Active Dye Staining

Bacterial metabolism can also be assessed by measuring the activity of the electron transport system, which is based on the reduction of tétrazolium salts. Electron transport system activity has been shown to be directly related to respiration. Active dye staining is a technique that allows the differentiation of metabolically active and inactive cells by their ability to combine with certain unnatural substances. The substance forms a complex with part of the cell which can be detected by microscopy. The complex forms with either the active or inactive cell population, not both.

Epifluorescence Microscopv

Epifluorescence microscopy is a form of active dye staining which uses fluorescent dyes to differentiate between metabolising and non-metabolising cells (Hobble et al., 1977). The direct count method using a fluorescent or epifluorescent dye has been widely reported. Bacteria are stained with a fluorochrome, retained on a membrane filter and counted under epifluorescence illumination (Fry, 1988). The criteria for a successful direct-counting technique on a filter are simple:

• All the bacteria must be retained by the filter • All the bacteria must be visible at the filter surface

• Staining and optical conditions must produce high contrast between the bacteria and the background

There are a variety of stains currently in use the intensity of fluorescence varies between the stains and has been ranked as DAPI > mithramycin > ethidium bromide > acridine orange. Although most of these stains are supposedly specific to DNA, other living and non-living material is commonly visible as well. DAPI and acridine orange are the most widely used because they stain bacteria and other particulate debris differentially (Fry, 1990).

Acridine orange (3,6-bis(dimethylamino)acridinium chloride) is the most commonly used stain. Bacteria grown at high growth rates in batch or continuous culture will fluoresce red-orange due to the predominance of RNA. The random coil of the RNA allows so many acridine orange molecules to attach and interact that the acridine orange fluoresces as a dimer. In contrast inactive bacteria have mostly DNA and fluoresce green, the rigid structure of the double helix allows fewer acridine orange molecules to attach, they do not interact and the acridine orange fluoresces as a monomer. In living bacteria the DNA fluorescence is always present but is always masked by the great amounts of RNA. The fixation by aldehyde does not change the nucleic acids, so the green-red distinction will continue after cell death

A fluorescence-based viability assay for the determination of mammalian cells using the carboxyfluorescein derivative BCECF (2'-7'-biscarboxyethyl-5(6)- carboxyfluorescein) by Feeder et al. (1988).

Other Methods Of Active Dve Staining

Due to the fact that respiration is closely bound to active metabolism the cytochemical detection of dehydrogenase activity was combined with a technique for direct counting of aquatic bacteria by Zimmerman et al. (1978). Zimmerman has pioneered an active dye staining technique for the determination of numbers o f viable bacteria occurring in marine ecosystems. The active electron transport system of respiring organisms reduces 2(p-iodophenyl)-3-(-p-nitrophenyl)-5-phenyl tétrazolium chloride (INT) to INT-formazan. INT acts as a hydrogen acceptor and respiring (active) bacteria will accumulate water-insoluble INT-formazan crystals intracellulary as dark red spots. Corresponding to electron transport activity these

Chapter 3 - Analytical development Introduction

deposits attain a size and a degree of optical density which allows them to be examined by light microscopy. If polycarbonate filters and epifluorescence microscopy are applied to analyse an INT-treated sample, it is possible to differentiate between respiring and non-respiring bacteria. This differentiation, which permits determinations of the total number of bacteria and the proportion involved in respiration, is seen directly in one microscopic image.

Using similar techniques, a method was developed by Bitton and Koopman (1982) to assess the activity of filamentous bacteria in activated sludge. It was previously necessary to identify the bacteria in the sample by epifluorescence microscopy and then switch to bright field microscopy to observe the formazan crystals. Bitton and Koopman set out to develop a technique that would allow observation, in the same field, active (with red formazan crystals) and inactive filamentous bacteria. Malachite green was found to be the most suitable counter stain that would dispense with epifluorescence dying. The activated sludge was incubated with INT followed by staining with malachite green. Both cells and INT-formazan crystals can then be observed by using bright field microscopy.

The dye NPN (1-A^-phenylnaphthylamine) was used to investigate the effects of heat stress on E. coli by Tsuchido et al. (1989). NPN is an uncharged lipophilic dye which fluoresces weakly in aqueous environments and strongly in hydrophobic and non polar ones, eg cell membranes. Membranes are among the critical targets of heat stress in bacterial cells, as shown by the leak of intracellular substances and loss of membrane components. Following heat stress the outer membrane's permeability to the dye increased and was reduced following repair. This appears to provide information about the state of damage to the cell following this form of damage.

Active dye staining is a technique which is potentially useful for this type of work however the disadvantages have to be taken into account. These include the potential for operator error and the safety problems with the toxicity of the reagents. The main advantage of this method is the fact that there is no reliance on growth kinetics and is therefore more rapid than plate counts.

3.1.1.5 Summary

Many technological advances have been made for enumeration of viable cells in recent years. In spite of these, the Standard Plate Count is still the most widely used method for the following reasons:

• As the standard technique used throughout the world for many years all novel techniques have to be calibrated against it. When a new method is tested SPC's still have to be carried out as a confirmation for publication purposes • No complex equipment is required and it is therefore inexpensive. Training

time is short and inexperienced operators can rapidly produce acceptable results

• Information concerning sterility of the culture and changes in colony morphology are rapidly apparent

• The methods and materials pose no hazard to the health of the operator. Unlike the use of active dye staining and radiolabels

All of the techniques that have been discussed in previous chapters have fundamental drawbacks. The plate count, although far from an ideal choice, appears to be the most acceptable form of enumerating viable cells. A possible approach would be to supplement the information provided by plate counts with another test.

As stated at the beginning of this review, cell viability is generally regarded as the ability of an organism to reproduce itself and metabolise. It has been shown that organisms are capable of metabolising without possessing the ability to reproduce themselves. The reverse does not hold true. Cells that are no longer intact are capable of neither. The damage caused to cells during downstream processing appear to be mainly caused by the action of mechanical stresses which lead to cell lysis. There is therefore a strong possibility that remaining, intact cells will retain viability. If this is the case then there is a chance that the processing techniques do not have to be assessed by viability tests and can be examined by testing for structural damage. The following section examines methods of testing for cell lysis.