Why is a needle used to isolate colonies

Multiple cultures are easily placed into test tube racks and stored under refrigeration. Bacteria are inoculated onto a slant using a loop and grow in the surface of the agar. This type of culture medium gives the ability to grow bacteria in both an aerobic, oxygen-rich, environment surface of the slant and an anaerobic, oxygen deficient, environment butt of slant. DEEP: solid medium made with agar and various nutrients and indicators.

This type of culture is used for the growth of anaerobic bacteria which grow in the absence of oxygen and are inoculated by stabbing the media with a needle.

BROTH: liquid medium made with various nutrients and indicators. Allows for the growth of large volumes of bacteria, the level of growth can be assessed based on the turbidity cloudiness of the culture. Bacteria are inoculated into a broth using a loop. Durham tubes are used to detect the production of gases, such as CO2 or N2, by microorganisms. The tube is initially filled with the medium and then collects gas as the bacteria grow, creating a bubble.

PLATE: solid medium made with agar and various nutrients and indicators. Can be made in Petri dishes of various sizes. Plates are particularly helpful in isolating a specific species of bacteria, which is not possible in a liquid medium. Using the SFIC technique bacteria can be diluted until individual colonies are formed. Bacteria are inoculated onto a plate using a loop. Please be aware that a loop will collect much higher concentrations of bacteria from a plate than from a broth.

You will be provided with Staphylococus epidermidis in a broth and on a plate. These will be shared with the table and will be found at the front of each table in a white plastic rack. Aseptically transfer bacteria from your BROTH culture to a broth, a slant, and a plate using your loop. All tubes will be placed in the provided racks at the end of your table. Bacteria are described by three basic criteria: size, shape, and arrangement.

A micrometer is one-millionth of a meter. The three most common shapes are the rod bacillus , the sphere coccus , and the spiral type vibrio. Arrangement is the manner by which groups of bacteria appear together.

Some common arrangement types are paired diplo , grape-like clusters staphylo or chains strepto. An appropriate physical environment must be created, where important factors such as temperature, pH, and the concentration of atmospheric gases particularly oxygen are controlled and maintained. The nutritional needs of bacteria can be met through specialized microbiological media that typically contain extracts of proteins as a source of carbon and nitrogen , inorganic salts such as potassium phosphate or sodium sulfate, and in some cases, carbohydrates such as glucose or lactose.

Bacteriological culture media can be prepared as a liquid broth , a solid plate media or slant media , or as a semi-solid deeps as illustrated in Figure 1.

Solid and semi-solid media contain a solidifying agent such as agar or gelatin. Agar, which is a polysaccharide derived from red seaweed Rhodophyceae is preferred because it is an inert, non-nutritive substance. The agar provides a solid growth surface for the bacteria, upon which bacteria reproduce until the distinctive lumps of cells that we call colonies form. Koch, Pasteur, and their colleagues in the 19th and early 20th centuries created media formulations that contained cow brains, potatoes, hay, and all sorts of other enticing microbial edibles.

Today, bacteriological media formulations can be purchased in powdered form, so that all the preparer has to do is to measure out the correct amount, add the right amount of water, and mix. After the basic formula has been prepared, the medium is sterilized in an autoclave, which produces steam under pressure and achieves temperatures above boiling. Once sterilized media has cooled, it is ready to be used.

A population of bacteria grown in the laboratory is referred to as a culture. A pure culture contains only one single type; a mixed culture contains two or more different bacteria.

If a bacterial culture is left in the same media for too long, the cells use up the available nutrients, excrete toxic metabolites, and eventually the entire population will die. Thus bacterial cultures must be periodically transferred, or subcultured , to new media to keep the bacterial population growing. Microbiologists use subculturing techniques to grow and maintain bacterial cultures, to examine cultures for purity or morphology, or to determine the number of viable organisms.

In clinical laboratories, subculturing is used to obtain a pure culture of an infectious agent, and also for studies leading to the identification of the pathogen. Because bacteria can live almost anywhere, subculturing steps must be performed aseptically , to ensure that unwanted bacterial or fungal contamination is kept out of an important culture. In microbiology, aseptic techniques essentially require only common sense and good laboratory skills.

First, consider that every surface you touch and the air that you breathe may be contaminated by microorganisms. Then think about the steps you can take to minimize your exposure to unwanted invisible intruders.

You should also be thinking about how to prevent contamination of your bacterial cultures with bacteria from the surrounding environment which includes you. To maintain an aseptic work environment, everything you work with should be initially free of microbes. Thus, we begin with pre-sterilized pipettes, culture tubes, and glassware.

Inoculating loops and needles made of metal wire can be used to transfer bacteria from one medium to another, such as from the surface of an agar plate to a broth. Metal tools may be sterilized by heating them in the flame of a Bunsen burner.

Standard aseptic techniques used for culturing bacteria will be demonstrated at the beginning of lab. One very important method in microbiology is to isolate a single type of bacteria from a source that contains many. The most effective way to do this is the streak plate method, which dilutes the individual cells by spreading them over the surface of an agar plate see Figure 2.

Single cells reproduce and create millions of clones, which all pile up on top of the original cell. The piles of bacterial cells observed after an incubation period are called colonies. Each colony represents the descendants of a single bacterial cell, and therefore, all of the cells in the colonies are clones.

Therefore, when you transfer a single colony from the streak plate to new media, you have achieved a pure culture with only one type of bacteria.

Different bacteria give rise to colonies that may be quite distinct to the bacterial species that created it. Therefore, a useful preliminary step in identifying bacteria is to examine a characteristic called colonial morphology , which is defined as the appearance of the colonies on an agar plate or slant. Ideally, these determinations should be made by looking at a single colony; however, if the colonial growth is more abundant and single colonies are absent, it is still possible to describe some of the colonial characteristics, such as the texture and color of the bacterial growth.

By looking closely at the colonial growth on the surface of a solid medium, characteristics such as surface texture, transparency, and the color or hue of the growth can be described. Texture —describes how the surface of the colony appears. Common terms used to describe texture may include smooth, glistening, mucoid, slimy, dry, powdery, flaky etc.

Transparency —colonies may be transparent you can see through them , translucent light passes through them , or opaque solid-appearing. Color or Pigmentation —many bacteria produce intracellular pigments which cause their colonies to appear a distinct color, such as yellow, pink, purple or red.

Many bacteria do not produce any pigment and appear white or gray. As the bacterial population increases in number, the colonies get larger and begin to take on a shape or form. These can be quite distinctive and provide a good way to tell colonies apart when they are similar in color or texture.

The following three characteristics can be described for bacteria when a single, separate colony can be observed. It may be helpful to use a magnifying tool, such as a colony counter or dissecting microscope, to enable a close-up view of the colonies.

Colonies should be described as to their overall size, their shape or form, what a close-up of the edges of the colony looks like edge or margin of the colony , and how the colony appears when you observe it from the side elevation. Figure 4 shows a close-up of colonies growing on the surface of an agar plate. In this example, the differences between the two bacteria are obvious, because each has a distinctive colonial morphology.

Using microbiology terms, describe fully the colonial morphology of the two colonies shown above. A full description will include texture, transparency, color, and form size, overall shape, margin, and elevation. A culture medium must contain adequate nutrients to support bacterial growth.

Minimally, this would include organic compounds that can provide the building blocks necessary for cellular reproduction. In many cases, predigested protein, such as hydrolyzed soy protein, serves this purpose and will support the growth of many different bacteria. These media formulations are generally referred to as complex media , to indicate that it is a mixture with many components. Many media contain additional substances such as an antibiotic that may be selective for a particular type of bacteria by inhibiting most or all other types.

Differential media will have additional compounds that permit us to distinguish among bacterial types based on differences in growth patterns. We will eventually use selective and differential media in our experiments, but the focus of this lab is to learn the basic culturing techniques, and therefore, the media used will be Tryptic Soy medium, a complex medium formulated with hydrolyzed soy protein. The media you use in this lab and in all of the future labs will have already been prepared, but it is important for you as a budding microbiologist to understand and appreciate how culture media is prepared.

With this in mind, your instructor may have you watch a brief video that demonstrates the art of media making. Pre-sterilized glass or plastic graduated pipettes Figure 5 are used to transfer specific volumes of sterile liquids accurately. It is important that you learn how to use these tools correctly, since it may be necessary to transfer sterile and sometimes contaminated liquids among various bottles and tubes.

Their appropriate use will be discussed and demonstrated in lab. Some tips to remember:. A portion of a 10 mL graduated pipette is shown in Figure 6. What is the volume of liquid in this pipette? Solid and semi-solid media. Growing cultures of bacteria on solid media agar plate or slant permits us to view and identify colonial characteristics, and also provides a way to separate bacteria in a mixed culture.

Bacteria may be grown in agar slant or stab media in tubes if the purpose is to maintain them in a longer term culture. Generally, bacteria grown on slants will remain viable for a few weeks to a few months, and sometimes longer if stored in a refrigerator. In this laboratory, you will be introduced to aseptic techniques and basic lab skills needed to grow and maintain bacteria in culture. You will be applying these skills often, so mastery is important.

Below, write the names of the two bacteria in the mixed culture and the appropriate BSL, as specified by your instructor:. The techniques needed will first be demonstrated by your instructor. Using aseptic technique, use a 10 ml graduated pipette to transfer 2 ml of broth to each tube. As demonstrated, use a flame-sterilized inoculating loop to pick up from the surface of the M. Note how the broths look immediately after you inoculate them they should still look mostly clear.

Bacterial growth in broths is indicated by the development of a cloudy appearance. If the newly inoculated broth looks cloudy at the start, you will have no way to determine if this is due to bacterial growth during the incubation period.

If your broth looks cloudy, discard it and make another broth using less bacteria. Place the broth subcultures in an incubator at the temperature and time specified by your instructor. Separation of a mixed culture into individual colonies that can be subcultured to make pure cultures depends on how well the streak plate is prepared.

The goal of streak plate method is to dilute the cells by spreading them out over the surface of the agar. This is accomplished in stages, as will be demonstrated in lab before you try it yourself. Use the simulated agar surface below to practice the streak pattern using a pen or pencil. Also write M. As demonstrated, use a sterilized inoculating loop to pick up one M. Spread the bacteria over approximately a quarter of the plate, edge to edge.