Medical Microbiology. Microscopic Techniques :

Transcription

1 ! Lecture 2 Dr. Ismail I. Daood Medical Microbiology Microscopic Techniques : Several types of microscopes are used in study of microbiology one of the most important tools for studying microorganisms is the light microscope for observing objects. ÏÓ- -

2 Types of microscopes are : 1. Light microscopes - Bright-field microscope - Phase-contrast microscope - Interference microscope - Dark-field microscope - Fluorescence microscope. 2. Electron microscopes - Transmission E.M. - Scanning E.M. 3. Scanning probe microscope - Scanning tunneling - Atomic force. Dyes and Staining : Stain combine chemically with bacterial protoplasm if the cell is not dead the staining process itself kill it. The basic dyes stain bacterial cells. Special staining techniques can be used to differentiate flagella, capsules, cell walls, cell membrane granules, nucleotides and spores. Types of Dyes and Staining : 1. Simple stain. 2. Differential stains. a. Gram stain : An important taxonomic characteristic of bacteria is their response to gram stain it divided the bacteria to gram positive and gram negative depend on the cell wall. b. Acid-fast stain : Acid-fast bacteria are those that retain carbolfuchsin even when decolorized with hydrochloric acid in alchohol. Use heated on a steam Ô - -

3 bath that a smear on a slide is flooded with carbolfuchsin. e.g. Mycobacterium (Tb), some of Actinomycetes. 3. Special stain : This stain specific structures inside or outside of the cell : a. Capsule stain b. Flagella stain c. Endospore stain. 4. Negative staining : This procedure staining the background with the acidic dye, leaving the cell colorless. 5. Fluorescent dyes and tags : This stain specific molecules or proteins as Abs. steps in gram stain. Steps in staining Step 1 : Crystal violet (primary stain) Step 2 : Iodine Step 3 : Alcohol (decolizer) Step 4: Safranin (counter stain) State of bacteria Cell stain purple Cell stain purple Gram (-ve) cells become colorless Gram (+ve) cells remain purple Gram (-ve) cells appear red Extracellular : Fimbriae Flagella Pili, pili F (sex) pili Surface layer Cell wall (rigid) (proides protection & shape). - Gram (+ve) Peptidoglycan (murein) Õ - -

4 - Gram (-ve) outer and inner, lipid bilayer protein, lipopolysaccharide, porins. - Capsule complex polysaccharides - Slime layer. Cytoplasmic membrane (cell membrane or plasma membrane) is the physical and metabolic barrier between interior and exterior of bacterial cell doing as selective permeability. Intracellular : - Cytoplasmic components - Nucleic acids - DNA as purines (A,G), pyrimidine (C,T,U) - RNA (mrna, rrna, and trna) - Plasmids, DNA persist as extra-chromosomal elem. Chromosomes - Ribosomes (active center of protein synthesis 70S two subunits, (50S & 30S). - Storage granules metabolic activity. Molecules of bacterial cell : All bacterial cell contain a variety of small organic an inorganic molecules, many of which occurred in the form of ions, other are very large molecules the type of molecules : 1. Small molecules (Micromolecules) : This as forms of ions, organic molecules contain carbon atoms bonded to each other or to hydrogen atoms, also contain ions principally Na +, K +2, Mg +2, Ca +2, Fe +, Cl -, PO4-3 and SO Large molecules (Macromolecules) : Ö - -

5 Macromolecules are large consisting of several thousand atoms each four major class of biologically important macromolecules : a. Proteins : Proteins constitute more than 50% of the dry weight of cell, proteins are responsible for : 1. catalyzing all reaction as enzymes 2. the structure and shape as Ribosomes 3. cell movement such as flagella 4. taking nutrient into the cell as protein 5. turning genes on and of as gene regulations 6. structure of membrane as outer and inner 7. amino acid as subunit of protein. b. Nucleic acids c. polysaccharides (carbohydrate) d. lipids. Bacterial physiology :. Nutritional requirements : Bacteria grow best in an environment that meet their nutritional requirement, some species are able to grow under a wide range of conditions but others especially the more strictly parasitic such as gonococcus pathogenic bacteria suitable artificial culture media, some are requirements specific organic compounds and inorganic. What are these requirements? The main elements required for growth are carbon, hydrogen, oxygen and nitrogen with sulphur and phosphorus required in smaller amounts. Other element such as sodium, potassium, magnesium, iron and manganese in smaller amount. 1. Carbon : - -

6 a. Autotrophy bacteria use CO 2 as sour of carbon. b. Heterotrophy bacteria require more complex organic compound such as carbohydrates and amino acid. 2. Inorganic ions : Such as PO 4, K +, Mg =2, Mn, N, S. 3. Organic nutrients: a. carbohydrate b. amino acid c. vitamins, purines, pyrimidine. 4. Electron donors : such as ammonia, nitrites, H 2 S. 5. Electron acceptors : such as oxygen, pyruvate, lactate. 6. Oxygen : Bacteria can be divided into five groups on the basis of their oxygen requirements : 1. Obligate aerobes e.g. Vibrio cholera, Bordetella pertussis 2. Obligate anaerobes e.g. Clostridium tetani, Cl. welchii 3. Facultative a anaerobes e.g. Bacillus anthraces, Enterobacteriaceae 4. Microaerophilic bacteria e.g. Campylobacter species, Helicobacter 5. Aerotolerant bacteria e.g. some of species produce enzymes term superoxide dismutase. (See figer down) - -

7 Bacteria requirement also divide on the basis of Energy sources in : 1. Phototrophic : Which get energy from photochemical reaction. 2. Chemotropic : Which get energy from chemical reaction. Influence of temperature : The optimal temperature for growth of some human pathogens is rather unique and can be a simple way to select those organisms, for example Campylobacter species grow best at 42 o C, Staphylococcus aurous at 37 o C this optimum for growth of the most other human pathogens at 37 o C. According temperature (Bacteria) divide into : 1. Mesophilic bacteria: Growth best at temp. ranging from o C. Most human pathogens are Mesophilic at 37 o C. 2. Thermophilic bacteria : Growth best at o C - -

8 3. Psychrophilic bacteria : Growth best at temp. ranging from 0 o -10 o C. 4. Hyperthermophilic bacteria : can grow a well above the temp. of boiling water. Influence of hydrogen ion cone (PH) : Most organisms of bacteria have a fairly narrow optimal ph range. The optimal ph must be empirically determined for each species as : 1. Neutralophilic : Grow best at a ph of Acidophilic : grow and have optima as low as ph Alkaliphilic : bacteria have optima as high as ph 10-5 Influence of ionic strength : Factors as osmotic pressure and salt concentration may have to be controlled in media or in environment some of bacteria adapted to grow in strong sugar solution bacterial divide : 1. Halophilic bacteria : requiring high salt concentration. 2. Osmophilic bacteria : requiring high osmotic pressure. Ð - -

9 Glossary Acidophilic : Preferring or requiring an acid environment. Amino acids : The subunits of a protein molecule. Anaerobe : An organism that can only grow in the absence of oxygen (O 2 ) Autotrophic : An organism that can use CO 2 as its main source of carbon Halophilic : An organism that prefers or requires a high salt (NaCl) medium Lipid A : The toxic portion of lipopolysaccharide outer membrane in gram (-ve) bacteria. Medium : Any material used for growing organisms Obligate aerobes : Those organisms that have absolute requirement for oxygen Obligate anaerobes : Those organisms that can only be grown in absence of oxygen (O 2 ). Some members of this group are killed by traces of oxygen gas. Purine : A component of RNA and DNA. There are two major purines Adenine (A) and Guanine (G). Ñ - -