Please note, you must be an educator in higher ed or maybe high school to qualify to recieve the MCI
Trying to study this mixed population is often difficult and in the tradition of the scientific method; researchers dissect a system and study each piece in isolation. For microorganisms this means separating the organisms and getting them into pure culture. A pure culture is defined as a growth of microorganisms (a culture) that contains one cell type. It is essential in microbiology to be able to obtain and preserve pure cultures. Over 100 years ago, Robert Koch devised methods to achieve this goal and the methods he developed are essentially still used today. The protocols used to maintain pure cultures are a major part of aseptic technique and are the subject of this chapter.
The goals of aseptic technique are two-fold. The first objective is to obtain pure cultures and secondly to prevent cross-contamination. Microorganisms in culture must not escape into the environment, and microbes in the environment must not get into the cultures we are studying. It is essential that aseptic technique be understood and practiced correctly. Contaminated cultures are worthless for diagnosis or for doing research on, because it is unclear what microbe is performing any action that is being observed.
Aseptic methods commonly used are flame sterilization, tube transfer, streak plates, spread plates and pour plates. Flame sterilization is an easy method to insure sterile transfer of a culture from a source to a growth medium. Tube transfer is useful for moving inocula from one tube to another. Mechanical dilution by making streak plates is the preferred method for obtaining a pure culture of a microorganism. Finally, spread plates and pour plates are common methods for enumerating microorganisms and are sometimes useful for obtaining isolated colonies.
Flame sterilization is a very quick simple method of killing microorganisms on an inoculating loop or needle. The loop or needle is held inside a flame for a few seconds to bring it to redness and then cooled. Once cool, the loop or needle can be used for various culture manipulations. Make sure that the area that contacts the culture is flamed to redness. Also, be patient and let the loop cool down, this usually takes about 15-30 seconds. Learning this technique is essential to everything else you do in microbiology.
Transfer of culture from agar plates to tubes, or from tube to tube, is a common, simple procedure. It is important to perform these transfers in a consistent and rapid manner. The following protocols have been found effective.
To transfer a culture from an agar plate to a broth or agar slant:
In tube to tube transfers by loop or straight wire, both the tube containing the inoculum source and the tube to be inoculated are usually in the hand at the same time. An easy procedure which prevents hand fatigue and the danger of dropping the tubes is illustrated in Figure 2-1.
Figure 2.1. Tube to tube transfer. The standard method for transferring microbes from one medium to another. Each of the steps is described in the text.
At first, this procedure will be awkward, but after some practice it becomes second nature.
The above video demonstrates the technique of flame sterilization and tube transfer.
Making medium is as simple as cooking and a crude medium can be made in almost any kitchen with a few utensils and a source of heat. Below is described the production of a chicken broth medium that will grow many common microorganisms. The medium will be sterilized by tyndalization. Simply boiling a medium once, while it will kill most vegetative cells, does not kill endospores. However, heating encourages the endospores to germinate, and a second boiling kills these microbes. The boiling process is repeated a third time to ensure that all spores have germinated and been killed.
The streak plate method is a rapid and simple technique of mechanically diluting a relatively large concentration of microorganisms to a small, scattered population of cells. The goal is to obtain isolated colonies on a large part of the agar surface, so that desired species can then be brought into pure culture. Proper streaking of plates is an indispensable tool in microbiology. In most cases a closed inoculating loop is used for streaking plates. The wire loop should not be badly oxidized or pitted or it will fail to dilute the inoculum and will scratch the surface of the agar. Streak plates can be made from a broth culture, an agar slant or from an agar plate. It is sometimes convenient to suspend a bit of growth from a solid surface in sterile saline and use this as a source of inoculum. Resuspension of colonies or cultures grown on solid surfaces dilutes the culture and makes streak plating easier. A loopful of inoculum is transferred from the source and put on the agar surface. When using a large inoculum (a turbid culture or growth from a solid surface), a small spot is spread during the initial transfer. If the inoculum is from a lightly turbid suspension, the first phase of the streaking pattern is begun. Several basic patterns are illustrated in Figure 2-3. The three-phase streaking pattern is recommended for beginners because it is most likely to give satisfactory results with suspensions having a wide range of microbial density.
Figure 2.3. Streaking patterns. There are a number of different methods for mechanically diluting microbes on a streak plate. The most common method is spreading microbes across a plate as shown in the first four figures. As the concentration of microbes increases so do the number of phases. Irrespective of the number of phases, loop is flamed between each one. The fifth plate shows an alternative method, where the streaks are not continuous, but are a series of parallel lines. foobar
Choosing a streaking pattern is a matter of individual preference and depends upon the number of microorganisms in the sample. Figure 2-3 demonstrates the most common patterns, but they are not the only methods. The object of any streaking pattern is the continuous dilution of the inoculum to give many well isolated colonies. For multi-phase streaking it is crucial to flame the loop before starting the next phase. Note the slight overlap into the previous phase to pick up a small inoculum. To streak a plate...
The above video demonstrates the streak plates technique.
An absolute requirement for a microbiologist is to be able to determine the concentration of microorganisms in a given sample. Various particle-counting devices, spectrophotometric methods and microscopic techniques have been used to count cells. However, one drawback to these methods is that they count dead as well as living cells. The most common method of enumerating viable cells is the plate-count method. Diluting microorganisms and placing them into petri plates (or plates) for incubation is another essential technique for working with microorganisms.This method suffers from some problems. First, only those organisms which can grow on the medium, and at the temperature and atmospheric conditions of incubation, will divide and develop into colonies. Second, each colony may not represent the progeny from one cell, as two or more cells (those in clusters, chains or otherwise close to one another) can give rise to one colony. For these reasons the counts obtained from the plate-count method are given as the number of colony-forming units (CFU's) per ml (or gram) rather than the number of cells per ml (or gram). Despite these drawbacks, the plate-count method is a powerful means by which concentrations of viable organisms may be estimated. Also, if it is desirable to count a specific subgroup of microorganisms in a sample, selective media or special incubation conditions can often be used to encourage the growth of only this class of organism. More information is available in the chapter on quantitative microbiology.
Due to the possibility of ingesting pathogens and toxic liquids, mouth-pipetting is forbidden in the laboratory! Pipettes are filled and subsequently emptied by the use of propipettes or other pipette bulb. Pay close attention to the demonstration of their use.
Important Safety Consideration: When fitting the pipette and pipette bulb together, use very gentle pressure!! Do not jam these items together! (Force is usually not the answer, a good general rule to live by in this lab and in life for that matter.) The glass pipette will probably break and possibly cause severe injury. Handle the pipette only at the top inch or so.
For volumes of 5 ml of less, micropipettes are often the tool of choice. Instruments are available that are capable of dispensing 5 ml all the way to less than 1 µl (one one-millionth of a liter). Micropipettes have made it possible to miniaturize many experiments and greatly decrease the cost of running them. They are also easy to use and can dispense volumes quickly, increasing the number of experiments that can be performed in a set amount of time. Micropipettes are the tool of choice for small volumes.
A practical and common laboratory technique used in isolating pure cultures or enumerating the living microorganisms in water, milk, foods, and other materials is the pour plate technique.
To aseptically transfer liquid into a pour plate, raise one side of a Petri plate lid only just enough to allow access of the sample (from a tube or pipette). Transfer a known amount of the sample to the dish and cover immediately with the lid. Then pour 15-20 ml of sterile agar culture medium which has been melted and cooled to 45-50°C into the plate as shown in Figure 2-5. The inoculum and medium are mixed by gentle rotation ten times in one direction and ten times in the other direction. The agar must be allowed to solidify completely before the plates are inverted for incubation. After incubation both surface and subsurface colonies will be observed.
Figure 2.5. Pour plates. Pour plates allow the addition of larger amounts of liquid (1-5 ml) to an agar dish. The sample is added to the bottom of a sterile Petri plate. Molten agar is then added to the plate aseptically. It is important to only open the cover enough to allow the pouring of the agar. This prevent contamination from the environment.
Above is a movie demonstrating the pour plate technique.