Laboratory procedure manuals

IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS:
Antifungal drug dilutions are used to determine the MIC or MFC for an isolate when grown in thepresence of an antifungal agent. Serial dilutions of antifungal agents are dispensed intoappropriately labeled tubes. Each tube is then inoculated with a standardized nutrient brothsuspension of the yeast being tested. The primary advantage of the broth dilution test is that itpermits a quantitative estimate of both the inhibitory and fungicidal activities of the antifungal agent. Antifungal susceptibility tests should be performed (i) when the patient is failing therapy with anantifungal agent(s) that is known to be active against the infecting organism; (ii) to learn ofpotentially efficacious alternative drugs when the pathogenic yeast is one with well-known resistanceto the drug of choice, e.g., Candida lusitaniae and amphotericin B (AMB); (iii) when one is treatingwith 5-fluorocytosine (5-FC), an agent to which yeasts may be innately resistant or to whichresistance rapidly develops; (iv) to ascertain antifungal activity with new agents for which nosubstantial or previously published data base exists; (v) for yeasts recovered from severelyimmunocompromised hosts with systemic disease, e.g., those with neutropenia; and (vi) forprospective studies of in vitro or in vivo correlation. The methods described here are those currentlybeing used in multicenter collaborative studies by the Subcommittee on Antifungal SusceptibilityTesting of the NCCLS. To date, no NCCLS standards have been published, but this procedure isbased upon the December 1992 proposed standards by the NCCLS.
B. Specimen: Five isolated colonies of similar colony morphology at least 1 mm in diameter grown for 24-48 h on Sabouraud dextrose agar (SDA).
C. Materials: Indicate the expiration date of reagents on the container label and in the work record, or on the manufacturer's label. Label all reagents and media when first placed in use.
1. Media and Reagents (storage conditions; shelf life) a. SDA plates, Emmons modification, pH 7 (2-8 C) b. SDA slants, Emmons modification, pH 7 (2-8 C) c. RPMI-1640 medium buffered with 0.165 M MOPS [3-(N-morpholino) propanesulfonic acid] containing L-glutamine and lacking sodium bicarbonate; supplied dehydrated by AmericanBiorganics, Inc., Niagara Falls, N.Y.; catalog no. R63165 (2 to 8 C; shelf life, 12 months);dehydrated RPMI 1640; sterile distilled reagent-grade water Add 1 package of RPMI-1640 to 1l reagent-grade sterile water and then stir to solubilize.
Filter sterilize through two 500-ml-capacity filtration units with 0.22-µ-pore-sizeMillipore filters.
Store the RPMI-1640 until needed (2 to 8 C; shelf life, 3 months).
d. Sterile distilled reagent-grade water (25 C) f. Dimethyl sulfoxide (DMSO) (25 C), dimethylformamide (DMF) (25 C), and polyethylene IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
g. Macrobroth dilution tubes (sterile, 12x75 mm, polystyrene, snap cap) containing 0.1 ml volumes of the following antimicrobial agents (diluted in RPMI-1640) except for amphotericinB which requires M-3) at 10 times the suggested final concentrations (see Section V)maintained at -70 C h. Potato Dextrose Agar Slants (PDA) (2-8 C) i. Bacto Antibiotic Medium 3 (M3) (2-8 C). Follow instructions on label for preparation.
j. 25% ethanol a. Sterile wooden applicator sticks or sterile swabs c. 500 ml capacity filtration units with 0.22-µm-pore-size Millipore filters d. Sterile 1, 5, and 10 ml disposable serological pipettes and pipette bulb c. Micropipette, 100 µl capacity with sterile disposable tips Repetitive dispensing pipette, 100 µl capacity with sterile disposable syringes Maintain permanent stock cultures at -70 C on an PDA slant.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
With sterile wooden applicator stick, chip off a small portion of the frozen surfaceof an SDA slant, and streak for isolation.
Weekly working stock cultures are prepared by subculturing three to five coloniesof similar colony morphology from the previous week's working stock plate to afresh SDA plate.
Subculture from working stock plate to another SDA plate on the day before use, and testas would be done for patient isolates. Candida albicans (ATCC 90028)
b. Positive growth control should demonstrate good growth and be free of contaminating c. Negative growth control should be free of growth.
d. Inoculum count verification plate should show 100 to 500 colonies.
MIC's for QC strains are within acceptable limits.
Growth and inoculum controls show appropriate growth.
Results are appropriate for isolate tested (see table above).
f. See troubleshooting tips section below.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
a. Preparation of antifungal drugs and dilution schemes: Because antifungal drug preparation is a critical step in the performance of reproducible assays, commercially prepared macrobrothdilution tubes are unavailable, and techniques for preparation of the tubes vary from thosegenerally employed with antibacterial antimicrobial agents, we have outlined this procedure indetail. Potency of pure assayed powder may vary from lot to lot.
Amphotericin B (AMB) - AMB is a polyene antifungal agent. Polyene drugs formcomplexes with ergosterol which open channels in the fungal membrane that causeleakage of critical intracellular constituents and subsequent cell death. 5 mg of Amphotericin B1 ml dimethylsulfoxide (DMSO)30 ml M-3C x V 5000 µg/ml x 1 ml = 160 µg/ml x V2V = (5000 µg/ml x 1 ml)/160 µg/ml = 31.25 ml Weigh out 5 mg of amphotericin B powder using the Mettler balance.
Dissolve the powder with 1 ml of DMSO.
NOTE: Keep the amphotericin B in darkness as much as possible becauselight degrades this drug.
Dilute 1 ml (5000 µg/ml) amphotericin B in 30.25 ml of M-3 to make aninitial concentration of 160.0 µg/ml.
NOTE: 30 ml of amphotericin B of 160 µg/ml is enough to dispense into 15
racks of tubes. However, instructions are for setting up 12 racks.
NOTE: Fungizone is also available as an alternative for the powder. Dissolve a
50 mg lyophilized cake with 10 ml of sterile water. Aliquot into 1 ml portions.
Add 1 ml to 30.25 ml of M-3 and continue as with the powder.
Prepare 12 racks of 12x75 Falcon tubes labeled 16-0.03 µg/ml (i.e., 16, 8, 4,2, 1, 0.5, 0.25, 0.125, 0.06, 0.03). Label tube 16 with the name of the drug AmB Label 10-50 ml Falcon centrifuge tubes starting with tube 1 labeled at 160µg/ml. Place 8 ml of M-3 in tube 2 through tube 10. Place 16 ml of 160µg/ml drug concentration in tube 1. Perform a serial dilution of 8 ml fromtube 1 to tube 2, continue the serial dilution to tube 10.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Starting with the Falcon centrifuge tube containing the 0.03 µg/mlconcentration of amphotericin B, pour the concentration into a reservoir. Setthe Eppendorf repeating pipette to dispense 100 ul (0.1 ml). Draw up 5.0 mlof the concentrate with the Eppendorf repeating pipette into the 5.0 ml tip. Starting with the 0.03 µg/ml culture tubes, uncap one culture tube anddispense 100 ul into the culture tube; recap, and continue until all the 0.03tubes receive 100 ul. Discard the reservoir. Pour out the contents of thecentrifuge tube containing 0.06 µg/ml into a clean reservoir and repeat theprocess. Continue repeating the process until all ten sets of tubes receive 100ul of the desired concentrations.
Place the tube racks into the -70 C freezer.
Each set of tubes should demonstrate an MIC of 0.25 µg/ml to 1.0 µg/mlrange for Candida albicans (ATCC 90028).
Fluconazole (FLU) - FLU is a new difluorophenyl bis-triazole derivative that appears topossess a broader spectrum of activity than MON or KETO and is additionally muchmore water soluble. This latter attribute results in the attainment of substantial levels ofFLU in biological fluids in contrast to other azole antifungal agents.
10 mg of Fluconazole1 ml dimethylformamide (DMF)15.6 ml RPMI-1640 mediumC x V V = (10,000 µg/ml x 1 ml)/640 µg/ml = 15.63 ml Weigh out 10 mg of fluconazole using the Mettler balance.
Dilute 10,000 µg/ml) fluconazole in 14.63 ml of RPMI medium to make aninitial concentration of 640.0 µg/ml.
NOTE: This will dispense 11 racks of fluconazole MIC tubes.
Make up 11 racks of 12x75 Falcon tubes labeled 64-00.125 µg/ml (i.e., 64,32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125). Label tube 64 with the name of the drug Label 10-50 ml Falcon centrifuge tubes starting with tube 1 labeled as 640µg/ml, tube 2 labeled as 32 µg/ml, and so on to tube 10 labeled at 0.125µg/ml. Place 8 ml of RPMI in tube 2 through tube 10. Place 16 ml of 640µg/ml drug concentration in tube 1. Perform a serial dilution of 8 ml fromtube 1 to tube 2, continue the serial dilution to tube 10.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Starting with the Falcon centrifuge tube containing the 0.125 µg/mlconcentration of fluconazole, pour the concentration into a reservoir. Set theEppendorf repeating pipette to dispense 100 ul (0.1 ml). Draw up 2.5 ml ofthe concentrate with the Eppendorf repeating pipette into the 2.5 ml tip. Starting with the 0.125 µg/ml culture tubes, uncap one culture tube anddispense 100 ul into the culture tube; recap, and continue until all the 0.125tubes receive 100 ul. Discard the reservoir. Pour out the contents of thecentrifuge tube containing 0.25 µg/ml into a clean reservoir and repeat theprocess. Continue repeating the process until all ten sets of tubes receive 100ul of the desired concentrations.
Place the tube racks into the -70 C freezer.
Each set of tubes should demonstrate an MIC of 0.125 µg/ml to 0.5 µg/mlfor Candida albicans (ATCC 90028).
Fluorocytosine (5-FC) - 5-FC (fluorocytosine; Ancobon; Roche Laboratories, Nutley,N.J.) is a fluorinated pyrimidine that acts as a true antimetabolite. In addition toinhibiting cellular multiplication, 5-FC may exert a fungicidal effect dependent on thedrug concentration and period of exposure. Cytosine permease transports 5-FC acrossthe fungal membrane and into the fungal cell, where it is deaminated by cytosinedeaminase to 5-fluorouracil. Fungi that lack cytosine deaminase are resistant to 5-FC. 5-Fluorouracil results in faulty protein synthesis and has an effect on fungal nucleic acids. The 5-FC used in susceptibility testing is pure assayed powder with a potency of 100%unless specified otherwise. 5 mg of 5-Fluorocytosine1 ml sterile water7.8 ml of RPMI-1640 mediumC x V V = (5000 µg/ml x 1 ml)/640 µg/ml = 7.81 ml Weigh out 5 mg of 5-fluorocytosine powder using the Mettler balance.
Dissolve in 1 ml of sterile water.
NOTE: When placed in a 56 C water bath, it will dissolve faster and moreefficiently.
Dilute 1 ml (5000 µg/ml) 5-FC in 6.8 ml of RPMI medium to make an initialconcentration of 640.0 µg/ml.
Prepare 6 racks of 12x75 Falcon tubes labeled 64-0.125 µg/ml (i.e., 64, 32,16, 8, 4, 2, 1, 0.5, 0.25, 0.125). Label tube 64 µg/ml with the name of thedrug 5-FC.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Label 10-50 ml Falcon centrifuge tubes starting with tube 1 labeled as 640µg/ml, tube 2 labeled as 320 µg/ml and so on to tube 10 labeled as 0.125µg/ml. Place 7.8 ml of 640 µg/ml drug concentration in tube 1. Perform aserial dilution of 4 ml from tube 1 to tube 2; continue the serial dilution totube 10.
Starting with the Falcon centrifuge tube containing the 0.125 µg/mlconcentration of 5-FC, pour the concentration into a reservoir. Set theEppendorf repeating pipette to dispense 100 ul (0.1 ml). Draw up 2.5 ml ofthe concentrate with the Eppendorf repeating pipette into the 2.5 ml tip. Starting with the 0.125 µg/ml culture tubes, uncap one culture tube anddispense 100 ul into the culture tube; recap, and continue until all the 0.125tubes receive 100 ul. Discard the reservoir. Pour out the contents of thecentrifuge tube containing 0.25 µg/ml into a clean reservoir and repeat theprocess. Continue repeating the process until all ten sets of tubes receive 100ul of the desired concentrations.
Place the tube racks into the -70 C freezer.
Each set of tubes should demonstrate an MIC of 1.0 µg/ml to 4.0 µg/ml rangefor Candida albicans (ATCC 90028).
Ketoconazole (KETO) - KETO is a dioxolane-imidazole derivative with broad-spectrumantifungal activity. Like MON, its mode of action is the inhibition of ergosterolsynthesis. Additional mechanisms of action have also been identified, e.g.,respirationeffects involving cytochrome P-450. KETO (Janssen Pharmaceutical, Beerse, Belgium)is available as a pure assayed powder. Store in a desiccator at 2 to 8 C.
5 mg of Ketoconazole1 ml of dimethylsulfoxide (DMSO)31 ml of RPMI-1640 mediumC x V V = (5000 µg/ml x 1 ml)/160 µg/ml = 31.25 ml Weigh out 5 mg of ketoconazole powder (USPA) using the Mettler balance.
Dilute 1 ml (5000 µg/ml) ketoconazole in 30.25 ml of RPMI 1640 to make aninitial concentration of 160.0 µg/ml.
Prepare 12 racks of 12x75 Falcon tubes labeled 16-0.03 µg/ml (i.e., 16, 8, 4,2, 1, 0.5, 0.25, 0.125, 0.06, 0.03). Label tube 16 with the name of the drug KETO.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Label 10-50 ml Falcon centrifuge tubes starting with tube 1 labeled as 160µg/ml, tube 2 labeled as 80 µg/ml and so on to tube 10 labeled at 0.03 µg/ml. Place 8 ml of RPMI in tube 2 through tube 10. Place 16 ml of 160 µg/mldrug concentration in tube 1. Perform a serial dilution of 8 ml from tube 1 totube 2; continue the serial dilution to tube 10.
Starting with the Falcon centrifuge tube containing the 0.03 µg/mlconcentration of ketoconazole, pour the concentration into a reservoir. Set theEppendorf repeating pipette to dispense 100 ul (0.1 ml). Draw up 5 ml of theconcentrate with the Eppendorf repeating pipette into the 5 ml tip. Startingwith the 0.03 µg/ml culture tubes, uncap one culture tube and dispense 100 ulinto the culture tube; recap, and continue until all the 0.03 tubes receive 100ul. Discard the reservoir. Pour out the contents of the centrifuge tubecontaining 0.06 µg/ml into a clean reservoir and repeat the process. Continuerepeating the process until all ten sets of tubes receive 100 ul of the desiredconcentrations.
Place the tube racks into the -70 C freezer.
Each set of tubes should demonstrate an MIC of 0.03 µg/ml to 0.125 µg/mlfor Candida albicans (ATCC 90028).
Miconazole (MON) - Miconazole is one of several synthetic antifungal compounds. Theimidazoles have broad-spectrum activity. MON is a phenethyl imidazole derivativewhose mode of action with the synthesis of ergosterol via inhibition of sterol C-14demethylation, thus exerting major effects on fungal cell membranes. Additionalmechanisms of action involve cytochrome P-450.
5 mg of Miconazole1 ml of dimethylsulfoxide (DMSO)18 ml of RPMI-1640 mediumC x V V = (2900 µg/ml x 1 ml)/160 µg/ml = 18.13 ml Weigh out 2.9 mg of miconazole powder with the Mettler balance.
Dissolve the powder in 1 ml of DMSO.
Dilute 1 ml (2900 µg/ml) miconazole with 17.13 ml of RPMI-1640 to makean initial concentration of 160.0 µg/ml.
Prepare 12 racks of 12x75 Falcon tubes labeled 16-0.03 µg/ml (i.e., 16, 8, 4,2, 1, 0.5, 0.25, 0.125, 0.06, 0.03). Label tube 16 with the name of the drug MON.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Label 10-50 ml Falcon centrifuge tubes starting with tube 1 labeled as 160µg/ml, tube 2 labeled as 80 µg/ml and so on to tube 10 labeled as 0.03 µg/ml. Place 4 ml of RPMI in tube 2 through tube 10. Place 8 ml of 160 µg/ml drugconcentration in tube 1. Perform a serial dilution of 4 ml from tube 1 to tube2; continue the serial dilution to tube 10.
Starting with the Falcon centrifuge tube containing the 0.03 µg/mlconcentration of miconazole, pour the concentration into a reservoir. Set theEppendorf repeating pipette to dispense 100 ul (0.1 ml). Draw up 2.5 ml ofthe concentrate with the Eppendorf repeating pipette into the 2.5 ml tip. Starting with the 0.03 µg/ml culture tubes, uncap one culture tube anddispense 100 ul into the culture tube; recap, and continue until all the 0.03tubes receive 100 ul. Discard the reservoir. Pour out the contents of thecentrifuge tube containing 0.06 µg/ml into a clean reservoir and repeat theprocess. Continue repeating the process until all ten sets of tubes receive 100ul of the desired concentrations.
Place the tube racks into the -70 C freezer.
5 mg of Itraconazole1 ml of polyethylene glycol (PEG)30 ml of RPMI-1640 mediumC x V 5000 µg/ml x 1 m l = 160 µg/ml x V2V = (5000 µg/ml x 1 ml)/160 µg/ml = 31.25 ml Weigh out 5 mg of itraconazole powder with the Mettler balance.
Dissolve in 1 ml of PEG.
NOTE: Heat in 56 C water bath to dissolve powder. Add drug to RPMI atroom temperature.
Dilute 1 ml (5000 µg/ml) itraconazole in 30.25 ml of RPMI medium to makean initial concentration of 160.0 µg/ml.
Make up 12 racks of 12x75 Falcon tubes labeled 16-0.03 µg/ml (i.e., 16, 8, 4,2, 1, 0.5, 0.25, 0.125, 0.06, 0.03). Label tube 16 with the name of the drug ITRA.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Label 10-50 ml Falcon centrifuge tubes starting with tube 1 labeled as 160µg/ml, tube 2 labeled as 80 µg/ml and so on to tube 10 labeled as 0.03 µg/ml. Place 8 ml of RPMI in tube 2 through tube 10. Place 16 ml of 160 µg/mldrug concentration in tube 1. Perform a serial dilution of 8 ml from tube 1 totube 2; continue the serial dilution to tube 10.
Starting with the Falcon centrifuge tube containing the 0.03 µg/mlconcentration of itraconazole, pour the concentration into a reservoir. Set theEppendorf repeating pipette to dispense 100 ul (0.1 ml). Draw up 5 ml of theconcentrate with the Eppendorf repeating pipette into the 5 ml tip. Startingwith the 0.03 µg/ml culture tubes, uncap one culture tube and dispense 100 ulinto the culture tube; recap, and continue until all the 0.03 tubes receive 100ul. Discard the reservoir. Pour out the contents of the centrifuge tubecontaining 0.06 µg/ml into a clean reservoir and repeat the process. Continuerepeating the process until all ten sets of tubes receive 100 ul of the desiredconcentrations.
Place the tube racks into the -70 C freezer.
Each set of tubes should demonstrate an MIC of 0.03 µg/ml to 0.5 µg/mlrange for Candida albicans (ATCC 90028).
Formulation2.9 mg of Clotrimazole1 ml of ethanol (25%)C x V V = (2900 µg/ml x 1 ml)/160 µg/ml = 18.13 ml Preparation - Follow instructions for Miconazole.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Potency Factor x Concentration ( g/ml) Sufficient material is weighed to prepare a solution of 5000 µg/ml.
V = (5000 µg/ml x 1 ml)/185 µg/ml = 27 ml Weigh enough drug to make a 5000 µg/ml using solution.
Dilute 1 ml (5000 µg/ml) nystatin in 27 ml of RPMI medium to make aninitial concentration of 185.0 µg/ml.
Prepare 6 racks of 12x75 Falcon tubes labeled 18.5-0.07 µg/ml (i.e., 18.5,9.25, 4.6, 2.3, 1.15, 0.58, 0.29, 0.14, 0.07). Label tube 18.5 with the name ofthe drug NYS.
Label 10-50 ml Falcon centrifuge tubes starting with tube 1 labeled as 185µg/ml, tube 2 labeled as 9.25 µg/ml and so on to tube 10 labeled as 0.07µg/ml. Place 4 ml of RPMI in tube 2 through tube 10. Place 8 ml of 160µg/ml drug concentration in tube 1. Perform a serial dilution of 4 ml fromtube 1 to tube 2; continue the serial dilution to tube 10.
Starting with the Falcon centrifuge tube containing the 0.07 µg/mlconcentration of nystatin, pour the concentration into a reservoir. Set theEppendorf repeating pipette to dispense 100 ul (0.1 ml). Draw up 5 ml of theconcentrate with the Eppendorf repeating pipette into the 5 ml tip. Startingwith the 0.07 µg/ml culture tubes, uncap one culture tube and dispense 100 ulinto the culture tube; recap, and continue until all the 0.07 tubes receive 100ul. Discard the reservoir. Pour out the contents of the centrifuge tubecontaining 0.14 µg/ml into a clean reservoir and repeat the process. Continuerepeating the process until all ten sets of tubes receive 100 ul of the desiredconcentrations.
Place the tube racks into the -70 C freezer.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Each set of tubes should demonstrate an MIC of 0.57 µg/ml to 4.6 µg/mlrange for Candida albicans (ATCC 90028).
Determine the amount of Natamycin to weight by using the followingformula: Potency Factor x Concentration ( g/ml) EXAMPLE:Desired volume = 3 mlDesired concentration = 6400 µg/mlPotency = whatever is on the label of the powdered drugX = [3 ml x 6400 µg/ml]/960 µg/ml Within 30 minutes, neutralize with 0.5N HCl to a pH of 6.0 to 7.0.
Bring the volume to 10 ml with sterile water. Resulting concentration is 1922µg/ml. Store at 4 C away from light.
Dilute 1:10 (1 ml of solution and 9 ml sterile water) for a concentration of192 µg/ml.
Prepare racks of 12x75 mm Falcon tubes labeled 19.2, 9.6, 4.8, 2.4, 1.2, 0.6,0.3, and 0.15. Write on tube 19.,2, the name of the drug NATA.
Label 8-50 ml Falcon centrifuge tubes staring with tube 1 labeled 19.2, tube 2labeled 9.6, and continuing until tube 8 is labeled 0.15. Add 4 ml of RPMI totubes 2-8.
To tube 1, add 1 ml of 192 µg/ml solution to 10 mls of RPMI; serial diluteby transferring 4 mls.
Dispense 0.1 ml of antifungal solution in the 12x75 Falcon tubes labeled withappropriate concentration.
Storage conditions: -70 C freezer for 1 year Solubility in H O and most organic solvents is extremely low.
At pH 3 or 9, natamycin solution is less stable.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Streak QC strain C. albicans and test isolates on SDA. Incubate overnight in anambient-air incubator at 35 C, and check for purity.
Pick three to five isolated colonies of similar colony morphology, and passage again toSDA. Use this plate for the initial inoculum preparation.
Using the tip of a sterile applicator stick, pick five isolated colonies of similar colonymorphology at least 1 mm in diameter, and add to 5 ml of sterile 0.85% NaCl.
Adjust the suspension to 85% transmittance at 530 nm using a spectrophotometer byadding sterile 0.85% NaCl as necessary. Resulting suspension = 1 x 10 to 5 x 10 Add 1 ml of suspension to 9 ml of RPMI-1640. Resulting suspension equals 1 x 10 to 5 Tubes of this suspension may be held at 2 to 8 C for up to 3 h.
Remove appropriate number of previously prepared drug concentration tubes from -70 Cstorage, and allow them to thaw at 25 C.
Arrange drug concentration tubes for each antifungal agent in ascending order, with thehighest concentration on the left.
Include two empty tubes designated as the positive and negative growth controls, at thefar right.
Calculate the volume of standardized inoculum-broth suspension needed for the QCstrain and each isolate (1 ml of 1 x 10 - to 5 x 10 RPMI required, or a 1:10 dilution). Final concentration is 1 x 10 to 5 x 10 Using a 5-ml serological pipette, add 0.9 ml of the final inoculum to each drugconcentration tube. This dilutes the drug concentration 1:10 to obtain the concentrationindicated on the tube. After setting up each ten tubes, vortex the inoculum suspension toresuspend the yeast.
For positive growth control, add 0.9 ml of final inoculum to 0.1 ml of broth.
For negative growth control, add 1 ml of broth to tube.
Purity plate (inoculum count verification) Using a 100 ul pipette, place 0.01 ml (10 µl) of broth inoculum on an SDA plate,and streak evenly over the entire surface.
Invert plate, and incubate it at 35 C for 24 to 48 h, or until colonies are visible foraccurate counting. Record the colony count on worksheet.
Solvent control: Place 0.01 ml of inoculum into tube containing solvent diluted in RPMIor M-3.
Incubate purity plates and MIC tubes in 35 C ambient-air incubator. Shake the tubesafter inoculating them.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
Read MICs at 24 and 48 h, and score as follows.
= prominent reduction in turbidity compared with that of the drug-free 3+ = slight reduction in turbidity compared with that of the drug-free growth 4+ = no reduction in turbidity compared with that of the drug-free growth Beginning with the lowest concentration and working toward the highest concentrationfor each drug, grasp the drug-free control plus one or two drug-inoculum tubes by thecaps, and hold them up to view by transmitted light.
Using the thumb and forefinger of the opposite hand, gently flick each tube, anddetermine its score. Using a vortex will help homogenize the organism suspension.
The MICs for AMB, NATA, and NYS are the lowest concentrations with a score of0.
The MICs for all other drugs are the lowest concentrations with a score of 2+. Thiswill correspond to 50%-80% inhibition.
Using a micropipette, remove separate 100-µl samples from the MIC tube, each higher-concentration tube, and the positive growth control tube.
Using the tip of the micropipette, spread each 100-µl sample over half the surface of anSDA plate. Incubate the plates at 35 C.
Read plates when colonies on the growth control plate are visible, usually 24 h, and againat 48 h.
Any plate with five colonies or fewer is negative.
The lowest concentration for which the subculture is negative is the MFC.
a. Interpretation - Providing QC isolate MIC values is acceptable, interpret results accordingly.
b. Reporting - Providing QC is acceptable, report accordingly.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
As infections caused by yeasts continue to increase, resistance to antifungal agentscontinues to occur, in vitro antifungal susceptibility testing will continue to increase in asignificant manner. Critical factors influencing test variability include inoculumpreparation, medium composition, pH, length of incubation, and the method of endpointdetermination. This macrobroth dilution antifungal susceptibility testing protocolincorporates data regarding these variables.
It is advisable for most antifungal testing to be done at reference laboratories.
The majority of clinically significant yeast isolates are amenable to testing by thisprocedure. One exception is Malassezia furfur, which requires an oil overlay for growth,precludes testing by this method.
Controls are set up with each drug tested to ensure that the test is providing reproducibledata. Each susceptibility test must include a QC strain that provides known andreproducible MICs. The laboratorian must monitor the MIC results with the QC strain asa means to monitor drug potency.
The growth control tube ensures that the medium is functioning appropriately andthe inoculum is actively growing.
The negative broth control provides evidence that the medium is sterile.
The inoculum verification plate confirms that the inoculum is properlystandardized.
Out-of-control results indicate that the test must be repeated.
The method of Pasarell and McGinnis (108) of storing isolates at -70 C is a simple,effective method for the long-term preservation of most fungi.
Actively growing yeast colonies 14 to 48 hrs old are required for testing. Most clinicalisolates have adequate growth within 24 h, but Cryptococcus neoformans var.
neoformans may require 48 h.
To provide an adequate volume of inoculum plus RPMI at the desired final concentration(1x10 to 5x10 ) for inoculation of all drug and control tubes, count the tubes to be used and divide by 9 to determine the amount of RPMI plus standardized inoculum required.
The test should be incubated at 35 C. Incubation at a lower temperature often results inlower MICs.
A yeast that does not produce adequate growth after 24 h of incubation is read at 48 and72 h.
When reading antifungal MFCs, the cutoff of five colonies or fewer is roughly equivalentto 99.9% killing.
Report results based upon 48 h of incubation.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF YEASTS: (cont’d)
a. The methods described here are similar to those being used in multicenter collaborative studies by the Subcommittee on Antifungal Susceptibility Testing of the NCCLS. To date, no NCCLSstandards have been published.
b. Problems with trailing endpoints for the azoles in this macrobroth dilution procedure exist. Agar dilution procedures reportedly eliminate this problem, but MFCs cannot be determined bythat method.
II. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
Antifungal drug dilutions are used to determine the minimal inhibitory concentration (MIC) forantifungal agents. Serial dilutions of antifungal agents are dispensed into appropriately labeledtubes. Each tube is then inoculated with a standardized nutrient broth suspension of the fungusbeing tested. Susceptibility testing of moulds is being evaluated in a multicenter collaborative studyby the Subcommittee on Antifungal Susceptibility Testing of the NCCLS. This procedure could beused for dermatophytes only if an appropriate control isolate is used. No proposed method has beendeveloped.
B. Specimen: Growth from three to five isolated colonies of similar colony morphology grown for 3-5 days on potato dextrose agar (PDA) slants.
C. Materials: Date and label all reagents and media when prepared and placed in use.
1. Media and Reagents (storage conditions) a. SDA plates, Emmons modification, pH 7 (2-8 C).
b. SDA slants, Emmons modification, pH 7 (2-8 C).
c. RPMI-1640 medium buffered with 0.165 M MOPS [3-(N-morpholino) propanesulfonic acid] containing L-glutamine and lacking sodium bicarbonate; supplied dehydrated by AmericanBiorganics, Inc., Niagara Falls, N.Y.; catalog no. R63165 (2 to 8 C; shelf life, 12 months).
d. Bacto Antibiotic Medium 3 (M-3) (2-8 C; 3 months) e. Sterile distilled reagent-grade water (25 C).
g. Dimethyl sulfoxide (DMSO) (25 C), polyethylene glycol (PEG) (MW 200), and h. Macrobroth dilution tubes (sterile, 12x75 mm, polystyrene, snap cap) containing 0.1 ml volumes of the following antimicrobial agents (diluted in RPMI-1640 except AMB which is inM-3) at 10 times the final concentrations (see Section V) maintained at -70 C.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
MOULDS: (cont’d)
i. Potato Dextrose Agar Slants (PDA) (2-8 C) a. Sterile wooden applicator sticks or sterile swabs c. 500 ml capacity Millipore filtration units with 0.22-µm-pore-size d. Sterile 1, 5, and 10 ml disposable serological pipettes and pipette bulb c. Micropipette, 100 µl capacity with sterile disposable tips d. Repetitive dispensing pipette, 100 µl capacity with sterile disposable syringes a. Candida albicans (ATCC 90028) and Paecilomyces variotii (ATCC 36257) b. Maintain permanent stock cultures at -70 C on PDA slants.
With sterile wooden applicator stick, chip off a small portion of the frozen surface of anSDA slant and streak for isolation.
Weekly working stock cultures are prepared by subculturing three to five colonies ofsimilar colony morphology from the previous week's working stock plate to a fresh SDAplate. Moulds are subcultured to a fresh PDA slant.
d. Subculture from working stock is plated to another SDA plate or PDA slant on the day before IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
MOULDS: (cont’d)
Candida albicans (ATCC 90028)
Paecilomyces variotii (ATCC36257)
2. Positive growth control should demonstrate good growth and be free of contaminating organisms.
3. Negative growth control should be free of growth.
4. Inoculum count verification plate should show several colonies.
5. Solvent growth control should demonstrate good growth.
6. Test considered in control when: a. MIC's for QC strains are within acceptable limits.
b. Controls show appropriate growth.
c. Results are appropriate for isolate tested.
1. Preparation of antifungal drugs and dilution schemes: 5 mg of Amphotericin B1 ml dimethylsulfoxide (DMSO)31 ml M-3 mediumC x V = C x V 5000 µg/ml x 1 ml = 160 µg/ml x V2V = (5000 µg/ml x 1 ml)/160 µg/ml = 31.25 ml Weigh out 5 mg of amphotericin B powder using the Mettler balance.
Dissolve the powder with 1 ml of DMSO.
NOTE: Keep the amphotericin B in darkness as much as possible because light
degrades the amphotericin B.
Dilute 1 ml (5000 µg/ml) amphotericin B in 30.25 ml of M-3 medium to make an
initial concentration of 160.0 µg/ml.
NOTE: 30 ml of amphotericin B at 160 µg/ml is enough to dispense into 15 racks
of tubes. However, instructions are for setting up 12 racks.
Make up 12 racks of 12x75 Falcon tubes labeled 16-0.03 µg/ml (i.e., 16, 8, 4, 2, 1,0.5, 0.25, 0.125, 0.06, 0.03). Label tube 16 with the name of the drug AMB.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
MOULDS: (cont’d)
Label 10, 50 ml Falcon centrifuge tubes starting with tube 1 labeled as 160 µg/ml,tube 2 labeled as 80 µg/ml, and so on to tube 10. Place 8 ml of M-3 in tube 2through tube 10. Place 16 ml of 160 µg/ml drug concentration in tube 1. Perform aserial dilution of 8 ml from tube 1 to tube 2, continue the serial dilution to tube 10.
Starting with the Falcon centrifuge tube containing the 0.3 µg/ml concentration ofamphotericin B, pour the drug solution into a reservoir. Set the Eppendorfrepeating pipette to dispense 100 µl (0.1 ml). Draw up 5.0 ml of the concentratewith the Eppendorf repeating pipette into the 5.0ml tip. Starting with the 0.03µg/ml culture tubes, uncap one culture tube and dispense 100 µl into the culturetube; recap, and continue until all the 0.03 tubes receive 100 µl. Discard thereservoir. Pour out the content of the centrifuge tube containing 0.6 µg/ml into aclean reservoir and repeat the process. Continue repeating the process until all tensets of tubes receive 100 µl of the desired concentrations.
Place the tube racks into the -70 C freezer.
Each set of tubes should demonstrate an MIC for C. albicans (ATCC 90028) of0.25 µg/ml to 1.0 µg/ml and P. variotii (ATCC 36257) of 0.25 µg/ml to 1.0 µg/ml.
20 mg of Fluconazole1 ml dimethylformamide (DMF)15.6 ml RPMI-1640 mediumC x V = C x V 20,000 µg/ml x 1 ml = 1280 µg/ml x V2V = (20,000 µg/ml x 1 ml)/1280 µg/ml = 15.63 ml Weigh out 20 mg of fluconazole using the Mettler balance.
Dilute 1 ml (20,000 µg/ml) fluconazole in 14.63 ml of RPMI to make an initial
concentration of 1280 µg/ml.
NOTE: This will dispense 11 racks of FLU MIC tubes.
Make up 11 racks of 12x75 Falcon tubes labeled 128-0.125 µg/ml (i.e., 128, 64,32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125). Label tube 128 with the name of the drug FLU.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
MOULDS: (cont’d)
Label 11, 50 ml Falcon centrifuge tubes starting with tube 1 as 1280 µg/ml, tube 2labeled at 640 µg/ml, and so on to tube 11 labeled at 1.25 µg/ml. Place 8 ml ofRPMI in tube 2 through tube 11. Place 16 ml of 1280 µg/ml drug concentration intube 1. Perform a serial dilution of 8 ml from tube 1 to tube 2, continue the serialdilution to tube 11.
Starting with the Falcon centrifuge tube containing the 1.25 µg/ml concentration ofFluconazole, pour the drug solution into a reservoir. Set the Eppendorf repeatingpipette to dispense 100 µl (0.1 ml). Draw up 2.5 ml of the concentrate with theEppendorf repeating pipette into the 2.5 ml tip. Starting with the 0.125 µg/mlculture tubes, uncap one culture tube and dispense 100 µl into the culture tube;recap, and continue until all the 0.125 tubes receive 100 µl. Discard the reservoir. Pour out the content of the centrifuge tube containing 0.25 µg/ml into a cleanreservoir and repeat the process. Continue repeating the process until all 11 sets oftubes receive 100 µl of the desired concentrations.
Place the tube racks into the -70 C freezer.
Each set of tubes should demonstrate an MIC for C. albicans (ATCC 90028) of 0.125 µg/ml to 0.5 µg/ml and P. variotii (ATCC 36257) of 1.0 µg/ml to 4.0 10 mg of Itraconazole1 ml of polyethylene glycol (PEG)30 ml of RPMI-1640 mediumC x V = C x V 10,000 µg/ml x 1 ml = 320 µg/ml x V2V = (10,000 µg/ml x 1 ml)/320 µg/ml = 31.25 ml Weigh out 10 mg of itraconazole powder using the Mettler balance.
Dilute 1 ml (10,000 µg/ml) itraconazole in 30.25 ml of RPMI to make an initialconcentration of 320 µg/ml.
Make up 12 racks of 12x75 Falcon tubes labeled 32-0.03 µg/ml (i.e., 32, 16, 8, 4,2, 1, 0.5, 0.25, 0.125, 0.06, 0.03). Label tube 32 with the name of the drug ITRA.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
MOULDS: (cont’d)
Label 11, 50 ml Falcon centrifuge tubes starting with tube 1 labeled as 320 µg/ml,tube 2 labeled as 160 µg/ml and so on to tube 11 labeled as 0.3 µg/ml. Place 8 mlof RPMI in tube 2 through tube 11. Place 16 ml of 320 µg/ml drug concentrationin tube 1. Perform a serial dilution of 8 ml from tube 1 to tube 2; continue theserial dilution to tube 11.
Starting with the Falcon centrifuge tube containing the 0.3 µg/ml concentration ofITRA, pour the drug solution into a reservoir. Set the Eppendorf repeating pipetteto dispense 100 µl (0.1 ml). Draw up 5 ml of the concentrate with the Eppendorfrepeating pipette into the 5 ml tip. Starting with the 0.03 µg/ml culture tubes,uncap one culture tube and dispense 100 µl into the culture tube; recap, andcontinue until all the 0.03 tubes receive 100 µl. Discard the reservoir. Pour out thecontent of the centrifuge tube containing 0.6 µg/ml into a clean reservoir and repeatthe process. Continue repeating the process until all 11 sets of tubes receive 100 µlof the desired concentrations.
Place the tube racks into the -70 C freezer.
Quality control: Each set of tubes should demonstrate an MIC for C. albicans (ATCC90028) of 0.03 µg/ml to 0.5 µg/ml and P. variotii (ATCC 36257) of 0.03 µg/ml to 0.5µg/ml.
d. Ketoconazole and Miconazole (KETO and MON) The tests are conducted as outlined in In Vitro Antifungal Susceptibility Testing ofYeasts.
Paecilomyces variotii (ATCC 36257) and C. albicans (ATCC 90028) are used ascontrols.
a. Streak QC strain of C. albicans on SDA. Incubate overnight in an ambient-air incubator at 35 C and check for purity. Test isolates and P. variotii (ATCC 36257) will be grown on PDAslants. Two slants per mould are required. Some isolates of Blastomyces dermatitidis andParacoccidioides brasiliensis may need to be subcultured to Emmons modification of SDAslants, rather than PDA slants in order to obtain better growth.
b. Pick three to five isolated colonies of similar colony morphology and subculture again to SDA. Use this plate for the initial inoculum preparation. The QC isolate C. albicans is 24-48 h old,the test moulds are 48-72 h old. Slow growing moulds should be incubated up to 7 days beforetesting.
c. Using the tip of a sterile applicator stick, pick five isolated colonies of similar colony morphology and add to 5 ml of sterile 0.85% NaCl. For moulds growing on slants, add 5 ml ofsterile 0.85% NaCl to each tube, rub surface with a sterile wooden applicator stick, and thentransfer suspension to a sterile tube.
e. Adjust suspension to a McFarland 0.5 turbidity standard.
f. Add 1 ml of suspension to 9 ml of RPMI 1640 or M-3 for amphotericin B (1:10 dilution).
g. Tubes of this suspension may be held at 2 to 8 C for up to 3 h.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
MOULDS: (cont’d)
a. Remove appropriate number of previously prepared drug concentration tubes from -70 C storage, and allow them to thaw at room temperature.
b. Arrange drug concentration tubes for each antifungal agent in ascending order, with the highest concentration on the left.Include two empty tubes, designated as the positive and negativegrowth controls, at the far right, as well as 2 tubes labeled DMF, DMSO, or PEG control.
c. Calculate the volume of standardized inoculum-broth suspension needed for QC strains and each isolate (1 ml McFarland 0.5 standard suspension for every 9 ml of either RPMI or M-3 isrequired).
d. Using a 5-ml serological pipette, add 0.9 ml of the final inoculum to each drug concentration tube. This dilutes the drug concentration 1:10 to obtain the concentration indicated on the tube. Uncap each tube one at a time and recap. Every ten tubes vortex the inoculum suspension toresuspend the mould in the solution. Never leave the inoculum tube uncapped.
For positive growth control, add 0.9 ml of final inoculum to 0.1 ml of broth.
For negative growth control, add 1 ml of broth to tube.
For DMF, DMSO, or PEG controls, add 0.1 ml of the 0.5% McFarland calibratedinoculum.
f. Purity plate (inoculum count verification) Using a 100 µl pipette, place 0.01 ml (10 µl) of broth inoculum on an SDA plate, andstreak evenly over the entire surface.
Invert plate, and incubate it at 35 C for 48 to 96 h (or until colonies are visible foraccurate counting). Record the colony count on worksheet.
g. Incubate purity plates and MIC tubes in 35 C ambient-air incubator. Shake the tubes after a. The test is read when the growth control shows adequate growth, which is typically 24-48 hours for most moulds, but it could be up to 96 hours.
b. Read MICs the first day that the growth control shows growth and then 24 hours later. Score = prominent reduction in turbidity compared with that of the drug-free growth 3+ = slight reduction in turbidity compared with that of the drug-free growth control 4+ = no reduction in turbidity compared with that of the drug-free growth control c. Beginning with the lowest concentration and working toward the highest concentration for each drug, grasp the drug-free control plus one or two drug-inoculum tubes by the caps, and holdthem up to view by transmitted light.
I. IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF DIMORPHIC FUNGI AND
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING MOULDS: (cont’d)
d. Using the thumb and forefinger of the opposite hand, gently flick each tube, and determine its The MICs for AMB are the lowest concentrations with a score of 0.
The MICs for azoles are the lowest concentrations with a score of 2+. This willcorrespond to 50-80% inhibition.
1. Interpretation - Providing QC is acceptable, interpret results accordingly.
2. Reporting - Providing QC is acceptable, report accordingly in the worksheets.
1. Principle - Although testing is currently nonstandardized, preliminary data being generated from multicenter studies by the Subcommittee on Antifungal Susceptibility Testing of the NCCLSsuggest that acceptable inter- and intralaboratory agreements can be obtained. Critical technicalfactors influencing test variability include inoculum preparation, medium composition, pH, lengthof incubation, and the method of endpoint determination.
a. Controls are set up for each drug tested on a daily basis to ensure that the test is providing b. Many methods for maintaining stock culture collections of fungi can be used. The method of Pasarell and McGinnis (108) of storing isolates at -70 C is an effective method for thepreservation of fungi.
a. Actively growing fungal colonies are required for testing; thus the requirement for a 72- to 96- h subculture. Owing to slow growth rates, some moulds may require more than 96 h foradequate growth to be present.
b. The test described here is incubated at 35 C. Incubation at lower temperature (e.g., 30 C) often results in lower MICs. Lower incubation temperature may be required for moulds that donot actively grow at 35 C.
c. A fungus that does not produce adequate growth after 72 hrs of incubation may be encountered. When this occurs, read results at 96 h.
d. Report on the worksheet an initial (usually at 48 h) and second (72 h later) reading of MICs to provide information on antifungal activity over time. The optimum time for reading endpointsfor various antifungal agents is currently being addressed by an NCCLS subcommittee.
e. The concentration marked on the tubes reflect the final drug concentration that they will 4. Forms - See the following tables.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING REFERENCES:
1. McGinnis, M.R., and M.G. Rinaldi. 1985. Antifungal drugs: mechanisms of action, drug
resistance, susceptibility testing, and assays of activity in biological fluids, p.223-281. In V.
Lorian (ed.), Antibiotics in Laboratory Medicine. The Williams & Wilkins Co., Baltimore.
2. Rinaldi, M.G., and A.W. Howell. 1988. Antifungal antimicrobics: laboratory evaluation, p. 325-
356. In B. Wentworth (ed.), Diagnostic Procedures for Mycotic and Parasitic Infections, 7th ed.
American Public Health Association, Washington, D.C.
3. Walsh, T.J., G.P. Melcher, M.G. Rinaldi, J. Lecciones, D.A. McGough, P. Kelly, J. Lee, D.
Callender, M. Rubin, and P.A. Pizzo. 1990. Trichosporon beigelii, an emerging pathogen
resistant to amphotericin B. J. Clin. Microbiol. 28:1616-1622.
4. Rex, J.H., C.R. Cooper, Jr., W.G. Merz, J.N. Galgiani, and E.J. Anaissie. 1995. Detection
of amphotericin B-resistant Candida isolates in a broth-based system. Antimicrob. Agents
Chemother. 39:906909.
5. Pfaller, M.A., M. Bale, B. Buschelman, M. Lancaster, A. Espenel-Ingroff, J.H. Rex, M.G.
Rinaldi, C.R. Cooper, and M.R. McGinnis. 1995. Quality control guidelines for National
Committee for Clinical Laboratory Standards recommended broth macrodilution testing of
amphotericin B, fluconazole, and flucytosine. J. Clin. Microbiol. 33:1104-1107.
IV. SUPPLEMENTAL READING:
Dick, J.D., B.R. Rosengard, W.G. Merz, R.K. Stuart, G.M. Hutchins, and R. Saral. 1985. Fatal
disseminated candidiasis due to amphotericin B-resistant Candida guilliermondii. Ann. Intern. Med.
102:67-68.
Doern, G.V., T.A. Tubert, K. Chopin, and M.G. Rinaldi. 1986. Effect of medium composition on
results of macrobroth dilution antifungal susceptibility testing of yeasts. J. Clin. Microbiol. 24:507-
511.
Drutz, D.J., and R.I. Lehrer. 1978. Development of amphotericin B-resistant Candida tropicalis in a
patient with defective leukocyte function. Am. J. Med. Sci. 276:77-92.
Galgiani, J.N. 1987. The need for improved standardization in antifungal susceptibility testing, p. 15-
24. In R.A. Fromtling (ed.), Recent Trends in the Discovery, Development and Evolution of
Antifungal Agents. J.R. Prous Science Publishers, S.A., Barcelona, Spain.
Guinet, R., J. Chanas, A. Goullier, G. Bonnefoy, and P. Ambroise-Thomas. 1983. Fatal septicemia
due to amphotericin B-resistant Candida lusitaniae. J. Clin. Microbiol. 18:443-444.
McGinnis, M.R. 1980. Susceptibility testing and bioassay procedures, p. 412-446. In Laboratory
Handbook of Medical Mycology. Academic Press, Inc., New York.
Merz, W.G. 1984. Candida lusitaniae: frequency of recovery, colonization, infection, and
amphotericin B resistance. J. Clin. Microbiol. 20:1194-1195.
NCCLS. 1986. Antifungal Susceptibility Testing. Committee report, vol. 5, no. 17. NCCLS,
Villanova, Pa.
IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING SUPPLEMENTAL READING: (cont'd)
Pasarell, L. and M.R. McGinnis. 1992. Viability of Fungal Cultures Maintained at -70 C. J. Clin.
Microbiol. 30(4): 1000-1004
Pfaller, M.A., Burmeister, M.S. Bartlett and M.G. Rinaldi. 1988. Multicenter evaluation of four
methods of yeast inoculum preparation. J. Clin. Microbiol. 26:1437-1441.
Pfaller, M.A., M.G. Rinaldi, J.N. Galgiani, M.S. Bartlett, B.A. Body, A. Espinel-Ingroff, R.A.
Fromtling, G.S. Hall, C.E. Hughes, F.C. Odds, and A.M. Sugar. 1990. Collaborative investigation
of variables in antifungal susceptibility testing of yeasts. Antimicrob. Agents Chemother. 34:1648-
1654.
Powderly, W.G., G.S. Kobayashi, G.P. Herzig, and G. Medoff. 1988. Amphotericin B-resistant
yeast infection in severely immunocompromised patients. Am. J. Med. 84:826-832.
ANTIFUNGAL SUSCEPTIBILITY TESTING QUALITY CONTROL DOCUMENTATION
Inoc. Count
MIC (µg/ml)
MLC (µg/ml)
(100 - 500)
24 hr/48 hr
24 hr/48 hr
ANTIFUNGAL SUSCEPTIBILITY TESTING WORKSHEET
INOCULUM STANDARDIZATION (1-5 x 10 CFU/ml)
MACRO BROTH DILUTION
Antifungal Agent
(µg/ml)
Drug "Set"
ANTIMICROBIAL STOCK SOLUTION QC
QC OF BACHARACH MODEL 35 SPECTROPHOTOMETER
With Didymium filter at 530 nm, adjust to read 50-60% T with oa/100% Coarse knob. Set at 520 and move to 540. Watch for lowest %T and record.
Record also Wavelenth at the lowest %T which should be 530 ± 1.
Procedure should be performed weekly.
Zero %T adjustment whould be performed with daily use.
Refer to Operating instructions of Model 35

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