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Background

• Local info
• Water issues
• Projects
• Characterization
• Sample article (pdf)
• How to use Bergey's Manuals

Methods

• Rules
• Media/culture
• Aseptic technique
• Staining/interpretation
• Isolation strategies
• Assays/characterization
• Microscopy
• Cultural characteristics

Media preparation
and training

Assays and Characterization

For over fifty years the identification of bacteria was based on the classification scheme given in Bergey's Manual of Determinative Bacteriology, a compilation of the characteristics of thousands of bacterial species, contributed by numerous authors. The original Bergey's Manuals relied on an empirical classification system that separated all bacteria into 19 groups on the basis of morphology, biochemistry, physiology, and growth requirements. Now that data have been accumulated from nucleic acid hybridization and sequencing studies and evolutionary relationships have become better established (although bacterial systematics is still a nightmare), the current edition of the Manual, retitled Bergey's Manual of Systematic Bacteriology, has been reorganized to reflect evolutionary relationships. Bergey's Manual will be the guide to characterizing your pure isolates.

Each volume of the new edition cost a minor fortune, so please treat them with care ! Some people in past labs have actually written in them! Don't even think about doing that if you actually plan to graduate. Other materials, including texts and laboratory manuals are available as well. Species that you receive will all be found in the first two volumes of the manual, and none of them will be exotic, unusually fastidious or pathogenic bacteria, or those living exclusively in very weird places (the armpits of left-handed Tibetan monks, for example).

Use the following criteria to characterize each of your isolates

• Ensure that you start with a pure culture
• Start with the broadest categories and work down to smaller more specific categories of bacteria
• First, positively determine the Gram stain properties and cell shape
• Second, establish the relationship to oxygen
• Third, determine whether or not the cells are capable of motility
• Continue to obtain data that are most effective in discriminating among the categories that remain. While doing so,
• Use common sense as you proceed
• Use the minimum number of tests needed to make an identification
• Be aware of possible complications associated with each test
• Compare the isolate to the appropriate type strain that is available in the laboratory

Study the recommendations under "Identification of Bacteria" in volume 1 of the Manual. They are reproduced, with additional details, here. ***IMPORTANT*** Please note that the results of any attempt at characterization may depend on size of an inoculum, temperature, time of incubation, type of medium, gas content of the medium, and a certain amount of subjectivity (positive and negative tests are not always readily distinguishable). The results from one laboratory to the next are therefore not always consistent. Furthermore, different strains of some species may give different results. Frequently it is more important to recognize the pattern of results than to rely completely on results of specific tests. Furthermore, once identification has been narrowed to a few candidate species, the individual descriptions of species may be useful in making a final determination.

In the tables in the manual, the most discriminatory tests are listed first, so it is advisable to work from the top down once a category is established. In the tables, the symbols mean the following:

Relationship to oxygen

Airtight jars with gas generator packages will be used to produce anaerobic conditions. Facultative anaerobes can use alternate metabolic pathways that do not require oxygen as a final electron acceptor, while obligate aerobes absolutely require oxygen. Even facultative anaerobes grow very slowly in the absence of oxygen, so the presence of numerous colonies on a plate so incubated is evidence of a facultative anaerobe, even if the colonies are small. The presence of a few large scattered colonies on a mostly sterile surface suggests that a facultatively anaerobic contaminant was introduced. Lack of any growth is indicative of an obligate aerobe, however all plates must be compared with identical, aerobically incubated controls, to establish that anaerobic conditions are responsible for the growth pattern and not a failure to obtain a viable sample.

It is critical that plates be prepared shortly before the jars are sealed. Aerobic growth can start prior to removal of the oxygen and continue for some time in the jar until most of the oxygen is removed, giving a false positive result.

For standardization, two plates can be streaked with Alcaligenes faecalis (obligate aerobe) on one side, and any member of family Enterobacteriaceae on the other.

Motility

Many species lose their flagella when incubated on an agar surface, although it is worthwhile to make a quick wet mount of a dry culture if a liquid culture is not available. Once motility is established for a pure culture, there is no need to double check with another method. If a wet mount of the plate or agar slant culture shows no translational motion, two tests are suggested.

First, try a wet mount or hanging drop mount of a young broth culture as suggested under staining methods. Consult the microscopy materials for suggestions for making a wet mount and observing with a microscope.

Standardize by comparing Staphylococcus epidermidis (nonmotile) with Escherichia coli, incubating at 37 degrees.

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Motility agar

We use Motility GI medium (not GI motility medium - that's another thing altogether!), which consists of a dilute nutrient agar medium that barely gels at room temperature.Tubes containing motility agar to a depth of 2 inches or so are inoculated by a stab method and incubated at an appropriate temperature. A needle is simply jabbed straight into the agar to the bottom and pulled straight back out. There is sufficient liquid so that motile bacteria usually maintain their flagella and can swim through the medium, spreading away from the site of the inoculum. Nonmotile bacteria are stuck along the original track.

Complications - Some motile bacteria, especially the larger ones, simply won't cooperate. Aerobic bacteria may grow only at the surface, giving the illusion that they are nonmotile. If the needle is not pulled straight out, it may make a second track with growth extending from the first track to the second. Growth may be very slight, so that only comparison with an uninoculated tube can confirm that bacteria are motile.

Test for catalase activity

Even though we depend on oxygen, molecular oxygen itself produces toxic compounds. The enzyme catalase helps detoxify cells that are exposed to oxygen by converting hydrogen peroxide to water and oxygen. If a species produces catalase, then the material will evolve oxygen in the presence of hydogen peroxide. Two methods are suggested for performing the test.

• Add a drop or two of 3% hydrogen peroxide to an isolated colony and observe for the formation of oxygen. Vigorous bubbling indicates a strong catalase reaction.
• If the direct application method produces no visible response, a glass capillary tube can be partly filled with hydrogen peroxide and the end of the tube jabbed into a colony so that it plugs the end. If the species is even weakly catalase positive, some bubbles should be seen rising from the plug.

Most species are catalase positive, but negative reactions are not uncommon.

Test for oxidase activity

The capacity of the cytochrome system of an organism to oxidize an artificial electron donor such as dimethyl-phenylenediamine to form a chromogenic product is a common distinguishing feature of Gram negative bacteria. All of the Enterobacteriaceae (Gram negative enteric bacteria) are negative for this oxidase reaction, while the Pseudomonas are all oxidase positive. Any two common species, one from each family, can be used for standardization. Alcaligenes faecalis, used for an earlier test, is also oxidase positive.

Oxidase test reagent, consisting of 1% dimethyl-p-phenylenediamine, can be purchased from a supplier in kits. The procedure for the oxidase test is quick and easy, however reagents are expensive. Any test using reagent kits should be conducted only if it appears to be useful as a distinguishing characteristic, and only on a confirmed isolate. Dropping vials frequently contain enough reagent for more than one test.

The tube is activated by squeezing the outer (plastic) tube with fingers or the tool provided, to crush the inner glass tube. A drop is squeezed out onto a piece of filter paper, and a bit of colony from an overnight culture is aseptically placed on the spot using a glass rod. We do not let the drop dry out as recommended by the ASM (http://www.microbelibrary.org/library/laboratory-test/3229-oxidase-test-protocol). Please do not return partially emptied vials to the assay bench; share them if possible; discard any vial in which the liquid has begun to turn purple.

A positive reaction for cytochrome oxidase is indicated by the appearance of a dark purple reactant. An immediate purple color (appearing within 10 to 30 sec) is positive for cytochrome oxidase. If the purple color appears but is delayed up to 3 min, you have a weak positive. No color after 3 min means oxidase negative. The reagent itself eventually turns purple in the presence of oxygen, therefore color development after 3 min does not indicate a positive result.

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Indole

Some bacteria hydrolyze tryptophan to pyruvic acid, which is then metabolized in a pathway similar to the familiar Krebs pathway. A byproduct of that hydrolysis is indole, which is excreted by the organism. The presence or absence of the responsible enzyme, tryptophanase, is important in the differentiation of enteric bacteria such as Eschericia coli. To promote tryptophan hydrolysis culture the organism in 1% Casitone in a 13 x 100 mm culture tube. Kovacs' reagent (5% para-dimethyl-amino-benzaldehyde in 75% amyl alcohol, 25% concentrated hydrochloric acid), purchased from a supplier, is one reagent that can be used to complete the assay for indole production.

An indole tube is incubated 24 hr at the appropriate temperature and a milliliter of culture is aseptically removed to a disposable culture tube. An equal volume of Kovac's reagentis added, mixed well, and leftt for about 5 min. A positive test is indicated by a red color in the alcohol (upper) layer. Conduct the test again at 48 hours if the first test is negative. Citrobacter freundii gives a negative response, and Escherichia coli is positive for indole. Kovac's reagent stinks -assay tubes should be capped and kept in the hood until disposed.

In order for the test to be valid, there must be significant growth in the indole broth tube, and that is not always the case. A negative control in the form of uninoculated broth or a negative standard should be conducted at the same time. With some tests the positive reaction produces a pronounced pink color, while others barely change the color but are nevertheless positive.

The indole assay and the methyl red/Voges-Proskauer tests are primarily for distinguishing members of the family Enterobacteriaceae, but do have limited applicability to the characterization of some other species.

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Methyl Red/Voges-Proskauer

The fermentation of glucose by bacteria results in end products that vary from species to species depending on metabolic pathways that are available to them under the culture conditions. A number of genera of Gram negative bacteria ferment glucose to produce lactic, acetic, succinic, and formic acids. They also typically produce large amounts of CO2, H2, and ethanol. Many of us greatly appreciate that last end product, by the way. Acid accumulation can reduce the pH to 5 or lower before the acid accumulation stops all growth.

Methyl red, a pH-sensitive dye, turns red at low pH, indicating that the organism produces mixed acids as end products of glucose fermentation. Many Gram-negative and some Gram-positive genera (e.g., Enterobacter, Serratia) yield a negative methyl red test because they produce a great deal of 2, 3 butanediol and ethanol rather than acids. While there is no procedure available for direct assay of 2, 3 butanediol, a precursor called acetoin is detectable using a reagent consisting of alpha-napthol and potassium hydroxide (Barrittÿs reagent). In the presence of acetoin addition of reagent will cause the medium to turn pink or red after standing for a period of time. The procedure is called the Voges-Proskauer test, and requires addition of two reagents in steps.

Methyl red/Voges-Proskauer (MR/VP) medium consists of 0.7% peptone (a meat digest), 0.5% glucose, and 0.5% anhydrous dibasic potassium phosphate in water, prepared and distributed in capped 13 x 100 mm culture tubes. The same medium is used to grow cultures for either assay. Frequently, an investigator will conduct both on the same culture. Methyl red reagent is prepared by dissolving 0.1 g methyl red dye in 300 ml 95% alcohol, then adding 500 ml distilled water (this formula can be scaled down for our lab, of course). Voges-Proskauer solution A consists of 5% alpha-napthol in absolute ethanol. Solution B consists of 40% KOH. Both reagents can be purchased from a supplier in kits.

For either procedure a tube of MR-VP medium is inoculated and incubated 24 h (minimum). For the methyl red test, 0.5 ml of culture is added (aseptically) to a 13 x 100 mm tube, followed by 10-15 drops of methyl red reagent. The appearance of a red color is positive for strong acid production on glucose. Yellow is negative. The Voges-Proskauer (V-P) procedure is conducted by removing 1 ml culture to a test tube and adding 15 drops VP reagent A, followed by mixing and the addition of 5 drops reagent B. After shaking gently to aerate, the tubes are examined for the appearance of a red color within 20 minutes. A negative test should be repeated at 48 h.

Bacterial growth in the MR/VP tube must be obvious, and sufficient medium must be available to perform each test twice (3 ml minimum). A positive methyl red test usually yields a red color right away, while the pH change causes the red color of the reagent to disappear as it is added. The VP test frequently requires the whole 20 minutes to show a positive response, and we have often had to wait even longer. A positive test must be compared with a negative control.

Enterobacter aerogenes is negative for MR, positive for VP. Citrobacter freundii is positive for MR, negative for VP.

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Physiological (metabolic) tests

Different taxonomic groups can be differentiated by differences in metabolic pathways. Once a family is established for a pure isolate, the ability to use different combinations of carbohydrates and amino acids is often the only practical way to distinguish bacteria at the species level.

Phenol red broth assays

The metabolism of specific carbohydrates can be demonstrated by incubation of the test organism in minimal medium with a high concentration of the appropriate sugar. This is done in the presence of a pH indicator for indication of acid production and/or a device called a Durham insert for measurement of gas production. Usually only one isomer of a molecule can be metabolized, which for carbohydrates is, with an exception or two, the D-isomer.

To prepare assay tubes, 0.5 g of the appropriate isomer, or 1 g of a racemic (DL) compound is mixed per 100 ml phenol red broth. Four ml per tube is distributed into 13 x 100 ml culture tubes and scaled up if larger tubes are needed. A Durham tube insert is filled with the same medium with syringe or pipet, inverted, and placed in the culture tube. Placement without admitting air can sometimes be tricky, but thank goodnes for surface tension! The Durham insert can be omitted if the test is for acid production only. The pH is adjusted if necessary, so that the broth is an orange color.

Note that with assays using high concentrations of carbohydrates or amino acids (decarboxylation assay - see the following section) the pH must be near neutrality upon inoculation otherwise many bacteria wonÿt grow. Unfortunately, the pH may be right prior to sterilization, then change during the process. With phenol red broth, red indicates too alkaline a condition, yellow indicates acidity. If the tubes come out of the autoclave bright red or yellow, the pH must be adjusted and the medium re-sterilized.

To perform the assay a tube is inoculated and incubated at the appropriate temperature. Gas and acid production results from fermentation. Since conditions near the bottom of the tube are close to anaerobic, anaerobic incubation should not be necessary. Yellow is positive for acid (check at 24 and 48 h); red or no change is negative, unless there is simply no growth, in which case the assay was not successful. Gas production is indicated by a bubble in the insert. The tube should be agitated to release any trapped gas before recording an assay as negative for gas production.

Serratia marcescens is negative for acid production on lactose and Enterobacter cloacae is positive. Staphylococcus epidermidis is negative for l-arabinose and Citrobacter freundii is positive. Gas production may also be present.

Warning! Tests for acid production on carbohydrates have been notoriously unreliable. No growth at all is a frequent result. As stated earlier, the pattern of test results and the species descriptions in the Manual are of greater importance to an identification than the results of any one test.

Decarboxylase/deaminase/dihydrolase assays

Decarboxylation refers to the process of removing the carboxyl group from an amino acid, producing an amine and carbon dioxide. Bacteria are cultured with an appropriate amino acid plus glucose and the pH indicating dye bromcresol purple. If the species is capable only of glucose fermentation, the medium will turn yellow and remain yellow as the increasing acidity prevents further growth. If the appropriate decarboxylase enzyme is inducible, the low pH will induce its production, initiating metabolism of the amino acid, and the medium will turn purple due to accumulation of alkaline end products.

Assay tubes consist of 1 gm of the l-isomer of the appropriate amino acid per 100 ml decarboxylase broth, sterilized as usual. Durham inserts should not be needed. The pH may require adjustment, especially for basic amino acids. The color should start off as dirty brownish green, definitely not purple and not yellow.

These assays must be monitored carefully, and growth confirmed, before making a decision. To perform the assay, a tube is inoculated and incubate at the appropriate temperature. Development of yellow color followed by purple is definitely positive for alkaline end products. Yellow is negative. A change from neutral to purple without witnessing the initial yellowing of the medium is still an acceptable positive, provided the change is obvious and there was good growth in the tube.

Enterobacter aerogenes is negative for arginine decarboxylase and Serratia marcescens is positive.

Some assays produce a yellow color with strong purple color at the top of the tube. This false alkalinization is due to oxidation of the color reagent, and must not be interpreted as a positive response. The medium can be overlaid with a sterile mixture of paraffin wax and petroleum jelly or with sterile mineral oil to prevent such artifacts and to promote anaerobic conditions. Obviously this won't work with obligate aerobes.