MCB 4322 Analytical Microbiology & Quality Control Notes (2023/2024) PDF

2023/2024 second semester Dept. of Microbiology, UMYUK MCB 4322: Analytical Microbiology and Quality Control ANALYTICAL MICROBIOLOGY The term “Analytical Microbiology” may be defined as that branch of microbiology in which micr...

2023/2024 second semester Dept. of Microbiology, UMYUK MCB 4322: Analytical Microbiology and Quality Control ANALYTICAL MICROBIOLOGY The term “Analytical Microbiology” may be defined as that branch of microbiology in which microorganisms are used as reagents for the quantitative or qualitative determination of certain chemical compounds. Such quantitative and qualitative determinations are referred as Assays and if living system are used (animals, plants, microorganism), it is called Biological Assay. A bioassay is an analytical method to determine the concentration or potency of a substance by its effect on living animals or plants (in vivo) or on living cells or tissus (in vitro). BASIC PRINCIPLES/ASPECTS OF BIOLOGICAL ASSAYS -In simple terms assay refer to the qualitative or quantitative chemical analysis of an agent/compound. -Assay can be done using physical, chemical or biological methods. -Biological assays (or bioassays) involve the qualitative or quantitative detection of an agent (stimulus/analyte e.g. antibiotic) upon application on biological system which generates a measurable response (e.g. inhibition zones). This can be summarized as follows: STIMULUS (analyte) + BIOLOGICAL SYSTEM  RESPONSE (standard/test samples) (plants, animals, tissues, cells, macromolecules) (observed change +/-) -The applied stimulus can be standard or test samples containing the biologically active substance or analyte. -The biological system which receives the stimulus may be i. plants or animals or isolated organs or tissues from multicellular organisms, ii. whole cells (i.e. microorganisms, especially bacteria, yeasts, fungi and protozoa), iii. biologically active macromolecules (such as antibodies, enzymes, e.t.c.) that are produced by living organisms. -The response which is normally a change observed in some aspects of the biological system upon the application of the analyte is indicative of a biological activity that is usually attributed to the analyte. i. It may be a positive response associated with increased growth or metabolic activity or ii. A negative response that is inhibitory or even lethal (killing) to the biological system. 1 2023/2024 second semester Dept. of Microbiology, UMYUK TYPES OF BIOASSAY PROCEDURE There are three principal types of bioassay procedure, namely, i. Assays using animals or their tissues ii. Assays using microorganisms, and iii. Assays using macromolecules such antibodies referred to as Immunoassays i) ASSAYS USING ANIMALS/THEIR TISSUES -Living animals can be used for bioassay which is carried out by applying an anlyte on the living organism and observation of a particular physiological response which is measurable. -Example, assessing the activity of a product containing vitamin D (analyte) on animals (biological system) by its ability to prevent the development of rickets (response) in mice fed on vitamin D- deficient diet. -Tissues are sites at which particular biological responses take place in an animal and could be used provided that they are freshly removed from the animal so that they will still respond to the active constituents within the sample. -Example is the assay of histamine (analyte) using its contracting effect (response) on smooth muscles (biological system)). In the assay contractions of intestinal smooth muscles of guinea pig on exposure to test samples are compared with contractions of the tissues following exposure to standard samples with known histamine activities. Advantage and disadvantages of bioassays using whole animal or animal tissues -The main advantage of Bioassays using whole animal or animal tissues is that they are usually very sensitive and highly specific for the biologically active analyte contained in a sample. Disadvantages are: - They are more expensive and their precision (accuracy) is poor compared with other assay methods. -The provision, maintenance and staffing of facilities for experimental animals is often extremely expensive. -Moreover, the animal right issues aimed at reducing and elimination of any mental or physical suffering to the animals also restrict the use of animals in laboratories. -Providing strict controlled conditions and regular inspections of laboratories also limit the use of animals. - Genetic variations exist, which may give rise to different responses Bioassays using animals should, therefore, be regarded as a last resort- i.e. only when no valid alternatives exist. 2 2023/2024 second semester Dept. of Microbiology, UMYUK (ii) ASSAYS USING MICROORGANISMS (MICROBIOLOGICAL ASSAYS) -Microbiological assay is an assay in which microorganisms are used as reagents for the qualitative/quantitative determination of certain chemical compounds thus giving rise to the concept known as Analytical microbiology. -In Analytical microbiology a suitable microorganism will react with to a certain chemical entity a with measurable response thus making it possible for the qualitative/quantitative estimation of the substance. -For instance, the activity of an antibiotic such as penicillin (analyte) can be determined and quantified by its ability to inhibit the growth (response) of a susceptible bacterium (biological system) in an artificial culture medium -Many chemicals can be assayed including a wide range of substances, from simple mineral elements to complex organic compounds involving a variety of growth promoting substances (GPS) or growth inhibiting substances (GIS). Advantage and disadvantage of Microbiological assays Advantages 1- There is no moral debate concerning the rights and wrongs of experimentation with microoganisms unlike with animals. 2- Microbiological assays can be performed without highly specialized and expensive equipment, thus considerable replication of test is possible. 3- The effect of genetic variations on microbiological assays is negligible because asexual reproduction gives rise to populations with minimal differences in the genetic constitution of individuals; in contrast, assays using animals are more prone to differences in response to same doses of analytes because of genetic variations among test animals. Some of the disadvantages of microbiological assays are: 1- It’s limited when measuring analytes which inhibit or promote microbial growth. 3 2023/2024 second semester Dept. of Microbiology, UMYUK 2- No other substance(s) must be present in the test samples which either inhibit or promote growth or modify the response to the analyte. iii) ASSAYS USING MACROMOLECULES SUCH AS ANTIBODIES OR IMMUNOASSAYS -In immunoassays, antibodies are used because they can bind foreign substances with a remarkable degree of specificity. -Numerous immunological methods are used for the identification and measurement of a very wide range of substances of biological and medical importance. -They are extensively and increasingly being used in forensic, toxicology, clinical biochemistry as well as non-pathology laboratories - Their main advantage lies in their simplicity, specificity, and sensitivity when compared with other analytical methods. DISADVANTAGE -Their main disadvantage is that they require the use of relatively, expensive kits. THE TEST ORGANISM (BIOLOGICAL SYSTEM) Requirements for an Ideal Test Organism In order to perform a microbiological assay, it is necessary to select a microorganism that can be used as a reagent. As with the reagents used in a chemical assay, there are specifications that this microbial reagent must meet. The ideal test organism should have these basic characteristics: (a) it must be sensitive to the substance being assayed; (b) it must be easily cultivated; (c) it must have some metabolic function or response that is measurable; (d) it must not be susceptible to variation in sensitivity. (e) it should have specificity, if possible; (f) it should be, preferably, nonpathogenic. Also, the choice of a particular microorganism depends on: i) The need of the researcher ii) The type and amount of material to be assayed iii) What response is desired iv) Time available for the assay v) Accuracy of the results required 4