TECHNIQUES EMPLOYED IN THE ISOLATION AND CHARACTERIZATION OF BACTERIA

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The microbiology laboratory is unique and different from the laboratory of other biological and medical sciences in that it has some fundamental techniques which the microbiologists use to manipulate microorganisms for improved productivity. Some of these techniques are what distinguishes the microbiologist from other scientists; and these techniques must be dutifully followed in order to achieve better research results. It is also important that students and tutors of microbiology acquaint themselves of these techniques so as to always be on top in their chosen discipline. Microbiology like the other biological sciences is a practical discipline, and it cannot do without some modus operandi which is universally acceptable and central to any research performed in the microbiology laboratory. These microbiology techniques including isolation, inoculation, culturing, incubation and microscopy amongst others shall be discussed in this section. Students of microbiology are advised to ensure continuous practice of these techniques in order to be familiar with them.  

  • STAINING TECHNIQUE: Staining is any microbiological process which increases the contrast of organisms when certain dyes or stains are applied to them prior to their examination under the microscope. It is generally the process of colouring specimens and microorganisms so that they can be easily observed and distinguished under the microscope. Staining in the microbiology laboratory is usually carried out on dead microbial cells which must have undergone a series of smearing during smear preparation. In some scenarios, microbial cells are only stained once but staining can involve a series of process in which bacterial cells are stained several times using different types of dyes or counterstain meant to reveal certain features of the organism during examination under the microscope. Some commonly used stains in the microbiology laboratory include crystal violet, methylene blue, lactophenol cotton blue, Giemsa stain, eosin stain, carbol fuchsin, acridine orange and fluorescent dyes amongst others. Gram staining, negative staining and acid fast staining are some of the commonly encountered staining techniques used in the microbiology laboratory. Staining technique generally helps microbiologist to observe microbial cells under the microscope and describe them appropriately based on their ability to react with certain stains or dyes.
  • ASEPTIC TECHNIQUE: Aseptic technique refers to all the quality control and precautionary measures taken by microbiologists in the laboratory in order to ensure that all working apparatuses are germ-free. It involves numerous processes taken to reduce or prevent the contamination of microbial cultures, reagents and culture media. Aseptic techniques also include all other precautionary measures taken to protect the laboratory personnel from direct or indirect contamination from harmful microorganisms. Working inside the biological safety cabinet or hood, flaming the inoculating loop prior to the transfer of microbes, disinfecting the work bench with antimicrobial agents, sterilizing or autoclaving used plates and cultures before disposing them, flaming the neck of reagent bottles prior to dispensing and flaming the surfaces of forceps and other working tools amongst others are some of the important aseptic techniques observed in the microbiology laboratory. The mouth or neck of reagent bottles and tubes or flask containing culture media should be properly flamed over a Bunsen burner flame (Figure 1) prior to dispensing in order to prevent contamination of the medium. In summary, the main aim of ensuring aseptic technique in the microbiology laboratory is to prevent contamination.
Figure 1. An illustration of blue flame (arrow) from a Bunsen burner. Passing the neck of culture media flasks or vessels through the flame from a Bunsen burner is a routine aseptic technique performed in the microbiology laboratory. Photo courtesy: https://www.microbiologyclass.com
  • INOCULATION TECHNIQUE: Inoculation is a microbiology technique which is used to introduce or place specimens and microbial cultures on or into a culture medium. The organism to be transferred into or onto the surface of the culture media (inclusive of solid and liquid or broth media) is known as inoculum. Inoculation is performed in the microbiology laboratory with an important piece of apparatus known as the inoculating loop (Figure 2). Inoculation technique allows microbiologists to obtain pure cultures of microorganisms through a process known as streaking.
Figure 2. Inoculating loop. Microorganisms or specimens are picked up with the loop (arrow) and transferred onto sterile culture media or other reagents. A loopful or speck of the microbial culture or specimen is obtained with the loop or tip of the wire instead of taken up a mass of the sample. Photo courtesy: https://www.microbiologyclass.com
  • ISOLATION TECHNIQUE: Isolation technique is a microbiology procedure which is used to obtain pure cultures of microorganisms especially from a mixed culture or specimen. It includes culturing microorganisms or specimens on selective media which allow the growth of particular organisms. After culturing, bacterial growth is often expressed on solid culture media as colonies which may contain contaminants and representative colonies of the organism of interest (Figure 3). But through subsequent subculturing onto fresh culture media, pure cultures of the organism of interest can easily be obtained via isolation as one kind of bacterium known as pure cultures. Pure cultures in microbiology are cultures or microbial growth in which all the microorganisms on solid culture media are of the same strain or species. Pure cultures are also known as axenic cultures; and they are mostly used for further studies in the laboratory such as in performing antimicrobial susceptibility testing (AST) and other biochemical studies. They usually appear as discrete colonies on culture media plate; and isolated colonies of bacteria are often the best for further microbiological tests in the laboratory especially for performing antimicrobial susceptibility studies and other molecular studies (e.g. amplification of bacterial gene by PCR).
Figure 3. Illustration of isolated or discrete colonies (arrows) of bacteria on growth media plate. Discrete colonies of bacteria are pure cultures of microorganisms that are of the same species or strain. Pure cultures are different from mixed cultures which are usually made up of diverse microbial community. Isolated colonies (i.e. pure culture) appear on culture media plate after incubation. Photo courtesy: https://www.microbiologyclass.com
  • STREAKING: Streaking is a microbiological technique that is used to obtain pure cultures of microorganisms (particularly bacteria) in the laboratory. It usually involves a series of drawing a loop (carrying an inoculum of the test organism or sample) back and forth on dried solid culture media. As the streaking of the microbe on the dried solid culture media continues, the inoculum is thinned out along the streak lines as it is distributed along the culture media (Figure 4). By streaking or spreading the inoculum on the surface of a dried solid culture medium as shown in Figure 4, single colonies of the inoculum will be obtained at some parts of the culture media plate during incubation. Streaking is just one method of obtaining pure isolated colonies of bacteria. Other methods of obtaining pure cultures in the microbiology laboratory are the spread plate and pour plate methods. In spread plate method, the test organism or sample is serially diluted in broth culture media before they are transferred onto dried solid culture media and then spread using the inoculating loop. Pour plate methods involve serially diluting the test organism or sample in liquid culture media and then transferring same into molten agar. The molten agar containing the diluted inoculum or sample is then poured onto sterile Petri dishes and allowed to set or gel. Streak plating, spread plating and pour plating are the three known methods used to obtain pure cultures in the microbiology laboratory.
Figure 4. Illustration of streaking pattern in the microbiology laboratory. Streaking is usually started at a point known as the primary inoculum on the solid media from where further streaks are made. As the streaking continues, the number of microorganisms on the loop reduces, and this ensures that a pure culture of the organism is obtained after the entire streaking process and incubation of the agar plate. 1= flame the inoculating loop over a Bunsen burner flame, and streak a loopful of the broth, sample or test organism at point 1 as shown in the figure; 2= re-flamethe inoculating loop and allow it to cool, then streak the organism or specimen from point 1 to spread the original inoculum over more of the agar surface; 3= re-flame the loop again and continue the streak to point 3; 4= re-flame the inoculating loop again and allow to cool before streaking the inoculum from point 3 to point 4. Finally, label the culture plate and incubate it at the appropriate temperature condition while making sure that the Petri dish plate is inverted. Photo courtesy: https://www.microbiologyclass.com
Figure 5. A nutrient agar culture plate. The organism is a clinical isolate of Klebsiella pneumoniae. Notice the mucoid colonies on the culture plate, a characteristic feature of Klebsiella. Photo courtesy: https://www.microbiologyclass.com
  • INCUBATION TECHNIQUE: Microorganisms are incubated in the incubator at different temperatures and time interval depending on the oxygen requirement of the organisms amongst other vital conditions required for optimum growth. Incubation technique is a microbiological procedure which is used to maintain microbial cultures of culture media at a particular ambient temperature at different time intervals. Microbial cultures or culture media can be incubated for hours, days, weeks or for months depending on the time limit required for the propagation of the microorganisms being cultivated. The main reason for this technique is to provide optimum conditions required for the growth of the organism. Some microorganisms (e.g. fungi) are incubated at room temperatures (e.g. 25-28oC); and this implies that the microbial cultures are left to thrive in the normal conditions of the laboratory without putting them in the incubator (where microbial growth occurs at 37oC). This technique allows microbiologists to grow or propagate microbes under optimal growth conditions such as in the presence or absence of oxygen for aerobic and anaerobic bacteria respectively amongst other environmental conditions required for growth (e.g. pH).
  • MICROSCOPY: Microscopy is an important microbiological technique which is used by microbiologists to observe the unseen world of microbes too small to be seen by the naked eyes. This technique makes use of the microscope, an instrument used for examining objects that are too small to be seen by the unaided eyes. By forming an enlarged distinct image of the microbe or object being examined, the microscope enables microbiologist to describe and understand the intracellular components of microorganisms. Examples of microscopes used for microbial examination include bright field microscope, phase-contrast microscope, fluorescence microscope and electron microscope amongst others.
  • STERILIZATION TECHNIQUE: Sterilization technique is used to make microbial culture media, reagents and other objects or apparatuses in the microbiology laboratory sterile. To make an object sterile simply means to make it germ-free. Several sterilization methods are used in the microbiology laboratory to decontaminate materials in the microbiology laboratory. The autoclave is one of the most important equipment used in the microbiology laboratory to achieve sterilization; and it uses moist heat under pressure to achieve this purpose. Another piece of equipment used for sterilization in the laboratory is hot-air oven, which uses dry heat to sterilize materials especially glass wares and other heat-stable materials. Sterilization can also be achieved through heating, filtration, radiation and by other chemical and physical means; but the type of sterilization method used is largely dependent on the nature of the material to be sterilized and the level of sterilization to be achieved. This technique is also applied in the food and pharmaceutical industry to ensure the safety and sterility of manufactured goods or products.    
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  • DIRECT PLATE COUNTING: Direct plate counting is a microbiological technique that is used to evaluate the actual bacterial content of a product or specimen; and they give an estimate of viable or living cells present in a sample. It is usually carried out by plating an aliquot of the sample on solid agar medium that supports the growth of the bacteria being sought for. The number of viable cells in the sample of interest is assessed from the number of colonies which develop on the solid agar medium after incubation. The colony counts of bacteria may range from < 1000 to > 106 colonies per ml (m-1) of the diluted sample or product. And the counted organisms or colonies on the solid agar plate is usually expressed as colony forming units per ml (cfu/ml); and cfu is used to express a single cell of bacteria that is capable of producing a single colony because it is generally assumed that each colony on the agar plate developed from a single bacterial cell. CFU is the microbiological expression of the number of bacteria or fungi in a microbial population (which is usually seen as distinct colonies on solid agar plates); and it is a measure of the viable cells (bacteria and fungi inclusive). While direct microscopy of cells under the microscope may give an estimate of both viable cells and dead cells; CFU (which is obtained via direct plating on solid culture media) measures only viable or living bacterial cells, and the theory behind it is based on the fact that a single bacterium can grow on agar medium and become a colony especially through binary fission. Direct plate count can also be used for counting fungi and other microbial cells excluding viruses. Electron microscopy is used for viral counts, and the counted viral particles are expressed as plaque forming units (pfu). Spread plating and pour plating are the two methods of direct plate counting techniques for microorganisms (with the exception of viruses). But the pour plate method is more preferable to the spread plate technique when counting microbes because the later (in which the diluted sample is spread over an agar plate) usually produces colonies that only form on the surface of the agar medium. In the pour plate technique, colonies do not just form on the surface of the agar but throughout the agar medium (i.e. in the substratum of the agar medium). The pour plate technique involves the dispensing of an aliquot of the test sample (usually in a diluted form in the range of 0.1 – 1.0 ml) on a clean Petri dish; and then the sterile molten agar is poured into the plate and the Petri dish is swirled in order to ensure an even mixture of the medium and the inoculum and/or diluted sample. The plate is allowed to set or dry prior to incubation at the appropriate temperature conditions. Other techniques available for the counting of microbes in the microbiology laboratory include microscopy and turbidometric method.   
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  • IDENTIFICATION TECHNIQUE: Identification technique is used to discover and categorize isolated microbial cultures in the microbiology laboratory. This technique is used to identify microorganisms to their precise species or strain level. Identification of microorganisms in the microbiology laboratory is often carried out using specific biochemical tests which is unique for a particular organism. Some biochemical tests or identification techniques employed in the microbiology laboratory for the identification of microbial pathogens include indole test, citrate test, urease test, motility test, Gram staining, sugar fermentation test, Voges Proskauer (VP) test, coagulase test, catalase test, optochin test, nitrate reduction test, oxidase test, starch hydrolysis test, determination of hydrogen sulphide production and methyl red test amongst others. Identification tests help microbiologists to identify a particular microorganism from a mixture of organisms; and they also assist microbiologists to put a name to a given pathogen. 
  • MOLECULAR TECHNIQUES: Molecular techniques used in the microbiology laboratory include polymerase chain reaction (PCR) based tests which are employed for the identification of microorganisms from clinical specimens and other samples based on the identification of nucleotide sequences peculiar to each microbe. Molecular detection techniques usually employ DNA probes, gene chips and other gene amplification and sequencing techniques that allow microbiologists to detect pathogen-specific nucleotide sequences directly from clinically relevant specimen and other samples. They are important tool for the amplification of genes responsible for antimicrobial resistance in pathogenic microorganisms. Molecular techniques can also be used for the prompt identification of microorganisms especially bacteria from culture plates. Other molecular methods used for microbial characterization include nucleic acid sequencing and restriction fragment length polymorphism (RFLP) amongst others. 

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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