The Use of Serial Dilution in Determining Bacterial Toxin Concentration - Report Example

Analyze the Use of Serial Dilution in Determining Bacterial Toxin Concentration" Introduction Bacteria are tiny microscopic organisms that are not visible through naked eyes. They are visible through the microscope. Direct count of these microorganisms is time consuming and tedious, moreover it requires a lot of expertise. Various methods are developed over the years to count the microbes; encompassing colony count, serial dilution followed by plating the sample, pour plate method etc (Davis, 2011; Ford, 2010). Bacterial are living organisms and they are present in nature as a large population. In order to estimate their number, plating is done but plating cannot give the countable number on the culture plate and therefore the sample is diluted in a sequential manner to get the countable number of microbial cells. This process is called serial dilution (Schmitt). A serial dilution is therefore a process in which a sample taken either from the nature or from the animal or human. This sample is diluted in a sequential manner to find out the probable number of microbial cells in the given sample (Davis, 2011; Ford, 2010). Bacteria release certain metabolic products in its environment. These products could be useful for the humans or they may be harmful. The metabolites could be certain enzymes, toxins or chemical agents that prevent the growth of other microbes in its vicinity. Certain species of bacteria synthesize toxins that act as the chief virulence factors. When these toxins encounter eukaryotic cells they affect the cells in varied ways. Some of the toxins released by bacteria comprise- 1. Staphylococcus aureus α- toxin 2. Shiga toxin 3. Cytotoxic necrotizing factor type 1 4. Escherichia coli heat-stable toxin 5. Botulinum neurotoxin 6. Tetanus neurotoxins 7. S. aureus toxic- shock syndrome toxin Toxins are known to alter the signal pathways in eukaryotic cells. It is therefore essential to analyze bacterial toxin and the potency which works as a toxic dose for the eukaryotes. Bacterial toxins could be defined as the substance that amend or modify the normal metabolism of the host cells while they cause harmful effects on the host cells. Some of the bacterial species namely, Corynebacterium diphtheriae, causing diphtheria; Bordetella pertussis, causing whooping cough; Vibrio cholerae, causing cholera; Bacillus anthracis, anthrax; Clostridium botulinum, causing botulism; Clostridium tetani, causing tetanus, and enterohemorrhagic Escherichia coli, causing bloody diarrhea and hemolytic uremic syndrome; all these diseases are the resultant of the toxins that are released by the respective micro-organisms (Schmitt). The altered eukaryotic metabolism includes damage to the cell membrane resulting in the cell lysis, damage caused by the alpha-toxin brings lysis of human monocytes, lymphocytes, erythrocytes, platelets and also the endothelial lining cells; inhibition of protein synthesis could be seen in Shigella toxicity as it directly affects the elongation of chain eventually leading to the cell death (Schmitt). In certain cases, bacterial toxins affect the protein synthesis and does not kill the cell, the secondary messenger pathway is inhibited, neurotransmitter release is inhibited or the host immune response is also altered. Such toxins are being released by Escherichia coli, called CNF type 1 and 2. They potentially binds with the Rho, a GTP- binding protein that is capable of regulating the actin cytoskeleton. Rho inactivating toxin is also released by Clostridium botulinum and also by Bordetella sp (Schmitt). Bacterial toxin thus plays an imperative role in altering the normal functioning of the eukaryotic cells. In certain cases such damages may be irreparable. On the other hand bacterial toxins could bring devastating implications. Constant research is going on to exploit these metabolic products for beneficial uses and to combat the diseases. An appropriate method is required to analyze bacterial toxicity (Schmitt). Serial Dilution Serial dilution is the process which is used for approximating matter deliberations and lowest inhibitory concentrations. Serial dilution is an assay which is being exploited in biological and medical science. It is based on Poisson-Bernoulli model, although it does not hold good for the continuous measurements. The motive to analyze the toxicity by means of serial dilution is to procure the serum concentration of the antibody and also to estimate the minimum inhibitory concentration (MIC) of a substance that is sufficient to incite the particular reaction. Serial dilution method is adopted to assess the least concentration or the minimal concentration of the a chemical or the biological agent capable to display the reaction. The dilutions are procured either in agar or in broth (Davis, 2011; Ford, 2010). In case of bacterial infection it is necessary to estimate the quantitative dose of the antibiotic that is required to be used to combat the disease or to reduce the effect of bacterial toxin. This also determines the "susceptible" and "resistant" nature of the organism releasing toxin, towards the proposed antibiotic. MIC is one such method that aids in determining the concentration of antibiotic required to fight or neutralize the effect of toxin being produced by the organism (Davis, 2011; Ford, 2010). Methodology Serial Dilution Methods for determining MIC a. Broth dilution method- It aids in assessing a small number of isolates. Method encompasses a sterilized set of approximately 10 tubes/ bottles with 9 ml of the broth in each tube. From the original inoculum of the bacterial culture capable of producing toxin, 1ml was added to the first tube and now this becomes 1/10th of the dilution of the first tube, shake it well so that it becomes homogeneous. From the first tube (which now becomes 10 ml), again 1 ml of the broth was taken and added to the second tube, the process was repeated till the tenth tube to get the dilution of 10-10 dilution. On the other hand the application could be performed with the blood serum sample of the infectious individual and it is serially diluted. The sample from the dilutions was plated as shown below- The colonies formed after incubation of the plates are calculated as the "Colony Forming Units" CFU/standard unit volume = no. of colonies x dilution factor x standard unit volume/aliquot plated b. Agar Dilution It is performed in the case of M. tuberculosis. In order to determine the effective concentration of the bacterial culture that is capable of producing the toxin and that could not be nullified with the antibiotic, the following method is adopted (Davis, 2011; Ford, 2010). The broth is incorporated with the antibiotic and then the bacterial inoculum is incorporated as mentioned above for the serial dilution process. The sample is then plated. Appearance of the bacterial colonies for various dilutions could be estimated. Nin-appearance of bacterial colonies at higher dilution indicates the efficacy of the antibiotic to combat the microbial population. The antibiotic solution could be incorporated in micro ml (µl). Discussion Various factors govern the toxin production by the microorganisms. These factors could be environmental, physiological, biological, pH, metabolic, nutritional or physical factors like temperature, oxygen content or the humidity. Variation in these conditions could result in the phases of no toxin production to higher level of toxin production. It is therefore essential to understand the conditions where the particular microorganism is capable of producing the toxin. Various studies have been carried out that envisage the toxin production by a particular micro-organism. Bioassays conducted using serial dilution serves the best purpose to determine the efficacy of the antibiotic against the toxin producing microbe (Schmitt). Serial dilution serves the best purpose to determine and analyze the toxin production by the microbes. It is the method where the concentration of the microbial culture could vary keeping the other variable i.e. the concentration of the antibiotic constant while on the other hand the concentration of the antibiotic could vary keeping the concentration of the microbial population constant (Schmitt). These parameters enable one to determine the minimal inhibitory concentration of the antibiotic required to prevent the growth of microbial population under the given conditions. The method is highly efficient if performed in a specified manner. Although if care is not taken then some errors may occur and therefore now-a-days more accurate methodology called ELISA (Enzyme Linked Immuno- Sorbent Assay) is preferred (Schmitt). References 1. Davis, W., Pritchett, 2011. General Microbiology, 1st Edition,Kendall Hunt Pub Co. 2. Ford, M., 2010. Medical Microbiology, 1st Edition, OUP Oxford. 3. Schmitt, C. K., Meysick, K. C., OBrien, A. D. Bacterial Toxins: Friends or Foes?. Available on http://www.cdc.gov/ncidod/eid/vol5no2/schmitt.html. [Accessed on 7th September 2011]. Read More