Determining Transmittance and Absorbance

Determining Transmittance and Absorbance - To assure that spectrophotometer readings indicate only the concentration of the solute we wish to measure, a reading must first be obtained using a blank, a sample that contains all the components of the solution except the absorbing molecule. For instance, if you are using a reagent that changes color when mixed with a certain solute molecule, a blank should contain all the components of the test solution, including the colorimetric reagent, except the substance (solute) to be measured.

Transmittance 

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Example A 1-ml sample of substance X is mixed with 5 ml of water and 1 ml of colorimetric reagent to give a volume of 7 ml in a sample tube. A blank is prepared by mixing 5 ml of water with an additional 1 ml of water (as a substitute for substance X) and 1 ml of colorimetric reagent. The volume in the blank tube is 7 ml. Note that the volume of the blank should always be the same as the volume of the sample.

With the blank inserted into the spectrophotometer, the instrument is adjusted to 100% transmittance

(zero absorbance). This step is similar to taring a balance: the transmittance of light through the blank will be less than 100% because of substances (including the colorimetric reagent) present in the blank. However, the instrument can be adjusted to accept this reading as 100% transmittance, so that when the blank tube is replaced by the sample tube to measure absorbance of the sample, the only thing absorbing light will be the sample molecule of interest (the solute that reacts with the colorimetric reagent).

Procedure

• Prepare a sample tube. Place 10 drops of albumin solution into a spectrophotometer tube and add 1 ml of distilled water. Add 5 ml of Coomassie brilliant blue (a colorimetric reagent used to identify protein) and allow the tube to stand until a blue color develops.
• Prepare a blank: place 10 drops of water into a spectrophotometer tube and add 1 ml of distilled water and 5 ml of Coomassie blue.
• Turn the power switch on, and allow a 5-minute warm-up period. The on/off switch is operated by the zero control knob on the left.
• Use the wavelength control knob to adjust the spectrophotometer to any wavelength between 550 and 600 nm. The selected wavelength is indicated on the wavelength readout in the window next to the knob.
• When using the Spectronic 20, the meter must be adjusted to read across its full scale—0% transmittance to 100% transmittance. With no sample tube in the machine, use the zero (left-hand) control knob to set the scale to 0% transmittance (infinite absorbance). (With no sample tube, the light path is automatically blocked, and no light reaches the phototube; thus, 0% transmittance and infinite absorbance are simulated.) Be sure the cover on the sample holder is closed when you perform this step.
• Insert your blank (be sure it is clean and dry on the outside) into the sample holder, and turn the right-hand control knob to set the meter scale to 100% transmittance, zero absorbance. This adjustment regulates the amount of light reaching the phototube in the absence of the absorber. Whenever the wavelength is changed, the 100% transmittance adjustment must be reset. Also, when operating at a fixed wavelength for an extended period of time, periodically check the 100% and 0% transmittance readouts and adjust if necessary.
• If you are beginning an experiment, repeat steps 5 and 6 to make sure the machine is stable.
• Insert the sample tube into the chamber; read absorbance directly on the absorbance scale (lower scale). The reading on the absorbance scale is proportional to the concentration of your sample substrate. Note: The absorbance scale reads from right to left, opposite to the direction of the transmittance scale. Record your data: wavelength nm, absorbance , transmittance .      Do not discard your sample and blank tubes!
• The steps that follow provide a

Principles of Spectrophotometry

Principles of Spectrophotometry - Molecules either absorb or transmit energy in the form of electromagnetic radiation. White light (normal daylight) is made up of all the wavelengths of electromagnetic radiation in the visible spectrum. How objects or chemical substances absorb and transmit the light that strikes them determines their color.

Principles of Spectrophotometry

What we see as the color of an object, or a solution, is determined by what wavelengths of light are “left over” to be transmitted or reflected by the object after certain wavelengths are absorbed by its constituent molecules. For example, the pigment chlorophyll, present in the leaves of plants, absorbs a high percentage of the wavelengths of light in the red and violet to blue ranges. Green light, not absorbed by chlorophyll molecules, is reflected from the surface of the leaf—thus most plants appear to be green. A solution of chlorophyll extracted from a leaf would also be green. The spectrophotometer can be used to measure the amount of light absorbed or transmitted by molecules in a solution. The spectrophotometer operates on the following principle. When a specific wavelength of light is transmitted through a solution, the radiant light energy absorbed, absorbance (A), is directly proportional to (1) the absorptivity of the solution—the ability of the solute molecules to absorb light of that wavelength; (2) the concentration of the solute; and (3) the length of the path of light (usually 1 cm) from its source, through the solution, to the point where the percentage of light energy transmitted or absorbed is measured by a phototube.

Spectrophotometers that employ ultraviolet or visible light are the types most often used to study

biological structures and reactions. The investigator selects a wavelength of light that will be maximally absorbed by a particular solute in solution. (If visible light is used and the molecule of interest does not absorb light, it is often possible to set up a chemical reaction that will yield a colored product.) After passing through the solution, the light energy received at the phototube is expressed as the ratio of transmitted light IT (the light that passes through the sample) to incident light I0 (the intensity of light at the source before it enters the sample). The light received at the phototube is measured as percent transmittance (T), or as the log of its inverse, absorbance (A): 

By measuring the absorbance (or transmittance) it is possible to determine the concentration of the absorber (molecule) in solution. Concentration can be calculated directly if the molar absorptivity of the molecule (the amount of light at a specific wavelength absorbed by a specified concentration of solute in moles per liter) is known. Usually, however, molar absorptivity is not known and absorbance readings indicate only relative concentrations—a higher absorbance (A) resulting from a higher concentration. In such cases, the concentration can be found by locating the absorbance reading of the unknown concentration on a graph of the absorbances of known concentrations (standard curve).

Using the Spectrophotometer

Using the Spectrophotometer - The Bausch & Lomb Spectronic 20 Colorimeter is an extremely versatile instrument that is useful for the spectrophotometric, or colorimetric, determinations of solutions. Within the optical system of the spectrophotometer, rotation of a prism (diffraction grating) allows the investigator to select specific wavelengths of light in a range from 375 to 625 nm. Light of a selected wavelength is passed through the sample and is picked up by a measuring phototube, where the light energy is converted to a reading on the meter of the spectrophotometer.

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Most spectrophotometers have two scales—one is a linear scale (the transmittance scale) given as percent transmittance, the other is a logarithmic scale with the same gradations as the percent transmittance scale (the absorbance scale). (Since transmittance is related to absorbance as the log of the inverse, values of 0.0 absorbance occur at 100% transmittance and values of infinite absorbanceoccur at 0% transmittance.) Examine the readout meter on the spectrophotometer you will be using. Note that the scales run in opposite directions.

An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. In order to determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through. (source: en.wikipedia.com)

Microcins E. coli

Microcins E. coli - There has been far less work on the frequency of microcin production in E. coli or other Gram-negative bacteria. All the microcins characterised to date are secreted from the cell, rather than being released as a consequence of cell lysis (Braun et al. 2002). It has also been suggested that as much as 90% of the microcin produced by a cell may be retained within the cell (Braun et al. 2002). Consequently, there is no reliable and simple phenotypic method for assaying microcin production.

The E. coli isolated from Australian humans and mammals were screened for seven microcins, using a PCR-based approach. Of the human isolates screened, 32% were microcin producers whereas, among the isolates from mammals, 9% were microcin producers. Why microcin production is less common in E. coli isolated from mammals compared to humans is unknown. In the isolates from humans, microcin production is significantly more prevalent among genetic group B2 strains (47%) than among A (16%), B1 (18%) or D strains (9%). The frequency of microcin production in both of these E. coli collections has almost certainly been underestimated, as a PCRscreening approach can be used only for those microcins which have been genetically characterised.

Interaction Of Colicins

Interaction Of Colicins - Typically, a third of E. coli strains produces a mitomycin C-inducible bacteriocin (Riley and Gordon 1996). For example, 24% of the E. coli isolates from humans and 33% of the isolates from mammals were colicin producers.

Interaction Of Colicins

Although there have been relatively few representative surveys of other members of the Gram-negative bacteria, similar frequencies of production are observed in these studies (Reeves 1972; Riley et al. 2003). However, the prevalence of colicinogenic strains may vary from 10 to over 70% among different E. coli populations (Riley and Gordon 1996; Gordon et al. 1998). Much of the variation in the frequency of colicinogenic strains in populations of E. coli is undoubtedly due to the dynamic interactions occurring between colicinproducing, resistant and sensitive cells (Riley and Gordon 1999), which result in temporal fluctuations in the relative frequencies of these three phenotypes. Such temporal fluctuations have been observed in vitro (Kerr et al. 2002) and in a population of E. coli isolated from house mice, in which the frequency of colicinogenic isolates declined from 71 to 43% over a 7-month period (Gordon et al. 1998).

Although the frequency of colicinogenic strains in a population is expected to vary naturally, theoretical considerations and empirical observation suggest that the environment in which the cells live also influences the frequency of colicinogenic cells. The predictions of mathematical models indicate that colicin-producing cells will have an advantage in benign habitats (resulting in high population growth rates) whereas harsher habitats will favour nonproducers (Frank 1994). There is some empirical evidence to support this prediction. Population growth rates and cell densities are significantly lower in the external environment than in the lower intestine of mammals and, in Australia, 9% of E. coli isolated from the environment were producers, a significantly lower proportion than the 30% observed for Australian faecal isolates. The frequency of colicin-producing E. coli also depends on the type of host from which the cells are isolated. In Australian mammalian carnivores, the frequency of colicin-producing strains is about 20% whereas colicin production is twice as frequent in strains isolated from herbivorous or omnivorous Australian mammals. For animals of similar body mass, the turnover rate of the carnivore gastro-intestinal tract is significantly faster than that recorded in herbivorous or omnivorous mammals (Hume 1999). The predictions of mathematical models support the observation that the cost of colicin production can result in colicinogenic strains being disadvantaged when living in hosts with high gut turnover rates (unpublished data). There is additional evidence for the importance of the host environment in determining the likelihood that a strain will be colicinogenic. There are two species in the genus Hafnia (Okada and Gordon 2003; Janda et al. 2005). A collection of Hafnia species 2 strains, isolated from fish, reptiles and mammals from across Australia, was screened for the presence of a bacteriocinogenic phenotype. The results of this screening showed that 4% of the isolates from fish, 64% of the isolates from reptiles and 29% of the isolates from mammals were bacteriocin producers.

Faecal isolates of E. coli can be assigned to one of four main genetic groups (subspecies), designated A, B1, B2 and D (Ochman and Selander 1984; Herzer et al. 1990). Strains of the four groups appear to occupy different ecological niches (Gordon and Cowling 2003; Gordon et al. 2005). For E. coli, it is well established that some traits, particularly virulence factors associated with extraintestinal disease, are largely confined to particular genetic groups (Johnson and Stell 2000). Genetic group membership also appears to predict the frequency of colicin production. Among the Australian mammal isolates, 46% of genetic group B2 strains produce a colicin, compared to only 23–27% of strains in the genetic groups A, B1 or D. The reasons for these differences are unknown.

The Diversity of Bacteriocins in Gram-Negative Bacteria

The Diversity of Bacteriocins in Gram-Negative Bacteria - Allelopathy is the production of chemical compounds which are toxic to other organisms but not to the producers of these compounds. For microorganisms, there is a wealth of data demonstrating that allelopathy is an important mediator of intra- and inter specific interactions and consequently, a significant factor in maintaining microbial biodiversity (Chap. 6, this volume). In bacteria,these allelopathic substances include metabolic by-products such as ammonia or hydrogen peroxide; the ‘classical’ antibiotics such as bacitracin and polymyxin B, lysozyme-like bacteriolytic enzymes and the bacteriocins.

The Diversity Bacteriocins (sumber: http://jb.asm.org/)

The bacteriocins produced by Gram-negative bacteria are diverse. Over 30 bacteriocins from Escherichia coli have been identified and, undoubtedly, more have yet to be discovered. The diversity present in other Gram-negative species, including other members of the Enterobacteriaceae, is largely unexplored. The molecular mechanisms by which this diversity has arisen, at least for that class of bacteriocins known as colicins, is well understood and is discussed elsewhere (Chap. 3, this volume). However, the factors influencing the frequency of bacteriocin production and the diversity of bacteriocins in populations of bacteria are largely unknown. Acquiring this knowledge is essential, not only if we are to understand the role bacteriocins play in shaping bacterial communities in natural environments but also because  there is an increasing desire to exploit bacteriocins to solve a range of applied problems.

As a consequence of the rising incidence of resistance to most traditional antibiotics, numerous research programs have been implemented aiming to explore the potential role which naturally produced and genetically modified bacteriocins might have as replacements for traditional antibiotics (Gillor et al. 2004). Other efforts focus on the use of antimicrobial toxins as food preservatives (Gillor et al. 2004). There is also an ever-increasing interest in he use of bacteria as biocontrol agents for the management of fungal and bacterial plant pathogens and, more recently, as the active agent in probiotic formulations. Probiotic therapy is a disease prevention strategy used in humans and domesticated animals, as well as a procedure considered to enhance the growth rate of livestock and poultry. The basis of the method is to ensure the establishment of ‘good’ bacteria in the gastro-intestinal tract in order to prevent the establishment of bacterial pathogens. One of the most important attributes of a ‘good’ probiotic strain is thought to be the strain’s ability to produce antimicrobial compounds. However, the successful use of bacteria as biocontrol agents will require a sound understanding of microbial ecology and the factors influencing the frequency of bacteriocin production and diversity in populations of bacteria. Thus, the aim of this chapter is not to describe the diversity of bacteriocins which have been characterised from Gram-negative bacteria but, rather, to identify and discuss some of the factors observed to influence bacteriocin diversity. As usual, much of the data relates to bacteriocin production in E. coli but reference will be made also to observations concerning other species of the Enterobacteriaceae. The material presented has largely not been published previously, and is based on phenotypic and genotypic surveys of bacteriocin production in three collections of E. coli. The first of these collections consists of 496 isolates taken from a variety of mammal species living throughout Australia. The methods used to isolate and characterise the strains in this collection have been described by Gordon and Cowling (2003). The second collection consists of 266 faecal isolates recovered from people living in the Canberra ACT region of Australia, the isolation and characterization of these strains having been described by Gordon et al. (2005). In addition to the human faecal isolates, the collection contains 353 isolates recovered from extra-intestinal body sites of people. The third dataset was collected and characterised using the same methods as those used for the other two, and consists of 208 strains recovered from soil, water and sediment samples from a variety of localities across Australia. All strains in the three collections have been screened using a PCR-based approach for the presence of 29 virulence-associated traits and to determine their E. coli group membership (A, B1, B2, D), as described by Gordon et al. (2005). The majority of the strains have been screened using a combination of phenotypic and genotypic approaches in order to determine the frequency of bacteriocin production and the types of bacteriocins present in these strains, using the methods described by Gordon and O’Brien (2006).