Laboratory Investigations in Microbiology

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Chapter 17: Respiratory pathways

As indicated in the last chapter, metabolism encompasses many different chemical reactions of a cell, including those of catabolism (breakdown) and anabolism (build-up). The major difference between bacteria and eukaryotes isn't only in the type of pathways bacteria can use, but in the enormous flexibility and self-sufficiency that many bacteria exhibit. Thus, many bacteria are able to grow with just a single carbon source (e.g. glucose). As long as all the biochemical pathways of a bacterial cell are complete, almost any sugar, fat, or amino acid is sufficient for growth. 

Last time we looked at the hydrolysis of macromolecules such as starch and protein. Today we will consider the next steps in metabolism: 

Citrate test. Citrate (citric acid) is a common metabolite of most cells. It is used and produced during the Krebs cycle, and most bacteria thus have the enzymes needed to break it down. However, not every bacterium can use citrate as a source of food. This is because some bacteria lack the enzyme citrate permease, a transporter that carries citrate across the plasma membrane into the cytoplasm. We can determine if a bacterium has this transporter by culturing bacteria on a medium that contains citrate as the only organic compound. Only bacteria that can use citrate will grow on this medium. Once inside the cell, citrate is metabolized as follows: Citrate pyruvate + CO2. As citric acid is used up and CO2 diffuses into the medium (combining with sodium to form sodium carbonate, a base), the pH of the medium rises, changing the color of the agar from green to blue (AF, KP). A negative citrate test is inindicated when the medium remains green in color and bacterial growth fails to take place on the slant (EC). 

SIM test. Once inside the cell, nutrients are processed to make them "fit" into the cells' metabolic machinery. This machinery usually consists of the biochemical pathways we know as glycolysis, respiration, and fermentation. Not all nutrients are ready to go into these pathways. For example, before amino acids can be used, the cell must remove their nitrogen and sulfur side chains. These reactions are called deamination and desulfurylation, respectively, and can be illustrated by the following examples:

Other side chains found in amino acids are also unusable by the metabolic machinery and must be removed:

These chemical reactions produce measurable quantities of hydrogen sulfide gas, indole and ammonium, and the appearance of such waste products indicate that the bacterium is able to use these amino acids as nutrients. Using a Sulfur-Indole-Motility (SIM) medium, we can detect the production of two of these compounds. Hydrogen sulfide gas reacts with iron in the agar to form an insoluble black precipitate (FeSO4) which is easily visible to the naked eye (PV). In the absence of H2S, the medium remains yellow (EC, KP). Indole side chains can be detected by adding a chemical reagent (Kovacs Reagent) to the top of the agar tube. It reacts with indole to form a pink-red color (EC). In the absence of indole, Kovacs Reagent remains green (KP, PV).

Oxidase test and Nitrate reduction test. When bacteria carry out respiration, nutrients such as glucose or amino acids are completely "burned" to carbon dioxide. The electrons and hydrogen gained from these metabolites are carried to the electron transport chain, which in turn converts their energy into ATP usable by the cell. At the end of the ETC, a final electron acceptor combines with the electrons (and often, H) to form a waste product which is then lost from the cell. The waste product generated depends on the type of respiration carried out:

Filter paper + bacteria after adding oxidase reagentFilter paper + bacteria before adding oxidase reagentThe oxidase test uses a reagent that is reduced by cytochrome oxidase. Bacterial colonies that have this enzyme turn purple or black.

The nitrate reduction test detects the presence of nitrite in the medium. Nitrite is only produced by bacteria that carry out anaerobic respiration using nitrate. Nitrite can be detected by adding two test reagents (A and B) to the medium. If nitrite was produced, the medium turns pink/red within 5 minutes (often faster). This indicates a positive test for nitrate reduction (EC). If nitrate was not reduced the color of the medium will not change color, indicating a possible negative test result. 

Nitrate test results after addition of reagents A and B and zincHowever, some bacteria, notably strains of Pseudomonas, carry out a process called denitrification. Instead of reducing nitrate to nitrite, these bacteria continue to add electrons to nitrite, eventually forming N2 gas. Since nitrite is therefore not detected in the medium, the test appears to be negative.  We can check whether denitrification has taken place by performing a zinc test. Adding zinc to a medium containing nitrate will change it to nitrite. A red color appears since the nitrite now reacts with the reagents (A + B). This confirms a negative test for nitrate reduction (AF). However, if a bacterium has carried out denitrification, no nitrate is available to be changed by the zinc, and adding zinc will not produce a red color. This indicates a positive test for denitrification (SOIL).


Materials & Methods

Materials per Lab Group
Citrate test
  1. Label 3 citrate slants (choose 3 from PA, EC, PV, EF)
  2. Inoculate each slant with a rich loop full of bacteria. Be sure to heavily inoculate the top of the slant.
  3. Bring slants up front to be incubated
  4. Next lab: Observe slants for a color change from green to blue.
SIM test

Reagents: Kovacs reagent (isoamyl alcohol, para-dimethylaminobenzaldehyde, hydrochloric acid)

  1. Label 3 SIM deeps (EC, PV, EF)
  2. Inoculate each deep with an inoculating needle dipped into the bacterial broth. Be sure to stab straight down into the deep, ~ 3/4 of the way to the bottom of the tube.
  3. Bring deeps up front to be incubated.

Next class:

  1. Observe deeps for a black precipitate, indicating a positive H2S test
  2. Add 3 drops of Kovacs reagent to the top of each tube. Observe for a red color, indicating a positive indole test
Nitrate reduction test

Reagents: Nitrate test reagents A (α- naphthylamine) and B (sulfanilic acid); zinc

  1. Label 3 nitrate broths (PA, EC, EF)
  2. Inoculate each broth with a loop full of bacteria. 
  3. Bring broths up front to be incubated.

Next class:

  1. Pipette 1 ml of each nitrate broth into separate plastic wells (well plate).
  2. Add 1 drop of Nitrate reagent A (sulfanilic acid) and 1 drop of nitrate reagent B (α- naphthylamine) to each well.
  3. Wait 5 minutes. A red or pink color indicates a positive test for nitrate reduction.
  4. If a broth is colorless after 5 minutes, add a pinch of zinc (tip of a toothpick) to the broth. Wait 5 minutes. 
Oxidase test 

Reagents: Oxidase test reagent (tetramethyl-p-phenylenediamine)

  1. Divide an agar plate into 3 sections.
  2. Inoculate the 3 sections with a single streak of: PA, EC, and EF
  3. After incubation (24+ hrs), transfer 1 loopful of each bacterium onto a piece of filter paper (label the filter paper!). Next, place a single drop of oxidase reagent near each bacterial mass.
  4. Observe bacteria for color change:
    1. Pink/purple/black color development = oxidase-positive
    2. no color (color same as background) = oxidase-negative
Anaerobic growth (demonstration only)


2003 - 2019 Jos de Ondarza, Ph.D.