Sterile autoclaved broth

To estimate the minimum fungicidal concentration (MFC), a loopful of biomass is taken from respective tubes containing the MIC and higher concentrations of antimicrobial compound and inoculated into tubes containing sterile (autoclaved) broth and incubated at 25 2 °C for 72 h (for fungi). Subsequently a loopful from these tubes is then streaked on to the surface of sterile agar plates and incubated at 25 2 °C for 72 h [29, 30]. 

PDCB was prepared by suspending cut-up unpeeled potatoes (100 g L ) and carrots (10 g L ) in purified water and heated to boiling in a microwave oven for 5-10 min. Dextrose (d-(- -)-glucose) was added (30 g L ). The medium was sterilized by autoclaving for 20 min at 121 °C and then decanting off the broth. The broth is clear to slightly opalescent and yellowish in colour. No pH adjustment was made. 

LB broth 10 g/L Bacto-tryptone, 5 g/L Bacto-yeast extract, 10 g/L NaCl, adjusted to pH 7.0 with NaOH and sterilized by autoclaving. 

LB agar add 1.5g Bacto-agar/100 mL LB broth. Sterilize by autoclaving... 

After the pH had been adjusted, 5 g of calcium carbonate, 5 ml of soybean oil antifoam and 0.020 g of Acridine Orange dye were added. The mixture was then autoclaved at 20 psi (250°F) for 15 minutes in order to sterilize the contents, before transferring the broth and mycelium thereto. 

Mueller-Hinton broth (Oxoid). This is prepared by dissolving 21 g of powder in one litre of distilled water and autoclaving at 121°C for 15 min. Alternatively, smaller amounts can be prepared and filter sterilized. 

Luria broth—Luria broth Prepare five 100-ml and five 10-liter solutions containing 10 g/liter Bacto-Tryptone, 5 g/liter Bacto-Yeast extract, 5 g/liter NaCl, 0.5 g/liter antifoam agent (e.g., Polyglycol P-2000), and 1 g/liter glucose (autoclave separately). Autoclave the main solution and separate solution of concentrated glucose for 20 min at 105 to 110°C and cool use sterile conditions to add the glucose solution to the main solution and use within 24 hr. 

All samples for study of the rate of manganese solution were prepared in a glucose enriched nutrient broth medium adjusted to a desired pH and poured into five replicate test tubes for each observation point. The sterilized tubes were taken from the autoclave as hot as possible for safe handling. Crystals of sterilized manganese dioxide were added. A vaspar plug was poured on top of the mixture and a screw cap tightened... 

Thioglycolate broth 3% thioglycolate broth (Becton Dickinson, Franklin Lakes, NJ) in sterile glass-distilled water. The bottle is placed in a boiling water bath to ensure complete dissolution of the powder. Autoclave the solution at 15 psi, 121°C for 15 min. Store at room temperature and use within 1 d. 

Tryptic soy broth (TSB) (Difco, Franklin Lakes, NJ), supplemented with 0.5% glucose after sterilization by autoclaving. 

Luna broth 5 g yeast extract (Difco), 10 g NaCl, 10 g Bactotryptone (Difco, Detroit, MI) dissolved in 1 L of distilled water. Sterilize by autoclaving. Add 0.01 vol of sterile 20% glucose before use. 

The pH of solution A is set to 7.0. 1 ml of solution B is added to solution A, which is then made up to 1000 ml with demineralized water. 5 ml quantities of the broth are put into test tubes. Sterilization is then carried out in an autoclave for 20 minutes at 121 C. 

The agar plate method was used to determine the minimum inhibition concentration (MIC) of CM, Q, and CMQ as follows the samples were prepared at a concentration of 1% (w/v) and then autoclaved at 121°C for 25 min. Duplicate twofold serial dilutions of each sample were added to nutrient broth (beef extract 5 g, peptone 10 g to 1000 mL distilled water, pH 7.0) for final concentration of 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, and 0.00313%. Some samples were prepared and diluted by the same way except for a final concentration of 0.00065% and 0.00033%. The culture of each bacterium was diluted by sterile distilled water to 105-106 CPU mL. A loop of each suspension was inoculated on nutrient medium with sample or control added. After inoculation, the plates were incubated at 37°C for 72 h, the colonies were counted, and the MIC values were obtained. The MIC was considered to be the lowest concentration that completely inhibited against on agar plates comparing, disregarding a single colony or a faint haze caused by the inoculum (Speciale et al. 2002). 

Media are prepared to defined formulations using requisite amounts of appropriate chemicals. Many of the more commonly used media, such as nutrient broth, tryptone soya broth, malt agar, and potato dextrose agar, are available in pre-mixed dehydrated form from various specialist suppliers. Any components of media that are heat-sensitive must be added to the remainder of the medium after it has been heat-sterilized by autoclaving this is normally done by syringing in these particular components as concentrated aqueous solutions through disposable sterile filter units available from various specialist suppliers. 

Flasks of broth or agar, complete except for any heat-sensitive components of the medium formulation, are sterilized in an autoclave under appropriate conditions (usually steam pressure of 15 pounds per square inch for 15 minutes). For small-scale biotransformations it is possible to use a domestic pressure cooker for steam sterilization. 

Sterilization of solutions, such as fermentation broths, containing thermally labile compounds is accomplished by sterile filtration. This requires the use of sterile membranes of porosity of <0.45 pm and a previously sterilized filter housing and flasks. Filter housings and clamps should be packaged prior to autoclaving and not opened until use. One may purchase special autoclavable wrappings of various sizes for this purpose alternatively, aluminum foil is frequently used. 

Some sugars (glucose and galactose) can be autoclaved. These are independently incorporated into the yeast extract (YE) broth before autoclaving. Others must be filter sterilized and are added, after autoclaving, to the YE broth/Durham tube setup (see section 3.5.4). 

Terrific Broth (TB) To 700 mL H2O, add 12 g Bacto Tryptone, 24 g yeast extract, 10 mL glycerol, and 2 g of glucose (tee Note 3). Stir to dissolve solutes then bring volume to 900 mL. Sterilize by autoclaving. Cool media to room temperature then add 100 mL of filter sterilized lOx phosphate buffer (100 mL 2.31 g KH2PO4,12.54 g K2HPO4). Store liquid TB at room temperature, add antibiotics just prior to use. 

Luria Broth liquid media per liter, 10 g tryptone, 5 g yeast extract, 10 g NaCl, dissolved in deionized water and sterilized by autoclaving. 

Because many sugars should not be autoclaved, prepare as 2% w/v solutions in the yeast extract broth and individually filter sterilize the solutions through 0.45 im membranes. For raffinose, prepare a 4% w/v solution because some strains only use part of the molecule (Yarrow, 1998). Transfer 10 mL aliquots of yeast extract broth to sterile 18 x... 

L plantarum is grown in Lactobacillus MRS broth (from DIFCO) at 37°C (shaking is not required) and kept at — 80°C in 25% glycerol, 50% reconstituted rnUk (12% milk powder) in MRS. Plates (245 X 245 mm Petri dishes) are prepared daily (2% agar in Biotin Assay Medium from DIFCO) autoclaved at 0.8 atm for 15 min and inoculated with 1% of a fresh overnight culture of L. plantarum previously washed twice with sterile water. The plate... 

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