Test Description Typical application
Observational
Conventional light microscopy Used for observation of cell morphology and size.
Various dyes and stains can be used to enhance visualization and identification. Direct counts can be obtained using a Petroff-Hauser counting chamber.
Cell counts, characterization of filamentous bacteria
Flow cytometry Light scattering and fluorescence detection with flow of single particles through detector allows for rapid cell counts. Number and size of cells related to change in electric conductivity. Common for algae enumeration.
Cell counts
Electron microscopy Electron microscopes include transmission and scanning types and are capable of magnification around 10,000,000x. However, the process of obtaining images with electron microscopes can be time consuming and expensive.
Visualization of microorganisms
Culture methods
Pour and spread plate method Diluted sample is mixed with agar and poured into culture dish. Agar is allowed to solidify and the dish incubated. After incubation, colonies formed on the agar are counted. Results are reported as colony forming units per milliliter (cfu/mL). In the spread plate method, diluted sample is spread on a surface culture dish containing a suitable culture medium.
Bacterial counts
Membrane filter technique Sample is passed through a membrane filter and the filter placed right side up in contact with an agar or other solid media. After incubation, colonies formed on the surface of the filter are counted.
Bacterial counts
Multiple-tube fermentation Sample is diluted serially and added to fermentation tubes and incubated. Positive tubes (cloudy) are counted.
Based on the principle of dilution to extinction, as illustrated on Fig. 2–33, the most probable number per 100 mL (MPN/100 mL) is computed using the Poisson distribution for extreme values.
Bacterial counts
Enzyme substrate coliform test Enzyme based methods used to simultaneously determine total coliform bacteria and E. coli. Bacterial enzymes present in total coliform group hydrolyze an added substrate resulting in a color change (yellow). E. coli cleave a fluorogenic substrate, resuling in the release of fluorogen, which fluoresces under ultraviolet light.
Total coliform bacteria and E. coli
Heterotrophic plate count (HPC) Pour plate, spread plate, or membrane filter method, as described above, can be used to determine HPC.
Colonies of bacteria, derived from pairs, chains, clusters, or single cells, are measured. The results are reported as colony forming units per milliliter (CFU/mL).
Bacterial counts
(continued )
(continued )
Table 2–23 (Continued )
Test Description Typical application
Culture methods (continued) Presence-absence
(P-A) test
A single 100 mL sample is tested for the P-A of coliform organ- isms using a selective media. The P-A test is used for highly treated samples such as effluent from a water treatment plant.
Presence of bacteria
Agar overlay method Sample is mixed with agar and E. coli. Solution poured onto solid agar plate and incubated. If colifphage are present, the bacterial cells will lyse resulting in the presence of clear spots. Clear spots, called plaques, are reported as plaque forming units (e.g., pfu/100 mL).
Coliphage countsb
Tissue culture (agar overlay method)
Virus assays are performed in the laboratory by inoculating sample concentrate onto monolayers of cultured cells (hence the name tissue culture). Buffalo Green Monkey Kidney (GBMK) is most common cell line for enteroviruses. Virus destroy the infect- ed cells. The destroyed cells appear as a hole or plaque in the cell monolayer. Each plaque (plaque forming unit or PFU) is the result of the presence of a single or a clump of viruses.
Virus counts
Physiological methods
Respiration gases Measurement of the rate of gas consumption or production, i.e., oxygen consumption, carbon dioxide evolution, and methane evolution.
Microbial activity and substrate conversion Microelectrodes Ulta-fine probes are inserted into a microbial sample
followed by continuous measurement of various cell activities, including oxygen uptake and nitrate reduction.
Microbial activity
Labeled constituents Introduction of a radiolabeled constituents into a microbial sample, including (a) labeled substrate followed by measurement of labeled carbon within the cell, liquid, and evolved as carbon dioxide and (b) labeled thymidine followed by the measurement of the rate of incorporation into DNA.
Microbial activity and substrate conversion
Cell products Methods include measurement of (a) proteins expressed under variable conditions, (b) enzyme activity through the production of fluorescent products generated by the hydrolysis of fluorescein diacetate, (c) dehydrogenase activity through the reduction of tetrazolium salts, and (d) metabolically active biomass through the adenylate energy charge, or ratio of ATP to total adenylates.
Microbial activity
Immunological methods
Fluorescent immunolabeling An antibody is tagged with a fluorescent dye. Once tagged, an antibody becomes attached to an antigen associated with a microorganism; the sample can then be examined using fluorescence microscopy. Fluorescein isothiocyanate (FITC) is the most commonly used fluorescent dye.
Spatial distribution of antigen, detection of bacteria, virus, protozoa, helminths
Enzyme-linked immunosorbent assay (ELISA)
An enzyme-antibody probe is added to a sample containing an antigen. After attachment, the substrate for the enzyme is added to the sample, resulting in a color change.
Quantification of biomass in biofilms, various assays
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Table 2–23 (Continued )
Test Description Typical application
Nucleic-acid methods
Cloning The process of cloning consists of the insertion of an isolated fragment of DNA of interest into a host cell, typically E. coli. The host cell, or clone, then generates identical replicates of the DNA fragment. The DNA fragments are typically analyzed by sequencing.
Replication of genetic material
Nucleic acid probes A nucleic acid probe is a molecule having a strong inter- action with a known complementary genomic sequence unique to the targeted organism(s) and possessing a means for detection. Typical methods include (a) fluores- cent in-situ hybridization (FISH), (b) detection of DNA or RNA on gels following electrophoresis, and (c) screening gene expression using an array of gene probes known as microarrays.
Identification of specific microorganisms, including spatial distribution in flocs and biofilms
Polymerase chain reaction (PCR) Amplification of the DNA of the genome of the microorgan- isms being tested by using complementary DNA fragments known as primers to bind to target DNA of virus The primer triggers a reaction which results in the production of many millions of copies of the microorganism DNA. Examples include reverse transcription PCR (RT-PCR), nested PCR, multiplex PCR, integrated cell culture PCR (ICC-PCR), and real-time quantitative PCR (qPCR).
Amplification of genetic material
Sequencing The coding of genetic material can be determined through the process of sequencing, which usually takes place at commercial laboratories. The DNA sequence can then be compared to databases to determine the relationship of the genetic material to other organisms that have had their DNA sequenced. The 16S rRNA region of the sequence has been found to be the most useful for determining the identity of isolated microorganisms.
Identification of isolated microorganisms
Restriction fragment length polymorphism (RFLP)
A method that uses enzymes to cut purified DNA or PCR products into small fragments at specific segments of the genome. The fragments are then analyzed by gel or capillary electrophoresis to obtain a microbial community fingerprint.
Microbial community fingerprint
Gel gradient electrophoresis A method that subjects PCR fragments to an increasing concentration of denaturant (DGGE) or temperature (TGGE) to allow for visualization of diversity in the genetic material resulting from differential melting or denaturing of the PCR fragments.
Microbial community diversity
Metagenomics The analysis of the collective genetic material recovered from an environment sample.
Microbial community diversity and metabolism
a Adapted from Ingraham and Ingraham (1995), Madigan et al. (2009), Maier et al. (2009),and Stanier et al. (1986).
b A bacteriophage is a virus that infects and replicates within bacteria. A coliphage is a type of bacteriophage that infects E. coli.
in 1893 (Smith, 1893). The method is based on the principle of dilution to extinction as illustrated on Fig. 2–32. Initially, the results obtained with the multiple tube method were identified as the indicated number. The name was changed to multiple tube method in the 1930s. Concentrations of total coliform bacteria are typically reported as the most probable number per 100 mL (MPN/100mL). The MPN is based on the application of the Poisson distribution for extreme values to the analysis of the number of positive and negative results obtained when testing multiple portions of equal volume and in portions constituting a geometric series. It is emphasized that the MPN is not the absolute concentration of organ- isms that are present, but only a statistical estimate of that concentration.
Enzyme Substrate Coliform Tests. In addition to the modified MPN test, several commer- cial enzymatic assays have been developed that can be used for the simultaneous detection of both total coliform bacteria and E. coli. In the enzymatic assays, powdered ingredients
Figure 2–30
Schematic of the technique used for the enumeration of coliphage:
(a) schematic, (b) development of coliphage on E coli lawn. The clear spots correspond to coliphage colonies (see footnote b, Table 2–23).
(a) (b)
Phage
dilution Bacterial
cells Lawn of host cells
develops on top of agar
Phage plaques Pour mixture onto
nutrient agar plate
Agar
Figure 2–29
Schematic of plate culture methods used for the enumeration of bacteria: (a) pour plate and (b) spread plate.
Place sample of bacterial dilution in empty petri dish
Bacterial dilution
Place sample of bacterial dilution on growth medium
Spread sample on surface
Bacterial colonies grow on surface of growth medium Add liquid
nutrient agar
Mix bacterial sample and agar by swirling
Bacterial colonies grow in and on solidified growth
medium (a)
(b)
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Figure 2–31
Membrane filter apparatus used to test for bacteria in relatively clean waters. After centering the membrane filter on the filter support, the funnel top is attached and the water sample to be tested is poured into the funnel. To aid in the filtration process, a vacuum line is attached to the base of the filter apparatus. After the sample has been filtered, the membrane filter is placed in a petri dish containing a culture medium for incubation and subsequent bacterial enumeration.
Figure 2–32
Schematic illustration of the methods used to obtain bacterial counts: (a) multiple tube fermentation technique using a liquid medium and (b) plate count method using a solid medium.
Samples not countable due to clumped growth
1 mL 1 mL 1 mL 1 mL
10–2
10–1 10–3 10–
30 10* 3 3 10* 4
Bacterial count
1 mL 1 mL 1 mL 1 mL
9 mL 9 mL 9 mL 9 mL
Presence of gas taken as a positive test
Inner fermentation tube (a)
(b)
9 mL 9 mL 9 mL 9 mL
4
comprised of salts and specific enzyme substrates that serve as the sole carbon source are added to various wastewater samples. When metabolized by total coliform and E coli, the specific enzyme substrates produce a yellow color and or fluoresce. After incubation, samples contain- ing coliform organisms turn yellow, and samples containing E. coli will fluoresce when exposed to long-wave UV illumination [see Fig. 2–33(a)]. The enzymatic test can be used in two different modes: presence/absence and quantification. In the presence/absence mode the chemical ingredients are added to 100 mL bottles containing the sample to be analyzed. The quantification mode can be carried out using the multiple-tube method or specialized apparatus such as the Colilert-18/Quanti-Tray method [see Fig. 2–33(b)]. The results are reported as pres- ent or absent in a 100 mL sample and as MPN/100mL in the quantification test.
Heterotrophic Plate Count. The heterotrophic plate count (HPC) is a procedure for esti- mating the number of live heterotrophic bacteria in wastewater samples. The HPC method
Figure 2–33
Schematic of the enzyme specific substrate coliform test for total coliform and E coli: (a) presence absence test using 100 mL bottles and (b) quantification test using Quanti-Tray apparatus. Note that sample dilutions can be prepared as necessary for a given sample (dilution shown is for example only). When using the Quanti-Tray, the number of total coliform or E. coli organisms present in the sample can be determined by counting the positive wells and then using IDEXX most probable number (MPN) tables.
Reagent chemicals added to 100 mL bottle
Enzyme specific substrate reagents
Water sample
Water sample added to 100 mL
sample/ reagent bottle
Water sample with reagents poured into
Quanti-Tray
Yellow cells indicate total coliforms
Yellow and flourescent cells
indicate E. coli Quanti-Trays are sealed and
placed in 35oC incubator for 24 h Bottles
incubated at 35oC for 24 h 100 mL
No color indicates total coliform absent
Yellow color indicates total coliform
present
Yellow color and flourescence indicates E. coli
present (a)
(b)
Reagent chemicals
added to each 100 mL
bottle Water
sample
10 mL
Enzyme specific substrate reagents
Water sample added to 100 mL sample/
reagent bottles (e.g., sample dilution is 10 mL added to 90 mL
deioniozed water)
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evolved from first plate count method which was included in the first edition of Standard Methods (Standard Methods, 1905). The HPC can be determined using the (1) pour plate method, (2) spread plate method, or (3) membrane filter method as described above. In the HPC test, colonies of bacteria, which may be derived from pairs, chains, clusters, or single cells, are measured. The results are reported as colony forming units per milliliter (CFU/mL).
Presence Absence Test. The presence-absence (P-A) test for coliform organisms is a modifica- tion of the multiple tube fermentation technique described above. The test is used intended for use for samples collected from water distribution systems or water treatment plants. Rather than using multiple dilutions, a single 100 mL sample is tested for the P-A of coliform organisms using lauryl sulfate tryptose lactose broth as used in the MPN test. Coliform organisms are pres- ent if a distinct yellow color forms, indicating that lactate fermentation has occurred in the sam- ple. The test is based on the rationale that no organisms should be present in 100 mL. It has been used in wastewater for highly treated samples. Enzymatic methods are also used in the P-A test.
Physiological Methods. Physiological methods are used to address the metabolic processes carried out by a microbial community, Measurement of microbial activity can be used to estimate the amount of active biomass, the response of a microbial system to a disturbance, and for determination of the status of an engineered biological process, such as biological wastewater treatment or composting of organic wastes. Common examples of physiological methods include measurement of the rate and type of substrate utilization, the rate of oxygen uptake, or the formation of respiration products.
Immunological Methods. Analytical methods that utilize an antibody for the detection or quantification of a target antigen are known as immunoassays. A key element of immunoassays is the visualization of the antibody-antigen interaction. The visualization step is accomplished typically through the use of signal molecules, which can be designed to allow for detection by various mechanisms, including color change, fluorescence, and radioactivity. Signal molecules can be attached to the antibody directly, known as direct labeling, or the signal molecule may be added after the antibody-antigen attachment has taken place, known as indirect labeling. Indirect labeling uses a secondary antibody to attach with the primary antibody, which is already attached to the target antigen. While nonspecific attachment is a key disadvantage for many immunoassays, the ability to observe the spatial arrangement of target microorganisms is a powerful advantage.
Nucleic-Acid-Based Methods. Molecular methods are based on the use of specific (DNA or RNA) sequences to identify microorganisms, and the use of DNA or RNA amplifi- cation procedures (e.g., polymerase chain reaction, PCR) to detect extremely low concentra- tions of nucleic acid. The PCR technique was developed by Dr. Kary B. Mullis in 1983 while he worked as chemist at the Cetus Corporation. He won the Nobel Prize in 1993 for his invention. Since 1983, the original procedure has been improved and modified to include a number of procedures such as those reported in Table 2–21. Additional details may be found in Madigan et al. (2009) and Maier et al. (2009). Application of some of these procedures to identify and follow specific microbial populations and activity are presented in Chap. 7.
Pathogenic Organisms and Prions
Pathogenic organisms and agents found in wastewater may be excreted by human beings and animals who are infected with disease or who are carriers of a particular infectious disease. The pathogenic organisms found in wastewater can be classified into four broad categories: bacteria, protozoa, helminths, and viruses. The principal pathogenic organisms found in untreated wastewater are reported in Table 2–24, along with the diseases and
Table 2–24