TITLE: Preparing 1/2 SŠ Algal Inorganic Nutrient Medium
AUTHORS: Jakub Nedbal, Lu Gao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes preparation of 1/2 SŠ inorganic nutrient medium for </span><span style = "font-style:italic;">C. vulgaris,</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">D. quadricauda,</span><span> and other fresh water microalgae. The medium is prepared out of sterile components inside sterile laminar flow hood. The medium recidpe is based on [</span><a href="https://doi.org/10.1007/BF02909100" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Zachleder & Šetlík, 1982</span></a><span>].</span></div></div>

[STEPS]
?. [Preparatory Steps]
Prepare the components listed int he following table. Make sure all constituents have been sterilized and that the deionized water has been autoclaved.Work in a sterile laminar flow hood.  ABCD1ComponentQuantityUnitNote2Final Volume503ml3Maximum Dry Culture Weight0.5g/l45Deionized Water500mlAutoclaved61/2 SŠ Stock KNO₃ (1.83 M)833μl71/2 SŠ Stock KH₂PO₄ (184 mM)417μl81/2 SŠ Stock MgSO₄·7H₂O (82.8 mM)417μl9Sodium Bicarbonate (500 mM)833μlDo not autoclave101/2 SŠ Stock Fe·Na·EDTA (9.1 μM)417μlDo not autoclave111/2 SŠ Stock CaCl₂ (66 μM)417μl121/2 SŠ Trace Elements (1 ml/l)42μl132M KOH20μlThe goal is to get pH 7.5
ABCD1ComponentQuantityUnitNote2Final Volume503ml3Maximum Dry Culture Weight0.5g/l45Deionized Water500mlAutoclaved61/2 SŠ Stock KNO₃ (1.83 M)833μl71/2 SŠ Stock KH₂PO₄ (184 mM)417μl81/2 SŠ Stock MgSO₄·7H₂O (82.8 mM)417μl9Sodium Bicarbonate (500 mM)833μlDo not autoclave101/2 SŠ Stock Fe·Na·EDTA (9.1 μM)417μlDo not autoclave111/2 SŠ Stock CaCl₂ (66 μM)417μl121/2 SŠ Trace Elements (1 ml/l)42μl132M KOH20μlThe goal is to get pH 7.5
?. [Preparatory Steps]
Autoclave 500 ml of deionized water in a glass clean bottle.Let it cool down.
?. [Preparatory Steps]
Prepare all the reagents and labware.
?. [Medium Preparation]
Work in a sterile laminar flow hood.Add components listed in table in STEP 1 into the autoclaved deionized water in the order listed in the table. Do not add KOH yet.
?. [Medium Preparation]
Adjust the pH to 7.5 using KOH.
?. [Medium Preparation]
Take a small aliquot (1.5 ml) of the medium into a 15 ml Falcon tube.
?. [Medium Preparation]
Measure the pH of the medium inside the Falcon tube using the pH meter.
?. [Medium Preparation]
Empty the Falcon tube.
?. [Medium Preparation]
Add a small aliquot (5μl) of 2 M KOH to the medium. Mix well by closing the bottle and shaking or turning up side down a few times.
?. [Medium Preparation]
Repeat by going back to STEP 5.1, until pH reaches a value of approiximatelly 7.5.
?. [Medium Preparation]
Label the bottle (1/2 SŠ, initials, date, pH).
?. [Medium Preparation]
Store at 4 °C in dark.Warm up to room temperature each time before use.Only open in a sterile cabinet.
?. [Summary]
This protocol describes the preparation of a medium for growth of freshwater algae on an orbital shaking incubator. The medium is to be used to dilute the growing cultures at regular intervals. The medium needs to be stored in a fridge at 4 °C and warmed up each time to room temperature to minimize the stress to the culture.Protocol for preparing the medium using filter sterilization: Preparing 1/2 SŠ algal inorganic nutrient medium​​1/2 SŠ inorganic nutrient medium: Recipe from the Culture Collection of Autotrophic Organisms​​Zachleder & Šetlík, 1982: The original article introducing this medium​