MGEM1003 Bacterial Culture and Sensitivity Tutorial Support PDF

 Infections and Defence MGEM1003 Microbiology Tutorial Week 6 Practical contents support Stan Ko PhD FHEA FIBMS Email: [email protected] Room 3008 Learning objectives 1. Establish the links between lecture materials to laboratory practical contents 2. Apply t...

 Infections and Defence MGEM1003 Microbiology Tutorial Week 6 Practical contents support Stan Ko PhD FHEA FIBMS Email: [email protected] Room 3008 Learning objectives 1. Establish the links between lecture materials to laboratory practical contents 2. Apply theories from lecture to practical microbiology work 3. Prepare for coursework tasks  Infections and Defence MGEM1003 Microbiology lecture Week 3 Classification of microorganisms Part I Stan Ko PhD FHEA FIBMS Email: [email protected] Room 3008  Bacterial cell wall Cell wall forms protective shields for bacteria Peptidoglycan layer thickness differentiates between two main groups of bacteria – Gram positive and Gram negative Gram positive bacterial teichoic acid forms rigid structure, exact role of lipoteichoic acid unknown Gram negative integral protein and lipoproteins strengthen cell wall structure Gram negative outer leaflet porins control molecules passage / permeability  Lab practical Week 5 Gram stain method to demonstrate peptidoglycan layer thickness  Gram stain method to demonstrate peptidoglycan layer thickness Thick peptidoglycan layer retain blue dye Bacteria with thin particles during Gram peptidoglycan layer lose staining procedure blue dye particles Bacteria with thick Subsequent addition of peptidoglycan layer red dye will stain these appear blue in colour bacteria red under microscope – Gram negative Gram positive  Gram stain principle Video on Moodle https://www.google.com/search?sca_esv=575127353&rlz=1C1 GGRV_enGB792GB792&sxsrf=AM9HkKnzHBdoeaDW6Fz37HKY 4IL3XDyTOw:1697791606720&q=gram+stain+principle&tbm=vi d&source=lnms&sa=X&ved=2ahUKEwjJy_vMnoSCAxXhQkEAHd coBfIQ0pQJegQICBAB&biw=1536&bih=750&dpr=1.25#fpstate= ive&vld=cid:4bef64e2,vid:aMU2euxpcyw,st:0  MGEM1003 Week 4 Microbiology lecture Microbial structure and growth requirements Stan Ko PhD FHEA FIBMS Email: [email protected] Room 3008  Microbiology laboratory testing request Microscopy Culture & Sensitivity (MC&S) Performed either Directly on samples or after overnight cultured e.g. Gram’s stain on urethral swab for suspected sexually transmitted infections to reveal white blood cells and phagocytosed bacteria e.g. Light microscopy on stool samples for intestinal parasite ova (eggs) Names of parasitic ova not required  Microbiology laboratory testing request Microscopy Culture & Sensitivity (MC&S) Base on growth requirements Samples inoculated on various culture media (agar supplemented with nutrients) and incubated at: Human body temperature Human pathogens are mainly mesophilic Incubation duration – mostly overnight More fastidious - Neiserria gonorrhoeae – may take 48 hours Mycobacterium tuberculosis (TB) – takes up to 7 or 8 weeks Aerobic + anaerobic atmospheric conditions for most samples. Microaerophilic (what is this?) if indicated by types of infections Different bacteria produce different characteristics growth (colonies) on various culture media Grey shaded bacteria name – not required to remember Green shaded bacteria name – need to remember Week 5 Practical  Microscopy Culture & Sensitivity (MC&S) Cultural medium – agar in a petri dish with different types of supplements e.g. Blood agar – solid agar with essential nutrients (proteins, ions, pH..) supplemented with blood Most common bacterial infections vs normal flora Normal flora Only focus on the colour- coded areas  Microscopy Culture & Sensitivity (MC&S) Many sample may have non-pathogenic bacteria e.g. from skin alongside the presence of pathogens A mixture of bacterial colonies appear after incubation Based on appearance of colonies on various culture media, suspicious bacterial colonies selected for further testing What should be done first? What is the most basic classification based on and which method is to be used?  Microscopy Culture & Sensitivity (MC&S) Bacteria utilise various metabolites – sugar / protein…etc Media contains sugar when fermented produce low pH zone. pH indicator will change colour Example Most E coli ferment lactose. Most enteric pathogens such as Salmonella do not ferment lactose Agar plate contains basic growth factors plus lactose with pH indicator bromophenol blue. e.g. CLED (Cysteine Lactose Electrolytes Deficient) agar plates Lactose fermentation produces low pH – indicator changes colour from green to yellow Yellow colonies = lactose fermenters Non yellow colonies = non-lactose fermenters Week 6 Practical Pathogen ID  Microscopy Culture & Sensitivity (MC&S) Biochemical testing - enzymes Some bacteria have cytochrome c oxidase for redox reactions in oxygen- dependent energy production – oxidase positive e.g. E coli oxidase negative Vibrios oxidase positive Catalase is produced by some bacterial species converting hydrogen peroxide to water and oxygen e.g. Staphylococci catalase positive Streptococci catalase negative  Microscopy Culture & Sensitivity (MC&S) Biochemical testing – metabolic profile Base on preferential utilisation of metabolites Carbohydrates (sugar) Peptides Organic compounds Positive reactions reveal by pH indicator colour change catabolite colour complex formation (sounds familiar?) Series of tests form a profile – check again database for similarities  Bacteria Classification of bacteria Antigenic structures (serotype) Some bacterial structures are used for the classification of certain types of bacteria O antigens – lipopolysaccharides H antigens – flagella K antigens – capsule F antigens – fimbriae e.g. E coli O157 Sometimes referred to as the ‘serotypes’ due to the use of antibodies (from animal / human serum) to test for the presence or absence of those antigens  Microscopy Culture & Sensitivity (MC&S) Recognition of unique structures / antigens by specific antibodies (serotyping) Upon recognition of unique antigens by specific antibodies, ‘clumping’ occurs and can be made visible by additional reagents added to the system e.g. Salmonella O and H antigens typing by specific antibodies Since antibodies from serum / sera of laboratory animals – this method is called serotyping Practical work to be carried out in Week 8 using Staph aureus agglutination test  Staphylococcus Staphylococcus Gram positive cocci, catalase positive Over 30 species, Clinically significant 4 species Staphylococcus (Staph or S) aureus S epidermidis S saprophyticus S lugdunensis Main differential characteristics Coagulase positive – S aureus Coagulase Negative Others Coagulase negative Staphyloccocus (CoNS)  MGEM1003 Infections and Defence Microbiology lecture Clinically significant microorganisms Week 7 Practical – Antimicrobial action Antibiotics sensitivity testing  Principle of Antibiotics sensitivity testing Kirby-Bauer method Filter paper disc impregnated with antimicrobial Antimicrobials (as chemical compounds) diffuse from the disc into agar only around the disc Solubility of the antimicrobial and molecular size determine size of infiltration around the disc When an organism will not grow in the area around the disc if susceptible to the antimicrobial agent Area of no growth around the disc - “zone of inhibition” Size (diameter) measured for comparison with known standards  Microscopy Culture & Sensitivity (MC&S) Sensitivity (anti-microbial sensitivity pattern) Testing of pathogens isolated against anti-microbials of choice Based on bacterial growth inhibition in areas diffused by effective antimicrobial agents Vital for targeting pathogenic microorganisms with appropriate types and dosage of antimicrobials Effectiveness of antimicrobials determined by clearance of microbial growth Also facilitates identification e.g. Methicillin Resistant Staph aureus  Bacteria Classification of bacteria Methicillin resistant Antimicrobial sensitivity profile Different species of bacteria have specific ‘patterns’ of antimicrobial sensitivity / resistance. Due to clinical relevance, also used for specific bacterial identification e.g. MRSA – Methicillin Resistant Staphylococcus aureus Methicillin sensitive  Week 8 tutorial Assessment support