Tools and Techniques of Experimental Microbial Ecology

Tools and Techniques of Experimental Microbial Ecology

Its important to take sample to examine or determine or to conduct any experimental examinaton. Sample should be the representive of whole unit. It must have all characteristic of the population. And first thing in any experiment is sample and process to collect sample is called sampling.

Sampling

Sampling procedure should ensure that the number or activity of microorganisms are not changed poditively or negatively during collecton or storage of the sample. The sample must be representative and not contaminated with foreign components, i.e., microorganisms should only be from the ecosystem being examined.

Sampling approaches in major natural ecosystem:

Tools and Techniques:

1. Direct examination or Microscopy

Microorganisms can be enumerated by direct observation of the samples under amicroscope. It is reliable, relatively fast and useful for studying the morphology of microorganisms.

The enumeration of bacteria, protozoa, yeast and algae can be done by using counting chambers that hold specified volumes such as Petroff-Hauser counting chamber.

The direct examination of fungi can be done by modified agar film technique. In this technique, the sample is mixed with agar media and pipetted on aglass slide to form a thin layer of film. The agar film is then stained and the slides are viewed under amicroscope. The length of the mycelia can be determined by counting intersections of a superimposed grid. Alternatively, the microscopic images can be projected on the screen and length of the mycelia can be estimated by adevice called 'map measure'.

Epifluorescence microscopy has been widely used in microbial ecology to observe small,unusually shaped microorganisms and those microorganisms that cannot be cultured. Moreover, epifluorescence microscopy can be applied to a wide variety of habitat. Epifluorescence microscopy with stains such as Acridine orange 4,6-Diamidino-2-Phenyl indole (DAPI), Fluorescein isothiocyanate (FITC) and Bisbenzamide are widely used in enumerating the bacteria.

Fluorescent staining methods can be used in combination with other procedures to determine the living microorganisms in a sample. Acridine orange direct counting (AODC) can be combined with 2-iodophenyl-3-paranitrophenyl-5-phenyl tetrazolium chloride (INT) staining to determine the viable count. The respiring microorganisms take INT and reduceinto INT-formazan which is red in colour. The red spot can be observed inside the viable cells.

Viable cells can also be enumerated by another method called direct viability count (DVC). In this method, the bacteria cells are incubated with nalidixic acid and the observed under a microscope by fluorescent staining. The viable cells are elongated. Nalidixic acid prevents DNA synthesis, which inhibits cross wall formation requires for cell division. Thus the cells elongate instead of dividing.

In agar film technique, diacetate can be used to estimate the living mycelia. The metabolically active hyphae absorb diacetate and degrade it to a compound which fluoresces when observed under the microscope.

Fluorescent antibody test (FAT) can be used to determine the specific types of microorganisms and their number in an environmental samole.

Limitations

• Unless specific procedures are applied, viable and non-viable cells can not be differentiated.
• Microorganisms may be underestimated in the samples containing high amount of debris.
• Inability to perform further studies for observed microorganisms

2. Culture Techniques/ Methods

Culturing of anenvironmental sample in an appropriate media is one of the widely used technique for detection of microorganisms (isolation) and for a viable count ( especially for bacteria). These techniques cannot be used to achieve total count. The cultivation of different bacterial, fungal, algal and protozoan species can be done by various methods.

1. Plate culture

Spread plate and pour plate techniques are widely used culture techniques for isolation and enumeration of viable microorganisms in a sample. The isolation can also be achieved by streak plate technique. It is assumed that each colony arises from a single colony. However, two or more cells clotted together may developa single colony and therefore, the number is enumerated as colony forming units (cfu) and not as number of cells. The plates with too many colonies (>300) and too few colonies (<30) are discarded to prevent errors.

In pour plate technique, the appropriately diluted environmental sample is mixed with molten agar (around 40-50°C) and is poured into the plates. It is inapplicable if the sample contains heat-sensitive microorganisms. In spread plate technique, the diluted sample is spread on the surface of solid media. The colonies that developed are subsequently counted. The plate counted method can be adjusted to a selective viable count and differential viable count with the use of selective and differential media respectively.

Limitations

• Plate count technique cannot be used to achieve total count.
• It cannot be used to enumerate filamentous fungi.

ii. Most Probable Number (MPN)

It is an alternative to plate count for determination of viable microorganisms using statistical analysis of successively diluted samples. The MPN procedure gives statistically based estimate of the most probable number of microorganisms in the sample within fixed confidence interval. The sample is successively diluted and growth is noted in tubes in the form of turbidity. the number of tubes showing positive growth is noted. the number of positive tube is tallied with standard MPN table and the number of microorganismsin the sample is determined in the form of MPT index.

Although MPN test is usually applied to enumerate bacteria, it can also be used to determine the number of protozoa and enteric virus in the sample.

3.Lipid Profile Analysis

Lipids are ubiquitous components of living cells. The lipid composition of diverse microorganisms vary and each microorganisms has a characteristic pattern of the lipid composition that can be used to identify a paryicular microorganisms. Such spcific pattern of lipid composition is called lipid profile and the examination of lipid profile allows identification of living organisms. The profiles of lipid composition can be used to describe the comopsitionof microbial community in an environmental sample.Lipid profiling can be done by fatty avid methyl ester (FAME) analysis and Phospholipid linked fatty acid (PLFA) analysis.

In FAME analysis, the lipids are esterified, separated, identified and quantified to detrmine the microbial species. FAME analysis has been done to distinguish Bradirhizobium spp.

PLFA analysis is used to describe bacterial communities. In this technique, fatty acid linked to polar lipid fraction which are characteristc to microbes are analysed. Branched fatty acids of bacterial origin whereas eukaryotic microorganisms are characterised by polyunsaturated fatty acids (especially 18:2). PLFA can also be used to determine the concentration of saturated, unsaturated, cis and trans fatty acids, which can be used to determine the stressed condition. Example,Micrococcus spp., can be distinguished by profiles of fatty acid.

Micrococcus varians- C₂₅, C₂₆, C₂₇

Micrococcus leuteus- C₂₇, C₂₈, C₂₉

Micrococcus sedentarius-C₃₀, C₃₁, C₃₂

4.Molecular Detection

The molecular detection rely on analysis of nuclei acid and is widely used for detection of specific microbial population and their activities without culturing microorganisms. for molecular detection, first the nuclei acids (DNA, RNA of mRNA) are recovered from environmental sample and purified. The analysis of nuclei acid is done by different molecular methods.

1. Gene probe detection/ Hybridisation

In this technique, a gene probe ( short nucloetide sequence) labeled to radioactive isotope( e.g., 32 P) or a fluorescent dye is used. The gene probe contains nucleotide sequence in the target DNA (microorganisms). The target DNA is first denatured to single strands on a solid support (hybridisation menbrane) to which the probe is added. If the target nuclei acid (microorganism's) is present in the sample, the probe binds (hybridizes) to the homologous sequence in the target nuclei acid. The gene probe that establishes double strand complementary target sequence can be detected by autoradiography or fluorescent microscopy. Hybridization, thus, allows detection of specific microbial population with specific genetic properties. In colony hybridization, the colonies from the medium are transferred to the solid support on which they are lysed releasing DNA. The DNA is then denatured to single strand and allowed to hybridize with gene probe.

ii. Polymerase Chain Reaction (PCR)

PCR has been applied to the environmental detection of microorganisms. PCR allows the in in vitro amplification of defined sequence of DNA. The amplification enhances gene probe detection of specific gene sequences. The rare sequence in the mixture of DNA can also be easily detected once they are amplified. In viteo amplification is achieved using automated thermocycler. The compnents of PCR reastion include a heat stable DNA polymerase (e.g., Taqpolymerase), oligomer primers, dNTSs, template DNA and Mg²⁺. The reaction proceeds in a sequence of denaturation/ melting (94°C-95°/ 30-60s); primer annealing (55°C-72°C) and extension.

iii. Genetic finger-printing

A genetic finger print is a specific pattern of DNA unique to each individual. In this technique, the microbial strains from various habitats are studied by analysing their DNA to determine a genetic or DNA finger print. Gnetic finger printing can be done by anumber of methods, for example, Restriction fragment length polymorphism ( RFLP).

5.Measurement of metabolic activityMeasurement of metabolic activity helps to determine the actual or potenial level of metabolic activities withinthe environment. Metabolic activity depend on a physical, chemical, biological and nutrient parameters of the environment. It also helps to undertsand the responsds of microbes to these parameters. The metabolic activities can be determined by the number of methods.

i. Heterotrophic potential ( V_max)

Heterotrophic potential is the measurement of rate at which a substrate uptaken and metabolised by heterotrophic microorganisms in an environmental sample. It is the determination of potential for utilising substrate. The procedure for determining hetertrophic potential consist of adding variuous concentration of radio-labeled substrate( for example, ¹⁴Ccontaining glucose, lactic acid, amino acid, etc) to the sample and icubating sample under the condiyion that are similar to the real environment. The amount of ¹⁴CO₂ produced and radioactivity in cooperated into the cells is then measured for its specific time.

The microorganisms uptake radio-labeled sunstrate as per the saturation kinetics( Michaelis- Menten kinetics) and the rate of uptake increase with the increas in concentration of substrate to teh maximum uptake rate( V_max). The V_max is a measurement of heterotrophic potential thatccan be determined by increased form of Michaelis- Menten equation. This method can aslo be used to calculate turn over time.

ii. Carbon respiration

This technique employes measurement of carbon dioxide released or O₂consumed during metabolism to determine respiratory activity. The CO₂ can be measured using radio-labeled CO₂(¹⁴CO₂) released after the degradtion of substrate can be measured to determine the decomposition rate. Alternatively, CO₂ can be measured by using specially designed flask , for example, biometers or using 'flow-through incubation system' such as 'gas trains'. The biometer or gas-train selectively trap CO₂ and measure it.The rate of oxygen consumption can be measured with the help of the oxygen electrodes, microprobes and respirometers.

iii. Photosynthesis (Primary productivity)

The rate of primary prouction/ photosynthesis can also be measured. One of the widely used method is by using radio-labeled bicarbonate. The radio-labeled bicarbonate can be used to determine autotrophic assimilation of CO₂ as well as heterotrophic assimilation of CO₂. The radio-labeled bicarbonate is incubated with the sample containing microbial community and then the amount of¹⁴CO₂ assimilated into cells is determined. In field measurement, a clear/ light and a covered/dark bottles are filled with radio-labeled bicarbonate sample. The bottles are incubated in situ for several hours . The difference in the incorporation of¹⁴C in light and dark bottle give net photosynthetic incorporation(rate).

6. Metagenomics or Environmental Genome

In this approach, random sequencing of total DNA from microbial community is used to reveal the entire gene complement of that community. Tthe goal of metagenome is not to generate total genome sequence but to detect as many genes as possible that code recognizable proteins. It gives the genetic picture of community and genetic relatedness.

References

Atlas, RM and R Bartha. Microbial Ecology:Fundamentals and Applications. The Benjamin Cummins Publication co. Inc., 1998.

Gordis, L. Epidemiology. third edition. 2004.

Maier, RM, IL Pepper and CP Gerba. Environmental Microbiology. Academic press Elsevier Publication, 2006.

park, K. Park's Text Book of social and prevention Medicine. 18th edition. 2008.