MICRO.PARA UNIT 2.pdf

AY 2024 – 2025 | Second Year | First Term | First Shifting UNIVERSITY OF SANTO TOMAS — Faculty of Pharmacy — Department of Pharmacy Pharmaceutical Microbiology and Parasitology FUNDAME...

AY 2024 – 2025 | Second Year | First Term | First Shifting UNIVERSITY OF SANTO TOMAS — Faculty of Pharmacy — Department of Pharmacy Pharmaceutical Microbiology and Parasitology FUNDAMENTAL FEATURES OF MICROBIOLOGY Asst. Prof. Marilyn A. Ngo, MSc OUTLINE I. FUNDAMENTAL FEATURES OF MICROBIOLOGY NON CELLULAR CELLULAR VIROIDS PRIONS EUKARYOTIC AND PROKARYOTIC CELLULAR BACTERIA - Types of bacteria Prokaryotic and eukaryotic FUNGI PROTOZOA II. NAMING OF ORGANISMS III. MICROBIAL METABOLISM CHEMOHETEROTROPHS REDOX POTENTIAL GLYCOLYSIS FERMENTATION METABOLIC PRODUCTS IV. MICROBIAL CULTIVATION CULTURE MEDIA COMPLEX MEDIA Note: Cellular refers to something that is made up of COMPONENTS cells, whereas non-cellular things are devoid of cells. TYPES OF CULTURE MEDIA While non-cellular organisms lack a cellular organization, SOLID MEDIA cellular organisms have their components organized SPECIAL PURPOSES structurally. CULTIVATION METHODS PLANKTONIC AND SESSILE GROWTH VIRUSES V. ENUMERATION OF MICROORGANISMS TOTAL COUNT These are intracellular parasites that infect all kinds of VIABLE COUNT organisms. They do not have a cellular structure TOTAL VIABLE COUNT containing both nucleic acid (ss and ds DNA or RNA) and TRADITIONAL METHODS protein(surrounds and protects the nucleic acid core). RAPID METHODS VI. MICROBIAL GENETICS BACTERIA EUKARYOTES GENETIC VARIATION AND GENE EXPRESSION VII. PRESERVATION OF MICROORGANISMS RATIONALE LONG-TERM STORAGE REFERENCE CULTURES FUNDAMENTAL FEATURES OF MICROORGANISMS There are different types of microorganisms: The envelope protein and envelope is the one responsible for the attachment to a cell for the virus to enter. NON CELLULAR Viruses, viroids, and prions 2B-PH Bicos, Gajutos, Lamac, Lazaro 1 PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY These are infectious particles comprising of small, single-stranded, covalently closed circular RNA. without associated protein existing as highly base-paired rodlike structures. These are also plant pathogens. size (20-400 nm), can only be seen with an electron microscope. PRIONS simplest infectious agent with no nucleic acid Viral nucleic acid redirects the host’s enzymatic machinery to functions associated with replication of the virus. This can only grow inside a host cell, incapable of independent replication. A typical form of a mammalian protein that can interact with a normal protein molecule and cause it to undergo conformational change to become a prion. agents responsible for transmissible spongiform encephalopathies, e.g. Creutzfeldt-Jacob disease (CJD) in humans, bovine spongiform encephalopathy (BSE; mad cow disease) in cattle, and scrapie in sheep. extreme resistance to conventional sterilizing agents like steam, gamma radiation and disinfectants. Difficult, time consuming and expensive to cultivate in the EUKARYOTIC and PROKARYOTIC lab using fertile chicken eggs or artificially cultured mammalian cells as hosts used as vectors (carriers) of genes to cells, as in gene Characteristics Eukaryotes Prokaryotes therapy. Size >10 micrometer 1-5 micrometer VIROIDS Chromosomes Within the nucleus In the cytoplasm also called virusoids Nuclear division Mitosis & meiosis Absent Nucleolus Present Absent Reproduction Asexual & Sexual Asexual 2B-PH Bicos, Gajutos, Lamac, Lazaro 2 PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY toxic to them), facultative anaerobes (can live with or Chromosomes >1 1 without the presence of oxygen), microaerophils (needs num oxygen to grow). Mitochondria Present Absent Chloroplast Present Absent Cell membrane Sterols present Sterols absent Cell wall Cellulose or chitin peptidoglycan Ribosomes 80S 70S Flagella Complex Simple Pili Absent Present These contaminate or causes spoilage to Fimbriae Cilia Present pharmaceutical materials. They have cell walls which resist osmotic stress, and drying. Storage Present Absent These are targeted by some antibiotics like penicillins Compound which achieve their selective toxicity. (Poly-B They grow well between ambient and human body hydroxybutyrate) temperature, and exhibit wide variations for or tolerance of oxygen. BACTERIA Ribosomes targeted by tetracyclines and erythromycin; are structurally different from eukaryotic cells. These are unicellular, rod-shaped (bacillus), spherical (cocci), curved or spiral cells that has a size of 0.5-5.0 mm. Their shape is what we called spirilla. Rickettsia and Chlamydia obligate intracellular parasites; like viruses. Mycoplasmas do not have cell walls Binary Fission in Bacteria TYPES OF BACTERIA Bacterias produce asexually. Genetic variation is more reliant on mutations. These leads to rapid There are two distinct types of bacteria the reproduction which the mutant gene can be selected Gram-positive and Gram-negative. Saphorphytes, quickly and become dominant. parasites, pathogens (disease causing bacteria).These are bacterias can be strict aerobes (which are abundant in the presence of oxygen, they depends on oxygen), strict anaerobes (can’t grow in the presence of O2, it is 2B-PH Bicos, Gajutos, Lamac, Lazaro 3 PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY FUNGI Able to form spores that are resistant to drying: considered to be non-photosensitizing plants. They contaminants of pharmaceutical raw materials of are structurally more complex and varies in appearance vegetable origin. These cause spoilage may cause than bacteria. severe illness in immunocompromised patients. Most are saprophytes (live on decaying organic matter). PROTOZOA Only few have pathogenic potential. Some may exhibit both yeast-like and mold-like predominantly unicellular microorganisms that are appearance depending on cultivation conditions. regarded as animals rather than plants. YEAST These are unicellular organisms that are larger than bacteria (5-10 μm). These can exhibit sexual reproduction but more commonly divide through binary fission or budding. Their sizes can vary from 10 to 50 μm many are free living motile organisms that occur in water and soil. Some are plant and animal parasites not found as contaminants of raw materials and manufactured medicines great majority are harmless; few are responsible for malaria and amoebic dysentery. NAMING OF ORGANISMS VIRUSES YEAST fungi that do not form fruiting bodies visible to the naked eye, thus excluding toadstools and mushrooms consist of a tangled mass printed in italics to designate their status as proper (mycelium) of filaments or threads names (hyphae) which vary from 1 to over 50 In old books scientific names are written in roman type μm wide but underlined most reproduce asexually through the formation of asexual spores often responsible for characteristic colors of fungal colonies. 2B-PH Bicos, Gajutos, Lamac, Lazaro 4  PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY Meanwhile if the used name is collective – it is not to be Indicates whether oxidizing or reducing conditions italicized or spelt with an uppercase initial letter prevail in particular situation Food should be oxidized to breakdown and release Note: Collective means isang buong grupo na ng microbes. energy Another example of this is bacilli. Since collective yung word na bacilli ganyan na lang itsura niya walang uppercase first letter, Anaerobic organisms and hindi nakaslant. - Prefer low redox potential environment - Note: they prefer low redox because they are MICROBIAL METABOLISM anaerobs meaning they don’t need oxygen. Aerobic organisms CHEMOHETEROTROPHS - Prefer high redox potential environment - Note: they prefer high redox because they are Obtain carbon, nitrogen, and energy by breaking aerobs meaning they need oxygen. down organic compounds. GLYCOLYSIS Catabolic reactions - Chemical reactions with liberated energy through digestion of food materials - Note: The process of breaking down complex molecules (complex to simple) Anabolic reactions - Making of complex cellular polymers, proteins, carbohydrates, and nucleic acids with use of liberated energy. conversion of glucose to pyruvic acid - Note: The process of making complex releases only a small amount of energy molecules (simple to complex) Note: glycolysis is the catabolism of glucose (breaking down of glucose) REDOX POTENTIAL Anaerobic respiration - nitrate and fumarate is the electron acceptor Aerobic respiration - oxygen is the electron acceptor - Note: this happens in Electron Transport Chain FERMENTATION process of releasing more energy from sugar in which final electron acceptor is an organic molecule production of ethanol and carbon dioxide from sugar (sucrose, etc.) 2B-PH Bicos, Gajutos, Lamac, Lazaro 5 PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY production of acids (lactic, acetic, and propionic), CULTURE MEDIA alcohols (ethanol, propanol, butanediol) and other materials like acetone and butanol 1. Simple glucose or salts media/truly synthetic media - chemically defined (e.g. simple carbon and nitrogen sources and mineral salts) Note: Fermentation is a process na ginagawa ng some organisms 2. Routine laboratory media/general purpose pag nagdeplete ang oxygen. Now when fermentation starts pero media/complex media – more commonly used biglang may exposure to oxygen nagkakaroon ng interference. Dito daw napoproduce yung maaasim na wine, nagiging maasim COMPLEX MEDIA sila due to exposure sa oxygen. MICROBIAL METABOLISM FOOD MATERIALS dilute solutions of mineral salts and sugar Rare carbohydrates proteins and non-carbohydrate foods Facultative anaerobes – switch from respiration to fermentation if oxygen supplies are depleted switching of metabolic pathways – reason why antibiotics do not work by interfering with chemical precise chemical composition is unknown, vary from reactions used by microorganisms batch to batch It is an aqueous solutions of animal or plant extracts that contain hydrolyzed proteins, B-group vitamins and METABOLIC PRODUCTS carbohydrates Primary metabolites – produced during the period when COMPONENTS a microbial culture is actually growing; e.g. ethanol, organic acids Secondary metabolites – produced after cell 1. PROTEIN multiplication has slowed or stopped, i.e. in the sources: meat extract (parts of animal carcasses), stationary phase; antibiotics, enzymes (amylases), milk, and soya toxins (botox – toxin of Clostridium botulinum), and hydrolyzed to give peptones (if partially hydrolyzed) or carbohydrates (dextran – plasma expander and used in amino acids by trypsin or other proteolytic enzymes gel filtration) e.g. tryptone soya medium or soya bean casein digest medium MICROBIAL CULTIVATION 2. B-group vitamins are provided by yeast extract to enhance microbial growth 2B-PH Bicos, Gajutos, Lamac, Lazaro 6  PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY SOLID CULTURE MEDIA Agar 1-1.5% conc. will provide firm gel that cannot be liquefied by bacterial enzymes sets at approx. 40°C but do not re-liquefy on until it is in excess of 90°C firm at 37°C during normal incubation still liquid at 45°C without killing microorganisms (important in pour plate counting methods) SPECIAL PURPOSES OF CULTURE MEDIA 3. Carbohydrates starch or sugar (glucose/dextrose), other sugar may 1. to restrict growth of certain types of organisms be added for diagnostic purposes antibacterial antibiotics added to fungal media bile to suppress organisms other than those grow at the GIT 2. to identify certain types of organisms 3. to permit a particular type of organism to grow in relative numbers - enrichment medium to grow anaerobic organisms a. add non-toxic reducing agents (solid medium) sodium thioglycolate or sulphur-containing amino acids (e.g. cysteine) to create low redox potential use redox indicator, e.g. methylene blue and resazurin, they are colored blue and pink, respectively, in oxidizing conditions and colorless TYPES OF CULTURE MEDIA when reduced redox b. boiling (liquid medium) 1. Liquid (broth) used in tubes (screw cap test tubes) 2. Solid used in petri dishes or plates 5. to grow fungi gelled by the addition of agar – a carbohydrate extracted a. add lactic acid to lower the pH (5.5-6.0) than bacterial from certain seaweeds (agar) culture media (pH 7.0-7.4) b. add antibacterial antibiotics (chloramphenicol or tetracyclines) 2B-PH Bicos, Gajutos, Lamac, Lazaro 7  PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY CULTIVATION METHODS 2. Sessile growth (biofilm or microcolony mode of growth) – attached to a surface like soil particles, glass, or for pathogens, epithelial surface of the body like lung or intestinal mucosa ENUMERATION OF MICROORGANISMS CONDITIONS 1. when measuring the levels of microbial contamination in a raw material or manufactured 1. Liquid Medium medicine from a single cell, bacteria can achieve 2. when evaluating the effects of an antimicrobial concentration of 109 cells/ml overnight chemical or decontamination process for concentrations more than 107 cells/ml, the 3. when using microorganisms in the manufacture of culture media become more cloudy (turbid) therapeutic agents as the concentration increases; turbidity is an 4. when assessing the nutrient capability of a growth indirect means of monitoring culture growth medium Some bacteria produce chains of cells and some produce elongated cells (filaments) TOTAL COUNT that may exhibit branching to create a tangled mass like a mould mycelium enumerating both living and dead cells yeasts will produce turbid culture too Applications: moulds if agitated may produce pellets 1. vaccine manufacture – dead and living cells may both produce an immune response 2. pyrogen testing – both dead and living cells can induce fever VIABLE COUNT number or concentration of the living cells TOTAL VIABLE COUNT 2. Solid Medium yeast colonies are larger and frequently viable count that records all the different species or colored types of microorganisms that might be present in a mould colony consist of mycelium with sample (bacteria plus fungi) central area (oldest, most mature region) with pigmentation associated with spore TRADITIONAL METHOD production, periphery is actively growing and non-pigmented Relatively labor intensive Not easy to automate PLANKTONIC AND SESSILE GROWTH Slow, they require incubation method May require relatively large volumes of culture media, many petri dishes, and a lot of incubator space TRADITIONAL METHODS - VIABLE COUNT POUR PLATE METHOD Viable count = 30-300 CFU/mL Addition of a small volume (1.0mL) of sample into molten agar at 45℃ which is then poured into empty sterile petri dishes, after incubation, colonies in and on the agar are counted and multiplied by the dilution factor 1. Planktonic growth – freely suspended cells in the culture medium, not normal situation 2B-PH Bicos, Gajutos, Lamac, Lazaro 8 PHA 6114 LEC FUNDAMENTAL FEATURES OF MICROBIOLOGY TRADITIONAL METHODS - ESTIMATED TOTAL COUNT TURBIDITY MEASUREMENT Use spectrophotometer or colorimeter (liquid form sample) then read the concentration from a calibration plot to standardize cell suspensions for use in antibiotic assays or other tests SURFACE SPREAD METHOD DRY WEIGHT DETERMINATION Viable count = 30-300 CFU/mL Estimates fungal biomes The sample is spread over the surface of agar which has previously set DIRECT MICROSCOPIC COUNTING For bacteria, yeasts, and fungal spores but not for moulds; use Helber or hemocytometer counting chambers ASSAY OF INSOLUBLE NITROGEN, PROTEIN, OR NUCLEIC ACIDS Indirect measure of biomass, used in research labs MILES MIERA (SURFACE DROP) METHOD RAPID METHODS Viable count = 30-300 CFU/mL Similar in principle but several individual drops of Enumerate viable organisms in a matter of hours sample are allowed to discrete areas of about 1cm Readily automated diameter on the agar surface Require expensive equipment, have limitation on For sample with approx > 100 CFU/mL so that the detection limits number of colonies is large to be statistically reliable Employ various means of indirect detection of living cells May give higher values than the traditional methods MEMBRANE FILTER METHOD EPIFLUORESCENT TECHNIQUES Viable count =