Microbiology The drinking water treatment plant has brought a new anthracite fil
ID: 54481 • Letter: M
Question
Microbiology
The drinking water treatment plant has brought a new anthracite filter on-line in parallel with their granular activated carbon (GAC) filter. Both are biologically active. The plant manager wants to know if the anthracite filter and the GAC filter have similar bacterial densities. Consulting the literature, describe a procedure for how you might release the attached biomass from the filter material for analysis. List two techniques that you might use to enumerate the bacteria, and discuss the benefits and drawbacks of each technique.Explanation / Answer
a) Detachment of Biomass
Detachment of biofilm is a common phenomenon, which always happens during the biofilm formation process. Biofilm detachment occurs through different processes.Detachment of biofilm by abrasion, erosion, sloughing, occurs when there is shear stresses, and lack of nutrient and oxygen in the biological filter. Abrasion and erosion leads to the removal of small groups of cells from biofilm while sloughing results in the detachment of a relatively large fraction of the biofilm. Porosity and roughness of the surface supporting the biofilm plays an important role in protecting the biofilm from hydrodynamic shear and abrasion (Wuertz et al., 2003). Human intervention can lead to the detachment process. Predator razing is also another factor that causes biofilm detachment with the involvement of protozoa, snails and worms (Percival et al, 2000).The detachment process has a significant impairment to the distribution of microorganisms within the biofilm and its structure. On the other hand, detachment removes dead microbes and creates free sites for new organisms to attach, thus microorganisms can quickly be replaced to retain the stability of the microorganism community and their activity (Percival et al., 2000; Wuertz et al., 2003).
The success of a biofilter highly depends on the efficient maintenance of biomass attached to the filter media. Biomass detachment is one of the most important mechanisms that can affect the maintenance of biomass in a biofilter. Erosion, abrasion,sloughing, grazing or predation and filter backwashing are the most common detachment mechanisms. Erosion of biomass occurs due to fluid shear whereas abrasion of the biomass is the process of scraping the biocell off the surface by collision with external particles. Similarly, large patches of biomass are detached by sloughing and a part of biomass especially on the outer surface of the biofilm may be lost due to the grazing of protozoa. Evaluation of the biomass lost due to filter backwashing is very important from an operational point of view. Backwash bed expansion, mode of backwash such as air scour or chlorinated water backwash may affect the biomass during backwashing. However, a previous study has shown that the effective biomass is not lost during normal filter backwash (Ahmad et al., 1998).
b)
Direct Microscopic count/ Total cell count
direct microscopic counts are possible using special slides known as counting chambers, consisting of a ruled slide and a cover slip. It is constructed in such a manner that the cover slip, slide, and ruled lines delimit a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted.
Bacteria can be counted easily and accurately with the petroff-Hausser counting chamber. This is a special slide accurately ruled into squares that are 1/400 mm2 in area; a glass cover slip rests 1/50 mm above the slide, so that the volume over a square is 1/20,000 mm3i.e.
1/20, 000, 000 cm3. If for example, an average of five bacteria is present in each ruled square, there is 5 x 20,000,000 or 108, bacteria per milliliter. A suspension of unstained bacteria can be counted in the chamber, using a phase-contrast microscope.
The formula used for the direct microscopic count is:
The number of bacteria per cc = The average numbers of bacteria per large double-lined square X The dilution factors of the large square (1,250,000) X The dilution factor of any dilutions made prior to placing the sample in the counting chamber, e.g., mixing the bacteria with dye
Advantage of Direct Microscopic count
Limitations of Direct Microscopic count
Dilution:
with both the spread plate and pour plate methods, it is important that the number of colonies developing on the plates not be too large because on crowded plates some cells may not form colonies and some colonies may fuse, leading to erroneous measurements. It is also essential that the number of colonies not be too small, or the statistical significance of the calculated count will be low. The usual practice, which is the most valid statistically, is to count colonies only on plates that have between 30 and 300 colonies. The number of bacteria in a given sample may be usually too great to be counted directly. To obtain the appropriate colony number, the sample be counted must almost always diluted in such a manner that single isolated bacteria form visible isolated colonies , the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. Several 10-fold dilutions of the sample are commonly used. In most cases, serial dilutions are employed to reach the final desired dilution.
However, if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria may be clumped together. The development of one colony can occur even when the cells are in aggregates. i.e. cocci in clusters (staphylococci), chains (streptococci), or pairs (diplococci), the resulting counts will be lower than the number of individual cells. Each colony that can be counted is called a colony forming unit (CFU). By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample rather than number of bacteria per milliliter. The assumption made in this type of counting procedure is that each viable cell can yield one colony.
There are two ways of performing a plate count: the spread plate method and the pour plate method.
Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find this, the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of times the original ml of bacteria was diluted (the dilution factor of the plate counted). For example, if a plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then the number of CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the formula below:
The number of CFUs per ml of sample = The number of colonies (30-300 plate) X The dilution factor of the plate counted
In the case of the example above 150 x 1,000,000 = 150,000,000 CFUs per ml
This method is used to count only live (viable) cells. A viable cell is defined as one that is able to divide and form off springs, and the usual way to perform a viable count is to determine the number of cells in the sample capable of forming colonies on a suitable agar medium. For this reason, the viable count is often called the plate count, or colony count. This method is used to enumerate bacteria in milk, water, foods, soils; cultures etc and the number of bacteria are expressed as colony-forming units (CFU) per ml.Advantage of plate count method
this method is used routinely and with satisfactory results for the estimation of bacterial populations in milk, water, foods, and many other materials.
Limitation of plate count technique
(1) Only living cells develop colonies that are counted;
(2) clumps or chains of cells develop into a single colony;
(3) colonies develop only from those organisms for which the cultural conditions are suitable for growth.
Types of Techniques; Pour plate technique, Spread plate technique and Streak plate technique