Identification of bacterial species

Microbiology is the study of bacteria, microscopic, single-celled organisms that can have many different sizes and shapes, or morphologies. Bacteria exist in every possible habitat on Earth – in soil, in water, and even on the skin and in the gastrointestinal tract of the human body. While most of the bacteria that live in our bodies are either harmless or beneficial, some bacteria are pathogens, meaning that they cause infection or disease. Correct identification of these unknown pathogenic bacteria is crucial in determining the appropriate type of antibiotic therapy that is necessary to eliminate bacterial infections when they occur.

Characteristics such as cell size and shape, relative thickness of cell wall, tendency to group, presence of flagella, and ability to form spores all help in the identification of unknown bacteria. Other distinguishing bacterial characteristics include hemolysis patterns (i. e. , ability to break down red blood cells) and ability to ferment lactose (i. e. , the ability to use lactose as an alternative source of energy under anaerobic conditions). Each bacterial species displays a specific morphology which is determined by its cell wall and cytoskeleton.

Most bacteria can be generally characterized as coccus (i. e. , spherical), bacillus (i. e. , rod-shaped), spirillum (i. e. , helical) and vibrio (i. e. , curvy rod-shaped). Because bacteria are microscopic in size, their classification based simply on morphology is not sufficient for identification. Therefore, other classification systems are needed. The bacterial cell wall is critical to the survival of bacteria, and its arrangement helps to differentiate bacteria into either Gram-positive or Gram-negative groupings, depending on the reaction of the cells to the Gram stain.

Gram-positive bacteria (which stain purple) have a very thick cell wall, while Gram-negative bacteria (which stain pink) have a thinner cell wall. Bacteria can also be identified by colony morphology, or their tendency to group together in pairs (e. g. Neisseria), chains (e. g. , Streptococcus), or clusters (e. g. Staphylococcus). Some bacteria have one or more flagella, rigid protein structures that can rotate and are used for motility. The presence or absence of flagella can be determined using a motility media, such as a semi-solid agar.

Bacteria are inoculated into the medium, and over time, bacteria with flagella will migrate away from the site of inoculation while non-motile bacteria will not. Other bacteria, such as Bacillus and Clostridium, produce dormant structures called endospores (or spores) which are highly resistant to environmental and chemical stresses and allow spore-forming bacteria to survive extreme conditions. The tough outer covering of spores makes them difficult to stain, but special procedures such as malachite green stain and safranin counterstain can be used for spore visualization.

The most common method of identifying bacteria involves isolating them, growing them in culture media, and then examining them closely under a microscope for characteristic features. Bacterial can also be identified by their response to differential staining, their growth rates in different selective media, serological methods that rely upon antibodies for bacterial identification, and gene sequencing. In the laboratory, bacteria may be grown using either solid or liquid media.

Solid growth media, such as agar plates, are used to isolate pure cultures of a single bacterial strain when the unknown sample contains multiple strains. Liquid media is usually reserved for situations where large volumes of cells are needed for bacterial identification. Selective media, such as Columbia-CAN agar, EMB agar, and milk agar, can be useful in the identification of bacteria. Columbia-CNA agar is a selective medium that contains two antibiotics which inhibit the growth of Gram-negative bacteria. Therefore, only Gram-positive bacteria can grow on Columbia-CNA agar.

Columbia-CNA agar also contains whole sheep’s blood which allows differentiation of bacteria based on their hemolysis patterns (i. e. , their ability to break down red blood cells). Partial hemolysis will appear greenish, while total hemolysis will exhibit clearing on the agar plate. Unlike Columbia-CNA agar, Eosin Methylene Blue (EMB) agar is a selective medium used to isolate and identify Gram-negative bacillus. EMB agar also contains two indicator dyes, eosin and methylene blue, which turn dark in the present of lactose fermenting bacteria.

Milk agar selects for bacteria based on their ability to catalyze the hydrolysis of the milk protein, casein, into amino acids for use as an alternative carbon source in anaerobic conditions (biosci. usc. edu). Bacteria that exhibit casein hydrolysis will appear as colonies with a zone of clearing around them on milk agar. There are many other laboratory tests for distinguishing bacteria based on their specific characteristics, such as their color, their ability to react with certain enzymes, the optimal temperature and pH for bacterial growth, their relative tolerance to salt, and their response to antibiotics.

Usually, multiple tests are used in combination for positive identification of unknown bacteria. Bacillus is a ubiquitous, rod-shaped Gram-positive bacteria that exhibits aerobic metabolism. It is approximately 1-6 ? m in size. In harsh environmental conditions, Bacillus is capable of forming endospores that remain dormant until conditions for survival are more favorable. The two medically significant Bacillus species are B. anthracis (which causes the current bio-threat, anthrax) and B. cereus (a food-borne illness that can be transmitted via improperly refrigerated rice).

When Bacillus is isolated, it forms irregularly-shaped, large colonies that spread across the culture medium (Barrow and Feltham 90). When viewed under the microscope, Bacillus looks like tiny rods and a substantial proportion of the bacteria will appear to bulge at one end due to the presence of an oval or round endospore (O’Leary 107). Bacillus will exhibit a zone of clearing when grown on milk agar because of its ability to hydrolyze casein, and it may or may not grow in motility media because while it is predominantly motile, some non-motile forms do occur.

Bacillus can be distinguished from Clostridia by its ability to form catalase (O’Leary 109). Escherichia is a Gram-negative rod-shaped bacteria that is facultatively anaerobic, or lactose-fermenting. Unlike Bacillus, it does not possess the ability to form spores. E. coli is a normal colonizer of the human gastrointestinal tract, and it is essential to the synthesis of vitamin K. Tens of millions of E. coli are excreted in every gram of human fecal matter. However, Escherichia can also be pathogen in humans, causing urinary tract infections, pneumonia, and diarrheal illnesses.

Different strains of Escherichia can be distinguished by either the rough or smooth appearance of their colony morphology. Escherichia will exhibit usually motility when grown in motility media, although there are some forms that do not have flagella. Lactobacillus is a Gram-positive rod-shaped bacteria that, like Escherichia, is a facultative anaerobe that is a normal part of the vaginal and gut flora in humans. It is approximately 0. 5-2. 0 ? m in diameter and when it is pigmented, it appears yellow or orange (O’Leary 137). Lactobacillus does not form endospores.

Because of its ability to ferment lactose, Lactobacillus is used industrially not only in the production of yogurt, cheese, and pickles, but also as a method of preventing spoilage in beer. Micrococcus is a Gram-positive spherical bacteria that ranges in size from 0. 5-3. 5 ?m. Micrococcus is an obligate aerobe, so it does not hydrolyze casein. The colony morphology of Micrococcus in culture media is clustered and individual organisms may organize in pairs, irregular clusters, or even cubical packets (O’Leary 95).

Even though this type of bacteria does not form spores, its cultures are quite hearty and can propagate for up to 10 years. Some species of Micrococcus have distinctive colors when grown on solid culture media – M. luteus forms yellow colonies on nutrient agar, while M. roseus forms pink colonies. M. luteus, which colonizes not only the mouth and upper respiratory tract, but also human skin, converts compounds into sweat into the unpleasant odor associated with human perspiration. Micrococcus is rarely pathogenic in humans, except in immunocompromised patients, such as those with HIV.

Alcaligenes is a Gram-negative rod-shaped bacteria that, like Micrococcus, is an obligate aerobe, so it will not exhibit a zone of clearing when grown on milk agar. It does not form endospores, and it has a variable response in motility media. Commonly found in soil, water, and fecal matter, Alcaligenes is usually considered to be a contaminant rather than a pathogen when isolated from humans. However, it is becoming more common as a cause of opportunistic infection in immunocompromised patients. A. eutrophus is a bacteria that is becoming increasingly useful in the production of biodegradable plastics.

Words Cited: Barrow, G. I. and Feltham, R. K. A, eds. Cowan and Steel’s Manual for the identification of medical bacteria, Third Edition. Cambridge: Cambridge University Press, 2004. Madigan, M and Martinko, J, eds. Brock Biology of Microorganisms, 11th edition. New York: Prentice Hall, 2005. O’Leary, William Michael. Practical Handbook of Microbiology. New York: CRC Press, 1989. ‘Types of Media for Microbiology. ’ biosci. usc. edu. 24 Nov 2006 <http://biosci. usc. edu/courses/2002-fall/documents/bisc300-lab_media. pdf>.

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