Aseptic Technique and Transfer of Microorganisms
In natural environments, microorganisms usually exist as mixed populations. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all organisms are descendants of the same organism.
Techniques for obtaining pure cultures from a mixed population will be described in Chapter ?.
In working with microorganisms we must also have a sterile nutrient-containing-medium in which to grow the organisms. Anything in or on which we grow microorganisms is termed a medium. A sterile medium is one which is free of all life forms. It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed.
Finally, in working with microorganisms, we must have a method of transferring growing organisms (called the inoculum) from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.
A. ASEPTIC TECHNIQUE
The procedure for aseptically transferring microorganisms is as follows:
1. Sterilize the inoculating loop.
The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way, all contaminants on the wire are incinerated. Never lay the loop down once it is sterilized or it may again become contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum.
2. Remove the inoculum.
a. Removing inoculum from a broth culture (organisms growing in a liquid medium):
1. Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop as if it were a pencil.
2. Remove the cap of the pure culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated.
3. Very briefly flame the lip of the culture tube. This creates a convection current which forces air out of the tube and preventing airborne contaminants from entering the tube.
4. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculum.
5. Again flame the lip of the culture tube.
6. Replace the cap.
b. Removing inoculum from a plate culture (organisms growing on an agar surface in a petri plate):
1. Sterilize the inoculating loop in the flame of the gas burner.
2. Lift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop.
3. Scrape off a small amount of the organisms and close the lid.
3. Transfer the Inoculum to the Sterile Medium.
a. Transferring the inoculum into a broth tube:
1. Pick up the sterile broth tube and remove the cap with the little finger of your loop hand. Do not set the cap down.
2. Briefly, flame the lip of the broth tube.
3. Place the loopful of inoculum into the broth, and withdraw the loop. Do not lay the loop down!
4. Again flame the lip of the tube.
5. Replace the cap.
6. Resterilize the loop by placing it in the flame until it is orange. Now you may lay the loop down until it is needed again.
b. Transferring the inoculum into a petri plate:
1. Lift the edge of the lid just enough to insert the loop.
2. Streak the loop across the surface of the  agar medium using a pattern shown in either Figure 2A or Figure 2B. 
Avoid digging into the agar by keeping the loop horizontal during streaking.
3. Remove the loop and close the lid.
4. Resterilize the inoculating loop.
every procedure in the your lab will be done using similar aseptic technique.
B. FORMS OF CULTURE MEDIA
1. Broth tubes are tubes containing a liquid medium. A typical nutrient containing broth medium such as Trypticase Soy broth contains substrates for microbial growth such as pancreatic digest of casein, papaic digest of soybean meal, sodium chloride, and water. After incubation, growth (development of many cells from a few cells) may be observed as one or a combination of three forms:
a. Pellicle : A mass of organisms is floating on top of the broth.
b. Turbidity:  The organisms appear as a general cloudiness throughout the broth.
c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube.
2. Slant tubes are tubes containing a nutrient medium plus a solidifying agent, agar-agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface.
3. Stab tubes (deeps) are tubes of hardened agar medium which are inoculated by "stabbing" the inoculum into the agar.
4. Agar plates are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stab and are commonly used in the culturing, separating, and counting of microorganisms.
C. OXYGEN REQUIREMENTS FOR MICROBIAL GROWTH
Microorganisms show a great deal of variation in their requirements for gaseous oxygen. Most can be placed in one of the following groups:
1. Obligate aerobes are organisms that grow only in the presence of oxygen. They obtain energy from aerobic respiration.
2. Microaerophiles are organisms that require a low concentration of oxygen for growth. They obtain energy from aerobic respiration.
3. Obligate anaerobes are organisms that grow only without oxygen and, in fact, oxygen inhibits or kills them. They obtain energy from anaerobic respiration or fermentation.
4. Aerotolerant anaerobes, like obligate anaerobes, cannot use oxygen for growth but they tolerate it fairly well. They obtain energy from fermentation.
5. Facultative anaerobes are organisms that grow with or without oxygen, but generally better with oxygen. They obtain energy from aerobic respiration, anaerobic respiration, and fermentation. Most bacteria are facultative anaerobes.
D. TEMPERATURE REQUIREMENTS
Microorganisms are divided into groups on the basis of their preferred range of temperature:
1. Psychrophiles are cold-loving organisms. Their optimum growth temperature is 0C to 15C with a maximum growth temperature of 20C. Psychrotrophs can, like psychrophiles, grow at 0C and even lower but can also grow above 20C.
2. Mesophiles are organisms that grow best at moderate temperatures. Their optimum growth temperature is between 25C and 40C. Most bacteria are mesophilic.
3. Thermophiles are heat-loving organisms. Their optimum growth temperature is between 50C and 60C and a few can tolerate temperatures as high as 110C.
E. COLONY MORPHOLOGY AND PIGMENTATION
A colony is a visible mass of microorganisms growing on an agar surface and usually originating from a single organism or arrangement of organisms. Different microorganisms will frequently produce colonies which differ in their morphological appearance (form, elevation, margin, surface, optical characteristics, and pigmentation).
Probably the most visual characteristic is pigmentation (color). Some microorganisms produce pigment during growth and are said to be chromogenic. Often, however, a formation of pigment depends on environmental factors such as temperature, nutrients, pH, and moisture. For example, Serratia marcescens produces a deep red pigment at 25C but does not produce pigment at 37C.
Pigments can be divided into two basic types: water insoluble and water soluble. If the pigment is water insoluble, as in the case of most chromogenic bacteria, it does not diffuse out of the organism. As a result, the colonies are pigmented but the agar remains the normal color. If the pigment is water soluble (as in the case of Pseudomonas aeruginosa) it will diffuse out of the organism into the surrounding medium. Both the colonies and the agar will appear pigmented.
Below is a list of several common chromogenic bacteria:
Staphylococcus aureus - gold; water insoluble
Micrococcus luteus - yellow; water insoluble
Micrococcus roseus - pink; water insoluble
Mycobacterium phlei - orange; water insoluble
Serratia marcescens - orange/red; water insoluble
Pseudomonas aeruginosa - green/blue; water soluble
MEDIA
Trypticase Soy Broth tubes (4), Trypticase Soy Agar slant tubes (4), Trypticase Soy Agar stab tubes (4), and Trypticase Soy Agar plates (7).
ORGANISMS
Trypticase Soy Broth cultures of Bacillus subtilis, Escherichia coli, and Micrococcus luteus, and Trypticase Soy Agar plate cultures of Mycobacterium phlei.
PROCEDURE (to be done in pairs)
1. Aseptically inoculate one Trypticase Soy Broth tube with B. subtilis, one with E. coli, one with M. luteus, and one with M. phlei. Remember to label all tubes with a wax marker.
2. Aseptically inoculate one Trypticase Soy Agar slant tube with B. subtilis, one with E. coli, one with M. luteus, and one with M. phlei.
3. Aseptically inoculate one Trypticase Soy Agar stab tube with B. subtilis, one with E. coli, one with M. luteus, and one with M. phlei by stabbing into the agar.
4. Aseptically inoculate one Trypticase Soy Agar plate with B. subtilis, one with E. coli, one with M. luteus, and one with M. phlei by using either of the patterns shown in Figure 2A or 2B. This procedure is termed streaking for isolation and has a diluting effect. The friction of the loop against the agar causes organisms to fall off the loop. Near the end of the streaking pattern, individual organisms become separated far enough apart on the agar surface to give rise to isolated single colonies after incubation.
5. Incubate all the tubes and plates inoculated with B. subtilis, E. coli, M. luteus, and M. phlei at 37C. Place the tubes in a plastic beaker to keep them upright. Incubate the plates upside down (lid on the bottom) to prevent condensing water from falling down on the growing colonies and causing them to run together.
6. In order to illustrate that microorganisms are all around us and to demonstrate the necessity for proper aseptic technique, contaminate three Trypticase Soy Agar plates as follows:
a. Remove the lid from the first agar plate and place the exposed agar portion in or out of the building for the duration of today's lab. Replace the lid, label, and incubate it at room temperature. Do this plate first.
b. Moisten a sterile cotton swab in sterile water and rub the swab over some surface in the building or on you. Use this swab to inoculate the second agar plate. Label the plate and incubate at room temperature.
c. Rub your fingers over the surface of the third agar plate. Label and incubate at 37C. Do this plate last.
Exercises
1. Draw and describe the growth seen in each of the four broth cultures.
Bacillus subtilis
growth = Escherichia coli
growth = Micrococcus luteus
growth = Mycobacterium phlei
growth =
2. Observe the growth in the slant cultures and stab cultures for pigmentation and purity.
3. Using the terms in Appendix A, compare a single colony of B. subtilis with a single colony of M. luteus. Use a hand lens or a dissecting microscope to magnify the colony.
characteristics B. subtilis M. luteus
the form of colony
elevation
margin (edge)
surface
optical characteristics
pigmentation
4. Observe the results of the three "contamination" plates and note the differences in colony appearances.
5. Observe the demonstration plates of chromogenic bacteria and state the color and water solubility of each pigment.
organism color solubility
Micrococcus luteus
Micrococcus roseus
Mycobacterium phlei
Serratia marcescens
Pseudomonas aeruginosa
Exercises
1. Draw and describe the growth seen in each of the four broth cultures.
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Bacillus subtilis
growth =
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Escherichia coli
growth =
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Micrococcus luteus
growth =
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Mycobacterium phlei
growth =
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2. Observe the growth in the slant cultures and stab cultures for pigmentation and purity.
3. Using the terms in the Appendix A, compare a single colony of B. subtilis with a single colony of M. luteus. Use a hand lens or a dissecting microscope to magnify the colony.
characteristics
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B. subtilis
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M. luteus
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form of colony
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elevation
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margin (edge)
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surface
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optical characteristics
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pigmentation
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4. Observe the results of the three "contamination" plates and note the differences in colony appearances.
5. Observe the demonstration plates of chromogenic bacteria and state the color and water solubility of each pigment.
organism
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color
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solubility
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Micrococcus luteus
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Micrococcus roseus
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Mycobacterium phlei
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Serratia marcescens
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Pseudomonas aeruginosa
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PERFORMANCE OBJECTIVES Aviculture Microbiology
After completing this Work Page, the Aviculturist will be able to perform the following:
1. Define the following terms: pure culture, sterile medium, inoculum, aseptic technique, andcolony.
2. State and define the three types of growth that may be seen in a broth culture.
3. Define the following terms: obligate aerobe, microaerophile, obligate anaerobe, aerotolerant anaerobe, and facultative anaerobe.
4. Define the following terms: psychrophile, psychrotroph, mesophile, and thermophile.
5. Define the following terms: chromogenic, water-soluble pigment, and water insoluble pigment.
PROCEDURE
1. Using an inoculating loop, demonstrate how to aseptically remove some inoculum from either a broth tube, slant tube, stab tube, or petri plate, and inoculate a sterile broth tube, slant tube, stab tube, or petri plate without introducing outside contamination.
2. Label all tubes and plates and place them on the proper shelf in the incubator.
3. Dispose of all materials when the experiment is completed, being sure to remove all markings from the glassware. Place all tubes and plates in the designated areas.
RESULTS
1. Recognize and identify the following types of growth in a broth culture: pellicle, turbidity, sediment, and any combination of these.
2. Consult with Avian Veterinarian for diagnosis with specific findings.
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