here is the website for Fisher scientific

to get the India Ink droplets for polysaccharide coating staining

and Lactophenol Cotton Blue - to use with microscope to simulate phase contrast and bring out more details on the floc, filaments and higher life forms

Identify microorganisms without mixing or measuring reagents and stains. Each dropper contains a one-day supply of reagent or stain, hermetically sealed inside an ampule. Strict quality controls assure uniformity and accuracy. To perform tests, squeeze the dropper to crush the ampule. Invert the dropper for convenient drop-by-drop dispensing. A clear dropper stand with cover is supplied to eliminate spills. Dispenser box blocks out light to prevent decomposition.

INDIA INK DROPPER 50/PK

14-910-56

Mfr. No.:261194 Pack of 50 for $57.97

14-910-15

BD No.:261188 Pack of 50 for $57.44

Other great sources:

VWR

USA Bluebook

Lactophenol Cotton Blue Stain is formulated with lactophenol, which serves as a mounting fluid, and cotton blue. Organisms suspended in the stain are killed due to the presence of phenol. The high concentration of the phenol deactivates lytic cellular enzymes thus the cells do not lyse. Cotton blue is an acid dye that stains the chitin present in the cell walls of fungi.

Ingredients per liter:*

* Adjusted and/or supplemented as required to meet performance criteria.

Lactophenol Cotton Blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. It can be used to also look at filaments and higher life forms with microscopic work. Just remember, the stain will slow down and or cause the higher life forms to die. This is great for taking photomicrographs, but not for a wastewater biomass analyses. Use a normal wet mount first for higher life form counts.

The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls. This also will bring out fine details in filaments.

Procedure for staining:

1. Place a drop of wastewater sample on a microscope slide.
2. Add one, or at most two drops of the Lactophenol Cotton Blue stain .
3. Folding the coverslip between forefinger and thumb, touch one edge of the drop of sample with the coverslip edge, and lower gently, avoiding air bubbles. The preparation is now ready for examination.
4. If desired, seal the edges of the coverslip with nail polish or permount to preserve the mount as a reference slide.

India Ink stain procedure is the exact same procedure, one drop of sample, one drop of India Ink on top of sample, cover and examine.

India Ink is basically carbon black particles. They block out all the light except the polysaccharide coating that the bacteria produce. The light that shows through in the microscope is a measure of how much polysaccharide coating present.

If high, it can indicate a lack of nutrients or a recent shock to the system. If there are tons of higher life forms present, you can probably rule out a shock and start to check your nutrient levels.

The photo on the bottom right shows very high polysaccharide coating.

REAGENTS/STORAGE: Reagent kits manufactured by BBL and Difco can be purchased from a local scientific supply house and contains four reagent bottles:

Crystal violet -- the primary dye

Gram’s iodine -- the complexing agent

Alcohol -- the decolorizer

Safranin -- the counter stain

The crystal violet and safranin can be stored indefinitely while iodine has to be replaced every 6-9 months. Storing in the dark will help to extend the life of the iodine solution.

1) Place a drop of the sample on the glass slide using a disposable Pasteur pipette and carefully spread the drop to form a thin film on the slide.

2) Allow the slide to air dry. Do not heat fix which may cause distortion of the filaments.

3) Flood the specimen with crystal violet. Leave it on for 30 seconds.

4) Tip the slide to drain off crystal violet and flood with gram’s iodine. Leave it on for 1 minute.

5) Drain off the iodine solution and rinse with the decolorizer to remove the excess purple color. Then let the slide sit for 10 seconds with alcohol in it and rinse the slide again with more decolorizer.

6) Wash gently with tap water for 5 seconds approximately to stop the action of the decolorizer.

7) Flood with safranin and leave it on the specimen for 1 minute.

8) Wash the slide very lightly with water and let it air dry.

9) Observe and examine under the microscope using the bright field oil immersion.

RESULTS: Gram positive bacteria appear blue/purple in color while gram negative bacteria are red. Since variation in gram reaction can occur due to the age of the culture. It is important to stain the samples within 24-48 hours of sampling. Types of ( i.e. chemical vs a papermill) can also effect the staining abilities of the filamentous bacteria. Polysaccharide coating and sheaths also effect coloration.

REAGENTS/PREPARATION: The staining procedure involves the use of a working solution and counter stain. The working solution is prepared by mixing two parts of solution 1 and one part of solution 2. Solution 1 and 2 have the following composition.

SOLUTION 1 SOLUTION2

Methylene Blue 0.1 g Crystal Violet 0.33 g

95% Alcohol 5.0 ml 95% Alcohol 10.0 ml

Glacial Acetic Acid 5.0 ml Distilled Water 100.0 ml

Distilled Water 100.0 ml

The counter stain consists of 0.333 grams of Bismarck Brown dissolved in 100 ml of distilled water.

PROCEDURE:

1) Prepare smears as in gram stain method and air dry.

2) Stain the specimen for 15 seconds with the working solution. Rinse briefly and shake off the excess water.

3) Stain for 1 minute with the Bismarck Brown counter stain. Thoroughly blot off the excess stain; do not rinse.

4) Let it dry and examine under the microscope with oil immersion and direct illumination.

RESULTS: Blue/gray cells ( sometimes purple in appearance) are considered positive and yellow/brown cells negative for this staining procedure. Types of environments that the filamentous bacteria are in can effect the staining abilities; Papermills, CPI, refineries, sheaths and polysaccharide coatings also.

How do I know if I have stained the filaments correctly or have identified the correct filament?

Wastewater Biomass Analyses Brochure