MCB 4322 Analytical Microbiology & Quality Control Notes PDF

2023/2024 second semester Dept. of Microbiology, UMYUK MCB 4322: Analytical Microbiology and Quality Control TYPES OF MICROORGANISMS USED FOR ASSAY (a) BACTERIA - Bacteria are used for the assay of AMINO ACIDS, ANTIBIOTICS, AND VITAMINS, and in the evaluation o...

2023/2024 second semester Dept. of Microbiology, UMYUK MCB 4322: Analytical Microbiology and Quality Control TYPES OF MICROORGANISMS USED FOR ASSAY (a) BACTERIA - Bacteria are used for the assay of AMINO ACIDS, ANTIBIOTICS, AND VITAMINS, and in the evaluation of ANTISEPTICS, DISINFECTANTS, AND CHEMOTHERAPEUTIC AGENTS. - The use of bacterial reagents, in general, poses relatively fewer problems than the use of other organisms. - Although many organisms are available for the assay of a variety of substances, certain bacteria have been found to be better suited as reagents. - Lactic acid-fermenting bacteria are used for the assay of vitamins and amino acids. - Commercially available antibiotics may be assayed using only five or six organisms. - Most official procedures for testing antiseptics and disinfectants specify Micrococcus pyogenes var. aureus or Salmonella typhosa as the test organism. - The interests of a particular laboratory will determine the number and variety of organisms used. In a laboratory where investigational work is of major importance, the number of organisms used will be greater. - Screening compounds for antibacterial activity requires the use of many organisms. - Special problems, such as the determination of substances in blood or natural products, might require an organism sensitive to small concentrations of the particular substance. (b) YEASTS - Assay procedures, using yeasts as the reagent, have been developed for Growth promoting substances (GPS) thiamine, nicotinic acid, pyridoxine, pantothenic acid, biotin, inositol, and cocarboxylase - Yeasts may also be used instead of lactic acid bacteria in the assay of vitamins from the standpoint of simplicity and minimum expenditure of time. DISADVANTAGES i) There main disadvantage is that there is need for caution against the use of yeasts for assay purposes on the basis test samples that contain stimulatory polypeptides it might invalidate the results of the assay where these substances increased the growth of the organism. ii) Another disadvantage in using yeast for assay is that in order that these organisms grow properly, aeration is necessary. This is usually accomplished by shaking the tubes or flasks. In many laboratories this would interfere with the processing of large numbers of samples. 5 2023/2024 second semester Dept. of Microbiology, UMYUK (c) MOLDS - Molds are used in a number of methods developed for the assay of antibiotics, trace metals, and vitamins, and in the evaluation of fungicidal and fungistatic materials. DISADVANTAGE - Molds, in general, have the disadvantage of being slow growers, thus increasing the time required for assay. - Molds do not have as wide an application as assay organisms as the bacteria do. They are used in a number of procedures where no other organism is available or where fungicidal or fungistatic evaluation is desired. (d) PROTOZOA - Protozoa have been found to have a variety of nutritional requirements for their growth such as vitamins, amino acids, nucleic acid derivatives and fatty factors, and this makes them useful in the assay of substances e.g. Vitamin B12 may be assayed using Euglena gracilis. - Cholesterol and hormones might be assayed microbiologically using protozoa as test organisms.. SOURCE OF TEST ORGANISM 1) Test organism for assay can be OBTAINED FROM INTERNAL INSTITUTES OR VARIOUS NATIONAL CULTURE COLLECTIONS New cultures usually arrive freeze-dried in a glass ampule, which may be under vacuum and some ampules have a separate small cotton wool-plugged tube inside Instructions for opening and culturing are usually supplied with the ampule, which should be followed strictly for successful growth of cultures. 2) Test organism for assay can also be SOURCED FROM THE ENVIRONMENT after undergoing primary and secondary screening procedures Commercially available dehydrated media are available and several companies specialize in supplying both maintenance and assay media in complete formulation together with full instructions for preparation. Microorganisms can be grown on media of very simple composition e.g. Escherichia coli or Bacillus subtilis Others require much richer media especially the Lactobacilli used in vitamin assay 6 2023/2024 second semester Dept. of Microbiology, UMYUK RECONSTITUTION AND CULTURING OF MICROORGANISMS FOR MICROBIOLOGICAL ASSAY After obtaining the test organism from the source the next step is to reconstitute and obtain cultured viable cells for the microbiological assay as follows: i- Open the ampul containing the Freeze-Dried Assay Cultures by rubbing the ampul all around with an ampul file above the label and apply a red-hot glass rod or electrically heated hot wire to the file mark until the glass cracks. ii- Allow time for air to enter the ampul and carefully remove the top of the ampul and discard it into a disinfectant solution. iii- Carefully tip out the small inner tube containing the culture and with the aid of forceps loosen the cotton wool plug. Discard the now-empty bottom half of the outer ampul into the disinfectant solution. iv- Using a sterile Pasteur pipette withdraw about 0.5 ml of recovery broth (e.g. Tryptone soy broth) and slowly introduce a few drops of the broth into the tube containing the Freeze-Dried Assay Cultures and reconstitute the culture by repeatedly filling and ejecting the broth with the pipette. v- Two or three drops of the re-suspended culture are then spread on the surface of solid agar (e.g. nutrient agar) in a petri dish and the remainder is added to 10 - 20 ml of broth and placed in an incubator. vi- Discard the cotton wool plug, the small tube, and the Pasteur pipette into disinfectant solution, Reconstituted cultures should be incubated for at least twice the usual growth period of the organism before pronouncing them nonviable. Every culture must be clearly identified with the species name and number and the date of inoculation. vii- As soon as turbidity is evident in the broth after incubation, subculture a small volume with a sterile Pasteur pipettes into fresh sterile medium and reincubate. viii- Some organisms will need at least three successive subculturings before they regain their full physiological characteristics after freeze-drying. ix- The inoculated agar plate should show distinct uniform colonial forms. x- If satisfactory growth was obtained in the broth, then the agar culture can serve to check the purity of the strain, and a single colony could be selected for culture maintenance. xi- If, on the other hand, the broth culture failed to grow up for some reason, the agar plate should provide a check for both viability and purity. 7 2023/2024 second semester Dept. of Microbiology, UMYUK CULTURE CHECKS Culture checks are performed to check the purity and confirm the identity of the cultured organism that it is the same as the one contained in the ampul. - This done by streaking out a loopful of the broth culture on the surface of a suitable solid medium in a petri dish. After incubation (16-48 hours) at the optimum temperature for the organism, developed colonies are examined. -Developed colonies should look uniform in appearance with regard to shape, color, size, etc. Slight variation in colony size may be permitted, but if more than one distinct colony form is visible the culture probably became contaminated and it will be best to start afresh with another freeze-dried ampul. -Then a Gram stain of a typical colony is performed and examined. Bacterial identification kits such as the API system of identification may be used. 8 2023/2024 second semester Dept. of Microbiology, UMYUK CULTURE MAINTENANCE Once the purity and identity of the cultured organism is confirmed, the next step will be to establish a system whereby the assay organism recovered from the ampul will be kept viable as long as possible. This is referred to as CULTURE MAINTENANCE which includes preparation of master cultures, submasters, and working cultures. -Master cultures only carry the strain. Their job is to preserve the culture with all its original characters, and only referred to in an emergency to provide another function, such as rescuing the strain in case of contamination or loss of strain. -Submasters provide the stock from which weekly or daily cultures are set up as needed. -Both master and submaster cultures are kept on sloped agar media, called "slopes" or "slants," in universal bottles or test tubes. -Appropriate assay media is recommended for each test organism. E.g. Blood agar base is satisfactory for most organisms, but some will require specialized media that contain special growth factors or antibiotic. -It is common practice to subculture (i.e. re-culture) master and submaster cultures at monthly intervals, except in few cases e.g. some of the more sensitive bacteria will need weekly subculturing while yeasts may not need to be subcultured more often than once or twice a year. 9