>Swab the cotton back and forth on the agar, rotate 60&#176; twice, then swab the perimeter for even coverage.</li>
		<li>Sterilize forceps over a flame for 1-2 s, allowing them to cool briefly, then use them to pick up the blank disks.</li>
		<li>Divide the plate into three equal sectors using a marker. Place one blank disk in the center of each sector.</li>
		<li>Add 12.5 &#181;L fluconazole stock (2 mg/mL, see Table of Materials) to each disk (25 &#181;g/disk), mix thoroughly to avoid bubbles, and incubate at 37 &#176;C for 20-24 h.</li>
		<li>Perform the TTC staining (see step 3). Scan plates at 1200 dpi using a 48-bit full-color optical scanner.</li>
	</ol>
	</li>
	<li>PDT petite colony induction 
	NOTE: Different fungi may produce different numbers of petite colonies after PDT. Some strains may not produce any at all.
	<ol>
		<li>Prepare the aPDT system. 
		NOTE: The setup procedure for the aPDT system device follows the method reported by Hung et al12. Briefly, the aPDT system consists of a green LED array with a peak at 520 nm (see Table of Materials), shining light from the bottom with good alignment with each well of a 96-well plate.</li>
		<li>Adjust the yeast cell suspension in the YPD medium to an OD600 of 0.65 (about 1 &#215; 107 cells/mL).</li>
		<li>Mix 1 mL of the yeast suspension with 111 &#181;L of 2% rose bengal (RB, Figure 1) (see Table of Materials) in a round-capped tube to give a final RB concentration of 0.2%. Incubate the mixture at 25 &#176;C for 15 min, rotating it at a 45&#176; angle at 155 rpm.</li>
		<li>Transfer the mixture to a 1.5 mL microcentrifuge tube and centrifuge at 16,100 x g for 2.5 min (at room temperature).</li>
		<li>Discard the supernatant and gently scrape the tube five times on the hood floor to resuspend the pellet.</li>
		<li>Wash the suspension with 1x PBS four times to remove all the RB. Each wash is followed by resuspension of the pellet with brief vortexing or pipetting for thorough resuspension.
		<ol>
			<li>After the final wash, add 1000 &#181;L of PBS. The solution is light pink in color. Transfer 200 &#181;L of the washed RB-loaded yeast suspension to each of three wells in a 96-well plate (triplicate).</li>
		</ol>
		</li>
		<li>Position the 96-well plate on the photodynamic light system with LED bulbs shining green light from below. Turn off the room lights to ensure uniform illumination and prevent interference. Activate the LED light system to deliver the PDT dose of 4.38 J/cm2 over 2 min; ensure the system is properly aligned with the wells.</li>
		<li>After irradiation, dilute the yeast suspension in a 96-well plate. Add 20 &#181;L of yeast suspension to a well containing 180 &#181;L PBS, creating a 10-fold dilution. Repeat for the remaining dilutions to achieve a range of 10-1 to 10-5.</li>
		<li>Choose four dilution factors (e.g., 10-2 to 10-5) based on the yeast cell concentration adjustment.</li>
		<li>For each dilution factor, plate 3 drops (20 &#181;L each) per quadrant on YPD agar plates.</li>
		<li>After the yeast suspension is completely absorbed, around 10 min by the agar plates, invert and incubate overnight at 37 &#176;C.</li>
		<li>Define T0 as the control parental C. glabrata without PDT treatment. Prepare T0 fungi in the log growth phase as described above and expose them to 4.38 J/cm2 green light in the presence of 0.2% RB. This PDT condition consistently inhibits 3 to 3.5 logs of fungal growth.
		<ol>
			<li>Denote the survived fungi as T1 and expose them to the same PDT dose again after they are expanded in vitro to a log growth phase. The survived fungi after the second PDT are denoted as T2 and so forth.</li>
		</ol>
		</li>
		<li>On the next day, choose quadrants with colony counts between 5 and 80. Count the number of colonies in each quadrant and calculate the titer with the following formula: Colony-forming unit (CFU)/mL = Numbers of colonies (average of triplicate) &#215; Dilution factor &#215; 50.</li>
		<li>Perform the TTC staining (see step 3). Scan the plate as described earlier (step 2.2).</li>
	</ol>
	</li>
</ol>
3. Mitochondrial function analysis (TCC staining test)
NOTE: TTC is a redox indicator and an electron acceptor. It turns red when white compounds are broken by electrons11. Note that not all cells necessarily form visible colonies within 24 h after culture on an agar plate. Colonies with functional mitochondria turn red, while those with non-functional mitochondria remain white. This allows differentiation between colonies with different mitochondrial functionality.
<ol>
	<li>After treatment, dilute Candida suspensions on YPD agar plates according to the desired cell density to obtain distinct colonies for easy visualization and staining.</li>
	<li>After 24 h of growth at 37 &#176;C, visible colonies are formed from a single cell. Pipette 20 &#181;L of 20% TTC directly onto the center of each colony.</li>
	<li>After complete absorption, around 10 min of TTC by the colonies, incubate at 37 &#176;C for 30-40 min.</li>
</ol>
4. Growth kinetics of normal and petite C. glabrata
NOTE: Three strains of yeast were compared: C. glabrata C2-1000907 T0 (clinical isolate without PDT treatment), T3n (C. glabrata C2-1000907 after 3 consecutive RB-PDT exhibiting colonies with an average diameter of 1.5 &#177; 0.8 mm similar to the parental cells), and T3p (petite colonies of C. glabrata C2-1000907 after 3 consecutive RB-PDT).
<ol>
	<li>Adjust the yeast cell suspension in YPD medium to an OD600 of 0.1 (5 &#215; 106 cells/mL) in a 3 mL tube.</li>
	<li>Automatically measure the OD600 of the statical culture every 20 min using the multi-mode microplate reader (see Table of Materials) for 24 h.</li>
</ol>

Analyzing Petite Colony Formation and Drug Resistance in Candida glabrata

<ol>
	<li>Soriano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+candidiasis:+current+clinical+challenges+and+unmet+needs+in+adult+populations.">Invasive candidiasis: current clinical challenges and unmet needs in adult populations.</a> J Antimicrob Chemother. 78 (7), 1569-1585 (2023).</li><li>Pappas, P. G., Lionakis, M. S., Arendrup, M. C., Ostrosky-Zeichner, L., Kullberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+candidiasis.">Invasive candidiasis.</a> Nat Rev Dis Primers. 4, 18026 (2018).</li><li>Meyahnwi, D., Siraw, B. B., Reingold, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiologic+features,+clinical+characteristics,+and+predictors+of+mortality+in+patients+with+candidemia+in+Alameda+County,+California;+a+2017-2020+retrospective+analysis.">Epidemiologic features, clinical characteristics, and predictors of mortality in patients with candidemia in Alameda County, California; a 2017-2020 retrospective analysis.</a> BMC Infect Dis. 22 (1), 843 (2022).</li><li>Whittaker, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+petite+mutation+in+yeast.">The petite mutation in yeast.</a> Subcell Biochem. 6, 175-232 (1979).</li><li>Hatab, M. A., Whittaker, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+respiration-deficient+mutants+from+the+pathogenic+yeast+Candida+albicans.">Isolation and characterization of respiration-deficient mutants from the pathogenic yeast Candida albicans.</a> Antonie Van Leeuwenhoek. 61 (3), 207-219 (1992).</li><li>Defontaine, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+resistance+to+azoles+associated+with+mitochondrial+DNA+deficiency+in+Candida+glabrata.">In-vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata.</a> J Med Microbiol. 48 (7), 663-670 (1999).</li><li>Brun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+respiration+and+susceptibility+to+azole+antifungals+in+Candida+glabrata.">Relationships between respiration and susceptibility to azole antifungals in Candida glabrata.</a> Antimicrob Agents Chemother. 47 (3), 847-853 (2003).</li><li>Bouchara, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+selection+of+an+azole-resistant+petite+mutant+of+Candida+glabrata.">In-vivo selection of an azole-resistant petite mutant of Candida glabrata.</a> J Med Microbiol. 49 (11), 977-984 (2000).</li><li>Badrane, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+diversity+and+unrecognized+antifungal+resistance+among+populations+of+Candida+glabrata+from+positive+blood+cultures.">Genotypic diversity and unrecognized antifungal resistance among populations of Candida glabrata from positive blood cultures.</a> Nat Commun. 14 (1), 5918 (2023).</li><li>Shantal, C. -. J. N., Juan, C. -. C., Lizbeth, B. -. U. S., Carlos, H. -. G. J., Estela, G. -. P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+glabrata+is+a+successful+pathogen:+An+artist+manipulating+the+immune+response.">Candida glabrata is a successful pathogen: An artist manipulating the immune response.</a> Microbiol Res. 260, 127038 (2022).</li><li>Gamarra, S., Mancilla, E., Dudiuk, C., Garcia-Effron, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+dubliniensis+and+Candida+albicans+differentiation+by+colony+morphotype+in+Sabouraud-triphenyltetrazolium+agar.">Candida dubliniensis and Candida albicans differentiation by colony morphotype in Sabouraud-triphenyltetrazolium agar.</a> Rev Iberoam Micol. 32 (2), 126-128 (2015).</li><li>Hung, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rose+bengal-mediated+photodynamic+therapy+to+inhibit+Candida+albicans.">Rose bengal-mediated photodynamic therapy to inhibit Candida albicans.</a> J Vis Exp. (181), e63558 (2022).</li><li>Cardoso, D. R., Franco, D. W., Olsen, K., Andersen, M. L., Skibsted, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivity+of+bovine+whey+proteins,+peptides,+and+amino+acids+toward+triplet+riboflavin+as+studied+by+laser+flash+photolysis.">Reactivity of bovine whey proteins, peptides, and amino acids toward triplet riboflavin as studied by laser flash photolysis.</a> J Agric Food Chem. 52 (21), 6602-6606 (2004).</li><li>Hall, R. M., Trembath, M. K., Linnane, A. W., Wheelis, L., Criddle, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+petite+induction+and+the+recovery+of+respiratory+competence+in+yeast+cells+exposed+to+ethidium+bromide.">Factors affecting petite induction and the recovery of respiratory competence in yeast cells exposed to ethidium bromide.</a> Mol Gen Genet. 144 (3), 253-262 (1976).</li><li>Chen, X. J., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+petite+mutation+in+yeasts:+50+years+on.">The petite mutation in yeasts: 50 years on.</a> Int Rev Cytol. 194, 197-238 (2000).</li><li>Piskur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+the+yeast+mitochondrial+genome.">Inheritance of the yeast mitochondrial genome.</a> Plasmid. 31 (3), 229-241 (1994).</li><li>Wong, T. W., Wang, Y. Y., Sheu, H. M., Chuang, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bactericidal+effects+of+toluidine+blue-mediated+photodynamic+action+on+Vibrio+vulnificus.">Bactericidal effects of toluidine blue-mediated photodynamic action on Vibrio vulnificus.</a> Antimicrob Agents Chemother. 49 (3), 895-902 (2005).</li><li>Wong, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green-mediated+photodynamic+therapy+reduces+methicillin-resistant+Staphylococcus+aureus+drug+resistance.">Indocyanine green-mediated photodynamic therapy reduces methicillin-resistant Staphylococcus aureus drug resistance.</a> J Clin Med. 8 (3), 411 (2019).</li><li>Warrier, A., Mazumder, N., Prabhu, S., Satyamoorthy, K., Murali, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+to+control+microbial+biofilms.">Photodynamic therapy to control microbial biofilms.</a> Photodiagnosis Photodyn Ther. 33, 102090 (2021).</li><li>Hung, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+photodynamic+therapy+against+fungal+keratitis.">Recent advances in photodynamic therapy against fungal keratitis.</a> Pharmaceutics. 13 (12), 2011 (2021).</li></ol>

The authors declare no conflict of interest.

This study unveils PDT as the first reported method to induce petite colony formation in Candida, surpassing the established effects of ethidium bromide and fluconazole. This novel observation necessitates further exploration to unravel its implications for both fungal eradication by decreasing virulence and the emergence of resistance mechanisms.
RB-mediated PDT effectively inhibits the growth of C. glabrata, suggesting a potential alternative treatment approach for Candida infection...

Fungal infections, particularly those caused by Candida albicans and increasingly drug-resistant Candida glabrata, pose a serious global threat1. These infections can be deadly, especially for hospitalized patients and those with weakened immune systems. Rising antifungal resistance threatens the control of invasive candidiasis, a severe fungal infection with high mortality, especially from Candida albicans2. Resistant strains hinder effective treatment, potentially increasing both complexity and death rates. In Alameda County, California, USA, C. glabrata has become the most prevalent invasive species3. This shift in the prevalence and distribution of Candida species may be influenced by local healthcare practices, patient demographics, the utilization of antifungal agents, and the prevalence of risk factors for Candida infections.
Petite mutants in Candida, lacking functional mitochondria, reveal how this organelle affects drug response, virulence, and stress resistance4,5. C. glabrata readily forms these colonies, gaining sensitivity to polyenes while losing it to azoles6. Azole sensitivity and respiratory function are intricately linked, with diminished respiration leading to resistance via mitochondrial DNA loss7. Petite colonies of C. glabrata with azole resistance have been isolated from human stool samples from a bone marrow transplant recipient undergoing fluconazole treatment8 and from blood culture bottles of patients with bloodstream infections9. Their potential implications in drug resistance, virulence, and stress response highlight their clinical significance. Additionally, their distinct properties make them valuable tools for investigating fundamental questions in mitochondrial biology5. As research into petite mutants continues, their applications in both clinical and basic research are likely to expand.
This study discovered that photodynamic therapy (PDT) can induce petite colonies in C. glabrata, expanding the range of methods beyond the traditional techniques of exposing C. glabrata to ethidium bromide or fluconazole.

<table><tbody><tr><td>0.22 &amp;mu;m filter</td><td>Merck, Taipei, Taiwan</td><td>Millex, SLGVR33RS</td><td></td></tr><tr><td>1.5 mL microfuge tube</td><td>Neptune, San Diego, USA</td><td>#3745</td><td></td></tr><tr><td>20% Triphenyltetrazolium chloride (TTC)</td><td>Sigma-Aldrich, MO, USA</td><td>T8877</td><td></td></tr><tr><td>5 mL polypropylene round bottom tube</td><td>Corning, AZ, USA</td><td>352059</td><td></td></tr><tr><td>5 mL round-bottom tube with cell strainer cap</td><td>Corning, AZ, USA</td><td>Falcon, #352235</td><td></td></tr><tr><td>96-well plate</td><td>Alpha plus, Taoyuan Hsien, Taiwan</td><td>#16196</td><td></td></tr><tr><td>Agar</td><td>BRS, Tainan, Taiwan</td><td>AG012</td><td></td></tr><tr><td>Blank disk</td><td>Advantec, Tokyo, Japan</td><td>49005040</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, UK</td><td>5415R</td><td></td></tr><tr><td>Ethidium bromide solution</td><td>Sigma-Aldrich, MO, USA</td><td>E1510</td><td></td></tr><tr><td>Fluconazole, 2 mg/mL</td><td>Pfizer, NY, USA</td><td>BC18790248</td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>Version 7.0</td><td></td></tr><tr><td>Green light emitting diode (LED) strip</td><td>Nanyi electronics Co.,Ltd, Tainan, Taiwan</td><td>5050</td><td>Excitation wave: 500~550 nm</td></tr><tr><td>Low Temperature. shake Incubators</td><td>Yihder, Taipei, Taiwan</td><td>LM-570D (R)</td><td></td></tr><tr><td>Mouth care cotton swabs</td><td>Good Verita Enterprise, Taipei, Taiwan</td><td>161357</td><td></td></tr><tr><td>Muller Hinton II agar</td><td>BD biosciences, California, USA</td><td>211438</td><td></td></tr><tr><td>Multimode microplate reader</td><td>Molecular Devices</td><td>SpectraMax i3x</td><td></td></tr><tr><td>OD&lt;sub&gt;600&lt;/sub&gt; spectrophotometer</td><td>Biochrom, London, UK</td><td>Ultrospec 10</td><td></td></tr><tr><td>Rose Bengal</td><td>Sigma-Aldrich, USA</td><td>330000</td><td>stock concentration 40 mg/mL = 4%, prepare in PBS, stored at 4 &amp;deg;C</td></tr><tr><td>Sterilized glass tube</td><td>Sunmei, Tainan, Taiwan</td><td>AK45048-16100</td><td></td></tr><tr><td>Yeast Extract Peptone Dextrose Medium</td><td>HIMEDIA, India</td><td>M1363</td><td></td></tr></tbody></table>

induction of petite colonies in candida glabrate via rose bengal-mediated photodynamic therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

The data is presented as the mean with &#177; standard error and was obtained from three independent experiments, with at least triplicates in each group. Experimental data, including colony counts, OD600 measurements, and TTC staining results, were graphed and statistically analyzed using graphing and statistical software (see Table of Materials). One-way ANOVA or t-test was used to analyze the data, and a p-value &#60;0.05 was considered significant. Scanning was performed ...

Watch this Scientific Journal Video about Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug-Resistant Candida for Antifungal Research at JoVE.com

Author Spotlight: Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug&#45;Resistant Candida for Antifungal Research

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

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662 Views.  National Cheng Kung University. Het onderzoek is gericht op het gebruik van fotodynamische therapie, ook bekend als PDT, om geneesmiddelresistente Candida-infectie te bestrijden. In deze studie proberen we te beantwoorden hoe effectief PDT de schimmels doodt, en hoe de virulentie van de schimmels verandert na de behandeling. We vonden kleine kolonies uit de na PDT.Huidige methoden voor het induceren van kleine kolonies zijn gebaseerd op ethidiumbromide of fluconazol, die via verschillende routes kunnen werken op resi...

Video: Auteur in de schijnwerpers: fotodynamische therapie als een nieuwe benadering om kleine kolonies te induceren in resistente candida voor antischimmelonderzoek

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Immunology and Infection

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

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