Principles and Methods of Different Microbiological Assay

A microbiological or microbial assay is a type of biological test that determines the potency of a compound by determining the amount needed to produce the expected effects on a test organism.

Microbial assay concepts are similar to those used in assays of higher plants or animals. The relative size of the experimental population is one existing distinction. When bioassays are conducted, the reaction of the test animal is monitored, and the findings are computed and analyzed after a few animals have been analyzed to determine the mean activity, standard error, etc. In a microbiological test, samples of a wide variety of organisms are used to measure the average response produced by a broad sample of organisms. In bioassays, the log dosage and response are frequently linear, but in microbiological assays, the dose and response (within specific limitations) are linear.

Using bacteria, yeast, and molds as microorganisms, the microbiological test is a biological test. These tests are more simpler than those used in animals to isolate important vitamins; for example, a Lactobacillus growth factor was shown to be identical to a haematopoietic factor in pernicious anaemic patients. Microbiological tests are used to standardize many medicinal drugs, such as antibiotics that limit microbial growth or critical growth factors (vitamins and amino acids). Molecular assays are used to identify the activity of antibiotics (their ability to inhibit microbial growth), vitamins, and amino acids (their ability to trigger microbial growth), while chemical assays measure the concentration of such substances. Therefore, the microbiological assessment of antibiotics, vitamins, amino acids, and other substances is extremely important.

Principles

It is believed that microbes grow in proportion to the amount of a particular compound when it is present in low concentrations. Microbiological tests generally follow the same underlying concept, but their circumstances differ.

Test substances are introduced to a liquid or gel medium, the test microorganisms are infused onto the medium, and the resulting reaction is monitored (depending on how they interact with the test organism). Growth response may be measured by counting, optical density, weight, or area (positive result for nutrition assays and negative for antibiotic tests); the growth result could be a clear end-point or an all-or-nothing result. A metabolic reaction can lead to the production of products or the change of a function. Some detectable metabolic reactions include acid generation, CO2 production, oxygen absorption, nitrate reduction, haemolysis of RBCs, anti-luminescent activity, and spore germination suppression.

In 1962, Cancellien and Morpurgo developed a method to determine amino acids, vitamins, and purines and pyrimidine bases. Substances similar to and antagonistic to normal metabolites limit the development of wild, prototrophic bacterial strains. The Nutrilite content may be determined by reactivating the growth with graded quantities of Nutrilite.

Merits

There are many merits of microbiological assay as given below -

• To make the assay of naturally occurring therapeutic agents, it is highly used.
• There is no need to use a high number of instruments and samplings.
• To standardize medical compounds accurately, it is highly used.
• Compounds that are hard to assay either by chemical or physical methods, it is highly used.
• It is used in determination of activity of the compounds as well as the concentration of the compounds.
• The entire procedure is automated to reduce the time it takes.
• As compared with bioassays, it is very rapid and simple.
• The mortality rate of animals has decreased.

Demerits

Given below are the demerits of microbiological assays -

• Within the laboratory, there is the need to maintain sterile conditions.
• It is very time-consuming to use this method
• People who are both proficient and well-trained are in high demand.
• Even a slight variation in incubation of temperature, the results may vary a lot.
• To carry out the assay of a particular compound, there is a need for a specific test organism.

Microbial Assay

• A wide range of bioassays has been performed using microorganisms, including:
• Trying to measure the levels of various compounds in complex chemical mixtures or in body fluids (such as amino acids, vitamins, and certain antibiotics).
• Making certain diagnoses.
• Tests to determine whether chemicals are mutagenic or carcinogenic.
• Immobilized enzymes are used for monitoring purposes.
• Testing for antibiotic sterility.
• The potency and quality of a product is determined by microbiological tests.
• Drug pharmacokinetics can be determined using these tests in humans and animals.
• To manage, control, monitor chemotherapeutic agents used in antimicrobial chemotherapy.

Microbial Assay of Antibiotics

• Potency of an antibiotic is determined by comparing its ability to inhibit sensitive microorganisms against one of the reference substances.
• Reference substances are substances in the assays that have been precisely determined to be active in accordance with the corresponding international standard or international reference preparation.
• Assays should be designed so that they will provide an opportunity to evaluate the appropriateness of the mathematical model used to calculate potency.
• The logarithmic dose-response curves of both the preparation to be studied and its reference preparation must be parallel, i.e., they should be linear across the dose range used in the calculation.
• Validity tests should be conducted for a given probability, usually P=0.05.
• The slope ratio model and other mathematical models may also be used, if they can be demonstrated to be valid.
• A confidence limit of 95 percent plus 105 percent applies to the potency assay (P=0.95), unless otherwise specified in the monograph.

Methods of Microbial Assays

Disc Diffusion Method

• Inoculate a suitable test organism with a suitable inoculum as described below in Petri dishes or rectangular trays filled to a depth of 3 or 4 mm with a medium prepared by inoculating the dishes or trays with 3 or 4 mm of inoculum.
• Two layers of nutrient agar can be used, but only one can be inoculated.
• At different concentrations of the standard, the inoculum concentration should be selected such that its zones of inhibition are severe and its dose-response is appropriate.
• A solution containing one milliliter of inoculum for every 100 milliliters of culture medium is recommended when using the inoculum.
• Molten agar medium cannot reach more than 48-50°C when inoculated with a suspension of vegetative organisms.
• A flat-bottomed dish or tray should be selected especially.
• An assay should be validated by comparing the concentrations of three different reference substances to the concentrations of at least three doses of the test substance, which is assumed to have the same activity as the solutions of the reference material.
• The dose levels used should be measured geometrically, for example using a 2:1 dilution ratio for successive dose levels.
• The logarithm of the concentration of the antibiotic and the diameter of the zone of inhibition can be linearly related when the system is linearly related to the concentrations and dilutions of the reference material and the test substance.
• An initial solution of the substance is prepared as directed in a monograph, then diluted with the appropriate sterile buffer as needed.
• It is preferred to arrange the references and test substances in the form of a Latin square on rectangular trays.
• A petri dish is alternated with reference and test solutions in such a way that the highest concentrations of both solutions are not adjacent.
• Pipettes are used to deliver uniform volumes of liquid into the cylinders or holes.
• To fill the holes almost completely with delivered volume, the holes should be used when holes are available.
• A suitable incubation temperature, maintaining the temperature between +/- 0.5 °C throughout, should be maintained for approximately 16 hours. The diameter or area of the zones of inhibition, generated by the varied concentrations of the standard and of the test substance, should also be precisely measured, preferably within 0.1 mm of the final zone size, by an appropriate measuring device.
• Test results provide an estimate of potency.

Turbidometric Method

• To test for resistance to an antibiotic, inoculate a suitable medium with a suspension of a microbe whose growth is significantly inhibited by the antibiotic under test.
• Utilize a known volume of the suspension whose opacity can be readily measured after 4 hours of incubation.
• Inoculate the medium as soon as it has been prepared.
• Using the solvent and buffer solution indicated, prepare solutions of the reference substance and the buffer solution to detect the antibiotic's activity.
• When assessing the validity of the assay, use a minimum of three doses of the reference substance and three doses of the antibiotic to be evaluated that have a presumed activity similar to that of the reference substance.
• In geometric progression, it is best to administer a series of doses. The reference substance and antibiotic to be examined may need to be selected from a large number of consecutive doses to achieve the required linearity.
• The same volume of each solution and 9ml of the inoculated medium should be placed in identical test tubes.
• Tyrothricin should be assayed with 0.1ml of the solution per 9.9ml of the inoculated medium.
• Prepare two identical tubes without antibiotic, one of which contains the inoculated medium and the other to which 0.5ml of formaldehyde is added as soon as possible.
• Use these tubes to establish the optical apparatus that will measure the growth.
• In a water-bath or suitable apparatus fitted with a means of rapidly warming all the tubes to the incubation temperature, arrange all the tubes randomly or in a Latin square or random block arrangement.
• Ensure that the temperature is uniform and the time of incubation is the same for 3-4 hours.
• Use an optical apparatus to measure 3 significant figures of opacity of the incubated cultures after stopping their growth with 0.5 ml of formaldehyde R or by heat treatment.
• Consider using a method that enables you to measure the opacity after exactly the same period of incubation for each tube.
• Estimate the potency by using the appropriate statistical analysis.
• Transformed or untransformed, linearity of dose-response relationships is often obtained over only a very narrow range.
• In order to allow for testing of linearity, at least three consecutive doses must be used in calculating the activity.
• If the linearity of the system can be demonstrated in a sufficient number of tests using a three-point assay, a two-point assay may be sufficient, assuming the competent authority has agreed.
• Nevertheless, all disputes must be assessed by a three-point method.
• Ensure that each assay is replicated sufficiently per dose to achieve the required precision.
• For statistical purposes, the results of the assay can be repeated and combined to obtain the required precision and to determine if the antibiotic's potency does not fall below the minimum required level.

Urease Assay

• Antibiotics that inhibit the synthesis of proteins are tested with this assay.
• Macrolides such as erythromycin, azithromycin, and clarithromycin as well as aminoglycosides such as streptomycin, amikacin, kanamycin, gentamicin, netilmicin.
• Proteus mirabilis produces urease, an enzyme found in certain microbes.
• Increased pH in the media indicates the presence of urease.
• The urea production in the body is inhibited by antibiotics that inhibit protein synthesis, so pH does not rise further.
• Urease is assessed in this way. The application of this test is relatively limited.

Luciferase Assay

• The luciferin-producing enzyme luciferase acts on luciferin, which is produced by different microorganisms, and gives off bioluminescence.
• Bacterial culture can be detected using this assay if there is very little ATP present.
• Inhibition of ATP or reduction of ATP levels is caused by Aminoglycosides.
• The absence of ATP and luminescence occurs when antibiotics inhibit microbial growth.

Radioenzymatic Assay

• The detection of antimicrobial resistance, especially against aminoglycosides and chloramphenicol.
• Antibiotic-resistant bacteria produce enzymes such as aminoglycoside acetyl transferase or aminoglycoside adenyl transferase, which enable them to continue living in the absence of antibiotics.
• In this instance, the radiolabeling agent 14C carbon isotope (as cofactor of [1-14C] acetyl coenzyme A) is used to detect the production by microorganisms of the aminoglycoside acetyl transferase (AAC), and for AAD and APH 3H (as cofactor of [2- 3H] ATP).
• When microorganisms produce AAC, AAD, or APH, their isotopes 14C/ 3H become radioactive and give rise to specific color changes.
• Resistance to aminoglycosides can be inferred from observing color changes.

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