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Culture media can be used to differentiate between different kinds of bacteria by detecting acid or gas production.
Show how microbial acid and gas production are detected
- Differential media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators.
- To measure acid production one can use a pH indicator in the media.
- The Durham tube method is used to detect production of gas by microorganisms.
- differential media: Differential media or indicator media distinguish one microorganism type from another growing on the same media.
Cultures and Differential Media
A microbiological culture, or microbial culture, is created using a method for multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine an organism’s type, its abundance in the sample being tested, or both. It is one of the primary diagnostic techniques of microbiology, where it is used as a tool to determine the cause of infectious diseases by letting the agent multiply in a predetermined medium. A throat culture, for example, is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a growing medium; this will allow analysis to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative agent of strep throat. The term “culture” can be used to refer to the process of culturing organisms, to the medium they’re grown in, and is more generally used informally to refer to “selectively growing” a specific kind of microorganism in the lab.
Differential media, also known as indicator media, distinguish one microorganism type from another growing on the same media. These types of media use the biochemical characteristics of a microorganism grown in the presence of specific nutrients or indicators that have been added to the medium to visibly indicate the defining characteristics of a microorganism. These indicators or nutrients include but are not limited to neutral red, phenol red, eosin y, and methylene blue. Differential media are used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.
The Durham tube method is used to detect production of gas by microorganisms. They are simply smaller test tubes inserted upside down in another test tube. This small tube is initially filled with the solution in which the microorganism is to be grown. If gas is produced after inoculation and incubation, a visible gas bubble will be trapped inside the small tube. The initial air gap produced when the tube is inserted upside down is lost during sterilization, usually performed at 121°C for 15 or so minutes
Escherichia coli (E. coli), a rod-shaped member of the coliform group, can be distinguished from most other coliforms by its ability to ferment lactose at 44°C in the fecal coliform test, and by its growth and color reaction on certain types of culture media. When cultured on an EMB (eosin methylene blue) plate, a positive result for E. coli is metallic green colonies on a dark purple media. Unlike the general coliform group, E. coli are almost exclusively of fecal origin and their presence is thus an effective confirmation of fecal contamination. Some strains of E. coli can cause serious illness in humans.
Sorbitol MacConkey Agar
Sorbitol MacConkey agar is a variant of the traditional MacConkey commonly used in the detection of E. coli O157:H7. Traditionally, MacConkey agar has been used to distinguish those bacteria that ferment lactose from those that do not.
This is an important distinction. Gut bacteria, such as Escherichia coli, can typically ferment lactose; important gut pathogens including Salmonella enterica and most shigellas are unable to ferment lactose. Shigella sonnei can ferment lactose, but only after prolonged incubation; it is referred to as a late-lactose fermenter.
During fermentation of sugar, acid is formed and the pH of the medium drops, changing the color of the pH indicator. Different formulations use different indicators; neutral red is often used when culturing gut bacteria because lactose fermenters turn a deep red when this pH indicator is used. Those bacteria unable to ferment lactose, often referred to as nonlactose fermenters (NLFs) metabolize the peptone in the medium. This releases ammonia, which raises the pH of the medium. Although some authors refer to NLFs as being colorless, in reality they turn neutral red a buffish color.
E. coli O157:H7 differs from most other strains of E. coli in being unable to ferment sorbitol. In sorbitol MacConkey agar, lactose is replaced by sorbitol. Most strains of E. coli ferment sorbitol to produce acid: E. coli O157:H7 can not ferment sorbitol, so this strain uses peptone to grow. This raises the pH of the medium, allowing the O157:H7 strain to be differentiated from other E. coli strains through the action of the pH indicator in the medium.
Negative Regulation of Cytosolic Sensing of DNA
In mammals, cytosolic detection of nucleic acids is critical in initiating innate antiviral responses against invading pathogens (like bacteria, viruses, fungi and parasites). These programs are mediated by multiple cytosolic and endosomal sensors and adaptor molecules (c-GAS/STING axis and TLR9/MyD88 axis, respectively) and lead to the production of type I interferons (IFNs), pro-inflammatory cytokines, and chemokines. While the identity and role of multiple pattern recognition receptors (PRRs) have been elucidated, such immune surveillance systems must be tightly regulated to limit collateral damage and prevent aberrant responses to self- and non-self-nucleic acids. In this review, we discuss recent advances in our understanding of how cytosolic sensing of DNA is controlled during inflammatory immune responses.
Keywords: Auto-inflammation Infertility Inflammasome Interferon NLRP14 Nucleic acid sensing Post-translational modifications RIG-I STING TBK1 cGAS.
© 2019 Elsevier Inc. All rights reserved.
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Separation techniques: Chromatography
Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification.
Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights [1, 2]. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster .
Based on this approach three components form the basis of the chromatography technique.
Stationary phase: This phase is always composed of a “solid” phase or 𠇊 layer of a liquid adsorbed on the surface a solid support”.
Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
The type of interaction between stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on separation of molecules from each other. Chromatography methods based on partition are very effective on separation, and identification of small molecules as amino acids, carbohydrates, and fatty acids. However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins. Paper chromatography is used in the separation of proteins, and in studies related to protein synthesis gas-liquid chromatography is utilized in the separation of alcohol, esther, lipid, and amino groups, and observation of enzymatic interactions, while molecular-sieve chromatography is employed especially for the determination of molecular weights of proteins. Agarose-gel chromatography is used for the purification of RNA, DNA particles, and viruses .
Stationary phase in chromatography, is a solid phase or a liquid phase coated on the surface of a solid phase. Mobile phase flowing over the stationary phase is a gaseous or liquid phase. If mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). Gas chromatography is applied for gases, and mixtures of volatile liquids, and solid material. Liquid chromatography is used especially for thermal unstable, and non-volatile samples .
The purpose of applying chromatography which is used as a method of quantitative analysis apart from its separation, is to achive a satisfactory separation within a suitable timeinterval. Various chromatography methods have been developed to that end. Some of them include column chromatography, thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, high-pressure liquid chromatography, and affinity chromatography .
Types of chromatography
Gel-permeation (molecular sieve) chromatography
Hydrophobic interaction chromatography
High-pressure liquid chromatography (HPLC)
Since proteins have difference characteristic features as size, shape, net charge, stationary phase used, and binding capacity, each one of these characteristic components can be purified using chromatographic methods. Among these methods, most frequently column chromatography is applied. This technique is used for the purification of biomolecules. On a column (stationary phase) firstly the sample to be separated, then wash buffer (mobile phase) are applied ( Figure 1 ). Their flow through inside column material placed on a fiberglass support is ensured. The samples are accumulated at the bottom of the device in a tme-, and volume-dependent manner .
Ion- exchange chromatography
Ion- exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved with ionic ties. Proteins are separated from the column either by changing pH, concentration of ion salts or ionic strength of the buffer solution . Positively charged ion- exchange matrices are called anion-exchange matrices, and adsorb negatively charged proteins. While matrices bound with negatively charged groups are known as cation-exchange matrices, and adsorb positively charged proteins ( Figure 2 ) .
Ion- exchange chromatography.
Gel- permeation (molecular sieve) chromatography
The basic principle of this method is to use dextran containing materials to separate macromolecules based on their differences in molecular sizes. This procedure is basically used to determine molecular weights of proteins, and to decrease salt concentrations of protein solutions . In a gel- permeation column stationary phase consists of inert molecules with small pores. The solution containing molecules of different dimensions are passed continuously with a constant flow rate through the column. Molecules larger than pores can not permeate into gel particles, and they are retained between particles within a restricted area. Larger molecules pass through spaces between porous particles, and move rapidly through inside the column. Molecules smaller than the pores are diffused into pores, and as molecules get smaller, they leave the column with proportionally longer retention times ( Figure 3 ) . Sephadeks G type is the most frequently used column material. Besides, dextran, agorose, polyacrylamide are also used as column materials .
Gel-permeation (molecular sieve) chromatography.
This chromatography technique is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins . A ligand which can make a complex with specific protein (dextran, polyacrylamide, cellulose etc) binds the filling material of the column. The specific protein which makes a complex with the ligand is attached to the solid support (matrix), and retained in the column, while free proteins leave the column. Then the bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution ( Figure 4 ) .
In paper chromatography support material consists of a layer of cellulose highly saturated with water. In this method a thick filter paper comprised the support, and water drops settled in its pores made up the stationary “liquid phase.” Mobile phase consists of an appropriate fluid placed in a developing tank. Paper chromatography is a “liquid-liquid” chromatography .
Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this method stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all solid substances used. in column chromatography (alumina, silica gel, cellulose) can be utilized. In this method, the mobile phase travels upward through the stationary phase The solvent travels up the thin plate soaked with the solvent by means of capillary action. During this procedure, it also drives the mixture priorly dropped on the lower parts of the plate with a pipette upwards with different flow rates. Thus the separation of analytes is achieved. This upward travelling rate depends on the polarity of the material, solid phase, and of the solvent .
In cases where molecules of the sample are colorless, florescence, radioactivity or a specific chemical substance can be used to produce a visible coloured reactive product so as to identify their positions on the chromatogram. Formation of a visible colour can be observed under room light or UV light. The position of each molecule in the mixture can be measured by calculating the ratio between the the distances travelled by the molecule and the solvent. This measurement value is called relative mobility, and expressed with a symbol Rf. Rf. value is used for qualitative description of the molecules .
In this method stationary phase is a column which is placed in the device, and contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Gas chromatography is a “gas-liquid” chromatography. Its carrier phase consists of gases as He or N2. Mobile phase which is an inert gas is passed through a column under high pressure. The sample to be analyzed is vaporized, and enters into a gaseous mobile phase phase. The components contained in the sample are dispersed between mobile phase, and stationary phase on the solid support. Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly applied technique for the extremely excellent separation of very minute molecules. It is used in the separation of very little amounts of analytes .
Dye- ligand chromatography
Development of this technique was based on the demonstration of the ability of many enzymes to bind purine nucleotides for Cibacron Blue F3GA dye . The planar ring structure with negatively charged groups is analogous to the structure of NAD. This analogy has been evidenced by demonstration of the binding of Cibacron Blue F3GA dye to adenine, ribose binding sites of NAD. The dye behaves as an analogue of ADP-ribose. The binding capacity of this type adsorbents is 10-fold stronger rhat that of the affinity of other adsorbents. Under appropriate pH conditions, elution with high-ionic strength solutions, and using ion-exchange property of adsorbent, the adsorbed proteins are separated from the column [20, 21].
Hydrophobic interaction chromatography (HIC)
In this method the adsorbents prepared as column material for the ligand binding in affinity chromatography are used. HIC technique is based on hydrophobic interactions between side chains bound to chromatography matrix [22, 23].
Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands because of their affinity especially for dehydrogenases, kinases, transferases, and reductases The mostly known type of this kind of chromatography is immobilized metal affinity chromatography (IMAC) .
High-prssure liquid chromatography (HPLC)
Using this chromatography technique it is possible to perform structural, and functional analysis, and purification of many molecules within a short time, This technique yields perfect results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active molecules, In HPLC, mobile phase passes throuıgh columns under 10 atmospheric pressure, and with a high (0.1𠄵 cm//sec) flow rate. In this technique, use of small particles, and application of high presure on the rate of solvent flow increases separation power, of HPLC and the analysis is completed within a short time.
Essential components of a HPLC device are solvent depot, high- pressure pump, commercially prepared column, detector, and recorder. Duration of separation is controlled with the aid of a computerized system, and material is accrued .
Application areas of chromatography in medicine
Chromatography technique is a valuable tool for biochemists, besides it can be applied easily during studies performed in clinical laboratories For instance, paper chromatography is used to determine some types of sugar, and amino acids in bodily fluids which are associated with hereditary metabolic disorders. Gas chromatography is used in laboratories to measure steroids, barbiturates, and lipids. Chromatographic technique is also used in the separation of vitamins, and proteins.
Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal pigments. With time its application area was extended considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective separation method. Column chromatography is one of the useful separation, and determination methods. Column chromatography is a protein purification method realized especially based on one of the characteristic features of proteins. Besides, these methods are used to control purity of a protein. HPLC technique which has many superior features including especially its higher sensitivity, rapid turnover rate, its use as a quantitative method, can purify amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, antibiotics, and steroids.
Some groups of microorganisms that are commonly occurring in the environment can cause serious problems in an industrial setting. For example, the proliferation of sulphate reducing prokaryotes (SRP) can influence corrosion rates in pipelines, vessels and machinery, and the conditions found within production facilities can provide the ideal environment for their growth.
The actions of different groups of microorganisms influence corrosion in different ways. For example growth of some groups of microbes can create conditions that allow proliferation of others, which in turn produce corrosive waste products such as hydrogen sulphide or acids.
Monitoring for the presence of the key groups of microorganisms known to influence corrosion in oilfield environments, allows remedial action to be taken if numbers start to rise. Continued regular monitoring can also determine the efficacy of steps taken to control these groups of organisms, helping you to make informed and timely decisions.
At NCIMB we can identify and quantify microorganisms known to influence corrosion using both the latest molecular methods, and traditional culture-based techniques. Analysis can be undertaken on most types of liquid and solid sample, including production fluids, scales and pig wax. We also offer analysis of the whole microbial community within the reservoir.
- qPCR: This technique is used to quantify groups of microorganisms without any requirement for growth. It therefore gives very rapid results, and ensures that microbes that do not grow under laboratory conditions are included in counts.
- Culture-based enumeration of:
- General heterotrophic bacteria
- Acid producing general heterotrophic bacteria
- Sulphate reducing bacteria (mesophilic, thermophilic and hyperthermophilic)
- Nitrate reducing bacteria
- Next-generation sequencing (metagenomics): This technique, which has revolutionised understanding of environmental microbiology in recent years, analyses the whole microbial population from a single sample. It identifies the groups of microorganisms present to give a comprehensive picture of the microbial ecosystem within the oilfield. This allows microbiological changes to be monitored in longitudinal studies as well as aiding understanding of the functional impact of the species observed. 16S community analysis is a powerful tool that can help operating companies assess the likelihood of reservoir souring and corrosion.
- NORM contaminated coupons: we can accept NORM contaminated coupons and can undertake microbial analysis as well as reporting on the NORM levels.
- NCIMB partners with corrosion monitoring specialists ICR: in the delivery of full microbiological audits and surveys of production facilities. Analysis of samples from retrieved corrosion coupons and probes allows sessile microbial growth on pipework and in vessels to be monitored. This data allows a more accurate evaluation on the threat of microbially influenced corrosion than monitoring planktonic populations alone. For more information about ICR's comprehensive corrosion monitoring and microbiological audit services visit their website.
We can advise on the most appropriate approach to monitoring and have participated in R&D projects as well as undertaking routine analysis.
We supply media kits for quantification of:
- Sulphate reducing bacteria
- General heterotrophic bacteria/acid producing general heterotrophic bacteria
- Nitrate reducing bacteria
We can also prepare other types of media on request, and store stocks of specific media for customers.
We can undertake lab-based biocide testing against our own in-house North Sea microbial consortium, or enrich from clients’ own samples for laboratory testing. We can also undertake direct enumeration from customer samples following treatment at the production facility to determine efficacy in situ.
We can use both traditional culture-based enumeration for determining the efficacy of biocides or take a molecular approach with qPCR. Culture based enumeration is likely to be the most appropriate approach for testing against lab-grown cultures, whereas qPCR can give faster results for testing the impact of biocides following on site treatment, as part of a longer term study.
For customers seeking more in depth understanding of the whole microbial community, we can undertake nextgeneration sequencing providing detailed metagenomic information on the microbes present, and their relative abundance. This information can then be used as part of an ongoing monitoring regime.
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Many cells are unable to carry out respiration because of one or more of the following circumstances:
- The cell lacks a sufficient amount of any appropriate, inorganic, final electron acceptor to carry out cellular respiration.
- The cell lacks genes to make appropriate complexes and electron carriers in the electron transport system.
- The cell lacks genes to make one or more enzymes in the Krebs cycle.
Whereas lack of an appropriate inorganic final electron acceptor is environmentally dependent, the other two conditions are genetically determined. Thus, many prokaryotes, including members of the clinically important genus Streptococcus, are permanently incapable of respiration, even in the presence of oxygen. Conversely, many prokaryotes are facultative, meaning that, should the environmental conditions change to provide an appropriate inorganic final electron acceptor for respiration, organisms containing all the genes required to do so will switch to cellular respiration for glucose metabolism because respiration allows for much greater ATP production per glucose molecule.
If respiration does not occur, NADH must be reoxidized to NAD + for reuse as an electron carrier for glycolysis, the cell’s only mechanism for producing any ATP, to continue. Some living systems use an organic molecule (commonly pyruvate) as a final electron acceptor through a process called fermentation. Fermentation does not involve an electron transport system and does not directly produce any additional ATP beyond that produced during glycolysis by substrate-level phosphorylation. Organisms carrying out fermentation, called fermenters, produce a maximum of two ATP molecules per glucose during glycolysis. Table 1 compares the final electron acceptors and methods of ATP synthesis in aerobic respiration, anaerobic respiration, and fermentation. Note that the number of ATP molecules shown for glycolysis assumes the Embden-Meyerhof-Parnas pathway. The number of ATP molecules made by substrate-level phosphorylation (SLP) versus oxidative phosphorylation (OP) are indicated.
Microbial fermentation processes have been manipulated by humans and are used extensively in the production of various foods and other commercial products, including pharmaceuticals. Microbial fermentation can also be useful for identifying microbes for diagnostic purposes.
Fermentation by some bacteria, like those in yogurt and other soured food products, and by animals in muscles during oxygen depletion, is lactic acid fermentation. The chemical reaction of lactic acid fermentation is as follows:
Bacteria of several gram-positive genera, including Lactobacillus, Leuconostoc, and Streptococcus, are collectively known as the lactic acid bacteria (LAB), and various strains are important in food production. During yogurt and cheese production, the highly acidic environment generated by lactic acid fermentation denatures proteins contained in milk, causing it to solidify. When lactic acid is the only fermentation product, the process is said to be homolactic fermentation such is the case for Lactobacillus delbrueckii and S. thermophiles used in yogurt production. However, many bacteria perform heterolactic fermentation, producing a mixture of lactic acid, ethanol and/or acetic acid, and CO2 as a result, because of their use of the branched pentose phosphate pathway instead of the EMP pathway for glycolysis. One important heterolactic fermenter is Leuconostoc mesenteroides, which is used for souring vegetables like cucumbers and cabbage, producing pickles and sauerkraut, respectively.
Lactic acid bacteria are also important medically. The production of low pH environments within the body inhibits the establishment and growth of pathogens in these areas. For example, the vaginal microbiota is composed largely of lactic acid bacteria, but when these bacteria are reduced, yeast can proliferate, causing a yeast infection. Additionally, lactic acid bacteria are important in maintaining the health of the gastrointestinal tract and, as such, are the primary component of probiotics.
Another familiar fermentation process is alcohol fermentation, which produces ethanol. The ethanol fermentation reaction is shown in Figure 1. In the first reaction, the enzyme pyruvate decarboxylase removes a carboxyl group from pyruvate, releasing CO2 gas while producing the two-carbon molecule acetaldehyde. The second reaction, catalyzed by the enzyme alcohol dehydrogenase, transfers an electron from NADH to acetaldehyde, producing ethanol and NAD + . The ethanol fermentation of pyruvate by the yeast Saccharomyces cerevisiae is used in the production of alcoholic beverages and also makes bread products rise due to CO2 production. Outside of the food industry, ethanol fermentation of plant products is important in biofuel production.
Figure 1. The chemical reactions of alcohol fermentation are shown here. Ethanol fermentation is important in the production of alcoholic beverages and bread.
Beyond lactic acid fermentation and alcohol fermentation, many other fermentation methods occur in prokaryotes, all for the purpose of ensuring an adequate supply of NAD + for glycolysis (Table 2). Without these pathways, glycolysis would not occur and no ATP would be harvested from the breakdown of glucose. It should be noted that most forms of fermentation besides homolactic fermentation produce gas, commonly CO2 and/or hydrogen gas. Many of these different types of fermentation pathways are also used in food production and each results in the production of different organic acids, contributing to the unique flavor of a particular fermented food product. The propionic acid produced during propionic acid fermentation contributes to the distinctive flavor of Swiss cheese, for example.
Several fermentation products are important commercially outside of the food industry. For example, chemical solvents such as acetone and butanol are produced during acetone-butanol-ethanol fermentation. Complex organic pharmaceutical compounds used in antibiotics (e.g., penicillin), vaccines, and vitamins are produced through mixed acid fermentation. Fermentation products are used in the laboratory to differentiate various bacteria for diagnostic purposes. For example, enteric bacteria are known for their ability to perform mixed acid fermentation, reducing the pH, which can be detected using a pH indicator. Similarly, the bacterial production of acetoin during butanediol fermentation can also be detected. Gas production from fermentation can also be seen in an inverted Durham tube that traps produced gas in a broth culture.
Microbes can also be differentiated according to the substrates they can ferment. For example, E. coli can ferment lactose, forming gas, whereas some of its close gram-negative relatives cannot. The ability to ferment the sugar alcohol sorbitol is used to identify the pathogenic enterohemorrhagic O157:H7 strain of E. coli because, unlike other E. coli strains, it is unable to ferment sorbitol. Last, mannitol fermentation differentiates the mannitol-fermenting Staphylococcus aureus from other non–mannitol-fermenting staphylococci.
Table 2. Common Fermentation Pathways Pathway End Products Example Microbes Commercial Products Acetone-butanol-ethanol Acetone, butanol, ethanol, CO2 Clostridium acetobutylicum Commercial solvents, gasoline alternative Alcohol Ethanol, CO2 Candida, Saccharomyces Beer, bread Butanediol Formic and lactic acid ethanol acetoin 2,3 butanediol CO2 hydrogen gas Klebsiella, Enterobacter Chardonnay wine Butyric acid Butyric acid, CO2, hydrogen gas Clostridium butyricum Butter Lactic acid Lactic acid Streptococcus, Lactobacillus Sauerkraut, yogurt, cheese Mixed acid Acetic, formic, lactic, and succinic acids ethanol, CO2, hydrogen gas Escherichia, Shigella Vinegar, cosmetics, pharmaceuticals Propionic acid Acetic acid, propionic acid, CO2 Propionibacterium, Bifidobacterium Swiss cheese
Think about It
- When would a metabolically versatile microbe perform fermentation rather than cellular respiration?
Identifying Bacteria by Using API Test Panels
Identification of a microbial isolate is essential for the proper diagnosis and appropriate treatment of patients. Scientists have developed techniques that identify bacteria according to their biochemical characteristics. Typically, they either examine the use of specific carbon sources as substrates for fermentation or other metabolic reactions, or they identify fermentation products or specific enzymes present in reactions. In the past, microbiologists have used individual test tubes and plates to conduct biochemical testing. However, scientists, especially those in clinical laboratories, now more frequently use plastic, disposable, multitest panels that contain a number of miniature reaction tubes, each typically including a specific substrate and pH indicator. After inoculation of the test panel with a small sample of the microbe in question and incubation, scientists can compare the results to a database that includes the expected results for specific biochemical reactions for known microbes, thus enabling rapid identification of a sample microbe. These test panels have allowed scientists to reduce costs while improving efficiency and reproducibility by performing a larger number of tests simultaneously.
Many commercial, miniaturized biochemical test panels cover a number of clinically important groups of bacteria and yeasts. One of the earliest and most popular test panels is the Analytical Profile Index (API) panel invented in the 1970s. Once some basic laboratory characterization of a given strain has been performed, such as determining the strain’s Gram morphology, an appropriate test strip that contains 10 to 20 different biochemical tests for differentiating strains within that microbial group can be used. Currently, the various API strips can be used to quickly and easily identify more than 600 species of bacteria, both aerobic and anaerobic, and approximately 100 different types of yeasts. Based on the colors of the reactions when metabolic end products are present, due to the presence of pH indicators, a metabolic profile is created from the results (Figure 2). Microbiologists can then compare the sample’s profile to the database to identify the specific microbe.
Figure 2. The API 20NE test strip is used to identify specific strains of gram-negative bacteria outside the Enterobacteriaceae. Here is an API 20NE test strip result for Photobacterium damselae ssp. piscicida.
Clinical Focus: Alex, Part 2
This example continues Alex’s story that started in Energy Matter and Enzymes.
Many of Alex’s symptoms are consistent with several different infections, including influenza and pneumonia. However, his sluggish reflexes along with his light sensitivity and stiff neck suggest some possible involvement of the central nervous system, perhaps indicating meningitis. Meningitis is an infection of the cerebrospinal fluid (CSF) around the brain and spinal cord that causes inflammation of the meninges, the protective layers covering the brain. Meningitis can be caused by viruses, bacteria, or fungi. Although all forms of meningitis are serious, bacterial meningitis is particularly serious. Bacterial meningitis may be caused by several different bacteria, but the bacterium Neisseria meningitidis, a gram-negative, bean-shaped diplococcus, is a common cause and leads to death within 1 to 2 days in 5% to 10% of patients.
Given the potential seriousness of Alex’s conditions, his physician advised his parents to take him to the hospital in the Gambian capital of Banjul and there have him tested and treated for possible meningitis. After a 3-hour drive to the hospital, Alex was immediately admitted. Physicians took a blood sample and performed a lumbar puncture to test his CSF. They also immediately started him on a course of the antibiotic ceftriaxone, the drug of choice for treatment of meningitis caused by N. meningitidis, without waiting for laboratory test results.
- How might biochemical testing be used to confirm the identity of N. meningitidis?
- Why did Alex’s doctors decide to administer antibiotics without waiting for the test results?
We’ll return to Alex’s example in later pages.
Key Concepts and Summary
- Fermentation uses an organic molecule as a final electron acceptor to regenerate NAD + from NADH so that glycolysis can continue.
- Fermentation does not involve an electron transport system, and no ATP is made by the fermentation process directly. Fermenters make very little ATP—only two ATP molecules per glucose molecule during glycolysis.
- Microbial fermentation processes have been used for the production of foods and pharmaceuticals, and for the identification of microbes.
- During lactic acid fermentation, pyruvate accepts electrons from NADH and is reduced to lactic acid. Microbes performing homolactic fermentation produce only lactic acid as the fermentation product microbes performing heterolactic fermentation produce a mixture of lactic acid, ethanol and/or acetic acid, and CO2.
- Lactic acid production by the normal microbiota prevents growth of pathogens in certain body regions and is important for the health of the gastrointestinal tract.
- During ethanol fermentation, pyruvate is first decarboxylated (releasing CO2) to acetaldehyde, which then accepts electrons from NADH, reducing acetaldehyde to ethanol. Ethanol fermentation is used for the production of alcoholic beverages, for making bread products rise, and for biofuel production.
- Fermentation products of pathways (e.g., propionic acid fermentation) provide distinctive flavors to food products. Fermentation is used to produce chemical solvents (acetone-butanol-ethanol fermentation) and pharmaceuticals (mixed acid fermentation).
- Specific types of microbes may be distinguished by their fermentation pathways and products. Microbes may also be differentiated according to the substrates they are able to ferment.
Which of the following is the purpose of fermentation?
- to make ATP
- to make carbon molecule intermediates for anabolism
- to make NADH
- to make NAD +
[reveal-answer q=″]Show Answer[/reveal-answer]
[hidden-answer a=″]Answer d. The purpose of fermentation is to make NAD + .[/hidden-answer]
Which molecule typically serves as the final electron acceptor during fermentation?
[reveal-answer q=″]Show Answer[/reveal-answer]
[hidden-answer a=″]Answer c. Pyruvate typically serves as the final electron acceptor during fermentation.[/hidden-answer]
Which fermentation product is important for making bread rise?
[reveal-answer q=″]Show Answer[/reveal-answer]
[hidden-answer a=″]Answer b. CO2 is important for making bread rise.[/hidden-answer]
Which of the following is not a commercially important fermentation product?
[reveal-answer q=″]Show Answer[/reveal-answer]
[hidden-answer a=″]Answer b. Pyruvate is not a commercially important fermentation product.[/hidden-answer]
Fill in the Blank
The microbe responsible for ethanol fermentation for the purpose of producing alcoholic beverages is ________.
[reveal-answer q=″]Show Answer[/reveal-answer]
[hidden-answer a=″]The microbe responsible for ethanol fermentation for the purpose of producing alcoholic beverages is yeast (Saccharomyces cerevisiae).[/hidden-answer]
________ results in the production of a mixture of fermentation products, including lactic acid, ethanol and/or acetic acid, and CO2.
[reveal-answer q=″]Show Answer[/reveal-answer]
[hidden-answer a=″]Heterolactic fermentation results in the production of a mixture of fermentation products, including lactic acid, ethanol and/or acetic acid, and CO2.[/hidden-answer]
Fermenting organisms make ATP through the process of ________.
[reveal-answer q=″]Show Answer[/reveal-answer]
[hidden-answer a=″]Fermenting organisms make ATP through the process of glycolysis.[/hidden-answer]
Match the fermentation pathway with the correct commercial product it is used to produce:
___acetone-butanol-ethanol fermentation a. bread ___alcohol fermentation b. pharmaceuticals ___lactic acid fermentation c. Swiss cheese ___mixed acid fermentation d. yogurt ___propionic acid fermentation e. industrial solvents
[reveal-answer q=″]Show Answer[/reveal-answer]
- Industrial solvents are produced by acetone-butanol-ethanol fermentation.
- Bread is produced by alcohol fermentation.
- Yogurt is produced by lactic acid fermentation.
- Pharmaceuticals are produced by mixed acid fermentation.
- Swiss cheese is produced by propionic acid fermentation.
Think about It
- Why are some microbes, including Streptococcus spp., unable to perform aerobic respiration, even in the presence of oxygen?
- How can fermentation be used to differentiate various types of microbes?
- The bacterium E. coli is capable of performing aerobic respiration, anaerobic respiration, and fermentation. When would it perform each process and why? How is ATP made in each case?
The oxidative-fermentative test determines if certain gram-negative rods metabolize glucose by fermentation or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the bromthymol blue indicator in OF media from green to yellow in the presence or absence of oxygen .
Certain nonfermenting gram-negative bacteria metabolize glucose using aerobic respiration and therefore only produce a small amount of weak acids during glycolysis and Krebs cycle. The decrease amount of peptone and increase amount of glucose facilitates the detection of weak acids thus produced. Dipotassium phosphate buffer is added to further promote acid detection.
Carbohydrate fermentation is the process microorganisms use to produce energy. Most microorganisms convert glucose to pyruvate during glycolysis however, some organisms use alternate pathways. A fermentation medium consists of a basal medium containing a single carbohydrate (glucose, lactose, sucrose, mannitol etc.) for fermentation. However, the medium may contain various color indicators. In addition to a color indicator to detect the production of acid from fermentation, a Durham tube is placed in each tube to capture gas produced by metabolism. The carbohydrate fermentation patterns shown by different organisms are useful in differentiating among bacterial groups or species.
Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate (glucose, lactose, or sucrose). Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed.
DMS: The Climate Gas You’ve Never Heard Of
For generations of mariners, a tangy, almost sweet odor served as a signal that land was nearby. What sailors called “the smell of the shore” had the opposite meaning to landlubbers, who would catch the same sweet scent wafting over the waves and think of it as “the smell of the sea.” Seabirds probably don’t have a name for it, but the odor means something to them, as well: the opening of an all-you-can-eat buffet.
Part of what they’re all smelling is a little-studied gas known as dimethylsulfide, or DMS. Some seabirds, possessing a keen olfactory sense, use the scent to track down its source: blooms of algae floating near the ocean’s surface, where the microscopic animals, krill, and other crustaceans that gather to graze on algae provide the birds with a hearty meal. (See Seabirds Use Their Sense of Smell to Find Food.)
DMS does far more than ring the birds’ dinner bell, though. Scientists believe it represents a large source of sulfur going into the Earth’s atmosphere. As such, it helps drive the formation of clouds, which block solar radiation from reaching the Earth’s surface and reflect it back into space.
If DMS production is speeded up by global climate change, as many scientists believe it will be, then it could provide a cooling effect. That means DMS could help offset greenhouse warming.
That hopeful claim has been made for more than two decades. In 1987, British chemist James Lovelock and several colleagues popularized an idea first proposed by others that algae might play a vital role in regulating the Earth’s climate.
Lovelock is famed as the originator of the Gaia hypothesis, which suggests that the Earth functions as a single living organism and maintains the conditions necessary for its own survival. By encouraging cloud formation, Lovelock theorized, DMS might help keep the Earth’s thermostat at a fairly constant temperature.
But scientists still understand very little about how and why marine algae make DMS, how it moves through the food web in the upper ocean, or how much of it gets into the lower atmosphere. Despite its potential impact on climate, the amount of attention focused on DMS remains relatively small, and scientists continue to be uncertain whether it can make a major difference in global climate change.
John Dacey, a biologist at Woods Hole Oceanographic Institution (WHOI), is one of the few marine scientists who have devoted a great deal of time to studying oceanic DMS over the past few decades. He says he’s amazed and dismayed that carbon dioxide receives so much research funding right now at the expense of other basic science, when other gases may have critical roles to play in countering or augmenting warming.
“Environmentally, understanding DMS is incredibly important,” said Dierdre Toole, a marine chemist at WHOI. “DMS just isn’t fashionable, but I think it could have a hugely important role to play—more important than a lot of things that are fashionable right now.”
Critical but difficult measurements
Dacey has long investigated the processes that control how environmentally important gases are exchanged between Earth, ocean, organisms, and the atmosphere. He explored the little-understood role that plants play in influencing greenhouse gases. Dacey was the first to show how vegetation transferred methane to the atmosphere, and he demonstrated that DMS is emitted from the leaves of certain species of marsh grass. Scientists had previously thought the gas was coming from the sediment around the grass.
For the ocean, Dacey has also developed ways to track the exchange of DMS from sea to air. He is also studying how a molecule called dimethylsulfoniopropionate, or DMSP—the source of DMS—concentrates in organisms in the marine food web.
These are critical but difficult measurements to make. Dacey’s work over the decades on the dynamics of dissolved gases has required him to develop better measuring tools, including automated devices that allow researchers to sample repeatedly in order to detect changes.
In Dacey’s ongoing efforts to find better ways to measure DMS, he had collected three years’ worth of measurements on DMS concentrations in the Sargasso Sea off Bermuda. But this unique set of open-ocean data remained unanalyzed until scientists such as Toole recognized its potential.
DMS attracted Toole precisely because so little was known about it. With degrees in chemistry, geography, and marine science, Toole wanted a specialty that spanned her many interests. DMS offered all of that, and the sweet smell of mystery, too.
In frigid waters, a great bloom
When spring comes to the Ross Sea, just off Antarctica’s largest ice shelf, the ice begins to melt, and sunlight reaches nutrient-rich waters that have welled up from the ocean’s depths to the surface. The result: One of the largest, most predictable algal blooms in the world.
“You get a huge explosion of phytoplankton,” Toole said. Phytoplankton are tiny, single-celled floating plants that live near the ocean’s surface where enough light reaches to support photosynthesis. They’re the base of the ocean’s food web.
Toole and several colleagues have sought out the Ross Sea algal bloom for the past three years aboard an icebreaking ship. For a couple of weeks, the scientists have the phytoplankton all to themselves. “It’s almost like working in the lab,” Toole said.
Every morning, Toole took samples from the ocean, snapping the tops on bottles at different depths to collect the algae. In their cells, the phytoplankton synthesize DMSP. Physiologists aren’t sure why the algae need DMSP, but they speculate that it could have something to do with regulating salinity or temperature inside algal cells. In cold environments, DMSP may act as a cryoprotectant, keeping the cell from freezing. Some researchers have guessed that DMSP acts as a chemical repellant that helps deter predators.
Toole is more convinced that light—particularly ultraviolet light—explains why the algae produce DMSP. Working with David Siegel, a professor of geography at the University of California, Santa Barbara, Toole found that phytoplankton appear to convert DMSP into DMS when they’re stressed by ultraviolet radiation from the sun. The DMS flushes out chemically reactive molecules that cause cellular damage, in much the same way that our bodies use antioxidants to bind to free radicals.
“Phytoplankton respond dramatically to UV radiation stresses,” Siegel said. “This response is incredibly rapid.” Siegel and Toole documented their research in May 2004 in the journal Geophysical Research Letters.
As long as the algae are left alone, the DMSP stays relatively intact inside their cells, although some DMS leaks out and begins to make that DMS smell. Soon enough, though, once the algae in the Ross Sea have reached full bloom, zooplankton arrive and begin chowing down on them. DMSP spills out of munched-on algae into the ocean.
This in turn provides a feast for bacteria, some of which split the DMSP into components such as DMS, said Naomi Levine. A graduate student in the MIT/WHOI Joint Program, Levine is studying how bacteria send DMSP down a divergent biochemical pathway, something called the “bacterial switch.”
DMS dissolved in seawater begins to waft into the air and register on seabirds’ and researchers’ nostrils. “The entire place reeks of DMS,” Toole said. The odor may be pleasant enough in small quantities (“it has actually been described as a ‘positive flavor component’ in Pacific oysters,” Dacey said), but large doses of it make Toole gag. “I think I’ve brushed my teeth enough in DMS water that it just turns my stomach.”
A global thermostat?
Once DMS has moved from the ocean to the air, it starts to play an entirely different role: cloud maker.
DMS is chemically reactive and can’t last long in the atmosphere. It quickly gets converted into a variety of sulfur compounds that serve as aerosols. They allow water vapor to condense around them. This is how clouds are made.
Clouds, of course, have a major impact on the Earth’s climate. They deflect solar radiation back into space, preventing sunlight from heating the Earth’s surface and providing a cooling effect. Clouds are even more important over oceans, which are both more extensive and darker than land and so absorb a majority of the heat hitting the planet, Toole said. So the question becomes, can algae produce enough DMS to increase cloud cover and keep the planet’s temperature from rising?
“DMS is undoubtedly part of the system of checks and balances that keeps the climate from taking wild swings,” said Ron Kiene, a professor of marine sciences at the University of South Alabama and one of the world’s leading DMS researchers. Putting sulfur in the atmosphere, as with DMS emissions, is a more efficient way of cooling the atmosphere than removing carbon dioxide. So it might be possible for the natural feedback mechanisms of the biosphere to use DMS to limit global warming, he said.
Recently, Dacey spent a week in Barrow, Alaska, calibrating an automatic system for measuring very low volumes of DMS in the air. He hopes the measurements made at Barrow will reveal whether a scenario that starts with melting sea ice and leads to more open water, more phytoplankton, more DMS in the atmosphere—and hence greater cloud cover—will offset an increase in solar radiation absorbed by dark open water, rather than reflected by white ice.
DMS also comes into play in less natural, more intrusive proposals to remedy greenhouse warming. Proponents of ocean iron fertilization—seeding the oceans with iron to increase the growth of marine plants that absorb the greenhouse gas carbon dioxide—could have a potentially beneficial side effect. (See Fertilizing the Ocean with Iron). More phytoplankton could produce more DMS and more clouds.
In the July 2007 issue of the journal Atmospheric Environment, scientists from Los Alamos National Laboratory, the University of California Irvine, and New Mexico Tech suggested that fertilizing two percent of the Southern Ocean could result in a cooling of 2°C and “set back the tipping point of global warming from about 10 years to about 20 or more years.”
Lovelock also continues to promote the potential benefits of DMS as a check on global warming. Last year he and Chris Rapley, director of London’s Science Museum, proposed building arrays of giant pipes that would suck nutrient-rich water from the deep ocean and promote algal growth, producing more DMS as a side effect. (See Proposals Emerge To Transfer Excess Carbon to the Ocean.)
Toole and Siegel’s research into the role of UV radiation could potentially strengthen the case for algae’s role in climate control. “Based on what we’ve seen and our research, there will be more DMS,” Toole said.
As the oceans warm, the upper layer of the ocean is expected to get shallower. That means phytoplankton will be trapped closer to the surface, where they’re exposed to more UV light, stimulating more DMS. “The shallower the layer they’re trapped in, the more DMS they’re going to make,” Toole said.
Toole and some of her colleagues are currently inputting their data from the Ross Sea and Bermuda into climate models to see if an increase in DMS production is enough to affect global temperatures. But the answers are anything but straightforward, because simply adding more clouds sends complex ripples through the entire system, she says.
“If you make more clouds, you get less UV radiation in the upper ocean, perhaps leading to less DMS production by phytoplankton,” Toole said. “You get less heat, which makes the ocean less stratified, changes wind patterns, and reduces mixing. That brings fewer nutrients to the surface for phytoplankton to grow. You might get different species of phytoplankton. If we don’t fully understand the DMS cycle, it’s hard to make predictions.”
Toole, Dacey, Levine, and others will continue to ask the questions, though. One thing that’s sure about DMS: There’s still a lot to learn.
Funding for this research came from the National Science Foundation grant OCE-0425166.
A health care professional will take a blood sample from a vein or an artery. To take a sample from a vein, the health care professional will insert a small needle into your arm. After the needle is inserted, a small amount of blood will be collected into a test tube or vial. You may feel a little sting when the needle goes in or out. This usually takes less than five minutes. Make sure you don't clench your fist during the test, as this can temporarily raise lactic acid levels.
Blood from an artery has more oxygen than blood from a vein, so your health care provider may recommend this type of blood test. The sample is usually taken from an artery inside the wrist. During the procedure, your provider will insert a needle with a syringe into the artery. You may feel a sharp pain as the needle goes into the artery. Once the syringe is filled with blood, your provider will put a bandage over the puncture site. After the procedure, you or a provider will need to apply firm pressure to the site for 5–10 minutes, or even longer if you are taking a blood-thinning medicine.
If meningitis is suspected, your provider may order a test called a spinal tap or lumbar puncture to get a sample of your cerebrospinal fluid.
126.96.36.199 Modification of Bile Acids by Gut Microbiota
Bile acids are saturated, hydroxylated C24 cyclopentanephenanthrene sterols. Primary bile acids, such as cholic and chenodeoxycholic acids, are synthesized from cholesterol in the liver, conjugated to either taurine or glycine, and then excreted through the canaliculi to the biliary system. 28,29 Following their excretion to the gastrointestinal tract, a portion of bile acids will be deconjugated and modified by bacterial metabolism, including oxidation, esterification, desulfatation, and epimerization, ultimately leading to the presence of over 50 different secondary bile acids in human feces, with subsequent minor modifications leading to numerous species of tertiary bile acid species ( Fig. 35.4 ). 30 The initial step in the production of secondary bile acids is to deconjugate them, which is mediated by bile salt hydrolase (BSH). All gut bacteria are thought to encode at least 1 BSH and many bacteria encode several leading to speculation that the ability to deconjugate bile acids is an essential fitness factor for gut bacteria due to primary bile acids being dangerous to bacteria, potentially destroying their membranes and interfering with various bacterial metabolic processes. Deconjugation of the polar conjugates makes bile acids more hydrophobic and, consequently, less dangerous to bacteria. Such increased hydrophobicity results in reduced re-absorption of bile acids and rather favors their loss on feces. Hence, germfree mice exhibit a complete loss of secondary bile acids resulting in increased levels of primary bile acids in liver and a stark reduction of bile acids in colon and feces, because primary bile acids are reabsorbed so efficiently. In the normal case wherein microbiota is present, deconjugated bile acids, some of which will escape enterohepatic recirculation, will be modified by bacteria in the lower GI tract. A range of modifications are possible including oxidation-reduction, amidation, esterification, and sulfation to result in a diverse repertoire of secondary with some analytic schemes reporting the quantitation of upwards of 50 bile acids. While the specific microbial enzymes that mediate such modifications have not been well defined, although some have been identified, overall, they appear to exhibit a high degree of variance among bile acid profiles that are very much impacted by microbiota composition. Conversely, bile acids clearly impact microbiota composition in that direct addition of bile acids to the gut and/or pharmacologic interference alters microbiota composition. Hence, the relationship between bile acids and microbiota composition is best thought of as being one of dynamic equilibrium. In any case, microbiota composition has a clear effect on bile acid levels, which in turn has a myriad of ways of impacting metabolism.
Fig. 35.4 . Overview of bile acid metabolism in gastrointestinal tract. Explained in text.
(Taken from Long SL, Gahan CGM, Joyce SA. Interactions between gut bacteria and bile in health and disease. Mol Aspects Med 201756:54–65.)