Chapter 1 - Basic Pure Culture Techniques

1 - 1 Aseptic technique

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.

1 - 2 Flame sterilization and tube transfer

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:

1. Place the Bunsen burner in front of you and assemble all necessary equipment with in arms reach. Position everything so that you will not burn yourself while trying to inoculate your tubes.
2. Label the tube of broth or agar to be inoculated with identifying marks. The culture, the date, and your initials for example. Place it in a rack in front of you.
3. Holding the inoculating loop handle, flame the entire wire to redness.
4. When the wire cools (about 15-30 seconds) remove the lid of the plate with your other hand and obtain an inoculum by removing a small portion of the surface growth on the agar plate. In most cases you will be picking an isolated colony. Choose a well isolated one. Do not dig into the agar. Replace the lid of the plate immediately.
5. Hold the tube to be inoculated with the free hand. Remove the cotton plug or cap of the tube with the little finger of the hand holding the needle holder. If a cotton-plugged tube is used, the mouth of the tube should be passed briefly through the flame to singe off dust and lint particles. (Dust or lint may fall into the tube and contaminate the medium.)
6. Introduce the inoculum into the tube. When inoculating a tube of broth, rub the wire against the glass just above the fluid level and then tip the tube slightly to wash the inoculum into the broth. The wire should not be rattled against the sides of the tube to shake an inoculum into the broth; this is unnecessary and may create a dangerous aerosol. If the transfer is made to an agar slant, a single mid-line stroke over its surface is made with the wire or loop.
7. Replace the cap or plug (the latter after reflaming the mouth of the tube).
8. Flame the inoculating wire again to redness, slowly to avoid spattering. Put the loop holder down after the wire cools.

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.

1. The tubes are positioned in the hand as shown in Fig. 2-1 B-E. Plugs and caps can be loosened by twisting them.
2. The needle holder is taken in the other hand and the wire flamed and allowed to cool (Fig. 2-1 A).
3. The plugs or caps are removed with the last two fingers of the hand holding the inoculating wire leaving the thumb and index finger free to hold and to manipulate the loop holder with the second finger as a guide and support. Flame the tops of the tubes.
4. Immerse the inoculating wire into the broth culture or scrape the wire across a portion of surface growth on an agar slant to obtain inoculum. Make the transfer from one tube to the other (Fig. 2-1 C).
5. Flame the tubes (Fig. 2-1 D).
6. Return the plugs or caps to the tubes (Fig. 2-1 E).
7. Flame the inoculating wire to sterilize it (Fig. 2-1 F).

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.

1 - 3 Making a medium - You try it

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.

1. Add 250 ml (about 1 cup) of water into a glass container or some other vessel that can stand boiling water. The container should be something you can cover. Glass bottles that can stand boiling or canning jars work well.
2. To this add 15 grams, about 1 tablespoon of instant chicken broth crystals. and stir until dissolved. Cover loosely so that steam can escape, but dust and dirt cannot enter.
3. Microwave the broth on high for 3 minutes or until it just begins to boil.
4. Let the broth cool and sit for at least 2 hours up to overnight at room temperature. Make sure that the medium vessel is covered to prevent contamination from the air. At this point the vegetative cells are dead and most endospores will germinate.
5. Repeat steps 3 and 4 a second time. This will kill the endospores that germinated on day 1.
6. Repeat steps 3 and 4 a third time. This will eliminate the remaining endospores, making the medium sterile.
7. Place the medium in a warm place overnight. If your tyndalization was done correctly, your medium should remain clear and free of microbes.
8. While you are waiting for your medium, search for a sample you would like to inoculate into it. Almost any sample should work, but natural samples are a good idea for this experiment. Samples coming from food or from your body may contain pathogens. Something you probably want to avoid.
9. Check your medium. If it is still clear and sterile, add a pinch of your collected sample to the medium. (The exact amount really does not matter). Again place the medium in a warm place and check it periodically. After a few days, you should start to see the medium become more turbid.
10. If you have the equipment, examine a sample of the medium under a microscope. What shapes of microbes are present? Is there more than one species?
11. When finished, carefully dispose of the borth. It is basically a spoiled food, but the properties of the spoilage microbe are unknown and should be treated with care.

1 - 4 Streak plates

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.

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...

1. Flame the loop to sterilize it and let cool.
2. Position the plate so that the spot of inoculum is nearest the hand not holding the loop (the opposite hand).
3. Lift the plate lid with the opposite hand; just enough to get the loop inside and touch the loop to the inoculum spot. It is often helpful to treat the inoculating loop as if it were a pencil - steadying the loop by resting the heel of the hand against the lab bench.
4. Move the loop back and forth across the spot and then gradually continue toward the center of the plate as you sweep back and forth. Use a very gentle and even pressure.
5. When creating each phase, do not worry about keeping each pass across the plate separate from previous ones.
6. When about 30% of the plate has been covered by the first streaking phase, remove the loop and flame sterilize it.
7. Repeat the above procedure for the second phase, but this time pick up some inoculum by crossing into the first phase 2-3 times and then not passing into it again (Figure 2-3).
8. Repeat as necessary for the third and fourth phases. After streaking the plate, flame sterilize the loop before setting it down.

The above video demonstrates the streak plates technique.

1 - 5 Spread plates and dilution plating

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.

As microbial quantitation involves the use of pipettes"(or micropipettes) in preparing dilutions and inoculating plates, the beginning student in microbiology must become familiar with their use.

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.

1 - 6 Pour Plates

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.

Above is a movie demonstrating the pour plate technique.