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Basic Microbiology Dr Hebat-allah Rashed , MD Professor Of Clinical Pathology (Clinical Microbiology Definition of Microorganisms Size: Bacteria are small – 2- Viruses are even smaller – nanometers (10⁻⁹) 5 microns (10⁻⁶ meters) Shape Classification Gram Reaction...

Basic Microbiology Dr Hebat-allah Rashed , MD Professor Of Clinical Pathology (Clinical Microbiology Definition of Microorganisms Size: Bacteria are small – 2- Viruses are even smaller – nanometers (10⁻⁹) 5 microns (10⁻⁶ meters) Shape Classification Gram Reaction based on Growth Requirements three things: Biochemical reactions Nucleic acid content The Basics - Terms Microorganism A microscopic living organism, such as a bacterium, virus, or fungus, that can only be seen under a microscope. Pathogen A microorganism that can cause disease in humans, animals, or plants. Infection The invasion and multiplication of pathogenic microorganisms in the body, leading to adverse health effects. Contamination The presence of undesirable microorganisms in or on a material or surface, potentially leading to health risks.  The Basics- Terms Bacteria can either grow or not grow in the presence of oxygen Oxygen: Aerobic (Pseudomonas, Bacillus) No Oxygen: Anaerobic (Clostridium, Bacteroides) Either: Facultative Anaerobe (E. coli) The Basics for Identification Microscopes show cellular morphology Growth on different media: the colony morphology Biochemical reactions: differentiate similar morphology Molecular techniques identify genes Cell Wall Composition The bacterial cell wall is a crucial structural component that provides shape, rigidity, and protection. Its composition varies across different bacterial species, which is the basis for the Gram stain classification. The cell wall is primarily composed of peptidoglycan, a polymer of sugars and amino acids. This creates a mesh- like layer that surrounds the cell membrane and gives bacteria their characteristic shape. The Basics for Identification Microscopic Media Growth Biochemical Testing Molecular Methods Analysis Observing how microbes Microscopes allow us to grow on different culture Performing biochemical Analyzing the genetic examine the cellular media can reveal distinct reactions helps makeup of microbes morphology and structure colony morphologies, a differentiate between through molecular of microorganisms, valuable identification microbes with similar techniques can provide providing key insights for tool. appearances, enabling definitive identification at identification. more precise the species level. identification. Identification of Bacterial Pathogens Microscopic Gram Staining Biochemical Profiling Antibiotic Sensitivity Examination The Gram stain helps Determining a pathogen's Examining bacterial samples classify bacteria into two Further tests on the susceptibility to various under a microscope is a main groups based on their bacteria's metabolic activities antibiotics is crucial for crucial first step in identifying cell wall composition, and enzyme production can guiding effective treatment pathogens based on their providing important clues for help narrow down the and preventing the spread of size, shape, and identification. specific pathogen species. resistance. arrangement. The Gram Stain Microscopic Technique The Gram stain is a widely used differential staining procedure in microbiology that helps classify bacteria based on their cell wall structure. Cell Wall Composition It distinguishes between Gram-positive bacteria, which have a thick peptidoglycan layer, and Gram-negative bacteria, which have a thinner peptidoglycan layer. Bacterial Identification The Gram stain is a crucial first step in the identification of unknown bacterial isolates, providing valuable information about their cell wall structure. Identification of Bacterial Pathogens The Gram Stain Developed in the late 1800’s by Dr. Gram, a pathologist Gram positive organisms are purple Gram negative organisms are red Based on cell wall composition Cell Wall Composition  Gram Stain Gives a quick look at the specimen Presumptive identification of microorganism & help direct antibiotic therapy Can interpret quality of specimen Number of “pus” (polymorphonuclear) cells Number of epithelial cells Number of bacteria present (and likely Genus) Normal vs. abnormal (colonization vs. infection)  Gram Stain Not so helpful if lots of normal flora present throats, stool, decubital ulcers Quite significant on sterile body sites CSF and other fluids Aspiration from petechiae Assists in the interpretation of culture results MCQ What is the primary purpose of the Gram stain in microbiology? A) To determine the metabolic activity of bacteria B) To identify the shape and arrangement of bacteria C) To differentiate bacteria based on their cell wall structure D) To assess the motility of bacteria  ) To differentiate bacteria C based on their cell wall structure  Which of the following best describes the outcome for Gram-positive bacteria after a Gram stain? A) They appear red or pink due to retaining the counterstain safranin. B) They appear purple or blue due to retaining the primary stain crystal violet. C) They do not retain any stain and remain colorless. D) They appear green due to the retention of the malachite green stain.  B) They appear purple or blue due to retaining the primary stain crystal violet.  Why do Gram-negative bacteria appear pink or red after a Gram stain procedure? A) They have a thick peptidoglycan layer that retains the primary stain. B) They have a thin peptidoglycan layer and an outer membrane that does not retain the primary stain. C) They are stained with a special red dye at the beginning of the procedure. D) They produce pigments that naturally appear red under a microscope.  B) They have a thin peptidoglycan layer and an outer membrane that does not retain the primary stain.  Metabolic & Biochemical Reactions Hemolysis Beta: complete destruction of the red blood cells in the (sheep) blood agar plate by haemolysins Alpha: partial destruction of the cells, leaving a greenish hue to the blood Gamma: old term, no hemolysis  Enzyme Activity Catalase Tests the organism’s ability to liberate oxygen from hydrogen peroxide Main distinguishing feature between Staphylococci and Streptococci / Enterococci Bacteria colonies placed into H₂O₂ – observe bubbles Sensitivity Testing This information guides clinicians in selecting the appropriate antibiotic therapy for their patients. Sensitivity testing, also known as antibiotic susceptibility testing, is a crucial step in identifying the most effective antimicrobial agents to treat bacterial infections. It helps determine which antibiotics a pathogenic bacterium is susceptible or resistant to. Sensitivity Testing Antibiotic Susceptibility Kirby-Bauer Disk Diffusion MIC Determination Sensitivity testing evaluates how This standardized method involves The minimum inhibitory concentration effective different antibiotics are at placing antibiotic disks on a bacterial (MIC) test precisely quantifies the inhibiting the growth of bacterial lawn and measuring the zones of lowest antibiotic concentration pathogens, crucial for guiding inhibition to determine susceptibility. needed to inhibit bacterial growth. appropriate treatment. Kirby-Bauer The Kirby-Bauer test is a standard method used to determine the antibiotic susceptibility of bacterial pathogens. It involves placing antibiotic-infused discs on a lawn of bacteria grown on agar, and measuring the zone of inhibition around each disc. This test provides crucial information about the most effective antibiotics to treat a specific bacterial infection, guiding appropriate and responsible antibiotic prescribing. Thank you