7-2 Classic strain testing

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Macroorganisms are identified most often by their outside appearance, for example, do they have bones, a spine, fur? Do they live as single cells or in a multi-cellular arrangement? Many other traits of large organisms can be cataloged and used for identification. Microorganisms are small, and frankly there are not many morphological traits than can be tracked. However, microbes are extremely versatile and varied in their metabolic traits. By growing them in various medium to test their metabolic capabilities, it is possible to identify microbes. For example, most Salmonella strains cannot metabolize lactose, while E. coli and many other enterics can. Also, Bacillus cereus, cause of food-borne illness, is know to be β-hemolytic on blood agar plates, while B. anthracis is not. By testing enough traits it is possible to identify an isolate to the species level and beyond.

Typical tests

Many biochemical and physiological tests have been developed to identify bacteria. The tests you will observe in this experiment (fermentation broths, starch hydrolysis, colony morphology, catalase test, motility agar and indole from tryptophan) are useful in microbial identification.

Gram stain

The Gram stain is a primary method for differentiating microbes. The reaction of any bacteria to the stain depends upon it cell wall structure. The Gram stain was covered in detail in Section 2-8

Figure 7-1 Gram reactions of strains

The Gram stain reactions of common microbes. E. coli, Gram negative (A), Staphylococcus epidermidis, Gram positive (B) and Bacillus cereus, Gram positive.

Fermentation

Fermentation broths contain a test sugar, peptone and beef extract (to provide secondary nutrients for fastidious microorganisms), and brom cresol purple (a pH indicator). If the test sugar is fermented, acid is usually produced. This drop in pH turns the brom cresol purple from blue to yellow. Also, the presence of a Durham tube, allows the detection of gas (H2) production from the fermentation. Results are reported as production of acid (+/-) and gas (+/-).

Figure 7-2 Reactions in fermentation broth

Example reactions of typical strains in fermentation broth. If a microbe does not ferment the test sugar, the indicator dye remains purple and no gas is produced (left). If fermentation does take place, acid is most often produced, lowering the pH and changing the color of the broth to yellow (middle). Some microbes produce hydrogen gas during fermentation and this will be trapped inside the inverted tube, called a Durham tube, and is observed as a bubble (right).

Starch Hydrolysis

The starch agar plate is used to test for extracellular starch hydrolysis. Starch is a high molecular weight polysaccharide, too large to be transported inside the cell without breaking it down into smaller units first. The ability to hydrolyze starch depends upon the production and secretion of several amylases that degrade the polymer. The breakdown of starch is detected after incubation by flooding the plate with iodine. Iodine complexes with intact starch to form a blue color. If the microbe is able to degrade the starch, none is available for reaction, and a zone of clearing will be seen around the colony.

Figure 7-3 Starch hydrolysis

Starch agar is a rich medium containing starch. Microbes capable of utilizing the starch will secrete amylases, which hydrolyze the starch. Starch breakdown is detected by flooding the plate with iodine, which forms a purple colored complex . Test microbes incapable of degrading the starch will be completely surrounded by a zone of purple. (left) Those that can degrade the starch, will have a zone of clearing around their streak, while the background of the plate will be purple (right).

Colony Morphology and Catalase Test

HIA is used as an all-purpose medium, that will allow the examination of colony morphology and catalase activity. The correct method for describing colony morphology is described in Figure 6-4. As mentioned in Experiment 3, some oxygen by-products, created during metabolism, are toxic to cells and special enzymes have evolved to detoxify those compounds. One of these enzymes, catalase, is responsible for the splitting of hydrogen peroxide (H2O2) into oxygen and water. It is easy to test for this enzyme in bacteria. A test culture is exposed to 3% H2O2. If catalase is present, H2O2 is broken down to H2O and O2. The oxygen is detected as a steady evolution of gas bubbles from the culture. Figure 7-17 shows a movie of the catalase reaction. The common descriptive names for colonies are described in Figure 6-4 and examples of colony morphology are shown in Figure 6-5.

Figure 7-4 Colony morphology descriptions

As microorganisms grow and divide on solid surfaces they form specific patterns of growth. Individual cells (or a few identical cells) will continue to grow and divide and form discrete units called colonies, the morphology of which is characteristic of that microbial species. An accurate description of an isolated colony can greatly aid in the identification of the microorganism. Microbiologists have developed standard words with specific meanings to describe colony form, elevation and margin. Scientists use this jargon to help accurately communicate their observations. This is a common theme in all disciplines of science. Imagine what would happen if everyone used different terminologies? There would be no effective communication between scientist.

Figure 7-5 Colony morphology

Examples of varous colony morphologies. The appearance of colonies on a plate is species specific and can be very helpful in identifying isolates.

Tryptophan Degradation to Indole

Tryptone broth contains a high concentration of the amino acid tryptophan. Some microorganisms are able to degrade tryptophan to indole and this ability is a useful tool for differentiating microbes. The test is performed by adding Kovac's reagent to a broth culture, which results in a red ring on the top of the broth if indole is present. Examples of positive and negative reactions in the indole test are shown in Figure 3-6

Figure 7-6 Tryptophan degrdation

A positive indole test will result in the formation of a red ring at the top of the medium (left), while a negative test remains tan/translucent (right).

Identifying of microorganisms through these types of biochemical tests has been a routine practice for many decades and the tests necessary to identify certain species of groups of related species have become standardized. To make these test more convenient, manufacturers have developed miniaturized versions that decrease the material cost and make inoculation of the tests much more convenient. However, the tests still need to be incubated to the prescribed period of time before being read. Usually 24-48 hours. One example of this type of test is shown in Figure 6-7, the API 20 strip.

Figure 7-7 API-20 strip test

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8101+++++++++++Escherichia
coli
5B++++++++++Enterobacter
agglomerans
8P44++++++++Edwardsiella
hoshinae
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8030++++++++++++++Klebsiella
pneumoniae
8068++++++++Proteus
vulgaris
8P14+++++++++Salmonella
sp.

The types of biochemical tests we have explored here can be miniaturized. The API-20 strip is one example of this type of test. Twenty tests are performed on this strip by a simple procedure, saving time and money. The first test is for the presence of the enzyme ?-galatosidase, an enzyme involved in lactose catabolism. The next three reactions (in order, arginine, lysine and ornithine) test for amino acid decarboxylation. Decarboxylation is shown by an alkaline reaction (red color of the particular pH indicator used). Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black color throughout the tube. A positive reaction for tryptophan deaminase (TDA) gives a deep brown color with the addition of ferric chloride; positive results for this test correlate with positive phenylalanine and lysine deaminase reactions which are characteristic of Proteus, Morganella and Providencia. The last nine tests are for carbohydrate fermentation. The carbohydrates tests are (glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose. Fermentation is shown by an acid reaction (yellow color of indicator).

In Experiment 7 the student will first become familiar with several diagnostic tests and the reactions of known organisms. You will then be given a virtual unknown and will apply these tests in the hope of identifying your microbe. By comparing the results of your tests to a virtual unknown you will identify your microbe. To simulate real life, the reactions of microbes in medium can sometime be ambiguous, and we will simulate this by sometimes having unclear or incorrect results. You have been warned.

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