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This section is designed to instruct you in
1. The types of laboratory equipment and culture media needed to develop and maintain
pure cultures.
2. The concept of sterility and the procedures necessary for successful sub-culturing of
microorganisms.
3. Streak-plate and spread-plate inoculations for separation of microorganisms in a mixed microbial population for subsequent pure isolation.
4. The serial-dilution-agar-plating technique for enumeration of viable microorganisms.
5. Cultural and morphological characteristic of microorganisms grown in pure culture.
Introduction
Microoganisms are ubiquitous. They are found in soil, air, water, food, sewage, and a body surfaces. In short, every area of our environment is replete with them.
The microbiologist separates these mixed population into individual species for study.
A culture containing a single unadulterated species of cells is called a pure culture.
To isolate and study microorganisms in pure culture, the microbiologist requires basic laboratory apparatus and the application of specific techniques.
Media
The survival and continued growth of microorganisms depend on an adequate supply of nutrients and a favorable growth environment. For the former, most microbes must use
soluble low-molecular weight substances that are frequently derived from the enzymatic
degradation of complex nutrients.
A solution containing these nutrients is a culture medium.
Basically, all culture media are liquid, semisolid, or solid. A liquid medium lacks a solidifying agent and is called a broth medium.
A broth medium supplemented with a solidifying agent called agar results in a solid or semisolid medium. Agar is an extract of seaweed, a complex carbohydrates composed mainly of galactose, and is without nutritional value.
Agar serves as an excellent solidifying agent because it liquefies at 100 degrees centigrade and solidifies at 40 degrees centigrade. Because of these properties, organisms, especially pathpogens, can be cultivated at temperatures of 37.5 degrees centigrade or slightly higher without fear of the medium liquefying. A completely solid medium requires an agar concentration of about 1.5 percent to 1.8 percent.
A concentration of less than 1 percent agar results in a semisolid medium. A medium solid medium has the advantage that it present a hardened surface on which on microorganisms can be grown using specialized techniques for the isolation of discrete colonies. Each colony is a cluster of cell that originates from the multiplication of a single cell represents the growth of a single species of microorganism. Such a defined and well-isolated colony is a pure culture. Also, while in the liquefied state, solid media can be placed in test tubes, which are then allowed to cool and harden in a slanted position, producing agar slants.
These are useful for maintaining pure cultures. Similar tubes that, following preparation, are allowed to harden in the upright position are designated as agar deep tubes. Agar deep tubes are used primarily for the study of the gaseous requirements of microorganisms.
However, they may be liquefied in a boiling water bath and poured into Petri dishes,
producing agar plates, which provide large surface areas for the isolation and study of microorganism.
In addition to nutritional needs, the environmental factors must also be regulated,
including proper ph, temperature, gaseous requirement, and osmotic pressure.
Sterilization
Sterility is the hallmark for successful work in the microbiology laboratory. To achieve sterility, it is mandatory that you use equipment and sterile techniques. Sterilization is the process of rendering a medium or material free of all forms of life. Although a more
detailed discussion is presented in Part IX, the section dealing with control of microorganisms.
Culture Tubes and Petri Dishes
Glass test tubes and glass or plastic Petri Dishes are used to cultivate microorganisms. A suitable nutrient medium in the form of broth or agar may be added to the tube, while only a solid medium is used in Petri dishes. A sterile environment is maintained in culture tubes by various types of closures.
Historically, the first type, a cotton plug, was developed by Schroeder and von Dusch in the nineteenth century. Today most laboratories use sleevelike caps made of metals, such as stainless steel, or heat ñresistant plastics.
The advantange of these closures over the cotton plug is that they are labor-saving and most of all slip on and off the test tubes easily.
Petri dishes provide a larger surface area for growth and cultivation. They consist of a bottom dish portion that contain the medium and a larger top portion that serves as a loose cover. Petri dishes are manufactured in various sizes to meet different experimental requirements. For routine purposes, dishes approximately 15 cm in diameter are used. The sterile agar medium is dispensed to previously sterilized dishes from molten agar deep tubes containing 15 to 20 ml of medium, or from a molten sterile medium prepared in bulk and contained in 250-ml to 500-ml flasks.
When cooled 40 degrees centigrade, the medium will solidify. Remember that after incoluation, Petri dishes are incubated in an inverted position (top down) to prevent condensation that forms on the cover during solidification from dropping down on to the surface of the hardened agar.
Transfer Instruments
Microorganisms must be transferred from one vessel to another or from stock cultures to various media for maintenance and study. Such a transfer is called subculturing and must be carried out under sterile conditions to prevent possible contamination.
Wire loops and needles are made from inert metals such as nichrome or plantinum and are inserted into metals shafts that serve as handles. They are extremely durable instruments and are easily sterilized by incineration in the blue (hottest) portion of the Bunsen burner flame.
A pipette is another instrument used for sterile transfers. Pipettes are similar in function to straws, that is, they draw up liquids. They are made of glass plastic drawn out to a tip at one end and with a mouthpiece forming the other end. They are calibrated to deliver different volumes depending on requirements. Pipette may be sterilized in bulk inside canisters, or they may be wrapped individually in brown paper and sterilized in an autoclave or dry-heat oven. Note: Pipetting by mouth is not permissible! Pipetting is to be performed with the aid of mechanical devices. The proper procedure for the use of pipette will be demonstrated by your instructor.
Cultivation Chambers
The specific temperatures for growth are needed. However, a prime requirement for the cultivation of microorganisms is that they be grown at their optimum temperature.
An incubator is used to maintain optimum temperature during the necessary growth period. It resembles an oven and is thermostatically controlled that temperature can be varied depending on the specific microorganisms. Most incubators use dry heat. Moisture is supplied by placing a beaker of water in the incubator during the growth period. A moist environment retards dehydration of the medium and thereby avoids spurious experimental results.
A thermostatically controlled shaking waterbath is another piece of apparatus used to cultivate microorganism. Its advantage is that it provides a rapid and uniform transfer of heat to the culture vessel, and its agitation provides increased aeration, resulting in acceleration of growth. The single disadvantage of this instrument is that it can be used only for cultivation of organisms in a broth medium.
Refrigerator
A refrigerator used for a wide variety of purposes such as maintenance and storage of stock cultures between subculturing periods and storage of sterile media to prevent dehydration. It is also used as a repository for thermolabile solution, antibiotics, serums and biochemical reagents.
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