Industrial Microbiology

Processes of Isolation, Characterization, and Preservation of Microbes

Written by MicroDok

The enrichment techniques for the isolation of microbes from their natural habitat are usually designed for the selective multiplication and/or isolation of only some of the microorganisms present in the sample; and the procedures for isolation of actinomycetes, algae, bacteria and fungi differ markedly, and they usually utilize specialized selective culture media for effective isolation of the desired microbes of interest. The procedures involved in the isolation, characterization and preservation of microbes of industrial importance are elucidated in this section (Figure 1).

  • Sample collection: Samples should be collected aseptically using the suitable sample collection containers. Collect soil samples from the appropriate site depending on the anticipated organism and/or desired product of interest. Samples should be collected from specific sites where organisms with desired traits are considered to be likely components of the natural microflora. A gram of soil contains between 106-108 bacteria; 104-106 actinomycete spores; and 102-104 fungal spores.
  • Serial dilution: After sampling, the next stage is to carryout serial dilution on the sample prior to culture. Two-fold or ten-fold serial dilutions are usually carried out to get diluted samples for culture.
  • Culture: The sample is cultured in enrichment media that supports the growth of certain organisms while inhibiting other undesired microbes. Several culture media exist for the growth of microorganisms. However, there is no single culture media that can support the growth of all kinds of microorganisms. For example, a soil sample plated on nutrient agar and incubated at 37oC for 24-48 hrs will only encourage the growth of non-fastidious and mesophilic organisms. Anaerobic microbes, thermophiles, psychrophiles, alkalophiles and acidophiles will not grow on such culture media and under the given environmental conditions. Culture media for bacteria differ from the growth media that supports the growth of fungal organisms; and this also applies to the cultivation of other microorganisms of industrial importance. Bacteria grow at 37oC while fungi grow at room temperatures (25-28oC). Most bacteria (both Gram positive and Gram negative bacteria) can grow on nutrient agar medium. However, antimicrobial agents such as cycloheximide (in concentration range of 50-100 mg/ml) can be added to the nutrient agar medium to make the growth medium selective for only bacteria. The cycloheximide suppresses the growth of fungi. MacConkey agar is usually used for the isolation of Gram negative bacteria because the growth medium contains bile salts which prevent the growth of Gram positive bacteria. To isolate spore forming bacteria, it is critical to first of all heat the sample to around 60-70oC for about 15 minutes. The essence of the heating is to kill vegetative cells present in the sample. Synthetic medium can be used for the isolation of bacteria that utilizes a given nitrogen or carbon source. Actinomycetes are preferably isolated from agricultural soils where they are usually found in abundance. There number is high in such soils during dry seasons than in rainy season; and this should be taken into consideration for the isolation of actinomycetes. Soil samples meant for the isolation of actinomycetes should be air dried for some couple of days prior to their plating on solid culture media. Actinomycetes are fungi-like bacteria that are filamentous in nature. Specialized culture media such as the arginine-glycerol-salt media are usually used for the selective isolation of actinomycetes from soil samples.

Figure 1. Flowchart showing the process involved in the isolation and usage of microbes for commercial purposes. MicroDok.

Arginine-glycerol-salt media is usually supplemented with several antimicrobial agents such as penicillin (100 mg/ml), nystatin (50 mg/ml), chloramphenicol (50 mg/ml) and cycloheximide (50 mg/ml) for the suppression of the growth of fungi and bacteria. Fungal isolation is carried out in selective media such as Sabouraud dextrose agar (SDA) and potato dextrose agar (PDA) which are usually supplemented with several antimicrobial agents such as penicillin, streptomycin and chloramphenicol for the suppression of bacterial growth.

  • Identification: The identification of specified microbes is crucial because it is aimed at separating the desired organism from the undesired ones. After the isolation of bacteria from the sample, it is important to identify the specified bacterium or bacteria from the whole lot of microorganisms on the growth media. The identification of bacteria is usually done using certain biochemical, microbiological, and molecular techniques aimed at typing the organisms down to the species or strain level. In addition, microbes can be identified using certain phenotypic characteristics such as microbial morphology, cultural/colonial features on growth media, staining techniques and microscopy.
  • Screening of isolated microbes for desired metabolites: The next step after isolation of microorganisms is their screening for the detection of the desired metabolite of industrial importance. Several rapid screening protocols exist for identifying the synthesis of novel products from microbes. The screening methods vary amongst the different metabolites sourced from microbes.
  • Preservation of culture: The isolated microbe of industrial importance should be preserved and protected from contamination – so that it will be readily and aseptically available for future research and other commercial purposes. Culture preservation techniques are also aimed at preventing the loss of a desired metabolite or metabolic pathway in the microorganism of industrial importance. Preservation techniques of microbial cultures should be either short-term or long-term. In short-term preservation, microbial cultures are preserved in the refrigerator from 0oC to 5o Microbial cultures could also be stored at very low freezing temperature such as ultra low temperature (-80oC) and in liquid nitrogen (at 196oC). Lyophilization is another long-term preservation technique that is used to preserve microbial cultures for future use. Lyophilization (freeze-drying) is defined as the process used for the long-term preservation of microbes and other products. In freeze-drying, volatile substances such as water are removed from deep-frozen samples or materials by sublimation under high vacuum. Lyophilization techniques dry microbial cells without disruption of the cell components.

References

Bader F.G (1992). Evolution in fermentation facility design from antibiotics to   recombinant proteins in Harnessing Biotechnology for the 21st century (eds. Ladisch, M.R. and Bose, A.) American Chemical Society, Washington DC. Pp. 228–231.

Nduka Okafor (2007). Modern industrial microbiology and biotechnology. First edition. Science Publishers, New Hampshire, USA.

Parek S (2004). Strain Improvement. In: the motherland. The Desk Encyclopedia of Microbiology. M. Schaechter (ed.). Elsevier Amsterdam. Pp. 960-973.

Das H.K (2008). Textbook of Biotechnology. Third edition. Wiley-India ltd., New Delhi, India.

Latha C.D.S and Rao D.B (2007). Microbial Biotechnology. First edition. Discovery Publishing House (DPH), Darya Ganj, New Delhi, India.

Nester E.W, Anderson D.G, Roberts C.E and Nester M.T (2009). Microbiology: A Human Perspective. Sixth edition. McGraw-Hill Companies, Inc, New York, USA.

Steele D.B and Stowers M.D (1991). Techniques for the Selection of Industrially Important Microorganisms. Annual Review of Microbiology, 45:89-106.

Pelczar M.J Jr, Chan E.C.S, Krieg N.R (1993). Microbiology: Concepts and Applications. McGraw-Hill, USA.

Prescott L.M., Harley J.P and Klein D.A (2005). Microbiology. 6th ed. McGraw Hill Publishers, USA.

Steele D.B and Stowers M.D (1991). Techniques for the Selection of Industrially Important Microorganisms. Annual Review of Microbiology, 45:89-106.

Summers W.C (2000). History of microbiology. In Encyclopedia of microbiology, vol. 2, J. Lederberg, editor, 677–97. San Diego: Academic Press.

Talaro, Kathleen P (2005). Foundations in Microbiology. 5th edition. McGraw-Hill Companies Inc., New York, USA.

Thakur I.S (2010). Industrial Biotechnology: Problems and Remedies. First edition. I.K. International Pvt. Ltd. New Delhi, India.

 

 

 

 

About the author

MicroDok

Leave a Comment