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Syllabus

• Agar slant tubes
• Aseptic technique
• Assays
• Assays Illustrated
• Colony morphology
• Gram stain
• Isolating colonies
• Making serial dilutions
• Culture media
• Preparing spread plates
• Relationship to oxygen
• Viewing Gram stains
• Viewing living bacteria

• Characterization writeup
• Lab responsibilities
• Project proposal guidelines
• Using Bergey's

Supplemental pdfs

• Inoculating sticks
• Microscopy procedures

Conducting and interpreting assays

This section describes assays for selected metabolic and physiological properties of bacteria, including the principles, how to prepare, conduct, and interpret each assay, and suggested applications. Suggestions for making direct observations can be found on the pages "Describing colonies," "Gram stain," and "Microscopic observation of bacteria." An asterisk next to the title indicates that the assay is described, with pictures, in our handout titled "Assays Illustrated."

Relationship to oxygen

Next to the Gram stain, relationship to oxygen is probably the most important piece of information toward identifying an isolate. Your species may be:

• an obligate aerobe (requires oxygen for growth)
• a facultative anaerobe (grows best in oxygen but can grow in the absence of oxygen by inducing alternate metabolic pathways)
• an obligate anaerobe (grows only in the absence of oxygen and will not be recovered from oxygen rich sources)
• a microaerophile (grows only in low oxygen environments)

To test for relationship to oxygen we spread duplicate agar plates with our unknowns and incubate one set aerobically and one set in an airtight jar with a gas generator package to produce anaerobic conditions, both at 35 degrees C. An obligate aerobe will show no growth on the anaerobically incubated plate. Facultative anaerobes, which can use alternate metabolic pathways that do not require oxygen as a final electron acceptor, should show sparse to moderate anaerobic growth. Obligate anaerobes, which absolutely require oxygen, will probably not be recoverable from our samples if they are there at all. Even under anaerobic conditions they typically grow very slowly. A microaerophile may or not grow under these conditions. A good assay for a microaerophile is to inoculate a broth culture and look for a band of turbidity, representing growth, starting a few mm below the surface where oxygen content is relatively low.

To avoid recording false positives or negatives we have to be careful when setting up and interpreting results. 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.

*Catalase test

Most of the time our attempt to identify an isolate is aided by knowing whether or not it produces catalase. Even though we depend on oxygen, molecular oxygen 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. We usually start with procedure 1 below, and if the test produces an apparently negative result we move to procedure 2.

1. 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.
2. If the direct application method produces no visible response, then fill the tip of a pasteur pipet with hydrogen peroxide and jab the end of the tube into a colony to obtain an agar plug with some culture material exposed to the solution. If the species is weakly catalase positive then you will see some bubbles rising from the plug.

*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 oxidase positive and Escherichia coli is oxidase negative. The oxidase test is good for differentiating many other taxonomic groups as well.

Because the procedure for the oxidase test is quick and easy, the reagent is good for only a few hours after an ampule is cracked open, and the test is useful more often than not for identifying an isolate, we recommend conducting it on all isolates as a matter of routine.

For our tests we use 1% dimethyl-p-phenylenediamine with added stabilizer, purchased in kits from a supplier. One vial contains enough reagent for a dozen or more tests. Activate a tube by squeezing the outer (plastic) tube with fingers or the tool that comes with the kit, to crush the inner glass tube. Squeeze a drop onto a piece of filter paper and then use a sterile glass rod or similar sterile instrument to pick up a bit of colony from an overnight culture and smear it onto the wet spot. We do not let the drop dry out as recommended by the ASM (http://www.microbelibrary.org/library/laboratory-test/3229-oxidase-test-protocol).

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. Please do not return partially emptied vials to the assay bench, share them if possible, and discard any vial in which the liquid has begun to turn purple.

*Indole assay

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

To promote tryptophan hydrolysis, culture the organism in 1% Casitone in a 13 x 100 mm culture tube. We prepare and sterilize the medium ourselves and purchase Kovacs' reagent (5% para-dimethyl-amino-benzaldehyde in 75% amyl alcohol, 25% concentrated hydrochloric acid) from a supplier. Inoculate an indole tube for 24 hr at 35 degrees C. Aseptically remove a milliliter of culture to a disposable culture tube, add an equal volume of Kovac's reagent, mix well, and leave it 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.

All broth tubes prepared for assays must be sterilized after filling and capping them. For any assay requiring growth of the culture in broth, make sure there is significant growth in the tube, indicated by significant turbidity. It is best to prepare a negative control in the form of uninoculated broth or a negative standard.

*Methyl red and Voges-Proskauer tests

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 (Barritt's 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 and sterilized. The same medium is used to grow cultures for either assay. Frequently, an investigator will conduct both on the same culture.

We prepare methyl red reagent 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. For either procedure, we inoculate a tube of MR-VP medium and incubate 24 h (minimum) at 35 degrees C.

For the methyl red test, we add 0.5 ml of culture (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. We conduct the Voges-Proskauer (V-P) procedure 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 and shaking gently to aerate the mixture. If the isolate is positve, a red color will appear within 20 minutes. If the test is negative, repeat it at 48 h.

Caveats: As with the indole test, 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 begins to turn the broth pink right away. If you have a negative test the low pH makes the red color of the reagent disappear as soon 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.

*Phenol red broth assays

We can look for the ability of an isolate to metabolize specific carbohydrates by incubating them in minimal media with a high concentration of the appropriate sugar. The broth includes pH indicator and we may invert and insert a smaller tube into the both tube to detect gas production. Usually only one isomer of a molecule can be metabolized. To prepare assay tubes we dissolve 0.5 g of the appropriate isomer, or 1 g of a racemic (DL) compound in 100 ml of phenol red broth. We distribute 4-5 ml per tube into 13 x 100 ml culture tubes and drop in an inverted tube if we need to assay for gas production. The inverted tubes self-fill when the tubes are sterilized in an autoclave.

You may have to adust pH with HCl or NaOH if after you add carbohydrate the color is very red (too alkaline) or very yellow (too acidic). Orange-red indicates a near neutral pH. To perform the assay, inoculate the tube and incubate at 35 degrees C. Fermentation of the carbohydrate typically produces gas (bubble in the tube), acid (broth turns yellow), or both. If you have significant growth (absolutely necessary, of course), and no gas, then agitate the tube to release any possible 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.

Phenol red broth supports a large number of assays – just add the appropriate substrate.

Decarboxylase assays

Decarboxylation refers to the process of removing the carboxyl group from an amino acid, producing an amine and carbon dioxide. The ability to decarboxylate arginine, lysine, and/or ornithine is an important differential characteristic of the Enterobacteriaceae and some other groups as well.

To prepare the assay we mix decarboxylase broth according to label instructions and split it into two batches. To one batch we add 1 gm of the l-isomer of the appropriate component (arginine, lysine, or ornithine) per 100 ml, and add nothing to the second batch. We distribute 4-5 ml per culture tube, cap, and sterilize as usual. We usually do not need to look for gas production - no inverted tube is necessary. 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.

To run the assay we inoculate two tubes with the isolate, one with the component (test) and one without (control), and incubate for 48h at 35 degrees C. Some sources recommend adding a half cm of sterile mineral oil to the surface to promote anaerobic conditions and prevent false alkalinization due to oxidation of the color reagent. The medium contains 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 decarboxylase pathway is inducible, the low pH will induce enzyme production, initiating metabolism of the amino acid, and the medium will turn purple due to accumulation of alkaline end products.

We have a positive test if there is growth in both tubes and the control tube is yellow, meaning the organism produced acid using glucose as the carbon source, and the test tube is purple, meaning that the decarboxylase pathway was induced when the glucose ran out. We have a negative test if both tubes are yellow. If the control tube is not yellow then we do not have a result.

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

Additional assays

Please inform an instructor if you need to conduct one or more assays listed below, or an assay not listed here. We can purchase additional media, media components, reagents, etc. to the extend that time and expense allow it.

• Citrate utilization (Simmons citrate agar)
• Hydrogen sulfide production (SIM medium)
• Urea hydrolysis
• Gelatin hydrolysis
• Growth in KCN
• Malonate utilization