Scientist Louis Pasteur Pasteur Institute, Paris Pasteurisation Liquid media Fatherof microbiology Fermentation Principle Autoclave Disapproved theory of abiogenesis Germ cell theory Vaccines: © Cholera coors © Anthrax © Rabies Robert Koch ‘+ Fatherofmodernmicrobiology © Koch postulates 41): ‘© Constant association of causative organisms with the disease (Mycobacterium TB causes TB). © Isolation inculture media possible © Culture growth inoculated in animals should produce the same lesion, ‘©. Re-isolation from the experimental animalsis possible. ‘© Whenever there is an antigen, the human should be able to, produce antibodies in serum. © Exception from postulates: > L-Mycobacterium leprae (armadillo) + P-Treponema Pallidum > G-Gonoc Koch Bacilli (Tuberculosis) Cholera organism Solid culture media Aniline Dye colour Hanging drop mot ooo 1y- motility test Paul Erlich Ehrlichia organism Father of chemotherapy Toxin-antitoxin standardisation (Nobel prize) Acid-fast stain/ Zieh! Neelsen stain Joseph Lister ‘¢ Father of antiseptic surgery ‘* First used carbolic acid GENERAL MICROBIOLOGY Anton Von Leeuwenhoek © Father of the light ‘microscope (Unilocular) ‘+ The first thing he Visuatised under his microscope was Animal cules. ‘© The Jansen duo gave us the compound microscope. Ernst Ruska Father of electron microscope Edward Jenner ‘+ First vaceine- smallpox © Prepared using cowpox Karry B Mullis PCR Sanger Sanger sequencing HC Gram Gram staining Kleinberger LL forms (cell wall deficient) ‘Alexander Fleming. Penicillin Barbara Mcclintock ‘Transposons (Jumping genes) Nobel prizes ‘© For Hepatitis c virus- To Michael Hougton, Harvey J. Alter, CharlesM. Rice © CRISPR Cas9- Emmanuelle Charpentier and Jennifer A Doudna. Stains Fixation ‘+ Toensure the sample stayson the slide © Heatfixation ‘© Chemical fixation (methanol) e200 Simple stains (one colour) © Methylene blue ‘+ Basic fuchsin (red) Negativestains (b/w) ‘Staining the background, so the sample is highlighted ‘+ Cryptococeus- causes eryptococcal meningitis (CSF) (© capsule that doesn't take up any colour© India Ink © Nigrosin Capsule ‘+ Deposit stain on the surfuce of the object to make it look thicker (forthin structures) ‘© Silverstains-black colour Thin structure © Flagella © Spirochetes (treponema- caused syphilis) Syphilis patienthas Genital uleers > Fontana stain- fluid > Levaditisstain-tssue Differential Stains © Gram stain (+/-): © Gram positive- contain a lot of peptidoglyeans, which relain thedyelonger © Gram-negative - contains lipopolysaccharides (lipid), which dissolvesinalcohol (decolourization). © Poorly gram staining:(gram stain doesn't work) smnemonic~MRCS > Mycoplasma Rickettsia > Chlamydia ~ Spirochetes Crystal/ MethyW/ Gentian Purple Purple Violet Iodine (Mordant) Makes Crystal violet penetrate Alcohol/ Acetone Purple Colourless (decolouriser-important step) Safranin Purple ——_Red/Pink # Acid-fast Stain: © Carbo! Fuschin (red)-organism colours © Heating mordant) © Acid Ai Sulphuric acid (decolourized) » Mycobacteria-20%H2S0, ~ TB - Alcohol (95% alcohol) and Acid fast (20% 11280.) ~ Atypical TB (M avium) Acid fast (20% H2S04) ~ Lepra-Acid Fast (5% H2SO4) - Fite Fora > Nocardia, Legionella- 1% H2S04 ~ Coccidian parasite family- COLD ZN stain (5% H2804) — Isopora, Cyclospora, Cryptosporidium ~ Spores, Head of .25:0.25-0,5% H2S04 > Hooklets of Hydatid > Eggs: Taenia Saginata © Methylene Blue/ Malachite green Background colour © COLD ZN stain- Kinyoun Stain/Gabbet stain — Instead of heating, increased concentration of phenol alcohol: incarbon fuchsin, > Used forthe family of coccidean parasites+ Albert ZN stain 00.50:58 © Albert solution 1 ~ Malachite Green- organism > Volutin granules-Toluidine blue (Metachromatic [Link]) > Glacial Acetic acid (© Albert solution 2: lodine (©. Used for Volutin / Babes Ernt granules ~ present in C-diphtheria ~ located as poles, itis also called bipolar Granules > Metachromatic Granules: > Also seen in Spirillum, yeast, MTB © Stains for Volutin granules: > Ponder's Stain + Loeffler methylene Blue (best) > Albert Stain Flagella Stains cer stains (impregnation method) © Leifsonand Ryustain ‘© Background - Methylene blue © Flagella-Basie fuschin Red © Mordant- Tannic acid iained by Toluidine blue. jandnerella, Yersinia pestis 10:46 SporeStain EUR ag ‘© Spore-resting/ dormant form of bacteria. ‘© Schaefferand Fulton stain/ Modified Ashby stain © Malachite green- spore © Heat-Dormant © Water-decolorizer © Safranin Red- organism MCQs Q. InGram staining, themordant is? A, Tannicacid B, Loeffer'smordant C, Lugol'siodine D. Bovine's fixative Q. After the primary strain and mordant has been added but before the decolourizing agent has been used, gram-positive organisms are stained __, and gram-negative organismsare stained. A. Purple, Purple B. Purple, colourless C. Purple, pink D. Pink, pink [Link] granules stained by all except? A. Albert stain B. Neisser C. Ponder D. Kinyoundemonstrate? A, Mycobacteria B, Spirochaetes C. Both of theabove D. None of theabove Q. Which of the followingiis acid-fast with 20% H2SO,? A. Meavium B, [Link] €. Actinomyces D. Nocardia ene eA NEE RA Deore A. Crystal violet B, Safianin C. Methylene blue D. Alcohol Q. Conect orderof gram staining? A, Carbol fuchsin-iodine-Acetone-methyl violet B. Crystal violet-iodine-Acetone-Safranin CC. Methyl violet-Acetone-iodine-Saffanin D. Crystal violet-Carbol fuchsin-Acetone-iodineMQ CROSS WORD PUZZLES i Crossword Puzzle Across 3. Father ofmodem microbiology 4. Game positive- contain a lot of peptidoglycans, which retain thedye longer Down 1. Silver stains (impregnation method) 2, Disapproved theory ofabiogenesis,MICROSCOPES Features of Microscopes ‘© Uses lens tomagnify ‘+ Resolution ~The ability to differen © Humaneye-0.2mm © LM-0.2micron © Blectron-0.2-0.5nm + Contrast-Dyes on0.26 ie two pointsas separate Light Microscope cos09 Objective Lens Stage Diaphragm Course Adjustmant Fine Adjustment Light Source ‘+ Transmitiedlightis used formicroscopes. ‘© Slides arekepton stage ‘+ Light should go only to the slide; there isa condenser that has iris, which regulates the light. ‘+ The condenseris placed Below the stage. ‘Components of Microscope + Twoknobs- © Bigknobis thecoarse adjuster (©. Small knob is for fine adjustment + Twolenses- © ByePiece lens > Magnitfies 10times © Objectivelens > Scanner lens -Magnifies for4 times, > Low Power-Magnifies 10 times > High Power-Magnifies for 40 times ~> Oil Immersion -Magnifies for 100 times © Theselenses revolve around withthe help of neck pieces. Scanner 4x. Low Power 10x 10x 100 022 High Power 40x 10x 400 065 Oil 100s 10x 1000 1.25 Immersion ‘© Max magnification offered by light microscope is 1000x. ‘© Asthe lenses increase, numerical apertures also increase, Inverse relationship of numerical aperture and working distance or NA=n sin) where n = 1.0 Q. What are Numerical Apertures? Ans: There is one angle forming between lens and slide. Numerical aperture is the angle by which light falls on the slide and getsreflected. Mathematically, NA=n(sind) where, mis the refractive index, sind is half of the angle between the object and lens. Dark Field Microscope + Lightisreflected ‘© Mluminates the objects Used for Thin structure © Flagetla © Spirochetes- spiral structuresBright field Dark etd objective y TE scattered 2. Specimen ——— lt 3. Condenser lens —centrat ALightsouce @ ° Interference Contrast Microscope * Revealscellsorganelles ‘+ Measurements of chemical constituents of ells, suchas: © Lipids © Protein © Nucleic acid Fluorescence Microscope \ nn Image ooasss fiter Exctaton —\ qemmnty~ Emision ‘iter = QQ Light Source poe LE semimen ‘© Light source: Ultraviolet light © Uses: © Cases of TB /Malaria/ pathology of kidney biopsy ‘* Immunoftuorescence + fluorescent dye Different dyes © ForTB-Auramine and Rhodamine © ForMTB-Auramine and Rhodamine is used © ForMalaria- Acridine Orange ‘© Fungus-Caleofluor White Flow Cytometry -FITC - Fluoro isothiocyanate ‘+ Initially the lightthat come has shorter wavelength, ‘© When the light falls on the specimen, it converts it into longer wavelength © A dichroic mirror is used in a fluorescence microscope alongside a strong light source, an excitation filter, and an emission filter, Autoffuorescence has their own shine, kept under UV lights © Spora Brothers © Cyclospora © Isospora Formalin © Skin biopsy forimmunoffuorescence in NS not formalin ‘+ NADPH-Used in fluorescent spot test in G6PD deficiency # Woodlamp: Ultraviolet light Phase Contrast Microscope oo2938 Phase-contrast 5 sese” (gy? Phase plate Ditracted ght Direct ight Specimen5 ee ee ee a ee ee bottom ‘* Annularphase plate -Added at the top ‘© Differences in refractive indices, it will be shown in different phases © These will be converted into a change in the amplitude of light Electron Microscope osm ‘© Invented by Emst Ruska + No light is used, « beam of an electron is used - electron is enclosed ‘+ Mediums vacuum ‘Types of Electron Microscope mission Elec piers Features tenn) Microscope Scattered Principle Transmitted electrons Electrons 3D Dimensional 2D More sample View and Less sample viewed in viewed in lesser Time _—_the same time time Surface details Details Internal Details —oooooooeaoeEoEoaeeeaeEeEeEeEeeyeEeEee—e—eEeeee Pr Perey Nie 22.5% Fixation 10% Neutral Glutaraldehyde Buffered Formalin, + Makes eye water Embedded in resin Embedding Embedding in paraffin wax Copper metal slides Slide Glass slides Electron Source Transmitted light Vacuum Medium Air 0.5 nm Resolution 0.2 micron MCQs Q. Scanning Electron Microscope is used toreveal what? A) Surface structure B) Intemal structure ©) Bothofthe above D) None ofthe above [Link] magnification strength attained by a light microscope is? A) 10x B) 40x ©) 100x D) 1000x Q-Inalight microscope, what function does a condenser serve? A) Inerease light inteasity B) Focusthe light on sample ©) Focusthe light on the eye D) Reduced the glare Q. A microscope that exposes specimens to UV LIGHT and forms an image with the emitted at a different wavelength is calleda microscope? A) Phase-contrast B) Dark-field C) Scanning Electron D) Fluorescentener c ee nn OOS OREO OOS microscope for taking thistypeofimage? EO ae B) Phase plate ©) Dichroic mirror D) Cathode ray tube Q.A microbiologist intern microorganisms. His senior advised him to use a light microscope, What is the arrangement from eye to light anted to study cells and souree ina light microscope? vyepiece lens — eyepiece ©) Eyepiece lens — objective lens — Condenser D) None ofthe aboveCROSS WORD PUZZLES Gs Crossword Puzzle Across 2. Specimen which has their own shine, keptunder UV lights 4, Illuminates the objects Down 1. Smallest lens - Magnifies ford times F 3. is used in a fluorescence microscope alongside a strong light ral LITT TT tt ttt) source, an excitation filter, and an emission filter, TTTT I 5 O11 1 DIT) OT CLOUT ITT Tt‘Capsule and slime layer ‘© Bacteria haseitherslime layer oreapsule '* Streptococcus salivarius-the only one to have both slime layer and capsule ‘© Capsule tough and demarcated ‘© Slime layer-loose /undemarcated ‘+ Virulence fuctor-prevents phagocytosis by preventing ‘opsonization 00026 Capsulated organisms Mnemonic-Pretty Nice Capsule ‘* Streptococcus Pneumonia Klebsiella Pneumoniae Bordetella Pertussis Vibrio Parahaemolyticus, Clostridium Perfringens Yersinia Pestis-F | peptide Neisseria meningococeus ‘Haemophilus Influenzae Cryptococcus Staphylococcus Aureus-microcapsule Bacillus Anthracis Allcapsules are made of polysaccharides, = Except © Yersinia pestis-F1 peptide © Bacillus anthracis polypeptide ‘© [Link] has microcapsule . pyogenes sometimes have capsule- made of hyaluronic acid Demonstration of eapsule 1, McFadyen'sreaction-Bacillusanthracis, © Addpolychrome methylene blue stain © Givespurple color 2. Quellung reaction Neufeld reaction o Example, pneumococeus © Bacteria capsule (Ag)and we add antisera(Ab)toit © There would be antigen antibody reaction — swelling of capsule Modification of slime biofilm ‘© Streptococcus mutans © Inmouth-cause dental caries ‘+ Staph. epidermidis-causes prosthetic heart valve endocarditis ‘+ Also shown by Pseudomonas aeruginosa BACTERIAL ANATOMY Biofilm advantage ‘© Helpsinadherence ‘© Anti-phagocytic © Antibiotic resistance Cell wall wore Gram positive Features Gram negative ppPr P-Positive P-Peptidoglycans P-Pentapeptide cross bridge T-Teichoie acid 100 layer thick Thickness 1-2 layer thickness 25% Lipid content 15-50% Peptidoglycans Composition Lipopolysaccharide Lipoproteins Lipoproteins Teichoie a Peptidoglycan Absent Outer present ‘membrane Absent Periplasmic Periplasmic space + Po space Porins + Porins Present Teichoic acid Absent Pentapeptide cross _Pentapeptide Pentapeptide cross bridge present cross brigde bridge absent present NAM-NAG present ; ae ‘© Peptidoglycan layer: © Thick-gram positive © Thin-gramnegative‘Teichoic acid- ‘© Present in gram positive organism ‘© Used forattachment Lipopolysaccharide OPA- from outside to inside + 0-Oantigen © Most variable part co Eg,salmonella typhi-widal test —> Westudy O antigen andHantigen ‘+ P-polysaecharide oA © Haveendotoxin © Presentonly in gram negative organism © Have lethal effect, pyrogeneticty aman ‘+ NAM-N-AcetylMuramic Acid o 4aminoacidsareattached tot 6 Different in gram negative and gram positive onganisms + NAG-N-Acetyl Glucosat fem im negative eon) (Mnemonic-AGMA) Alanine Alanine Glutamine Glutamine Lysine Meso diaminopimelic acid Alanine Alanine Neceeacouaing oJ N scymaraie scam “evapetoe Cos Lnting * Q. Identify the cell wall © AGLAinNAM ‘© Cross bridge-pentapeptide ‘So, thisis gram positive cell wall Demonstration of cell wall Mnemonie-Registered Medical Practitioner Dr # R-reactionofantibody ‘+ M-mechanical rupture of cell, microdissection + P-plasmolysis © Dr-differential staining © Gramstaining Endotoxins vs exotoxins onus Present in lipid - A Made of proteins In gram negative cell wall Gram negative bacteria In both GNB and GPC. Heat stabile Heat labile Low antigenicity High antigenicity Cannot ‘Toxoid production is possible Can be used for vaccine production ‘Needed in large amounts to Small amounts can mediate create effects, effects Produces constant effect Variable effect Released as secretions Except botulinum toxin ~ released on lysis of cell Release on lysis of cell Coll wat O-antigen core Polysaccharide Exotoxn oo Una core © GNB-Lysis—+ release endotoxin ‘© Gram positive or gram negative-exotoxins aresecreted that shows endotoxin - LISTERIA ‘© Botulinum toxin-the exotoxin are released by lysis of the cell——— ‘* Endotoxin binds o Toll like receptors (TLR)-4 Demonstration ‘Limulus ameboeytelysate assay-LALA Horseshoe crab -limulus Limulusamebocyte lysate mixed with clinical sample Ifthere:is gelling of lysate —> endotoxin is present (Cellwall deficient forms -Lform ‘© Identified in Lister institute-London ‘© Studied in Streptobacillus moniliformis ‘= Discovered by Kleinbeger Lform ‘+ Unstable L forms © Lose their ell wall when treated with penicillin (o Canrevert back to their original form © Protoplast > Gram positive organisms > Whole cellwall ost © Spheroplast ~ Gramnegative > Some part of cell wall remains + StableLforms © Mycoplasma © Permanently loss cell wall (© Unable to revert to original form Outermembrane ‘© Ingramnegative organisms Cellmembrane ‘© Dipin cell membrane -Mesosomes /Chondroids © Respiratory unitin bacteria © Seen in both gram positive and gram negative ~> But prominent in gram positive © Itisan invagination of cell membrane 031.26 Ribosomes * 70S-(50S+ 308) —— © Single circular ‘© Double stranded DNA Inclusions © Corynebacterium diphth: - Volutin granules Flagetla ‘© 3-20micronin length * 0.01 -0.013 micron thick © Locomotion ‘© Flagelta antigen -Hantigen © Widal test for typhoid © Oantigen-cell wall © Hantigen-flagella Endo flagella © Inspirochetes © Treponema © Borrelia © Leptospira ‘© Originates from periplasmic space © Ingramnegativeorganism Structure of flage! He — ter Membre tan PHA © Filament Flagelta in gram positive and gram negative differs by Basal body- © Gramnegative-4rings © Grampositive-2rings Based on Location -flagella can be eo O& @& toprootons & Morava angherchous eens© Oneflagella on side © Vibrio © Pseudomonas Amphitrichous © Flagellaon either side Lophotrichous co Entire tuft offlagella on oneside © Helicobacter © Campylobacter Peritrichous © Flagellaall around he periphery © PR-Proteus © Listeria pes of motility ‘Tumbling © Listeria Darting © Like shooting star © Vibrio © Campylobacter Corkscrew © Looks like a tight serew- treponema ‘Twitehing © Seen with Fikenella © Trichomonas vaginalis > Cause Strawberry cervix Fallingleaf © Giardia lamblia Differential motility © Motileat22-25°C © Nonmotileat 37°C (© Seenin Yersiniosis, Listerios ‘Swarming Consideredas growth pattern -in blood agar Mnemonic-PVCS, P- Proteus V- Vibrio parahaemolyticus = Vibrio alginolyticus oooo ° C-Clostridium tetani — Bacillus Cereus © S-Serratia Demonstration of motility <~ <——r cae’ ‘+ Hanging drop motility ‘© Organism on Covers © Putaslide ontopof © Andtheninvertit ‘© Youwill see something hanging from the coverslip © Semi-solid agar method © Agar-normally used at2% concentration © Atvery less concentration-0.5 % organism shows motility + Craige's tube INOCULATE BACTERUM — Isolated from surface RAGES TUBE aa© Bacteria inoculated in small tube (©. Andthen isolate from the surface ofthe big tube + Utube © Organism puton one endof Utube (© And then isolated from otherend Flagella Stain Dark Field Microscope is used Oruse LEIFSON and RYU'S stain Mordant-Tannicacid Fimbriae/ pili Hairlike Adhesion Antigenic Primary purpose -attachment This process iscalled conjugation Detection ‘+ Hemagglutination © Ecoli,[Link] Cannot be looked under light microscope-as this is very thin Ifused formating between male and female bacteria -Sex pl © Gonococcus -divided based on hemagglutination ‘+ Surface pellicle test © Thin layer is produced on the surface of the broth Exceptio ‘© Flagellamainly used. forlocomotion ‘© Piliused forattachment © TypelVpili~inneisseria © Used for locomotion © Flagella independent motility Spores + Resting/dormant tate ‘© Under unfavorable conditions, bacteria goes to dormant stage Central ‘Spherical Subtorinal Elliptical Teninal > ‘Types of spores ‘+ Based on bulge © Nonbulging > Bacillus 05328 Not Bulging Bulging © Bulging > Clostridium + Based on location © Subterminal-most common location Terminal > [Link] = Round terminal spore — Drumstick appearance — [Link] — Oval terminal spore ~ Tennis racket appearance -tertium © Central spores, © [Link] © [Link] Parts ofspore Inner Germ Cell Walt outer smemrane Interspace Exesporium © Center-core ‘© Made of dipicolin ieacid © Makes it heat resistant Ouiside the eore + Cortex + Coat (©. Makes chemical resistant © Exosporium Innermost to outermost © Core © Cortex © Coat © Exosporium Spore formation ReferImage3.1 Bacteria was dividing ‘© Butunder unfavorable conditions it forms spores © Tstageseee ae Stage2- divides into spore Stage3-engulfspore Matured in stage 4.5.6 Released in tage? Stains ZNstain-0.25%of H2S04 Modified Ashby Stain - Schaeffer and Fulton -used for spore staining © Greencolor ©. Malachite green © Safranin red Moellerstain Uses ofspores + Usedascontrols ‘+ Geobacillus Stearothermophilus © Control inautoclave ‘© Spores of bacillus subtilis /atrophaeus / clostridium tetani © Control in autoclave Difference between prokaryote and eukaryote a a Called nucleoid Nucleus All present No proper nucleus Single circular Chromosome Multiple tinear dsDNA Plasmids Extra chromosomal Mitochondrial DNA DNA. Absent Onganelles: Present NAM Cell wall Absent NAG Sterols absent Cll membrane Sterols + 1S Ribosomes 80S Present Mesosomes Absent Site of Respiration a * Prokaryote having sterolsin ell membrane- mycoplasma ‘© Prokaryote lacking muramic acid in cell wall -chlamydia, MCQs Q. While examining a bacterial slide under the microscope, you notice that the organism lacks a cell wall. The cell deficient organism that could have been on the slide is A. Chlamydia B. Mycoplasma C. Streptococcus D. Anacrobes QA sputum sample was sent for testing in the microbiology laboratory. The gram staining showed the presence of Gram positive cocci which correlates with bacterial cell wall properties. Which of the following statements is false regarding the bacterial cell wall? Cell wall of Gram positive bacteria is thicker than that of Gram negative bacteria ‘Teichoie acid is present in the cell wall of gram negative bacteria RegionIl of LPSis core polysaccharide LPS is responsible for antigen specificity of gram negative bacteria c Explanation: © More peptidoglycans ‘* Teichoicacid -cell wall of gram positive + RegionIl polysaccharideae Pe Vegitative Call division \ cycle Stage 1 ‘Axial Fiement Formation stage 7 supe Free Endospore ‘Asymmetric Cell Division / orulati Stage 6 Stages and Mother cell lysis. Mothers cell e@ oe Cytoplasmic membrane ‘Spore Coat Synthesis: Spore wall 2 @) wesBacterial Shapes (Cocei) Cocei in clusters (Grape like) Staphylococcus Aureus, 0.00.09 Cocei in chains Streptococcus Cocei in pairs (Diplococci) Pheumococcus Lens shaped (Diplococei) _ Neisseria meningitidis Kidney shaped (Diplococci) Neisseria gonorrhoeae Cocci in tetrads (Cluster in 4) ‘Micrococcus ‘Cocci in octate (Cluster of 8) Sarcina, Bacterial Shapes (Bacilli) 00:03:29 Tee ac BG See ([Link] in BAmboo) Diplobacilli Klebsiella Pneumoniae Branching filamentous, Nocardia, Actinomyces Curved GNB ‘Vibrio, bacters Spiral Spirochetes Pleomorphie Haemophilus Influenzae, Brucella Bacterial Physiology 0508 ‘+ Population doubling time © Example + [Link]-20 mins + MTB-20hours > MLeprae- 20 days ‘+ Total count: Living anddead combined ‘+ Viable count: Only living bacteria ‘+ Definitely require Oxygen © Some require and some don't ‘+ Capnophilic: Some that require CO2 is campylobacter = ples © Acidic pH: Acidophiles > Lacto: Lactobacillus || BACTERIAL SHAPES AND PHYSIOLOGY © Alkaline pH: Alkaliphiles + Vibrio (Causes vibrio cholera) ‘© Some needs light and some don't © Temperature © Psychrophite: <20°C + Psychrotrophs: they grow in normal and lower temperatures (eg: yersinia, listeria) > Grow betterat low temperatures (4°C) © Mesophiles: 25°-40°C © Thermophile: 55°-80°C > Thermus aquaticus (Tag polymerase) Q. Which arethe organisms that require 02? Ans. © Obligateaerobes ‘+ Mnemonic: Must Let Nagging Pets Breathe o MIB © Legionella © Nocardia © Pseudomonas © Bacillus © Brucella © Bordetella Q. Which are the organisms that don't require 02? © Obligate Anaerobes © Choked By Air © Clostridium © Bacteroides © Actinomyces Facultative anaerobes Staphylococcus # Streptococcus ‘© Enterobacteriaceae © Haemophilus * Vibrio Microaerophilic «© S%ofoxygenneeded © Thebacters © [Link] Quick Definitions + Bacterial Physiology © Autotrophs: Can synthesize all compounds by using Co2 © Heterotrophs: Cannot synthesize their metabolites, utilize CO2 and N2 from air© Lithotrophs: Use inorganic compounds Q. A 65-year old blind man living in a rural area was admitted © Phototrophs: Derive energy from from sunlight with @ respiratory infection, There was no improvement © Chemotrophs: Derive energy from Chemical reactions. despite treatment and the patient started showing septicaemia symptoms. Radio graphic images showed wide Bacterial Growth curve oos60s mediastinum. Bacillus anthracis along with some spores \were isolated from blood culture. In which phase of bacterial Stationary growth spores are formed? 3] Dead A, Declinephase Z| B. Lagphase 5 . Stationary phase i D. Logphase é Q. 9 year old boy was referred to the AIIMS NEW DELHI with a low-grade fever, cough and sore throat for S days. A. swab was taken and inoculated into a suitable liquid culture Bacterial Growth curve ‘medium and the growth of the bacterium was plotted. In @ + Phases bacterial growth curve, in which phase is the maximum cell © Lag size obtained? © Log A. Beginning oflagphase © Stationary B, End oflag phase © Dead C. Beginning of stationary phase + LagPhase D. Logphase © Nojnerease of number ofbacteria © Celladaptation © Increase insize, enzymesand metabolite Q. When is the maximum size of the bacteria seen? ‘Ans. End of the lag phase + LogPhase © Celldivision © Smallerinsize © Maximum metabolie act ity © More susceptible toantibiotics © We get a uniform gram stain because all of them are living(viable) ‘+ Stationary Phase © We see Some of the bacteria remain viable and some are dead © Bacterial growth cease due to depletion of nutrients © Viable bacteria=Non viable bacteria (Non uniform staining) © STAB Sporulation ~+ Exo Toxin production > Antibiotic production ~ Bacteriocin production © Antibiotic production happens in this phase © Dead © Decline phase © Celldeath © Involution formsCROSS WORD PUZZLES a Crossword Puzzle Across 3. Fatherofmodem microbiology 4. Game positive: contain a lot of peptidoglycans, which retain thedyc longer Down 1, Silver stains (impregnation method) 2. Disapproved theory of abiogenesisBasies Deoxyribose sugar Ribose sugar ae ane Uracil replaces thymine Held together by H bonds Held together by H bonds Sadie © Definition: Permanent irreversible change in DNA + Types: a, © Frameshift Point Mutations ‘+ Transition-Purine is replaced by purine’ pyrimidine replaced by pyrimidine (same family) © Transversion-Purine replaced by pyrimidine (opposite family) © 3types: © Silence > Noeffect © Missense ~ Ex: Sicklecell anemia (HbA changed to HbS) Sense has changed © Nonsense > Ex: Beta thalassemia (Hb production is stopped) + Nosenseat all — Results in formation of stop codons (UAA, UAG, UGA) Frame Shift CAG caG CAG GG Frame T Frame 2 Framed Frame 4 Frames © Frame=triplet © Deletion CAR cGG CAG ca Frame © Frame2 © Frames Framed © Deletion of nucleotide © Addition © Ifany nucleotide is added inthe sequence, frame is shifted 5 | BACTERIAL GENETICS CAG A _CGG CAG caG Frame! Frame2 ss Frame3— Frame 4, Addition of nucleotide © Ex: Thalassemia Gene Transfer 00720 © 3methods © Transduction: Bacteriophage isinvolved (© Conjugation: Connection formed ©. Transformation: Directentry to DNA & & Ge Cox 1000 (0) Tansformation (0 Conjugate (0) Teansduction Rediscovered: Avery, McLeod, McCarty Transfer of free DNA Uptake of soluble DNA fragments by bacteria © Throughcell wall (©. During log phase (division) (© Required competence factor Griffith Experiment Bacteria: S pneumoniae (capsulated-viuent* smooth strain) RoughStan Saath Sisin_Hesklled Rough Sin eos Ta ee, ; as as oe> *, 2a ear tg i eee ‘ee 0 ee pate‘+ Unencapsulated strain: Mouse lives ‘© Lessdangerous ‘+ Capsule + Heat (Heat killed capsulated strain): Mouse lives © Follow both lytic and lysogenic ‘+ Nocapsule+ Heat killed capsulated strain: Mouse dies © Ex: Lambda phage of [Link] co Reason: Virulence is transferred Generalized Transduction ‘Transduction Head (protein and DNA) 2 Tait (protein only) Tail sheath ‘© Tadpole shape/circular/ filamentous ‘+ Thisisa DNA virus (mediator) Generalized Transduction ‘© First demonstrated in salmonella i MG moterot dmg resetance ia 8 uve Bacteriophage injects DNA into Donor bacteria . 1 Racietlophage Cycle New bacteriophages are formed (Mispackaging is seen - took donor DNA) ! New bacteriophages give donor DNA to Recipient bacteria * Lyticeycleis followed ysis Fusion may get into vertical transfer (no separation) > The offsprings will have new properties ight Generalized vs Specialized Transduction Cycle Lysogenic Occurs due to Defective excision during induction of prophage Virulent or Mediated by Only temperate phage ‘temperate phages & Important Information + Virulents Lyticeycle ‘+ Temperate: Both Bacterial Toxins Coded by Lysogenic Conversion A-AandC toxins of [Link] B-Cand Dtoxinsof Botulinum C-Cholera D-Diphtheria E-Enterohemorthagic [Link] 3. Conjugation Demonstrated first by: Lederberg and Tatum Bacteria: [Link] K12 strain MC modeof MDR using plasmids is conjugation & Important Information + MC modeof MDR except: (© [Link]~Transduetion © [Link]-Transformation ‘© Allothers Conjugation dite Plasmid: Extrachromosomal cireulardsDNA Episome: Plasmids may combine with cell DNA 2 Types (based on transfer) © Conjugated: Both horizontal (bacteria-bacteria) and vertical transfer (bacteria-offsprings) © Non-conjugated: Only vertical bacteria-offsprings) 4 types (based on what they carries) ‘© Virulence: Code for virulence factors like toxins Fertility: Code for fertility factor (male or female) Resistance: Code forresistance ° ‘© Colplasmids: Codes for bacteri F-Plasmid Fertility factor © Male=Present(F’) © Female-Absent (F) Fwith Fpisxmid —Conhigaton plus Chromosome Oot Go > Oo - © | reat le Fret Conjugation tube formed between F’ and F> Othername: Bridge Formed by sexpili Fentlity factor iscopied Both has fertility factors (both males) Mfrwith F Cell DNA combined with F’= Hr ‘© Mifrcells: High Frequency Recombinant cells acer Fol / Crore HER cell DNAS F Hf Cells© Conjugation tube formed ‘+ Tube broken mid way Leads to incomplete transfer F receives only small amounts of donor DNA and donor plasmi HfrwithF =F (Sex Duction) OF Piasmia mc Hfr splits to donor plasmid and donor DNA. But donor plasmid carries some donor DNA with it ‘Thuscalled F prime cell (F') When the F' conjugates with F F will getacopy of F' plasmid Thus, F'xP=FandF Bacteriat chromosome Summary F (plasmid) F (ao plasmid) ‘Hfr(DNA + plasmid) F (plasmid separated along with DNA) FxF-FF HiexF PxF-FF) ‘Trick: Male dominance (patriarchal society) ‘Transferable Drug Resistance 0092.26 ‘* Resistance = Resistance Transfer Factor (RTF) + Resistance determinants ® ‘¢ RT cancarry many rat same time Te Rotasmid RTF segeme™ ‘* More dangerous © Difficultto treat Mutational Drug Resistance masse © Dueto single mutation * Basiertotreat ‘+ Ex: Rif mpicin resistance in TB is due to 1poB gene Restriction Endonucleases oats Produced by bacteria for defense against foreign bacteria ‘Every REneedsaco-factorMg” There are 4 typesofRE © Type2isused for © Genetic engineering © Drugs © Tests ‘Restriction site is identified by RE, which is very short ‘ Knownas the Palindromic sequence (Left to Right and Right toLeftaresame) & Important Information ‘* Bacteria protects its own genome from RE by Methylation CRISPR-Cas9 0049.00 Trick © Cas9-Scissors © C-Clustered + R-Regularly I-Interspersed (spaces) * S-Short © P-Palindromic * R-Repeats (0) Vir invades sediniegraedinto \ ” AdsPtsbon 1 CRSP Seavence wee eee —L perce Prana ‘© Virusiskilled and is saved in memory (interspersed - spaces) © Cas9 will kill the virus ee Important Information ‘© CRISPR-Cas9is bacteria's defense systemne Q. Transductionsis: A. Bacterial-medial viral recombination B, Viral-mediated viral recombination C. Viral-mediated bacterial recombination D. Bacterial-mediated bacterial recombination, Q. The cell in which F factor carries along with it some chromosomal genes are known as: A. F'cell B. Fell C. Freell D, Freell Q. F factor integrates with bacterial chromosome to form: A. Hr B. RTFr CF D. RTF [Link] 86-year-old male who is a known case of DM * 3 years presented to ER with a complaint of a non-healing foot ulcer, ‘The patient also complained that he is been taking different antibiotics for a long time for his foot ulver but the medications had no results, The doctor suspected drug resistance. Which of the following statements is false regarding drug resistance? he TEL EOE CNG COMEECS COD Gg TCS Se B. Virulence is decreased in transferable resistance C. Drug combination can prevent Mutational drug resistance D. High degree of resistance is seen in transferable drug resistance Q. The restriction endonuclease is a defence mechanism in the bacterial system against foreign DNA such as viruses. But how isitable to protectitsown DNA? A. By methylation of bacterial DNA B. Bymethylation of foreign DNA C. Byphosphorylation of bacterial DNA D. Byphosphorylation of foreign DNA Q. Function of CRISPR is? A. Bacterial genome editing to protect ag: infections B. Bacterial genomeediting to protect against human immunity C. Mechanism for viral immunity D. Genetiereaetivation- || CULTURE MEDIA +5] + History © Liquid culture media ~ By SirLouis Pasteur > Urine, meat broth © Solidmedia ~ Robert Koch Potato ‘+ Composition: Polysaccharide ‘+ Nutritive value: NONE ‘© Temperature © Melis 98°C. © Solidify 42 = Concentration: © SOLID:2% © Semi solid:0.5% © Firm5-6% ‘* Whenever we want to inhibit the Swarming motility it will inhibit the motility. Peptone Agar Water Electrolytes © All4willremainconstant, ‘+ Meatextract/ Yeast extract ‘© Serum/Blood ‘Simple Media / Basal Medium /Basic Medium ‘© Only Non-Fastidious organisms will grow ‘© Peptone Water © Peptone 1% © NaC10.5% Water = S =— : Seaweeds (red algae of gelidium and gracilaria). © Nutrient Broth © Peptone 1% © NaCl0.5% © Water © 1% Meat extract © Nutrient Aga > Peptone 1% © NaCl0.5% o Water © 1% Meat extract © 2% Agar Enriched Media © Used to grow Fastidious organisms. ‘+ Egg/Blood/Serum isadded to provide extra nutrition. ‘+ Blood Agar: © Autoclaved nutrient agar cooled to 50°C. 0 5-10%sterilesheep RBCS © Made rich by adding blood. © Thisisa differential media © Chocolate agar © Autoclaved nutrient agar cooled t0 70°C © 5-10% sterilesheep RBCs © Morenutritious (Releases many factors, + Vand X factors© Liquid media that is selective in nature & Important Information ‘+ Whenever there is word media itis soli. ‘+ Wheneverthere is the word broth itis liquid. © Enriched media: Adding something extra, © Enrichment broth: Liquid media with selective media characteristics. © Selective media: Adding something which doesn't let other organisms grow. ‘© Loeffter's serum slope ‘© Corynebacterium diphtheria richment Broth > Klebs Loffler's media ‘© Alkaline peptone water. ~> Enriched media © Vibrio — Shours (8 letters in Loffier and enriched) Selenite F broth © Tetrathionatebroth © Salmonella © Shigella Selective Media + MacConkey Agar © Made of (MNEMONIC- PLANT) = Peptone = Lactose = Agar —> Neutral Red — Fermentation of lactose is pink. = Ifnot itis ale. = Itsanindieator. > Inspissation is done. ‘+ Lowenstein Jensen medium: Tuberculosis © Composition is ike an English breakfast. > Coagulatedhen'segg, — Mineral salt solution > Asparagine ~ Malachite green iW > TAUROCHOLATE (Bile salt) (selective) © SELECTIVEmedium © DIFFERENTIAL medium © INDICATOR medium © Mannitol Salt agar © Selective Media (only lets S aureus grow), » Mannitol fermentation — Yelloweolor © Inhibits other media, © Differential Media aii2 nee Staphylococcus aureus Staphylococcus epidermidis LED © Cysteine lactose electrolyte deficient ~> Differential media Selective media > For lactose fermentation yellow colonies. © Indieator-bromothymol blue © Better than MacConkey- less inhibitory (non-selective) Thiosulfate citrate bile salt sucrose © TCBS © Vibrio © Bilesaltmakesitselective. Vibrio cholerae on TCBS Agar Vibrio parahaemotytics ‘on TCBS Agar © Indicator: Bromothymol blue. © Differential media. Mannitol Salt Phenol red MF (yellow) agar [Link] TCBS —_Bromothymol Sucrose fermentation Vecholera blue SF (yellow) CLED ——_Bromothymol Lactose fermentation blue Yellow McConkey Neutral red__Laetose fermentation Pink Only McConkey the fermentation is pink. CVBA Crystal violet Blood agar) © Streptococeus pyrogens ‘© Modified Thayer Martin Medium © NeisseriaM © NeisseriaG © Cetrimide Agar (© Pseudomonas + PLET (polymyxin Lysozyme EDTA thallousacetate). © Bacillus Anthracis ‘© MYPA (Mannitol egg yolk phenol red polymyxin) and PEMBA. © BCereus © DCA (deoxycholate citrate agar) + XLD (Xylose lysine deoxycholate) © Salmonella © Shigella Transport media: Sach’s Buffered Glycerol Saline «Potassium Tellurite Agar © Black color © [Link] —> Selective media: 48hrs00:30:49 ‘Transport Media (maintain the vitality of bacteria), ‘+ Stuar’smedium ‘+ Amiesmedium © Neisseria ‘+ VRmedium © Cary Blairmedium © Vibrio ‘+ Pike'smedium © Streptococcus pyogenes ‘+ Sach's Buffered Glycerol Saline © Salmonella and shigella sisters Anaerobic Media ‘© Clostridium ‘* RCMB (Robertson's cooked meat broth). + Proteolytic © Turmsmeat black © Clostridium tetani + SACCHAROLY ©. Tums pink, © Clostridium perfringens > Per Sac Pink Anaerobic methods ‘MeTntoshand Fildes jar Clamp screw Me intone anders tae haere ar ua Catalyst Cuture plates ak ip Incicater: Reduced meth bie (colors anerabically) ue # O20) id with ‘Oxrng gasket Clamp with damp screw z Pali catalyst patets Envelope containing | EE ‘odu' bicarbonate ‘and sochom Boromeiao Anaerobic indicator —]T] (rethytene blue i # Onthe culture plateapacketiskept © Methylene blue is placed too. ‘© Methylene blue will look bluc till 02 ispresent. ‘ Itwill become colorless if 02 is removed. Latest Gaspak ae 4 / ‘© Takes lots of manual effort Anaerobic methods © Anoxomat. ‘© Glove Box/Anaerobie chamber. © PRAS (Pre-Reduced anaerobic system). + GASPAK (method of choice for anaerobiosis). Culture Technologies Streak Culture ostias‘The streak plate isolation method beter iamacah ‘It is performed by a straight wire, charged with culture © © & material, by puncturing deep inside the agar. € © Wetake anichrome loop and sterilize it. © Dipinsample + Start streaking at 90 degrees. + Wecan obtain Pure colonies. © Gelatin liquefaction Stroke Culture ‘© Inverted Firtree: shown by B. anthrax. ‘If done ina test tube: Sterilize, pickup sample and stroke ino. Firtree:C. te Agar slope/slant © Napiform/turip: [Link] © Uses; Provide a pure growth for the bacterium of slide © Itisusedto: ‘agglutination and other diagnostic tests ‘© Demonstrate gelatin liquefaction. Soke Catsre = Stoke atures madein tubes (4 pr pate (2 Te erent meta containing ogo slope! slant. bee towoim ain 1 Provide a pure growth of bacteria for slide agglutination and other Igectte 1 rocate plate diogrese tests Tirite cong | ‘siamedun Lawn culture ‘© Lawn cultures are obtained by flooding the plate's surface? Aint with the bacterium’s suspension, aie © AKAcarpetculture. | ea . ; ora Snacded 7) A © Take the sample directly and it put on a culture plate,© Fourplate method. in Rio mibon fal 7 mi tt ‘mt Amt ibm 1. Addbacteria first. 2. Thenaddagar. © Quantification of bacteria + Spread plate method. 1. Youhaveagar. 2. Thenadd patients’ samples (Bacteria). Antibiotic Sensitivity Testing msi ‘© Minimal inhibitory concentration MIC. © lowest concentration of the antibiotic that inhibits the growth ofthe pathogen inthe medium. © Minimal bactericidal concentration MBC. © lowest concentration of antibiotic that kills the bacteria in amedium, ‘Methods © Dilutionmethod © Disediffusion method ‘¢ E-Test (combo of firsttwomethods) * Automated methods —_——— © Inoculum prepared. © AdJE. coli tosterile peptone water and incubate, ‘After growth measures the turbidity using a spectrophotometer/ Me Farlands standard media 0.5 Me Farlands = 1.5x 108 CFU/mlis the standard, co Thereare 1.5 x 108 colony-forming units + Micro broth © Done with 96 well plat. (o Saves medium and time used + Macro broth © Doneintest tubes, o Requires time, * Growth + At 4nigiml, bacterial growth is inhibited. For the tested bacterial sample, this is the MIC of the antibiotic. This is compared with the breakpoint concentration laid down by CLSland determines ithe strains sensitive or resistant. Procedure: Contra has no abiotic 1 mlofstondord inoculum is added to each test tbe and ‘neubated overnight ContetOSugm 1 2 4 8 16 32 64 ‘Sera ion of antic ater overightincubatin with turbity Observation: First 3 test tubes are turbid indicating microbial Dise Diffusion Method or Kirby Bauer Disc Diffusion Method Lawn culture J Incubate overnight. 4 Dispense antibiotics dises 25mm di 4 Overnight incubate at 37°C© Agarused: CAMHA. © Cationadjusted Mueller Hinton agar. ‘+ Sample spread out ‘© If the zone of inhibition is seen the bacteria are antibiotic sensitive. ‘+ Canmeasure the zone of ‘* Disadvantage: Cannot quantify (Minimum inhibition concentration). ‘* Zoncof inhibition present- Sensitive ‘+ Zoncof inhibition absent-Resistant, bition. Stoke's Dise Diffusion Method Zone of Inhibition Modified stokes disc difusion method © Controltells validity. ‘+ Can Compare (By control strain). Epsilometer Test MIC 0.25 git Aka E-test Weean see a zone of inhibition, Wecan see concentration marked. Wecan see MIC. Q. Not trucabout agars? A. Souree of nutrition B. long chain polysaccharide CC. Melis at 95-98°C D, Solidifies around 40°C QA patient suffering from cough, sore throat, fever, and chills, Visited the moming session ofthe OPD. His sample was sent for culture. The lab technician performed the culture method shown in the figure below. Identify the method. A. Streakeulture B. Lawnculture C. Stroke culture , Pour plate method. QA patient with complaints of @ buming sensation when urinating and lower abdominal pain presented to the outpatient department. She was on antibiotics fora few days but there was no improvement. A urine sample was collected and sent for antibiotic sensitivity testing in the laboratory. ‘What is the most commonly used Dise diffusion method in antibiotic sensitivity testing? A. Kirby Bauer method B. Etestmethod C. MIC method D. Stokes method QA 35-year-old male resident presented with a fever and cough, He was well until 3 days earlier when he suffered the onset of nasal stuffiness, mild sore throat, and a cough productive of small amounts of clear sputum. The physician sent him for @ sputum culture and the microbiologist used blood agar for culturing it, What type of media is blood agar’? A. Enriched media B. Indicatormedia C. Enrichment media D, Selective mediaQ. 40-year-old female presented to the OPD with a burning sensation while urination, pelvie pain, and an inerease in urge and frequency while urination. Antibioties given showed no results. So, the sample was collected and sent for an antibiotic sensitivity test. The inoculum prepared should ‘match which of the following Me farland standard? A. Mefarland standard 0.5 B, Mefarland standard | C. Me farland standard 2 D. Me farland standard3 Q.A.22-year-old female presents with complaints of frequency and some urgency associated with painful micturition, The doctor suspects UTI & sends her urine sample to the laboratory for testing. The microbiologist decides to grow the culture on MacConkey agar. Which of the following is a constituent of MacConkey agar? A, Lactose: B. Bilesalt C. Neutral Red D, Alloftheabove Antibiotic sensitivity testing is carried out to determine the appropriate antibioties to be used for a particular strain isolated from clinical specimens, The most commonly used ‘medium for thisis A. CLEDagar B. Nutrient Agar C. Mueller-Hinton agar D, Saltmilk agar