Bacteria grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike. In the identification of bacteria and fungi much weight is placed on how the organism grows in or on media.
This exercise will help you identify the cultural characteristics of a bacterium on an agar plate - called colony morphology. Although one might not necessarily see the importance of colonial morphology at first, it really can be important when identifying the bacterium. Features of the colonies may help to pinpoint the identity of the bacterium. Different species of bacteria can produce very different colonies.
In the above picture of a mixed culture, an agar plate that has been exposed to the air and many different colony morphologies can be identified. Nine obviously different colonies are numbered: some colony types recur in various areas of the plate note 3 and 4.
Not only are pigment differences seen, but also size, edge, pattern, opacity, and shine. 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. If you click on the graph and choose print, it will size graph to full page. Figure 1. Linear trendline added.
You can use the graph to determine the generation time. Choose two points on the graph between which the population doubled and determine the time it took for this to happen. For DNA, the sample is loaded onto an agarose gel and subjected to an electrical current. Numerous markers are commercially available, so DNA size may be determined. The 11 bands produced by this digest are shown in Figure 2.
These fragments are suitable for sizing linear double-stranded DNA from bp. When the gel is finished and a photograph of all bands made, the distance each band has traveled on the gel is graphed vs. The size of the DNA you have isolated can then be determined by comparing the distance it traveled to the standard curve.
One of the most common types of statistical analysis done on laboratory data is determination of standard deviation. Standard deviation is defined as the positive square root of the variance.
The formula is:. The sample deviation can be determined for an individual sample or for a population. In order to determine standard deviation, one must first calculate the sample mean and the deviation between each sample and the sample mean. The data for the experiment are listed in the first 2 columns of Table 3. Table 3. The results of Escherichia coli grown at 37 o C on nutrient broth one ml plated. The deviation for each sampling point column 3 is obtained by subtracting the sample mean.
The standard deviation for each sampling point column 4 is obtained by taking the positive square root of the deviation.
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