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 uses to manipulate microorganisms for improved productivity and better service delivery. Some of these techniques (as shall be discussed in this unit) are what distinguishes the microbiologist from other scientists; and these techniques must be dutifully followed in order to achieve better research results as a beginner. 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. The techniques outlined in this section are not conclusive of the microbiological modus operandi; however, they are basic and fundamental microbiological techniques that students of the subject should understand at first introduction. 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 to mention but a few are succinctly discussed in this section. Students of microbiology are advised to ensure continuous practice of these techniques and other emerging techniques or technologies in the field of microbiology in order to be familiar with them.
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 processes – in which bacterial cells are stained several times using different types of dyes or counter stains meant to reveal certain unique 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. 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 or to certain stains or dyes.
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 and conditions are germ-free and ready for use. It involves numerous processes taken to reduce or prevent the contamination of microbial cultures, reagents, working environment 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 (Figure 1), flaming the inoculating loop prior to the transfer of microbes, disinfecting the work bench or area 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 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 2) prior to dispensing in order to prevent contamination of the medium during its usage. In summary, the main aim of ensuring aseptic technique in the microbiology laboratory is to prevent contamination of the experimentation process by microorganisms and other contaminants.
Inoculation is a microbiology technique which is used to introduce or place specimens and microbial cultures on or into a culture medium for further experimentation. The organism to be transferred into or onto the surface of the culture media (inclusive of solid and liquid media or broth) is known as inoculum. Inoculation is performed in the microbiology laboratory with an important piece of apparatus known as the inoculating loop (Figure 3). Inoculation technique allows microbiologists to transfer and obtain pure cultures of microorganisms through a process known as streaking.
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 culture 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 4). But through subsequent subculturing onto fresh culture media that is selective for the organism, pure cultures of the organism of interest can easily be obtained through isolation technique. The isolated culture is known as pure culture. 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 or molecular studies. They usually appear as discrete colonies on culture media plate. Isolated colonies of bacteria that are pure cultures are often the best for further microbiological tests in the laboratory especially for performing antimicrobial susceptibility studies and further molecular studies (for example, amplification of bacterial gene by the polymerase chain reaction [PCR] technique).
Streaking is a microbiological technique that is used to obtain pure cultures of microorganisms (particularly bacteria and fungi) in the microbiology 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 in a regular (defined) pattern. 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 plate (Figure 5). 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. This allows the researcher to obtain pure cultures of the test bacteria which will further be characterized for proper identification and purity using other characterization and identification techniques.
Streaking is just one method of obtaining pure isolated colonies of bacteria from a mixed culture or sample. 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 plate and then spread-plated using the inoculating loop as shown in Figure 5. Pour plate methods involve serially diluting the test organism or sample in liquid culture media (broth) 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. After incubation, the culture plate is observed macroscopically for pure cultures of the organism of interest. Streak plating, spread plating and pour plating are some of the known methods used to obtain pure cultures in the microbiology laboratory; and they are the conventional methods through which the isolation of pure cultures can be carried out in the microbiology laboratory. However, there are molecular techniques such as polymerase chain reaction (PCR) and commercially available selective culture media such as chromogenic agar that can allow for the isolation of pure cultures of a given organism from different clinical and environmental samples. Chromogenic culture media are fast becoming the best medium for microbiologists to isolate and identify bacteria from a variety of samples because of the unique colonial features that they have. But after the isolation of the organism using chromogenic culture media, the isolated organism can still be subjected to further screening or characterization for proper identification. Chromogenic culture media employs a technology that is based on the use of chromogens (which are soluble colourless molecules). These culture media are composed of a substrate that targets a specific enzymatic activity in the organism of interest. The other component of the chromogenic media apart from the chromogens and substrate is the chromophore – which is released when the target organism of interest produces an enzyme that cleaves the colourless chromogen-substrate complex in the media. Upon cleaving, a variety of colours are released which is unique for a particular bacterium, and this allows for the easy identification of the organism from the culture.
Culturing technique is used for the propagation of microorganisms in the microbiology laboratory; and it is an important procedure required for studying the morphological (colonial) characteristics of microbes especially on solid culture media. Microorganisms are cultured in various ways and in various conditions using different growth media (inclusive of solid media or broth/liquid media). Bacterial growth on solid culture media is often the best approach for studying the physical appearance of a microbe in terms of its consistency, shape, texture, size and colour as produced on solid growth medium. Culturing technique involves the inoculation of growth media with the inoculum (for example, bacteria or fungi) or test sample suspected to contain a particular microbe of interest. It also involves the incubation of the culture plate under optimum condition that encourages the growth of the test organism. The cultured organism can appear in any of these forms on the solid culture media: as individual or discrete colonies and as confluent growth (i.e., film of surface growth).
Confluent growth is often required for performing antimicrobial susceptibility testing (AST) in the microbiology laboratory. In broth or liquid medium, bacterial growth is usually inferred by the presence of turbidity or cloudiness in the medium; and a loopful of the organism can be transferred onto a solid culture media to obtain pure cultures of the organism for further studies. Various culturing techniques are used for the propagation of microorganisms in the microbiology laboratory. Examples of some notable culturing procedure include stab culture (produced by deep inoculation of solid medium in a tube with a straight wire loop), slope or slant culture (produced on the gradient of a solid medium in tubes) and plate culture (Figure 6). Stab cultures are often used for biochemical tests and mycological investigations in which fungi or fungal samples are stabbed deep into solid media in tubes.
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 in culture media at a particular ambient temperature and at different time intervals required for unperturbed microbial growth. Microbial cultures or culture media ladened with the test isolate or sample 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 of interest. Some microorganisms (for example, fungi) are incubated at room temperatures (for example, 25-28oC); and this implies that the microbial cultures may be left to thrive in the normal conditions of the laboratory without putting them in the incubator (where microbial growth occurs at 37oC). The incubation 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 (for example, pH).
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 and/or extracellular components of microorganisms. Examples of microscopes used for microbial examination include bright field microscope, phase-contrast microscope, confocal microscopy, fluorescence microscope and electron microscope.
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 in order to prevent cross-contamination of the working environment and/or sample by unwanted organisms or contaminants. 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. Sterilization technique is also applied in the food and pharmaceutical industry to ensure the safety and sterility of manufactured goods or products.
DIRECT PLATE COUNTING
Direct plate counting is a microbiological technique used to evaluate the actual bacterial content of a product or specimen; and it gives 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 are usually expressed as colony forming units per ml (cfu/ml). The acronym ‘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 or living microbial cells (bacteria and fungi inclusive). While direct observation of cells under the microscope may give an estimate of both viable cells and dead cells; CFU (which is obtained through direct plating on solid culture media) measures only viable or living bacterial cells. 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 usually 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. Turbidometric methods of counting microbial cells measures the turbidity produced by bacterial cells grown in liquid medium (broth). Turbidity is the measurement of suspended solids (in this case, microbial cells) in a solvent (for example, water or normal saline). The turbidometric method makes use of a spectrophotometer for the measurement of bacterial cells in suspensions or broth (i.e., liquid medium).
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 technique, 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. 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. However, there are other commercially available rapid identification techniques for the prompt identification of microbes (bacteria and fungi) from cultures or samples. While some of these techniques make use of reagents or chemicals that help in the prompt identification of the target organism, others use molecular techniques such as PCR.
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, pulse field gel electrophoresis (PFGE), multilocus sequence typing (MLST), DNA microarray, gene probing techniques and restriction fragment length polymorphism (RFLP).
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