eamag/protocols-with-steps huggingface dataset:
TITLE: Microfluidic channels wax priming
AUTHORS: Jorge Fernández, Manuela de las Casas

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Procedure for printing  microfluidic hydrophobic channels in nitrocellulose membranes.</div><div class = "text-block">This protocol depicts the steps for easily printing wax channels using plastic stamps and molten wax.</div></div>

[STEPS]
?. Set the hot plate to 105ºC or at least warm enough to melt the wax. Cut a small amount from one of the wax pencils and place it  on a Petri dish. Set the Petri dish on the hot plate and wait for the wax to melt.
?. Cut to their appropiate size the nitrocellulose pieces to prime with the designed microfluidic path.
?. Once the wax is perfectly melted, take the plastic stamp and wet their sealing ends by immersion for 1-2 seconds in the molten wax. Lift the stamp and observe that the stamping surface is fully covered in wax.
?. As fast as possible place the stamp against the membrane, in order to release the wax on the surface of the nitrocellulose membrane. Apply mild pressure for 2-5 seconds, and lift the stamp. It is possible that the membrane goes up wtith the stamp. In that case, touch softly the outer region of the sealed membrane in order to detacht it from the stamp.
?. Cover the nitrocellulose strip with plastified paper that protects the membrane (one sheet on top, another underneath). Place the sandwich on the hot place with a Petri dish on top (this is just to apply soft pressure, any temperature-resistant flat object is valid here). Allow the strip to warm for 5 minutes. This will allow the wax to penetrate all through the strip.
?. Remove the strip from the hot plate and let it cool down again. You should see the  shape of the channel wax primed on the membrane.  Ussually it is vissible a thin "white shadow" near the border of the wax imprimation, that represents the limit of the primed channel.  If the channel is not wide enough, or it is not uniform, consider repeating the procedure.
TITLE: Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This collection describes the maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture.

Collection overview
Thawing of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Expansion of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Freezing of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Harvesting and irradiation of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Mitomycin C inactivation of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Preparation of mouse embryonic fibroblast (MEF) feeder plates for hPSC cultures
    A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional)
    B. Starting with fresh irradiated or Mitomycin C inactivated MEFs

General notes
1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. Either fresh or frozen stocks of irradiated or Mitomycin C inactivated MEFs can be used to prepare hPSC feeder cells. 
3. The indicated MEF densities are recommended starting densities and might have to be adjusted for each hPSC line and hPSC media formulation (KSR, serum-free versus serum-containing media). 
4. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)
 


[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocoll outlining the Preperation and transformation of chemo-competent cells for <em>Vibrio natriegens</em></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
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?. 
?. 
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?. 
?. 
?.
TITLE: Expression and purification for cryo-EM samples of FIP200NTD:ATG13(363-517)-ULK1(MIT) complexes
AUTHORS: Minghao Chen, Xuefeng Ren

[DESCRIPTION]
Expression and purification for cryo-EM samples of FIP200NTD:ATG13(363-517)-ULK1(MIT) complexes

[STEPS]
SECTION: Expression
1. Transfect HEK GNTi cells at concentration of 2 × 10^6 cells/ml
SECTION: Expression
2. Dilute PEI with Warm Hybridoma-SFM(1X)
SECTION: Expression
3. In a separate tube, dilute DNA with Hybridoma-SFM(1X)
SECTION: Expression
4. Add PEI to DNA dilution. Incubate mixture for 30 min  at 37 °C

SECTION: Expression
5. Add mixture to cells. Let cells grow for 2880 min

SECTION: Expression
6. Harvest Cells 500 rpm, 4 °C, 10 min

SECTION: Expression
7. Wash pellet with cold PBS. Store pellet at-80 °C until purification. 

SECTION: Purification
8. Cell pellets were lysed at room temperature for 20 min with lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 10% Glycerol) with 5 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific)  for 15 min

SECTION: Purification
9. Clarify lysate for 17000 rpm for 35 min at 4 °C.  




SECTION: Purification
10. Wash strep-tactin resin (IBA Lifesciences, Germany) into lysis buffer (without Triton). Load clarified lysate onto resin
SECTION: Purification
11. Rock supernatant with equilibrated Strep-Tactin Sepharose resin for 60 min at 4 °C
SECTION: Purification
12. Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)
SECTION: Purification
13. Elute with lysis buffer plus 4 mM desthiobiotin for STREP resin
SECTION: Purification
14. His6-TEV cleavage at 4 °C 60 min, 




SECTION: Purification
15. Concentrate elution and inject onto pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva) 
 (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM TCEP)
SECTION: Purification
16. Pool peak fractions, concentrate, snap freeze, and store at -80 °C
TITLE: Deriving steps per mm for an LVE system
AUTHORS: Robert B Jess, Jonathan Widdowson, Feihu Zhao, Christopher Wright

[DESCRIPTION]
Modification of off-the-shelf fused filament fabrication (FFF) 3D printers with a syringe pump, used as a large volume extruder (LVE) is a common method for the creation of low-cost laboratory 3D bioprinters. Open-source syringe pumps with accompanying firmware exist, however they require researchers to choose accurate control of the syringe pump over rich control over the 3D printer system. The combination of open-source syringe pump hardware controlled with 3D printer electronics and firmware gives the best of both worlds, however this firmware is not designed to control fluids, consequently some parameters may need to be calculated differently. FFF printer firmware (such as RepRapFirmware) uses E-steps-per-mm to translate the movement of the stepper motor into the extrusion of filament pre-nozzle. This method finds the value of this parameter for a bioprinter with a bowden-tube based LVE.

[GUIDELINES]
This method is for an LVE that is based around the MOST Open Source Syrigne Pump [1], or similar syringe pumps such as the Poseidon from Patcher Lab [2], connected to the printer carriage and print head via a bowden tube.

[1] Joshua M. Pearce, Bas Wijnen (2016). "Open-source syringe pump". Appropedia. Retrieved October 13, 2022.
[2] A. Sina Booeshaghi, Eduardo da Veiga Beltrame, Dylan Bannon, Jase Gehring and Lior Pachter, "Principles of open source bioinstrumentation applied to the poseidon syringe pump system", Scientific Reports 9, Article number: 12385 (2019)   

[STEPS]
SECTION: Take measurements
0.1. Take the following measurements:

 


SECTION: Deriving the revolutions per mm
2. The ratio between the syringe and the Bowden tube can be calculated as follows:

  
Where  is the distance travelled by the syringe plunger,   is the resultant distance the gel travels through the bowden tube,   is the internal radius of the syringe, and   is the internal radius of the bowden tube.

The ratio between the motor and the syringe is equal to the ratio between the motor and the linear distance travelled by the syringe plunger is:

  

From this we know, when ls = lead, the ratio between the lead screw and the Bowden tube, is:
  

This makes the revolutions per mm:
  






SECTION: Finding the E-Steps-per-mm
3. Multiply the motor steps by the micro-stepping by the length of filament produced through thr bowden tube with one revolution of the stepper motor
  
SECTION: Finding the thread&#x27;s lead
1. Most lead screws are single start, and the one recommended in the MOST OSSP is single start, this means that the lead (the distance the carriage travels with one rotation of the lead screw) is equal to the pitch (the distance between the crests of the thread).

If you are using a multi-start lead screw the following formula should be used to calculate the thread's lead:
TITLE: 10000x DNA gel stain: User protocol
AUTHORS: Stephane  Fadanka, Nadine    Mowoh

[DESCRIPTION]
Beneficial Bio DNA Gel Stain is a conventional nucleic acid staining reagent made of Thiazole Orange dye and DMSO. The DNA gel stain compares favorably to common staining methods, in that it is sensitive, excitable with both UV or blue light (to prevent sample damage), which make it suitable for downstream experiments and safer than ethidium bromide and therefore can be generally disposed of as common chemical waste.

Features:

Easy to Use: Classic DNA gel stain, View and document your results as you would with EtBr staining.
Support dual visualisation: Results can be viewed under UV light or blue light.
Safety: Safer alternative to toxic Ethidium Bromide (EtBr).

Applications
Preparation of agarose gels for visualisation of DNA after electrophoresis.

[BEFORE_START]
Make sure your pipette and all other reagents or equipment you would need are available and set.

[GUIDELINES]
Wear protective clothing and follow good laboratory practices although all the reagents for this experiment are generally safe to use.

[STEPS]
SECTION: User protocol
1. 
 
Preparing a 0.0013 mg/mL final concentration of DNA gel stain in agarose gel for electrophoresis from a 13mg/ml stock

Prepare a 1x TBE buffer from a 10x stock by diluting 10 mLof the 10x TBE buffer stock into 90 mLof distilled water.
Prepare a 1% agarose gel by dissolving0.25 g of Agarose into 25 mL of 1x TBE buffer, heat until it boils for about 1 min in a microwave .
 Pipette 2.5 µL of nucleic acid gel stain into 25 mLof pre- heated TBE gel, swirl for some seconds to completely and evenly distribute the stain in the gel (mix gently to avoid bubbles). 
Assemble the gel casting tray with combs and pour into the tray, leave it to stand for about 20 minutes to solidify. 
Remove the comb, load and run the gel following the standard protocol to finish.
After the electrophoresis, visualize the gel on UV or a visible light transilluminator.


SECTION: Notes and Troubleshooting
2. 
To achieve optimal results, cast the gel in the dark or cast in the light and use immediately. 
The gel stain has been tested for stability and was able to allow visualization after 6 hours of casting in light and leaving at room temperature.
If the DNA gel stain is not working, check to make sure it is stored at the right temperature, the right solvent was used to dissolve the dye and the right storage container is used (to avoid it loosing its color and ability to stain DNA. The solvent that has been able to completely dissolve the dye is DMSO and storing the dye stock at4 °C is also increases its shelve life).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The different kinds of stained histological sections are widely used to detect and observe physiological and pathological changes in various tissues.</p>

[BEFORE_START]
<p>Bouin's fixative was formulated by saturated solution of picric acid (75 ml), 40% aqueous formaldehyde (25 ml), and glacial acetic acid (5 ml), kept at room temperature.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Financing and Insurance (Part 12 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Part 12 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: U54 SCENT Olink® Target 96
AUTHORS: andrew.nixon, Chris Brady

[DESCRIPTION]
This protocol outlines the sample preparation and running of the Olink 96 panel

[STEPS]
SECTION: Preparation
1. Aliquoting and Day of Preparation 

For Aliquoting of EDTA plasma samples:
Thaw all EDTA plasma samples in a -4°C refrigerator until ready to use.
Keep Samples in a chilled rack whenever possible.
Match your parent tubes (Original tubes shipped) with a prelabelled 2mL
clarification tube.
Vortex all samples on a Vortex Genie with a vortex setting of 7-10.
Transfer samples from the parent tube to the clarification tube.
In a pre-cooled centrifuge set to -4°C centrifuge all samples for 5 minutes
on a setting of 20,000 rcf.
Carefully remove samples from centrifuge into a chilled rack.
Transfer liquid from the clarification tube to a prelabelled aliquot tube in
desired amounts.
Using Liquid Nitrogen, freeze back the tubes and place in a -80°C freezer
until ready to use.


For EDTA samples on day of use:
Thaw all EDTA plasma samples in a -4°C refrigerator until ready to use.
Keep Samples in a chilled rack whenever possible.
Vortex all samples on a Vortex Genie with a vortex setting of 7-10.
In a pre-cooled centrifuge set to -4°C centrifuge all samples for 5 minutes
on a setting of 20,000 rcf.
Return the samples to a chilled rack.
Your samples are ready for use.



 
SECTION: Running Olink Panel
2. Once samples are prepared use the following links to access the protocols:
Olink Target 96 Short Instructions
Olink Target 96 User Manual
TITLE: Double picking protocol for tracking
AUTHORS: Priota Islam

[STEPS]
?. [Imaging plates]
Prepare and pour low peptone NGM onto 35mm imaging plates  Put them in the cold room for at least 2 days before use Seed imaging plates with 50ul of freshly made 1:10 solution (OP50:M9 solution) the day before recording Leave on bench to dry with lid on overnight
?. [Day before recording]
Pick around 45 L4s with a hair pick onto seeded NGM plate from maintenance plates having a mixed population of wormsLeave the plates in a 20C incubator for ~24hrs
?. [Day of recording]
Using a hair pick – pick 5 young adults onto the seeded imaging plates  Put the plates on the tracker – agar side up with the lids off  Wait 30min for the worms to acclimatise  Record for 15min (See protocol Imaging on the multiworm tracker  for detailed tracking instructions)
TITLE: Transformation Protocol
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Quick Ligation products may be transformed by many different methods. The following protocol is recommended by New England Biolabs.

[STEPS]
1. Thaw competent cells on ice. 
2. Chill approximately 5 ng (2 µL) in a 1.5 ml microcentrifuge tube. 
3. Add 50 µL to the DNA. 
4. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
5. Place the mixture on ice for 30 min. Do not mix. 
6. Heat shock at 42 °C for 30 s. Do not mix. 
 

7. Add 950 µL to the tube. 
8. Place tube at 37 °C for 60 min. Shake vigorously (250 rpm) or rotate. 
9. Warm selection plates to 37 °C.
10. Spread 50 µL–100 µL  onto the plates.
11. Incubate 60 min at 37 °C.
TITLE: M9 Medium
AUTHORS: Fan Zhang, Jessica Weckhorst, Adrien Assie, Anastasia Khodakova, Mario Loeza Cabrera, Daniela Vidal, Christopher Ayoub, Buck Samuel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>M9 Buffer recipe, transposed from the WormBook chapter: Maintenance of</span><span style = "font-style:italic;">C. elegans</span></div><div class = "text-block">https://www.ncbi.nlm.nih.gov/books/NBK19649/ </div></div>

[STEPS]
?. Start with
[water]
?. of Na2HPO4 ()
6 g
?. of KH2PO4 ()
3 g
?. of NaCl ()
5 g
?. of MgSO4∙7H2O OR MgSO4
0.25 g
1 mL
?. Adjust water to
1000 mL
?. Autoclave
TITLE: REAGENTS AND SOLUTIONS (Support Protocol 7.2)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;font-weight:bold;">Primers</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">CLYBL insertion of hNIL </li><li style = "counter-reset:ol0;list-style-type:disc;">CLYBL insertion of mag-hNIL 
 
 
 
 
 
 </li></ul></div></div>

[STEPS]
TITLE:           ELISA for measurement of platelet-activating factor (PAF) in serum.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. An anti-human PAF coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human PAF present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-PAF antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-PAF antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of PAF present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human PAF standard dilutions and the human PAF sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Single-cell RNA sequencing
AUTHORS: Klaus H. Kaestner Lab, Suzanne Shapira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Single-cell RNA sequencing (scRNA-seq) allows for transcriptional profiling of individual cells within a heterogenous sample. This protocol describes a method for performing scRNA-seq using handpicked pancreatic islets from organ donors.</span></div><br/><br/></div>

[STEPS]
?. [Steps in pre-processing]
1. Transfer handpicked islets (approximately 5,000 IEQs) into  conical tube. 2. Add  of 1xPBS w/o Ca2+, Mg2+ (Rockland, MB-008). Centrifuge for 2 min at RT, 180 xg. Aspirate the supernatant.3. Add  of warm ( ) 0.05% Trypsin (Invitrogen, 25300054) to the islets. Pipette up and down with p1000. 4. Incubate at  for 9 min, or until cells are in single cells. Pipette up and down at t=7 min, 4 min, 2 min, 0 min. 5. Stop the trypsin reaction by adding  of 100% FBS (Hyclone, SH3091003) to the dissociated islets and pass cells through BD FACs tube with strainer top (Corning 352235)6. Use  of 100% FBS to rinse the tube and pass through the strainer.7. Transfer cells to  conical. Centrifuge 4 min, 400 xg. 8. Remove the supernatant and wash cells with PBS with 10% FBS. Centrifuge for 4 min, 400 xg. 9. Wash the cells with PBS with 10% FBS and centrifuge for 4 min, 400 xg. Remove the supernatant.10. Count cells using a countess chamber.11. For the scRNAseq, do the final resuspension in 10% FBS in PBS and adjust the volume to make the final suspension 1000 cells/microliter. Filter the cells one more time prior to loading them onto the 10X Genomics chip.
15 mL
10 mL
1 mL
37 °C
37 °C
1 mL
1 mL
15 mL
?. [Links to Kits used in post-processing]
1. Older HPAP samples (specifically donors HPAP-001 to HPAP-019) were processed using C1 Single-Cell mRNA Seq HT IFC and Reagent Kit v2 (product ID: 101-4964) which has been discontinued.2. All current samples (HPAP-019 onwards) were processed using the Chromium Single Cell 3’ Reagent Kit.3. Unfortunately, since 10x Genomics is going to stop manufacturing the above kit, we will be using the new kit Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1. For this protocol we target a 5000 cell recovery.
TITLE: Candida tropicalis filamentation assay with fluconazole
AUTHORS: Laboratorio de Microbiología Mr JORGE ENRIQUE PEREZ CARDENAS, Sebastian  Hernandez

[DESCRIPTION]
This protocol was used to obtain enough quantity of RNA of a nonsusceptible strain of Candida tropicalis, with the goal  to do a transcriptomic analysis to demonstrate the degree of differential expression of genes under filamentation and non-filamentation conditions and against a non-filamenting strain, susceptible to fluconazole (Candida tropicalis ATCC750)

[STEPS]
SECTION: RPMI PREPARATION
1. MEDIA PREPARATION
SECTION: RPMI PREPARATION
1.1. Medium to inhibit  filamentation (RPMI+NAC)
RPMI1640: 10g
3-[N-morpholino]propane sulfonic acid buffer (MOPS): 35 g
N-acetyl glucosamine: 20 g
Distilled water 500 ml
Adjust pH at 7.0
Adjust volume at 1000 ml
Esterilize by filtration
SECTION: RPMI PREPARATION
1.2. MEDIUM TO PROMOTE THE FILAMENTATION (RPMI+SFB)
RPMI1640: 10g
3-[N-morpholino] propane sulfonic acid buffer (MOPS): 35g
Glucose: 20 g
Distilled water: 500 ml
Adjust pH at 7.0
Adjust volume at 1000 ml
Add  10% of Fetal bovine serum 

2. Fluconazole preparation
Weigh 6.4 mg of Fluconazole (Cat Sigma: F8929)
Dissolve in 1 ml of Dimethyl sulfoxide (DMSO)
aliquot and freeze at -80°C

SECTION: Susceptibility test
6. 
SECTION: Susceptibility test
5. CULTURE OF C. tropicalis  TO HARVEST  YEAST FOR TRANSCRIPTOMIC ANALYSIS
Obtain a Fluconazole Strain and a certificate strain (ATCC750)
Establish the susceptibility grade of both strains
Culture both strains with filamentation (RPMI+SFB) and Non-filamentation (RPMI+NAC) media, using Fluconazole at subinhibitory concentrations
Incubate at 35°C by 24 hrs with constant agitation (200 rpm)
Check the growth degree and filamentation degree
Harvest the colonies with a sterile Pasteur pipet
Centrifuge at high speed 
Extract the supernatant
Add RNA later (Sigma Aldrich Ref0901)
Freeze at -80°C until the RNA extraction

SECTION: Susceptibility test
3. INOCOLUM DILUTION
Cultivate Candida tropicalis in Potato dextrosa agar o in Liquid Sabouraud
Incubate for 24 h at 35°C
Dilute one or two colonies in sterile saline solution
Vortex until homogeinity
Adjust the turbidity according to the  0.5 MacFarland Standard  (1x106-5x 106 CFU)

Make a 1:100 dilution of the inocolum using each media (filamentation and No fiamentation)
Vortex for 15 seg
Make a 1:20 dilution using the 1:100 dilution, using the same media (Final yeast concentration: 5x102 to 3x103 FCU
SECTION: Susceptibility test
4. MIC ASSAY USING MEDIA TO FILAMENTATION (SFB)  AND MEDIUM TO INHIBIT THE FILAMENTATION (NAC)

USE A  STERILE 24 WELLS PLATE
RPMI + NAC

 

RPMI+SFB

 
Incubate at 35°C for 24 hrs with constant agitation (200 rpm approx.)
Read using an inverted microscope, comparing controls with each well
The Minimal inhibitory concentration will be where was observed 50% or more of growth
Also, register the filamentation grade in each well
2.1. FLUCONAZOLE (FLU) DILUTION

USE A V or U bottom 96 wells plate