31: METABOLIC TESTING

Some of the most valuable tools for identifying microorganisms are tests based on their metabolic end products (the chemical compounds produced when organisms break down nutrients). Even when different organisms are provided with the same medium, they may metabolize the nutrients through entirely different pathways. This is because not all bacteria use the same enzymatic steps to process a given compound. As a result, the final products—or waste products—of metabolism can vary between species. These end products represent substances the organism cannot use further, or byproducts it must eliminate because they may be toxic if accumulated.

For example, animals eliminate excess nitrogen in the form of urea. Some bacteria, however, can use urea as a nutrient source. The ability to metabolize such compounds is determined by the specific enzymes an organism produces, which in turn are encoded by its DNA. Therefore, examining the metabolic waste products of a microorganism is, in effect, a way to study its genetic blueprint. Over the next several weeks you will

Metabolic tests are often performed using specialized culture media. Selective media are designed to support the growth of certain organisms while inhibiting others. This selection is achieved by including nutrients that only target organisms can utilize, or by incorporating compounds that are toxic to non-target species. If a microorganism cannot grow due to its inability to use the available nutrients or withstand inhibitory agents, it will not appear as a colony on the plate.

Selective Media

Selective media are highly sensitive to contamination. Introducing nutrients or other chemicals when transferring the inoculum can produce misleading results—appearing weakly positive or negative when the true reaction is more clear-cut. To avoid this, it is best to suspend a sample of the pure culture in sterile water before inoculating the test media. This reduces the risk of transferring additional nutrients or overwhelming the test with too many cells.

Simmons Citrate Agar is a common example of a selective medium used in metabolic testing. In this medium, citrate is the only available source of carbon. Only bacteria capable of transporting and breaking down citrate can survive and grow. As citrate is metabolized, the medium releases ammonia as a byproduct, which raises the pH. A pH indicator dye in the agar changes from green to blue when this alkaline shift occurs. Bacteria that cannot metabolize citrate will not grow and will not cause any color change in the medium.

By observing growth and color change, metabolic tests like the citrate test allow microbiologists to infer the presence or absence of specific enzymes and pathways, providing important clues for microbial identification.

Differential Media

Differential media are designed to support the growth of a wide variety of organisms while distinguishing between them based on their metabolic properties. Unlike selective media, which inhibit the growth of certain organisms, differential media contain a full complement of nutrients that allow most organisms to grow. What makes them useful for identification is the inclusion of indicators—compounds that visibly change in response to metabolic byproducts produced by the growing organisms.

As bacteria grow and metabolize nutrients in the medium, they may release acids, bases, gases, or other compounds. These byproducts can interact with the indicator, producing visible changes such as a color shift or changes in the appearance of the colony or the medium around it. If an organism does not produce the specific metabolic product being tested for, no change occurs.

A classic example is Eosin Methylene Blue (EMB) agar, which is both selective and differential. It allows the growth of Gram-negative organisms while inhibiting most Gram-positive bacteria. The differential component lies in its ability to detect lactose fermentation. Organisms that ferment lactose and produce significant acid will appear as colonies with a distinctive metallic green sheen. Those that do not ferment lactose, or do so with little acid production, form white, cream, or pale pink colonies without a green sheen. The dye itself responds to the acid level, creating this visual contrast.

Selective and Differential Media

Some media combine both selective and differential functions. A widely used example is Mannitol Salt Agar (MSA). This medium contains: A high concentration of salt (7.5%), which inhibits most organisms except for salt-tolerant species such as those in the genus Staphylococcus. The sugar mannitol, which may be fermented by certain bacteria. A pH indicator (phenol red), which changes color in response to acid production. If a bacterium can tolerate the high salt concentration and ferment mannitol, the acid produced during fermentation will turn the agar from red to bright yellow. If the bacterium grows but does not ferment mannitol, the agar remains red. If the organism cannot tolerate high salt, it will not grow at all, and no change will be observed.

MSA is commonly used to help identify Staphylococcus aureus, a clinically significant human pathogen. S. aureus can grow in the high-salt environment and ferment mannitol, causing a yellow color change in the agar. Therefore, the appearance of yellow growth on MSA from a clinical sample is a strong presumptive indication of S. aureus infection.

By combining metabolic profiling with selective growth conditions, selective and differential media provide powerful tools for identifying and differentiating microbial species based on their physiological characteristics.

Cell/Water Suspension

Some bacterial metabolic tests require making a cell and water suspension before performing the reaction. This step helps ensure that results reflect the bacteria’s enzyme activity based on only the nutrients in the test media without interference from nutrients from the nutrient agar. When bacterial cells are mixed in sterile water instead of nutrient media, the environment stays chemically neutral and doesn’t supply extra nutrient sources that could interfere with the test. The water suspension also helps standardize the number of cells making results easier to compare. To make a cell/water suspesion use a sterile loop to select an isolated colony from your T-streak plate then inoculte the cells into a test tube of sterial water. Only inoculate cells into the water once. Use the same sterial water suspension to set up all the metabolic tests.  

Exoenzymes

Many microorganisms have developed an efficient strategy for accessing nutrients that are too large to be transported across the plasma membrane. Some energy-rich substances in the environment—such as starches, proteins, and lipids—exist as polymers (large molecules made by joining many smaller building blocks). These large molecules are often too big to be directly absorbed by the cell.

To solve this problem, some bacteria possess the genetic ability to produce exoenzymes—enzymes that are secreted into the environment and function outside the cell. These enzymes are encoded by the bacterial DNA, synthesized within the cell, and then released into the surrounding medium. Once outside the cell, exoenzymes break down complex macromolecules into smaller subunits that can be transported through the membrane and used by the cell.

For example, starch is a carbohydrate polymer made of many linked sugar molecules. Starch cannot pass through the bacterial membrane in its full form. However, certain bacteria produce amylase, an exoenzyme that breaks the bonds between the sugar units, releasing individual glucose molecules that are small enough to be taken up by the cell and used as a carbon and energy source.

Bacteria can produce various exoenzymes to degrade other naturally occurring organic polymers:

Proteases break down proteins into amino acids

Lipases degrade fats and oils into fatty acids and glycerol

DNases and RNases digest nucleic acids

These enzymes enable bacteria to thrive in nutrient-rich environments by breaking down a wide range of organic materials. Some bacteria are even capable of degrading more complex or synthetic compounds. For instance, certain species have been shown to break down petroleum and toxic industrial chemicals, and research is ongoing to explore or engineer bacteria that produce exoenzymes capable of degrading plastics and other man-made polymers.

The production and activity of exoenzymes are important tools in microbial metabolism testing. By providing specific substrates in or on the culture media—such as starch, casein, or triglycerides—microbiologists can determine whether a bacterium is capable of producing the corresponding exoenzyme. The presence or absence of a visible reaction, such as a clearing zone around bacterial growth, helps identify metabolic capabilities and can contribute to bacterial classification and identification.

Before using metabolic tests to identify an unknown organism, you will first perform these tests on an assigned known organism. A Gram stain is completed first to determine the organism’s cell wall type, which helps guide the selection of appropriate tests. You will conduct all the metabolic tests outlined below on the assigned organism.  Once you have recorded the metabolic test results for your assigned organism, you will complete a written summary for each test. The class data for all the metabolic test results for all the assigned organisms will be compiled into two excel sheets, one for Gram negative and one for Gram positive bacteria. The data will then be used to create two flow charts—one for Gram-negative organisms and one for Gram-positive organisms. These charts will serve as road map to help you decide which tests to perform on the unknown organism in order to determine its genus and species. Not all the metabolic tests will be conducted on the unknown organism. Only the tests indicated in the flow chart you create.

Your top priority is to maintain the viability and purity of your assigned organism. If you notice anything unusual in your Gram stain or streak plate that suggests contamination or a mixed culture, consult your instructor immediately.