LAB 11: Unknown Lab

Introduction

We know how to isolate bacteria from the environment and how to select out certain species while not growing others (using selective and differential media). Bacteria are divided into species just like higher organisms. Members of the same species can still vary considerably; these are called strains. Consider the case of E. coli: some strains of E. coli may cause hemolytic uremia (destruction of red blood cells), some cause urinary tract infections, while another strain may be a harmless resident of your intestines. If these strains were on petri plates, they would all look similar. How would you tell these strains apart in the lab?

One way to determine the similarity of strains (called typing) is to test the biochemical reactions the bacteria can perform. Most of the biochemical tests are similar to those in differential media: consumption of a molecule changes the pH, leading to a colour change from a pH indicator dye.

When reading the tests, remember to distinguish between no growth and no reaction. A strain that cannot grow in a medium because it lacks an essential nutrient is much different from a strain that can grow but produces a negative reaction for the biochemical test.

In the microbiology laboratory, samples are often received and it the job of the technologist to identify the organisms in the sample. Typically, bacteria are identified by culture-based approaches (using selective and differential media), microscopy (e.g. Gram staining), and molecular approaches. In this lab, you will identify an unknown organism using microscopy and culture.

Many important human and plant pathogens are in the Enterobacteriaceae family. This family has four main traits:

• Gram negative
• Ferment glucose
• Test negative for oxidase
• Possess the enzyme catalase

Your Gram negative unknown may belong to the Enterobacteriaceae or be a non-Enterobacteriaceae. The non-Enterobacteriaceae group has three main traits:

• Gram negative
• Do not ferment glucose, but may oxidize it
• Many are oxidase positive

This lab is a summary of all the techniques you have learned to date. In addition, we will use a few new tests:

Carbohydrate Fermentation (Phenol Red Glucose, Sucrose or Lactose)

This media has one carbohydrate as the sole carbon source and phenol red, a pH indicator. If the bacteria consume the carbohydrate to produce acids (fermentation), phenol red turns from orange-red to yellow. If the bacteria consume the carbohydrate without fermentation, the medium does not turn yellow but will be turbid.

This medium is a broth and is performed in a test tube. Inside the test tube is an inverted miniature test tube called a Durham tube. If the bacteria produce gas, it will be captured by the Durham tube.

The results for this test could be the following:

• NG (no growth) (Figure 11.3, Tube A
• negative for consumption of the carbohydrate, but the strain grew (Fig. 11.3 Tube C)
• positive for consumption of the carbohydrate and no gas production
• positive for consumption of the carbohydrate with gas production (Fig. 11.3 Tube B)

Citrate Utilization (Simmon’s Citrate)

In this medium, citrate is the sole carbon source. If the bacteria possess citrate permease (a transporter to import citrate), they can grow on this medium. Simmon’s citrate also contains ammonium phosphate as the sole nitrogen source. As the bacteria grow, this is converted into ammonia and ammonium hydroxide, both of which alkalinize the agar. Bromothymol blue is the indicator; as the pH increases, it turns from green at pH 6.9 to blue at pH 7.6.

For this test, growth is linked to the reaction. An organism that grows and consumes citrate will also raise the pH of the agar (Fig. 11.4 Tube B). Strains that are unable to use citrate will be unable to grow (Fig. 11.4 Tube A).

Decarboxylase of Amino Acids (Lysine Decarboxylase)

This medium tests the ability of bacteria to decarboxylate an amino acid (remove the COOH-). It is used to differentiate enterics from other bacteria. The pH indicator, bromocresol purple, is purple at pH 6.8 and above and yellow at pH 5.2 and below (Fig. 11.5 Tube A shows an uninoculated tube). Degradation of amino acids increases the pH of the medium, causing it to turn purple (Fig 11.5. Tube B and Tube C show a weak positive). If the bacteria do not degrade the amino acid but they make acid from glucose, then the tube will turn yellow (Fig 11.5, Tube D)

This test must be conducted anaerobically to cause expression of the decarboxylase enzyme; thus mineral oil is overlaid on the tubes.

Indole production (tryptone broth)

The ability of bacteria to produce indole from tryptophan is a way of differentiating closely related strains. By including tryptophan in the growth medium, indole is produced if the organism is able to. SIM media also contains tryptophan, to allow the indole test to be completed.

Indole is detected after growth by adding Kovac’s reagent (containing dimethylaminobenzaldehyde (DMABA) and HCl in alcohol). The DMABA reacts with any indole present to produce a red colour.

Nitrate Reduction

This tests the ability of a strain to reduce nitrate to nitrite or nitrate to nitrogen gas. Nitrate respiration is a form of anaerobic respiration, with nitrate used in place of oxygen as the terminal electron acceptor. When nitrate accepts electrons, it becomes nitrite. If the bacteria possess the correct enzymes, nitrite can then be reduced into nitrogen gas.

This is a broth medium, and the test tube contains an inverted Durham tube. After growth, nitrate A and B (sulfanilic acid and a-naphthylamine) are added to the tube. If nitrite is present, the medium will turn red due to nitrite reacting with the reagents (Fig. 11.6 Tube A). If the nitrite has been reduced to nitrogen gas OR there is no nitrite present, the medium remains colourless (Fig. 11.6 Tube B). If this is the case, powdered zinc is added. The zinc will reduce any remaining nitrate into nitrite, which then reacts with nitrate A and B, causing the red colour (Fig. 11.6 Tube D). Thus, a red colour after the addition of zinc is negative for nitrate reduction, while a colourless result indicates nitrate was reduced to nitrogen gas or other nitrogenous compounds (Fig. 11.6 Tube C). Gas in the Durham tube indicates the presence of nitrogen gas.

Urease Production

This differentiates organisms based on their ability to degrade urea. If bacteria possess urease, urea is converted into ammonia and carbon dioxide. Phenol red is the pH indicator. Ammonia raises the pH, causing phenol red to turn from orange-red below pH 8.4 (Fig. 11.7 Tube A) to magenta pink at a pH greater than 8.4 (Fig. 11.7 Tube C). Bacteria that acidify the media below pH 6.2 cause phenol red to turn yellow (Fig. 11.7 Tube B).

IMViC Tests

Together, the indole, methyl red (MR), Voges-Proskauer (VP), and citrate tests comprise the IMViC tests. This panel of tests is important in distinguishing Enterobacteriaceae members from each other. Table 1 shows the commonly encountered species in the Enterobacteriaceae and the typical test results.

Table 11.1: Typical Enterobacteriaceae culture results.

Genus	Indole	MR	VP	Citrate	H2S	Motility	Lactose fermentation	Gelatin hydrolysis
Salmonella	–	+	–	+	+	+	–	–
Proteus	v	+	–	–	+	+	–	+
Klebsiella	–	–	+	+	–	–	+	–
Enterobacter	–	–	+	+	–	+	+	–
Citrobacter	+	+	–	+	+	+	+	–
Escherichia	+	+	–	–	–	+	+	–
Serratia	–	–	+	+	–	+	+	+
+ indicates a positive reaction – indicates a negative reaction V indicates a variable reaction for species within a genus

Once you have all the test results for your organism, you then need to figure out which species you have. The table above can be useful, as can the more extensive table and dichotomous key on FOL. The dichotomous key is simply a series of yes/no questions about your test results. As you work through the questions, you will eventually arrive at a species name. Once you have a name, you can search to see if this species fits with your other observations. Remember, you need to justify the species that you ultimately arrive at in your conclusions.