PRACTICAL 1 : Microalgal sampling equipments & Preparation of algal medium culture

OBJECTIVES

1.      Familiarize with the different microalgal sampling equipment

2.      Prepare different algal medium culture

INTRODUCTION

The sampling cycle can be subdivided into a presampling, sampling, field sample manipulation, transportation, laboratory manipulation and sample maintenance. Thus, sampling equipment are needed to make the sampling work is easy and efficient.

A culture can be defined as an artificial environment in which the algae grow. In theory, culture conditions should resemble the alga’s natural environment as far as possible; in reality many significant differences exist, most of which are deliberately imposed.

A)    Sampling equipments and their uses

Equipment	Uses
·         Water sampler	·         To collect water sample in pond or lake at different depth
·         Plankton net/ phytoplankton net 20 micron	·         for collecting a water sample that will be used to determine the presence of phytoplankton ·
·         Hydrolab	·         To measure multi parameter of water such as temperature, dissolve oxygen, conductivity, pH, salinity
·         Refractrometer	·         To check the salinity of water
·         DO meter	·         To check dissolve oxygen in the water
·         pH meter	·         to measure the amount of dissolved oxygen in water
·         Water depth sensor	·         To measure the depth in the water
·         GPS	·         Giving a location of sampling
·         Sampling bottles	·         To keep water sample before doing the analysis in laboratory.
·         Lugol’s solution	·         To preserve the water sample
·         Data sheets	·         To write the data, station, gps location, time, weather, depth, pH, temperature, and stratification

B)    Preparation of algal medium culture

There are many different types of algal culture media such as bold’s basal medium (BB), f/2 medium, f/2 + Si (Guillard’s medium for diatoms), provosali enriched seawater media (PES), von stosch enrichment (VSE) and macrophyte medium culture (Murashige  and skoog (MS) media formula.

Bold‘s Basal Medium (BB)

This medium is for freshwater algae. The stock that are needed for preparation this medium is NaNO_3, MgSO₄·7H₂O, NaCl , K₂HPO₄, KH₂PO_, CaCl₂·2H₂O. This stock solution is 10.0 ml each per litre. For agar, add 15.0 g/L Bacteriological Agar and autoclave at 15 psi for 15 minutes. Trace elements solutions that are required are ZnSO₄·7H₂O, MnCl₂·4H₂O, MoO_3, CuSO₄·5H₂O, Co(NO₃)₂·6H₂O, EDTA, KOH, FeSO₄·7H₂O, H₂SO_4. This stock solution is needed only 1.0 ml each per litre.

f/2 Medium

Guillard’s (F/2) Marine Water Enrichment Solution 50 X, is a concentrate of major nutrients, trace metals and vitamins that can be diluted in filtered seawater to support reconstitution of Guillard’s (F/2) Marine Enrichment medium. Guillard’s (F/2) Marine Enrichment medium is a widely used enriched seawater medium for growing costal marine algae (diatoms).

f/2 agar: Prepare 1 liter of f/2 medium and dissolve 9g Bacto-agar (heat and mix). For test tubes, dispense dissolved agar medium into tubes, autoclave, and then cool with tubes slanted at an angle. For Petri plates, autoclave in a flask, cool almost to the gelling point, and then aseptically dispense into sterile Petri plates

f/2 Medium + Si

Prepare as for f/2 Medium but omit Na₂SiO₃·9H₂O. Make final volume up to 1.0 L with filtered seawater. Autoclave after all additions. Extra Silicon and Selenium in this medium are beneficial to several diatom strains. Prepare as for f/2 Medium, then add 1.0 ml of Selenium Stock Solution (1.29 mg H₂SeO₃/L dH₂O) and 1.0 ml of Silicate Stock Solution (30.0 g Na₂SiO₃·9H₂O/L dH₂O). Make final volume up to 1.0 L with filtered seawater. Then after that autoclave.

Provasoli Enriched Seawater Media (PES)

For the preparation of (PES) it needed four solutions. Solution 1 is base solution which have deionized water, tris buffer, sodium hydroxide, glycerophosphate and thiamine (Vit. B1). Then solution 2 contain Fe act as EDTA complex; 1:1 molar. While for solution 3 is for metal which are include deionized water, disodium ethylenediamine tetraacetate, boric acid, ferric chloride, manganese sulfate monohydrate, cobaltous sulfate monohydrate and zinc sulfate, 7-hydrate. Last solution contains vitamins mixing with deionized water, vitamin B12, and biotin. All solutions are mixed together. The final solution pH is adjusted to 7.8 using HCl.

Von Stosch Enrichment (VSE)

To prepare medium (VSE) its needed six solutions. Solution 1 is nitrogen contain. It have deionized water and ammonium chloride. Then solution 2 is phosphate which are deionized water and sodium phosphate. Next solution 3 is based on iron. There are deionized water and ferrous sulfate. Solution 4 contain EDTA plus deionized water. Solution 5 manganese chloride and deionized water. Last solution is mix of vitamin thiamine, biotin B12 and deionized water. All solutions are mixed well.

Macrophyte medium culture- Murashige and skoog (MS) media formula

To prepare this medium it needed eight solutions. The first one is macronutrient stock solution. It contains ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulphate and potassiumdihydrogenorthophosphate. Then in micronutrient stock solution contain boric acid, manganous sulphate, zinc sulphate, potassium iodide and sodium molybdate. While for vitamins stock solution it have thiamine HCl, nicotine acid, pyridoxine HCl and glycine. In ferum stock solution it mixed between sodium EDTA  and ferrous sulphate. The others solution are sucrose, myo-inositol, gelrite and plant hormone.