PHB Stain

Poly-ß-Hydroxybutyrate Stain*

The poly-ß-hydroxybutyrate stain is used to stain granules present within the confines of the filamentous bacteria cells. These granules are storage components of the cells giving indication of the cell's ability to take advantage of an opportunity of "free" easily absorbable and available low molecular weight carbonaceous compounds present in the environment. Typically, PHB deposition within a cell occurs under specific operational conditions: High BOD:N:P ratios, anaerobic conditions present or created within a treatment process (insufficient DO, dead zones within an aeration basin which create anaerobic conditions which lead to production of low molecular weight organic acids such as acetic, propionic, butyric acids due to fermentation). There are several filamentous bacteria capable of PHB deposition and they include but not limited to filament type 021N, 1701, Sphaerotilus natans...

Preparation:

Solution 1: Sudan Black B (IV), 0.3% w/v in 60% Ethanol:

Solution 2: Safranin O 0.5% w/v aqueous

Procedure:

1. Prepare thin smears on microscope slides and thoroughly air dry. Do not heat fix.

2. Stain 10 minutes with solution 1; add more stain if the slide starts to dry out..

3. Rinse 1 second with water.

4. Stain 10 seconds with solution 2; rinse well with water; blot dry.

5. Examine under oil immersion at 1000x magnification with direct illumination (not phase contrast): Blue-violet is positive (either entire cell or intracellular granules; yellow-brown is negative.

*David Jenkins et al, “Manual on the Causes and Control of Activated Sludge Bulking and Foaming”, Water Research Commission and USEPA, April, 1986.

Photos of Filamentous Bacteria with PHB Granules
[]	Live Sample 1000x Phase Contrast. Small arrow pointing at PHB granules within Sphaerotilus natans filament. Two larger arrows are pointing at distinct type 1863 filaments.
	PHB stain 1000x Brightfield. Filaments extending out of floc have small black dots that represent PHB granules. The filamentous bacteria is type 1701.
	PHB stain 1000x Brightfield. Small black dots within filaments extending out of flocs are PHB granules. The filamentous bacteria are type 1701.

Poly-ß-Hydroxybutyrate Stain Using Nile Blue A

Preparation:

Prepare a 1% aqueous solution of Nile Blue A. Ensure that Nile Blue A is fully dissolved by using mild heat (around 50°C) and continuous stirring. Filter prepared solution.

Procedure:

1. Prepare thin smears on microscope slides and thoroughly air dry. Heat fix slides.

2. Immerse slides into a vesicle (Coplin Staining Jar) containing Nile Blue A stain. The slides are put into incubator set to 55°C for 10 minutes.

3. After incubation, wash slides with tap water.

4. Remove excess stain by using an 8% acetic acid solution on the slide for 1 minute.

5. Put cover slip on slide if observing with immersion oil. Oil will extract some of the fluorescent dye.

The Nile Blue Stain A fluoresces as an orange color and become excited at wavelength of approximately 362 nm but can still be visible at 460 nm.

Photos of Filament Type 1701 Under Epifluorescent Microscope
[]	Stained Sample, 100x, Epifluorescence at 460 nm. Notice orange dots following filament contour as they extend out of floc and into the bulk solution
	Stained Sample, 1000x, Epifluorescence at 460 nm. Several strands of filament type 1701 can be seen extending horizontally across the photo as well as two distinct Nocardia filaments (with distinct branching). Two orange aggregates within each 1701 cell can be clearly distinguished in this photo as the PHB granules.