Place a piece of absorbent paper at the opposite edge of the cover slip.

In normal activated sludge, the India ink particles penetrate the flocs almost completely, at most leaving clear centers, In activated sludge containing large amounts of exocellular polymeric material, large, clear areas with low cell density will be observedġ Alternatively, apply 1 drop of India ink to the edge of a cover slip under which a thin preparation of the sample has been made.

Place on the cover slip and observe at 100× using phase contrast.

Mix 1 drop of India ink and 1 drop of activated sludge sample on a microscope slide,1depending on the ink used, the sample volume may need to be reduced.

Reagent: India ink (water-based black drawing ink aqueous suspension of carbon black particles also known as China ink).

Observe at 1000× using phase contrast, as in Test A.

Shake the flask overnight at room temperature.

Add 1 mL of sodium thiosulfate solution to the flask (final sodium thiosulfate concentration is 2mM).

Add 1 to 2 mL of mixed activated sludge sample to the flask.

Allow activated sludge sample to settle transfer 20 mL of clear supernatant to a 100 mL Erlenmeyer flask.

Reagent: Sodium thiosulfate solution (NaSO.5HO), 1 g/100 mL prepare fresh weekly. (1981), Microscopic Sludge Investigation Manual, TNO Research Institute for Environmental Hygiene, Delft, The Netherlands. Note: If results are inconsistent, use the sulfur oxidation test A positive S test is the observation of highly refractive, yellow-colored intracellular sulfur granules inside the filaments (Figure 2.22b, Figure 2.23b, and Figure 2.23d).

Observe at 1000× using phase contrast.

Place a cover slip on the preparation and gently press to exclude excess solution remove expelled solution with a tissue.

Allow to stand open to the air for 10 to 20 min.

On a microscope slide, mix 1 drop of activated sludge sample and 1 drop sodium sulfide solution.

Reagent: Sodium sulfide solution (NaS.9HO), 1.0 g/L prepare fresh weekly. Much of the biological material will not be visible while nonbiological (especially inorganic) material is readily observedĭetermine the presence of bacterial colonies that appear to be composed of one type of microorganism. Measure 10 to 20 flocs and place them in the following size categories based on their minimum dimensions or diameters if they are approximately spherical:ĭetermine whether flocs are round or irregular and compact or diffuse determine whether texture is firm or weak, you can also determine the presence and types of protozoa and other macroorganisms (e.g., rotifers, nematodes), in addition Nonbiological Organic and Inorganic Particles can be identifies by observation under direct illumination. Blue-violet is positive (either entire cell or intracellular granules) yellow-brown is negative

Examine under oil immersion at 1000× magnification with direct illumination (not phase contrast).

Stain 1 min with Solution 2 rinse well with water blot dry.

Stain 30 s with Solution 1 rinse 1 s with water.Solution 2 : 33.3 mL Bismark Brown (1% w/v aqueous)+66.7 mL distilled water. Mix two parts by volume of A with one part by volume of B. Solution B: 3.3 mL Crystal Violet (10% w/v in 95% ethanol)+6.7 mL ethanol 95%+100 mL distilled water. Solution A: 0.1 g Methylene Blue+5 mL ethanol 95%+5 mL glacial acetic acid+100 mL distilled water. Solution 1 : Prepare Solutions A and B separately. Reagents: Prepare fresh every 3 to 6 months. Test each batch of Gram stains on cultures with known Gram staining reactions.Stain with Solution 3 for 1 min rinse well with water blot dry using bibulous paper.Hold slide at an angle and decolorize with 95% ethanol added drop by drop (not in a continuous stream) to the smear for exactly 25 s.Stain 1 min with Solution 2 rinse well with water.Stain 1 min with Solution 1 rinse 1 s with water.Prepare thin smears on microscope slides and thoroughly air dry.Solution 3 : 10 mL Safranin O (2.5% w/v in 95% ethanol)+100 mL distilled water. Solution 2: 1 g iodine+2 g potassium iodide+300 mL distilled water.

Solution B= 0.8 g ammonium oxalate+80 mL distilled water. Solution A= 2 g Crystal Violet+20 mL ethanol 95%. Solution 1: Prepare Solutions A and B separately, then combine them as following There are many stains used to define the most important structure of bacterial and flocs of activated sludge

India ink staining